COMPOSITIONS FOR MICROFLUDIC WORKFLOWS AND METHODS OF USE THEREOF

BACKGROUND

[0001] Microfluidic devices allow researchers to manipulate, categorize, and maintain micro-objects such as biological cells. The present disclosure relates to compositions, systems, and related methods for assaying, protecting and controlling micro-objects in a microfluidic device.

SUMMARY

[0002] In a first aspect, a composition for forming a hydrogel is provided, including: a first and a second polyethylene glycol polymer molecule, each comprising a respective polyethylene glycol moiety and a covalently linked reactive moiety R _x ; a crosslinker molecule comprising a first reactive moiety RXP disposed at a first end of a linker L moiety and a second reactive moiety RXP disposed at a second end of the linker moiety, wherein each of the first and the second crosslinker moiety RXP is configured to be activatable to react with the respective reactive moiety R _x of the first and the second polyethylene polymer molecules, wherein the first and the second polyethylene polymer molecule comprises different polyethylene glycol moieties or each comprises a same polyethylene glycol moiety. The first polyethylene glycol moiety and/or the second polyethylene glycol moiety may be, independently, a 1-arm, 2- arm, 4- arm or 8- arm polyethylene glycol moiety. In some embodiments, each arm of the first polyethylene glycol moiety and/or the second polyethylene glycol moiety may include the covalently linked reactive moiety R _x .

[0003] In other embodiments, when the first polyethylene glycol moiety and/or the second polyethylene glycol moiety is the 2-arm, 4- arm, or 8- arm polyethylene moiety, at least one arm of the first polyethylene glycol moiety and/or the second polyethylene glycol moiety may have the covalently linked reactive moiety R _x and at least one arm of the first polyethylene glycol moiety and/or the second polyethylene glycol moiety may lack a covalently linked reactive moiety R _x . The first polyethylene glycol moiety and/or the second polyethylene glycol moiety may independently have a molecular weight from about 500 Da to about 25K Da. In some embodiments, the reactive moiety R _x may be a norbomene reactive moiety.

[0004] In some embodiments, the composition comprises a crosslinker molecule having a vic-diol. The crosslinker may further include a first thiol moiety at a first end of the crosslinker, and a second thiol moiety at a second end of the crosslinker, each configured to react with, for example, a norbomenyl moiety of the first or the second polyethylene glycol polymer molecule.

[0005] In some embodiments, the crosslinker has a molecular formula:

HS-LB-CH ₂ -C(H)(OH)-C(H)(OH)-CH ₂ -LB-SH Formula (2) [0006] where each instance of linker backbone (LB) is independently selected to comprise 0 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms. In some embodiments, the crosslinker is dithiothreitol.

[0007] In some embodiments, the composition comprises a crosslinker molecule having a disulfide, moiety. The disulfide moiety may be disposed at a location other than the first end or the second end of the crosslinker. The crosslinker may further include a first thiol moiety at a first end of the crosslinker, and a second thiol moiety at the second end of the crosslinker, each configured to react with a norbomenyl moiety of the first or the second polyethylene glycol polymer molecule. In some embodiments, the crosslinker has a molecular formula:

HS-LB2-CH ₂ -S-S-CH ₂ -LB2-SH Formula (3)

[0008] where each instance of linker backbone LB2 is independently selected to comprise 0 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms.

[0009] In yet other embodiments, the composition includes a crosslinker having a peptide sequence configured to be a substrate to a protease. The crosslinker may further include a first thiol moiety at a first end of the crosslinker, and a second thiol moiety at the second end of the crosslinker, each configured to react with, for example, a norbornenyl moiety of the first or the second polyethylene glycol polymer molecule. In some embodiments, the crosslinker may have a formula

HS-PEPT-SH Formula (4)

[0010] wherein PEPT is a peptidyl moiety comprising about 4 to about 12 amino acids, wherein the peptidyl moiety is susceptible to enzymatic cleavage. In some embodiments, the peptide sequence comprises GCRDLPRTGGDRCG (SEQ ID NO: 1). In some embodiments, the crosslinker may have a peptide sequence configured to be a tryptase substrate.

[0011] In some further embodiments, the composition includes a crosslinker having a formula

HS-LB4-SH Formula (5)

[0012] where linker backbone LB4 comprises 3 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms. The crosslinker may further include a first thiol moiety at a first end of the crosslinker, and a second thiol moiety at the second end of the crosslinker, each configured to react with, for example, a norbomenyl moiety of the first or the second polyethylene glycol polymer molecule. In some embodiments, the crosslinker may be sodium 2, 3 -dimercaptopropanesulfonate monohydrate.

[0013] The composition may further include an inhibitor configured to inhibit reaction of the crosslinker with the reactive moiety of the first and/or the second polyethylene glycol polymer molecule. In some embodiments, the inhibitor may be sodium ascorbate, MEHQ, 4-hydroxy TEMPO, or the like. [0014] In some embodiments, at least one of the first and the second polyethylene glycol polymer molecules further comprises a functionalizable moiety configured to react with a moiety other than a thiol moiety or a norbomenyl moiety. The functionalizable moiety may include, for example, a biotin, an aldehyde, a succinimidyl moiety, or the like.

[0015] In another aspect, a hydrogel is provided, including: a first polyethylene glycol polymer moiety covalently linked to a first end of a crosslinker moiety, where a second end of the crosslinker moiety is covalently linked to a second polyethylene glycol polymer moiety, where the first and the second polyethylene polymer molecule may be structurally different polyethylene glycol moieties or may be structurally the same.

[0016] In some embodiments, the hydrogel comprises a structure of Formula I:

PEG1-CG1-L-CG1-PEG2 Formula (1)

[0017] wherein PEG1 is the first polyethylene glycol moiety and PEG2 is the second polyethylene glycol moiety; CGi is a coupled group covalently linking a polyethylene glycol moiety with a crosslinker (L) moiety; and L is the crosslinker moiety having a linear portion wherein a backbone of the linear portion comprises 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms.

[0018] In some embodiments, the first polyethylene glycol moiety and/or the second polyethylene glycol moiety may be, independently, a 1-arm, 2-arm, 4-arm or 8-arm polyethylene glycol moiety.

[0019] In some embodiments, when the first polyethylene glycol moiety is multi-armed (e.g., a 2-arm, 4- arm, or 8- arm polyethylene moiety), at least one arm of the first polyethylene glycol moiety is covalently linked to one end of a crosslinker moiety and at least one arm of the first polyethylene glycol moiety is not covalently linked to a crosslinker moiety, and/or when the second polyethylene glycol moiety is multi-armed (e.g., a 2-arm, 4-arm, or 8-arm polyethylene moiety), at least one arm of the second polyethylene glycol moiety is covalently linked to one end of a crosslinker moiety and at least one arm of the second polyethylene glycol moiety is not covalently linked to a crosslinker moiety. In some embodiments, the first polyethylene glycol moiety and/or the second polyethylene glycol moiety may independently have a molecular weight from about 500 Da to about 25K Da. In some embodiments, the hydrogel may include a mixture of hydrogels, each having a structure of Formula I.

[0020] In some embodiments, the hydrogel may have a crosslinker moiety including a vic-diol. The crosslinker moiety may have a molecular formula:

-LB-CH ₂ -C(H)(OH)-C(H)(OH)-CH ₂ -LB- Formula (7)

[0021] where each instance of linker backbone LB is independently selected to comprise 0 to 200 nonhydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms. In some embodiments, the crosslinker moiety may be derived from a threitol moiety. [0022] In some embodiments, the hydrogel may have a crosslinker moiety including a disulfide moiety. The disulfide may be disposed in a location other than the first end or the second end of the crosslinker moiety. The crosslinker moiety may have a molecular formula:

-LB2-CH2-S-S-CH2-LB2- Formula (8)

[0023] where each instance of linker backbone LB2 is independently selected to comprise 0 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms.

[0024] In some embodiments, the hydrogel may have a crosslinker moiety including a peptide sequence configured to be a substrate to a protease. In some embodiments, the crosslinker moiety may have a formula

[0025] -PEPT- Formula (9)

[0026] where PEPT may be a peptidyl moiety comprising about 4 to about 12 amino acids, where the peptidyl moiety is susceptible to enzymatic cleavage. In some embodiments, the peptide sequence may include the peptide sequence GCRDLPRTGGDRCG (SEQ ID NO: 1). In some embodiments, the crosslinker moiety may include a peptide sequence configured to be a tryptase substrate.

[0027] In some embodiments, the hydrogel may have a crosslinker moiety having a formula

-LB4- Formula (10)

[0028] where linker backbone LB4 comprises 3 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms. In some embodiments, the crosslinker moiety may be derived from sodium 2,3-dimercaptopropanesulfonate monohydrate.

[0029] In some embodiments, at least one of the first and the second polyethylene glycol polymer moi eties may further include a functionalizable moiety configured to react with a moiety other than a thiol moiety or a norborenyl moiety. In some embodiments, the functionalizable moiety may include, for example, a biotin, an aldehyde, a succinimidyl moiety, or the like. In some embodiments, the functionalizable moiety may be coupled to a coupling partner covalently linked to a detectable label. The detectable label may be, for example, a visible label, luminescent label, or a fluorescent label.

[0030] In another aspect, a method of forming a hydrogel barrier within a microfluidic device is provided, where the microfluidic device includes an enclosure comprising a base, a cover, and microfluidic circuit material defining a fluidic circuit therein, and further where the fluidic circuit includes a flow region and chambers opening to the flow region, the method including: introducing a photoactivatable flowable polymer composition into the flow region of the microfluidic device; diffusing the composition from the flow region into the chambers; activating crosslinking of the composition in a selected area of the microfluidic device, thereby forming a hydrogel barrier within the flow region or one or more of the chambers of the microfluidic device. In some embodiments, the photoactivatable flowable polymer composition may include modified polyethylene glycol moieties. In some embodiments, the photoactivable flowable polymer composition may be any composition as described herein. In some embodiments, the composition further comprises an inhibitor of crosslinking. In some embodiments, the inhibitor may be MEHQ, 4-hydroxy TEMPO, sodium ascorbate, or the like.

[0031 ] The method may further include introducing a photoinitiator into the flow region, and permitting the photoinitiator to diffuse into the chambers.

[0032] In some embodiments, when the composition comprises a single type of crosslinker molecule comprising no cleavable moieties, forming the hydrogel barrier further includes reducing a risk of loss of clonality for one or more biological micro-objects or daughter micro-objects thereof disposed within at least one of the chambers. Forming the hydrogel barrier may include forming the hydrogel barrier at an opening of a selected chamber. In some embodiments, the method further includes unpenning biological micro-objects from the remaining chambers that have no hydrogel barrier at a respective opening of the remaining chambers.

[0033] In another aspect, a method of introducing hydrogel barriers having different physical properties to a microfluidic device is provided, where the microfluidic device includes an enclosure comprising a base, a cover, and microfluidic circuit material defining a fluidic circuit therein, and further where the fluidic circuit includes a flow region and a plurality of chambers opening to the flow region, the method including: introducing a first photoactivatable flowable polymer composition into the flow region of the microfluidic device, where the first composition has a first selected set of characteristics defining physical properties of a first hydrogel formed therefrom; diffusing the first composition from the flow region into the plurality of chambers; activating crosslinking of the first composition in a selected area of the microfluidic device, thereby forming a first hydrogel barrier within the flow region or one or more of the plurality of chambers of the microfluidic device; introducing a second photoactivatable flowable polymer composition into the flow region of the microfluidic device, wherein the second composition has a second selected set of characteristics defining physical properties of a second hydrogel formed therefrom; diffusing the second composition from the flow region into the chambers; and activating crosslinking of the second composition in a selected area of the microfluidic device, thereby forming a second hydrogel barrier within the flow region or a sub-set of the plurality of chambers of the microfluidic device, wherein the first hydrogel differs from the second hydrogel, based on at least one different physical property. In some embodiments, the first composition and the second composition each may include modified polyethylene glycol moieties. In some embodiments, the first composition and the second composition may each be any composition as described herein. In some embodiments, a viscosity of the first composition and a viscosity of the second composition are substantially the same. In some embodiments, the composition further comprises an inhibitor of crosslinking. In some embodiments, the inhibitor may be MEHQ, 4-hydroxy TEMPO, or sodium ascorbate.

[0034] In some embodiments, one of the first hydrogel or the second hydrogel may be a reversible hydrogel and the respective other hydrogel may be a non-reversible hydrogel. In some other embodiments, the first hydrogel and the second hydrogel may each be a reversible hydrogel and require orthogonal conditions for reversing. In yet other embodiments, the first hydrogel may have a different permeability from a permeability of the second hydrogel.

[0035] In a further aspect, a method of reducing a risk of loss of clonality within a microfluidic device is provided, where the microfluidic device includes an enclosure comprising a base, a cover, and microfluidic circuit material defining a fluidic circuit therein, and further where the fluidic circuit comprises a flow region and a plurality of chambers opening to the flow region, the method including: introducing a biological micro-object into one of the plurality of chambers; assaying or culturing the biological micro-object or a daughter micro-object thereof, thereby identifying a biological micro-object or daughter micro-object thereof of interest; identifying the chamber containing the biological microobject or daughter micro-object of interest; introducing a photoactivatable flowable polymer composition into the flow region of the microfluidic device; diffusing the composition from the flow region into the plurality of chambers; activating crosslinking of the composition in a selected area of the microfluidic device, thereby forming a hydrogel barrier at the opening of chambers other than the identified chamber, thereby producing a subset of capped chambers of the plurality of chambers; and unpenning and exporting the biological micro-object or daughter micro-object thereof of interest, thereby preserving clonality of the biological micro-object or daughter micro-object thereof of interest.

[0036] In yet a further aspect, a method of reducing a risk of loss of clonality within a microfluidic device is provided, where the microfluidic device includes an enclosure comprising a base, a cover, and microfluidic circuit material defining a fluidic circuit therein, and further wherein the fluidic circuit comprises a flow region and a plurality of chambers opening to the flow region, the method including: introducing a biological micro-object into one of the plurality of chambers; introducing a first photoactivatable flowable polymer composition into the flow region of the microfluidic device; diffusing the first composition from the flow region into the plurality of chambers; activating crosslinking of the first composition in a selected area of the microfluidic device, thereby forming a first hydrogel barrier within a first sub-set of the plurality of chambers of the microfluidic device; assaying or culturing the biological micro-object or a daughter micro-object thereof, thereby identifying one or more biological micro-objects or daughter micro-objects thereof of interest; identifying one or more chambers containing one or more biological micro-objects or daughter micro-objects of interest; introducing a second photoactivatable flowable polymer composition into the flow region of the microfluidic device; diffusing the second composition from the flow region into the plurality of chambers; activating crosslinking of the second composition in a selected area of the microfluidic device, thereby forming a second hydrogel barrier at the opening of chambers other than the identified chambers, thereby producing a second capped subset of chambers of the plurality of chambers; and unpenning and exporting the one or more biological micro-objects or daughter micro-objects thereof of interest from the identified chambers, thereby preserving clonality of the biological micro-object or daughter micro-object thereof of interest.

[0037] In another aspect, a method of functionalizing an in-situ generated hydrogel within a microfluidic device, is provided wherein the microfluidic device includes an enclosure having a base, a cover, and microfluidic circuit material defining a fluidic circuit therein, and further wherein the fluidic circuit comprises a flow region and a plurality of chambers opening to the flow region, the method including: introducing at least one hydrogel barrier in at least some chambers of a plurality of chambers, wherein the at least one hydrogel barrier is any hydrogel barrier having a functionalized moiety as described herein, and the method of introducing the at least hydrogel barrier is performed according any method of introducing a hydrogel barrier as described herein; introducing a functionalization reagent comprising a functionalizing reaction pair moiety configured to react with the functionalizable moiety of the hydrogel barrier; contacting the functionalization reagent with the hydrogel barrier; and coupling the functionalization reagent to the hydrogel barrier.

[0038] In another aspect, a method for preparing a chamber of a microfluidic device to detect an analyte produced by a cell within the chamber is provided, the method including: disposing the cell into the chamber of a microfluidic device, the microfluidic device having a microfluidic circuit comprising a flow region and the chamber, wherein the chamber comprises an opening to the flow region; forming an in situ-generated barrier within the chamber, wherein the in situ-generated barrier creates a first area proximal to the opening of the chamber to the flow region and a second area distal to the opening; allowing the cell to secrete an analyte within the enclosed culture area; defining an assay area within the first area or the second area ; and detecting a detectable signal associated with the assay within the area of interest.

[0039] In another aspect, a kit for preparing a chamber of a microfluidic device to detect an analyte produced by a cell within the chamber is provided, the kit comprising: a flowable polymer configured to be controllably activated to form an in situ-generated barrier comprising a solidified polymer network; and an inhibitor.

[0040] In another aspect, a method for preparing a chamber of a microfluidic device to export a cell selectively from the chamber is provided, the method including: disposing each of a plurality of cells into a respective chamber of plurality of chambers of a microfluidic device, the microfluidic device having a microfluidic circuit comprising a flow region and the plurality of chambers, wherein each chamber includes an opening to the flow region; assaying the plurality of cells in the plurality of chambers; identifying a cell of interest; forming an in situ-generated hydrogel barrier across the opening of each chamber of the remainder of the plurality of chambers, thereby preventing each of the remainder of the plurality of cells from exiting the respective chamber of the remainder of the plurality of chambers; and exporting the cell of interest from the respective chamber of the microfluidic device.

[0041] In some embodiments, exporting the cell of interest includes selectively exporting only the cell of interest. In some embodiments, selectively exporting only the cell of interest further includes reducing clonality risk, e.g., reducing the risk of loss of clonality when exporting the cell of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIG.1A illustrates a microfluidic device and a system with associated control equipment according to some embodiments of the disclosure.

[0043] FIG. IB illustrates a microfluidic device with sequestration pens according to an embodiment of the disclosure. [0044] FIGS. 2A to 2B illustrate a microfluidic device having sequestration pens according to some embodiments of the disclosure.

[0045] FIG. 2C illustrates a sequestration pen of a microfluidic device according to some embodiments of the disclosure.

[0046] FIG. 3 illustrates a sequestration pen of a microfluidic device according to some embodiments of the disclosure.

[0047] FIGS. 4 A to 4B illustrate electrokinetic features of a microfluidic device according to some embodiments of the disclosure.

[0048] FIG. 5 A illustrates a system for use with a microfluidic device and associated control equipment according to some embodiments of the disclosure.

[0049] FIG. 5B illustrates an imaging device according to some embodiments of the disclosure.

[0050] FIG. 6A is a graphical representation of hydrogel barriers according to some embodiments of the disclosure.

[0051] FIGS. 6B to 6C are photographic representations of hydrogel barriers according to some embodiments of the disclosure.

[0052] FIGS. 7A to 7C are graphical representations of assay methods utilizing hydrogel barriers according to some embodiments of the disclosure.

[0053] FIG. 7D is a photographic representation of another configuration of hydrogel barrier providing an assay region and a culture region.

[0054] FIGS. 8 A to 8B are photographic representations of fluorescent images of microfluidic chambers at two different timepoints, according to some embodiments of the disclosure.

[0055] FIGS. 9A to 9B are photographic representations of fluorescent and brightfield images of a bead-based assay according to some embodiments of the disclosure.

[0056] FIGS. 10A to 10B are photographic representations of fluorescent images of chambers with barriers with size-dependent permeability according to some embodiments of the disclosure.

[0057] FIGS. 11 A to 11C are photographic representation of fluorescent images of chamber with size dependent permeability according to some embodiments of the disclosure.

[0058] FIGS. 12A to 12C are brightfield photographic representations of cells culturing according to some embodiments of the disclosure.

[0059] FIGS. 12D to 12F are photographic representations of fluorescent images of assays according to some embodiments of the disclosure. [0060] FIG. 13 is a graphical representation of results of productivity assays according to some embodiments of the disclosure.

[0061] FIG. 14 is a graphical representation of results of productivity assays according to some embodiments of the disclosure.

[0062] FIG. 15 is a graphical representation of the distribution of productivity assay results according to some embodiments of the disclosure.

[0063] FIGS. 16A to 16F are photographic representations with annotation showing the process of exporting cells from a chamber having a non-uniform hydrogel barrier according to some embodiments of the disclosure.

[0064] FIG. 17A is a photographic representation showing hydrogel barriers introduced near the proximal opening of the sequestration pen and extending within the pen.

[0065] FIG. 17B is a photographic representation of the same sequestration pens after the hydrogel barriers have been reversed and no longer remain within the pens.

[0066] FIG. 18A is a photographic representation of hydrogel barriers capping a plurality of pens containing growing cells.

[0067] FIG. 18B is a photographic representation of the same pens after culturing and reversal of the hydrogel barriers and shows viable cells continuing to grow.

[0068] FIG. 19A is a photographic representation of a plurality of pens, each having both a mid-pen reversible hydrogel barrier, and alternate pens having a second, non-reversible hydrogel barrier at the proximal opening of the pen to the channel.

[0069] FIG. 19B is a photographic representation of the same plurality of pens, after treatment to reverse the reversible hydrogel barriers.

[0070] FIGS. 20A to 20D are photographic representations of a time course of reversal of mid-pen hydrogel barriers in pens having no other hydrogel barrier at the proximal opening of the pen; having a less dense non-reversible hydrogel barrier at the proximal opening; and having a more dense non- reversible hydrogel barrier at the proximal opening.

[0071] FIG. 21 is a photographic representation of a plurality of twinned pens, one or each pair having a protective hydrogel barrier.

[0072] FIG. 22A is a schematic representation of a variety of hydrogel barriers for creating transport regions between twinned pens.

[0073] FIGS. 22B to 22D are photographic representation of several different kinds of hydrogel barriers for creating transport regions between twinned pens. [0074] FIG. 23 is a schematic representation of supportive population splitting hydrogel elements.

[0075] FIGS. 24A to 24D are photographic representations of hydrogels having increasing amounts of functionalization.

[0076] FIG. 24E is a photographic representation of fluorescence intensity for the hydrogels of FIGS. 24A to 24D after labelling the functionalized hydrogel barriers with fluorescent streptavidin.

[0077] FIGS. 25 A to 25D are photographic representations in either brightfield or fluorescence images, comparing nonfunctionalized hydrogel barriers to functionalized hydrogel barriers after exposure to a fluorescently labelled streptavidin.

[0078] FIG. 26 illustrates diffusion of assay reagents from a chamber to a flow region (e.g., a microfluidic device) and how an in situ-generated structure affects the diffusion rate.

[0079] FIG. 27A shows a fluorescent image at Time 0 after a permeability test mixture was introduced into a microfluidic device without an in situ-generated structure formed.

[0080] FIG. 27B shows a fluorescent image at Time 60 after a permeability test mixture was introduced into a microfluidic device without an in situ-generated structure formed.

[0081] FIG. 27C shows a brightfield image showing in situ-generated structures formed and sealed at the opening of each chamber.

[0082] FIG. 27D shows a fluorescent image at Time 0 after a permeability test mixture was introduced into a microfluidic device having an in situ-generated structure formed at the opening of each chamber.

[0083] FIG. 27E shows a fluorescent image at Time 60 after a permeability test mixture was introduced into a microfluidic device having an in situ-generated structure formed at the opening of each chamber.

[0084] FIG. 28 illustrates changes of diffusion coefficient along with the changes of concentration of hydrogel solution components including polymer component A, polymer component B, crosslinker, initiator, and inhibitors. Top: diffusion coefficient of AF488P-IgG. Bottom: diffusion coefficient of AF647-OVA. In each plot, X-axis indicates the change of concentration of the indicated component in percentage of a standard concentration; Y-axis indicates the diffusion coefficient. Gray area shows the diffusion coefficient measurement within one standard deviation.

[0085] FIG. 29A shows an image of six chambers of a microfluidic device. Chambers 2901 and 2904 had a first non-reversible hydrogel barrier 2921 and a reversible hydrogel barrier 2910 formed therewithin. Chambers 2902 and 2905 had a first non-reversible hydrogel barrier 2922 and a reversible hydrogel barrier 2910 formed therewithin. Chambers 2903 and 2906 had only a reversible hydrogel barrier 2910 formed therewithin. FIG. 29A is a brightfield image taken at Time 0 when a dissolution reagent had not yet been introduced. [0086] FIG. 29B shows a brightfield image taken at a time point after a dissolution reagent being introduced and the reversible hydrogel barriers 2910 within chambers 2903 and 2906 were dissolving.

[0087] FIG. 29C shows a brightfield image taken at a time point when the reversible hydrogel barriers 2910 within chambers 2901, 2903, 2904, and 2906 were dissolving.

[0088] FIG. 29D shows a brightfield image taken at a time point when the reversible hydrogel barriers 2910 within chambers 2903 and 2906 were dissolved and the reversible hydrogel barriers 2910 within chambers 2901 and 2904 were still dissolving.

[0089] FIG. 29E shows a brightfield image taken at a time point when the reversible hydrogel barriers 2910 within chambers 2901, 2903, 2904, and 2906 were dissolved and reversible hydrogel barriers 2910 within chambers 2902 and 2905 were still dissolving.

[0090] FIG. 29F shows a brightfield image taken at a time point when all the reversible hydrogel barriers 2910 were dissolved.

[0091] FIG. 30A is a brightfield image showing three hydrogel barriers of different orthogonality formed in the mid-region of every other three chambers. Three of each kind of hydrogel barriers (solid circle indicates hydrogel# 1, solid triangle indicates hydrogel#2, solid square indicates hydrogel#3) are shown in the field, and the empty chambers without hydrogels (indicated by hollow circles) are used as negative controls.

[0092] FIG. 30B is a fluorescent image showing the fluorescent signal detection of the same field as shown in FIG. 30A after a testing solution comprising fluorescent-labeled reporter molecules was perfused into the flow region of the microfluidic device. Brighter areas seen in the image indicates the existence of the reporter molecules while the darker areas indicates that the hydrogel barriers restricted the reporter molecules from entering the chamber (see the locations of the hydrogel barriers from the brightfield image in FIG. 30A).

[0093] FIG. 30C is a fluorescent image showing the fluorescent signal detection of the same field as shown in FIG. 30A after the testing solution comprising fluorescent-labeled reporter molecules was perfused for a period and equilibrium was reached within the microfluidic device. Brighter areas seen in the image indicates the existence of the reporter molecules while the darker areas indicates that the hydrogel barriers restricted the reporter molecules from entering the chamber (see the locations of the hydrogel barriers from the brightfield image in FIG. 30A).

[0094] FIG. 30D is a fluorescent image showing the fluorescent signal detection of the same field as shown in FIG. 30A after the residual testing solution comprising fluorescent-labeled reporter molecules in the flow region was flushed out the microfluidic device. Brighter areas seen in the image indicates the existence of the reporter molecules while the darker areas indicates that the hydrogel barriers restricted the reporter molecules from entering the chamber (see the locations of the hydrogel barriers from the brightfield image in FIG. 30A). [0095] FIG. 31 are photographic representations showing the in situ-generation of five different hydrogels, each was functionalized with different oligonucleotides or without oligonucleotides. The five different hydrogels were formed in every other four chambers one after one in an order of: (a) Control hydrogel, (b) Oligo A hydrogel, (c), Oligo B hydrogel, (d) Oligo C hydrogel, and (e) Oligo D hydrogel.

[0096] FIG. 32 are photographic representations showing images taken after a cocktail reagent comprising four kinds of probes (herein “Probe A,” “Probe B,” “Probe C,” and “Probe D”), each recognizes a respective hydrogel, was introduced, (a) Brightfield image, showing the locations of each hydrogel, (b) Fluorescent image, showing the signals of Probe A, (c) Fluorescent image, showing the signals of Probe B, (d) Fluorescent image, showing the signals of Probe C, and (e) Fluorescent image, showing the signals of Probe D.

[0097] FIG. 33A is a brightfield image showing a plurality of hydrogels formed at the side walls of the chambers of a microfluidic device. The plurality of hydrogels contains two different hydrogels each was functionalized with different oligonucleotides.

[0098] FIG. 33B is a fluorescent image showing the same field of FIG. 33 A after a cocktail reagent comprising two kinds of probes, each recognizes a respective hydrogel, was introduced.

DETAILED DESCRIPTION

[0099] This specification describes exemplary embodiments and applications of the disclosure. The disclosure, however, is not limited to these exemplary embodiments and applications or to the manner in which the exemplary embodiments and applications operate or are described herein. Moreover, the figures may show simplified or partial views, and the dimensions of elements in the figures may be exaggerated or otherwise not in proportion. In addition, as the terms "on," "attached to," "connected to," "coupled to," or similar words are used herein, one element (e.g., a material, a layer, a substrate, etc.) can be "on," "attached to," "connected to," or "coupled to" another element regardless of whether the one element is directly on, attached to, connected to, or coupled to the other element or there are one or more intervening elements between the one element and the other element. Also, unless the context dictates otherwise, directions (e.g., above, below, top, bottom, side, up, down, under, over, upper, lower, horizontal, vertical, "x," "y," "z," etc.), if provided, are relative and provided solely by way of example and for ease of illustration and discussion and not by way of limitation. In addition, where reference is made to a list of elements (e.g., elements a, b, c), such reference is intended to include any one of the listed elements by itself, any combination of less than all of the listed elements, and/or a combination of all of the listed elements. Section divisions in the specification are for ease of review only and do not limit any combination of elements discussed.

[00100] Where dimensions of microfluidic features are described as having a width or an area, the dimension typically is described relative to an x-axial and/or y-axial dimension, both of which lie within a plane that is parallel to the substrate and/or cover of the microfluidic device. The height of a microfluidic feature may be described relative to a z-axial direction, which is perpendicular to a plane that is parallel to the substrate and/or cover of the microfluidic device. In some instances, a cross sectional area of a microfluidic feature, such as a channel or a passageway, may be in reference to a x-axial/z-axial, a y- axial/z-axial, or an x-axial/y-axial area.

[00101] As used herein, "substantially" means sufficient to work for the intended purpose. The term "substantially" thus allows for minor, insignificant variations from an absolute or perfect state, dimension, measurement, result, or the like such as would be expected by a person of ordinary skill in the field but that do not appreciably affect overall performance. When used with respect to numerical values or parameters or characteristics that can be expressed as numerical values, "substantially" means within ten percent.

[00102] As used herein, “similar” means the properties at issue of the two or more entities are substantially the same or have no substantial difference. When the properties of interest can be presented in numbers, the difference of the two or more entities in the properties at issue is +/-30%, +/-25%, +/- 20%, +/-15%, +/-10%, or +/-5%, or lower.

[00103] The term "ones" means more than one. As used herein, the term “plurality” can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[00104] As used herein: pm means micrometer, pm ³ means cubic micrometer, pL means picoliter, nL means nanoliter, and pL (or uL) means microliter.

[00105] As used herein, “air” refers to the composition of gases predominating in the atmosphere of the earth. The four most plentiful gases are nitrogen (typically present at a concentration of about 78% by volume, e.g., in a range from about 70-80%), oxygen (typically present at about 20.95% by volume at sea level, e.g. in a range from about 10% to about 25%), argon (typically present at about 1.0% by volume, e.g. in a range from about 0.1% to about 3%), and carbon dioxide (typically present at about 0.04%, e.g., in a range from about 0.01% to about 0.07%). Air may have other trace gases such as methane, nitrous oxide or ozone, trace pollutants and organic materials such as pollen, diesel particulates and the like. Air may include water vapor (typically present at about 0.25% or may be present in a range from about lOppm to about 5% by volume). Air may be provided for use in culturing experiments as a filtered, controlled composition and may be conditioned as described herein.

[00106] As used herein, the term "disposed" encompasses within its meaning "located."

[00107] As used herein, a “microfluidic device” or “microfluidic apparatus” is a device that includes one or more discrete microfluidic circuits configured to hold a fluid, each microfluidic circuit comprised of fluidically interconnected circuit elements, including but not limited to region(s), flow path(s), channel(s), chamber(s), and/or pen(s), and at least one port configured to allow the fluid (and, optionally, micro-objects suspended in the fluid) to flow into and/or out of the microfluidic device. Typically, a microfluidic circuit of a microfluidic device will include a flow region, which may include a microfluidic channel, and at least one chamber, and will hold a volume of fluid of less than about 1 mL, e.g., less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 microliters. In certain embodiments, the microfluidic circuit holds about 1-2, 1-3, 1-4, 1-5, 2-5, 2-8, 2-10, 2-12, 2-15, 2-20, 5- 20, 5-30, 5-40, 5-50, 10-50, 10-75, 10-100, 20-100, 20-150, 20-200, 50-200, 50-250, or 50-300 pL. The microfluidic circuit may be configured to have a first end fluidically connected with a first port (e.g., an inlet) in the microfluidic device and a second end fluidically connected with a second port (e.g., an outlet) in the microfluidic device.

[00108] As used herein, a “nanofluidic device” or “nanofluidic apparatus” is a type of microfluidic device having a microfluidic circuit that contains at least one circuit element configured to hold a volume of fluid of less than about 1 pL, e.g., less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nL or less. A nanofluidic device may comprise a plurality of circuit elements (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, or more). In certain embodiments, one or more (e.g., all) of the at least one circuit elements is configured to hold a volume of fluid of about 100 pL to 1 nL, 100 pL to 2 nL, 100 pL to 5 nL, 250 pL to 2 nL, 250 pL to 5 nL, 250 pL to 10 nL, 500 pL to 5 nL, 500 pL to 10 nL, 500 pL to 15 nL, 750 pL to 10 nL, 750 pL to 15 nL, 750 pL to 20 nL, 1 to 10 nL, 1 to 15 nL, 1 to 20 nL, 1 to 25 nL, or 1 to 50 nL. In other embodiments, one or more (e.g., all) of the at least one circuit elements are configured to hold a volume of fluid of about 20 nL to 200nL, 100 to 200 nL, 100 to 300 nL, 100 to 400 nL, 100 to 500 nL, 200 to 300 nL, 200 to 400 nL, 200 to 500 nL, 200 to 600 nL, 200 to 700 nL, 250 to 400 nL, 250 to 500 nL, 250 to 600 nL, or 250 to 750 nL.

[00109] A microfluidic device or a nanofluidic device may be referred to herein as a “microfluidic chip” or a “chip”; or “nanofluidic chip” or “chip”.

[00110] A “microfluidic channel” or “flow channel” as used herein refers to flow region of a microfluidic device having a length that is significantly longer than both the horizontal and vertical dimensions. For example, the flow channel can be at least 5 times the length of either the horizontal or vertical dimension, e.g., at least 10 times the length, at least 25 times the length, at least 100 times the length, at least 200 times the length, at least 500 times the length, at least 1,000 times the length, at least 5,000 times the length, or longer. In some embodiments, the length of a flow channel is about 100,000 microns to about 500,000 microns, including any value therebetween. In some embodiments, the horizontal dimension is about 100 microns to about 1000 microns (e.g., about 150 to about 500 microns) and the vertical dimension is about 25 microns to about 200 microns, (e.g., from about 40 to about 150 microns). It is noted that a flow channel may have a variety of different spatial configurations in a microfluidic device, and thus is not restricted to a perfectly linear element. For example, a flow channel may be, or include one or more sections having, the following configurations: curve, bend, spiral, incline, decline, fork (e.g., multiple different flow paths), and any combination thereof. In addition, a flow channel may have different cross-sectional areas along its path, widening and constricting to provide a desired fluid flow therein. The flow channel may include valves, and the valves may be of any type known in the art of microfluidics. Examples of microfluidic channels that include valves are disclosed in U.S. Patents 6,408,878 and 9,227,200, each of which is herein incorporated by reference in its entirety.

[00111] As used herein, the term “transparent” refers to a material which allows visible light to pass through without substantially altering the light as is passes through. [00112] As used herein, “brightfield” illumination and/or image refers to white light illumination of the microfluidic field of view from a broad-spectrum light source, where contrast is formed by absorbance of light by objects in the field of view.

[00113] As used herein, “structured light” is projected light that is modulated to provide one or more illumination effects. A first illumination effect may be projected light illuminating a portion of a surface of a device without illuminating (or at least minimizing illumination of) an adjacent portion of the surface, e.g., a projected light pattern, as described more fully below, used to activate DEP forces within a DEP substrate. When using structured light patterns to activate DEP forces, the intensity, e.g., variation in duty cycle of a structured light modulator such as a DMD, may be used to change the optical power applied to the light activated DEP actuators, and thus change DEP force without changing the nominal voltage or frequency. Another illumination effect that may be produced by structured light includes projected light that may be corrected for surface irregularities and for irregularities associated with the light projection itself, e.g., fall-off at the edge of an illuminated field. Structured light is typically generated by a structured light modulator, such as a digital mirror device (DMD), a microshutter array system (MSA), a liquid crystal display (LCD), or the like. Illumination of a small area of the surface, e.g., a selected area of interest, with structured light improves the signal-to-noise-ratio (SNR), as illumination of only the selected area of interest reduces stray/scattered light, thereby lowering the dark level of the image. An important aspect of structured light is that it may be changed quickly over time. A light pattern from the structured light modulator, e.g., DMD, may be used to autofocus on difficult targets such as clean mirrors or surfaces that are far out of focus. Using a clean mirror, a number of self-test features may be replicated such as measurement of modulation transfer function and field curvature/tilt, without requiring a more expensive Shack-Hartmann sensor. In another use of structured light patterns, spatial power distribution may be measured at the sample surface with a simple power meter, in place of a camera. Structured light patterns may also be used as a reference feature for optical module/system component alignment as well used as a manual readout for manual focus. Another illumination effect made possible by use of structured light patterns is selective curing, e.g., solidification of hydrogels within the microfluidic device.

[00114] As used herein, the term “micro-object” refers generally to any microscopic object that may be isolated and/or manipulated in accordance with the present disclosure. Non-limiting examples of microobjects include: inanimate micro-objects such as microparticles; microbeads (e.g., polystyrene beads, glass beads, amorphous solid substrates, Luminex™ beads, or the like); magnetic beads; microrods; microwires; quantum dots, and the like; biological micro-objects such as cells; biological organelles; vesicles, or complexes; synthetic vesicles; liposomes (e.g., synthetic or derived from membrane preparations); lipid nanorafts, and the like; or a combination of inanimate micro-objects and biological micro-objects (e.g., microbeads attached to cells, liposome-coated micro-beads, liposome-coated magnetic beads, or the like). Beads may include moieties/molecules covalently or non-covalently attached, such as fluorescent labels, proteins (including receptor molecules), carbohydrates, antigens, small molecule signaling moieties, or other chemical/biological species capable of use in an assay. In some variations, beads/solid substrates including moieties/molecules may be capture beads, e.g., configured to bind molecules including small molecules, peptides, proteins or nucleic acids present in proximity either selectively or nonselectively. In one nonlimiting example, a capture bead may include a nucleic acid sequence configured to bind nucleic acids having a specific nucleic acid sequence or the nucleic acid sequence of the capture bead may be configured to bind a set of nucleic acids having related nucleic acid sequences. Type of binding may be understood to be selective. Capture beads containing moieties/molecules may bind nonselectively when binding of structurally different but physico- chemically similar molecules is performed, for example, size exclusion beads or zeolites configured to capture molecules of selected size or charge. Lipid nanorafts have been described, for example, in Ritchie et al. (2009) “Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs,” Methods Enzymol., 464:211-231.

[00115] As used herein, the term "cell" is used interchangeably with the term “biological cell.” Nonlimiting examples of biological cells include eukaryotic cells, plant cells, animal cells, such as mammalian cells, reptilian cells, avian cells, fish cells, or the like, prokaryotic cells, bacterial cells, fungal cells, protozoan cells, or the like, cells dissociated from a tissue, such as muscle, cartilage, fat, skin, liver, lung, neural tissue, and the like, immunological cells, such as T cells, B cells, natural killer cells, macrophages, and the like, embryos (e.g., zygotes), oocytes, ova, sperm cells, hybridomas, cultured cells, cells from a cell line, cancer cells, infected cells, transfected and/or transformed cells, reporter cells, and the like. A mammalian cell can be, for example, from a human, a mouse, a rat, a horse, a goat, a sheep, a cow, a primate, or the like.

[00116] A colony of biological cells is "clonal" if all of the living cells in the colony that are capable of reproducing are daughter cells derived from a single parent cell. In certain embodiments, all the daughter cells in a clonal colony are derived from the single parent cell by no more than 10 divisions. In other embodiments, all the daughter cells in a clonal colony are derived from the single parent cell by no more than 14 divisions. In other embodiments, all the daughter cells in a clonal colony are derived from the single parent cell by no more than 17 divisions. In other embodiments, all the daughter cells in a clonal colony are derived from the single parent cell by no more than 20 divisions. The term "clonal cells" refers to cells of the same clonal colony.

[00117] As used herein, a “colony” of biological cells refers to 2 or more cells (e.g., about 2 to about 20, about 4 to about 40, about 6 to about 60, about 8 to about 80, about 10 to about 100, about 20 to about 200, about 40 to about 400, about 60 to about 600, about 80 to about 800, about 100 to about 1000, or greater than 1000 cells).

[00118] As used herein, the term “maintaining (a) cell(s)” refers to providing an environment comprising both fluidic and gaseous components and, optionally a surface, that provides the conditions necessary to keep the cells viable and/or expanding.

[00119] As used herein, the term “expanding” when referring to cells, refers to increasing in cell number.

[00120] As referred to herein, “gas permeable” means that the material or structure is permeable to at least one of oxygen, carbon dioxide, or nitrogen. In some embodiments, the gas permeable material or structure is permeable to more than one of oxygen, carbon dioxide and nitrogen and may further be permeable to all three of these gases.

[00121] A "component" of a fluidic medium is any chemical or biochemical molecule present in the medium, including solvent molecules, ions, small molecules, antibiotics, nucleotides and nucleosides, nucleic acids, amino acids, peptides, proteins, sugars, carbohydrates, lipids, fatty acids, cholesterol, metabolites, or the like.

[00122] As used herein in reference to a fluidic medium, "diffuse" and "diffusion" refer to thermodynamic movement of a component of the fluidic medium down a concentration gradient.

[00123] The phrase "flow of a medium" means bulk movement of a fluidic medium primarily due to any mechanism other than diffusion and may encompass perfusion. For example, flow of a medium can involve movement of the fluidic medium from one point to another point due to a pressure differential between the points. Such flow can include a continuous, pulsed, periodic, random, intermittent, or reciprocating flow of the liquid, or any combination thereof. When one fluidic medium flows into another fluidic medium, turbulence and mixing of the media can result. Flowing can comprise pulling solution through and out of the microfluidic channel (e.g., aspirating) or pushing fluid into and through a microfluidic channel (e.g., perfusing).

[00124] The phrase "substantially no flow" refers to a rate of flow of a fluidic medium that, when averaged over time, is less than the rate of diffusion of components of a material (e.g., an analyte of interest) into or within the fluidic medium. The rate of diffusion of components of such a material can depend on, for example, temperature, the size of the components, and the strength of interactions between the components and the fluidic medium.

[00125] As used herein in reference to different regions within a microfluidic device, the phrase “fluidically connected” means that, when the different regions are substantially filled with fluid, such as fluidic media, the fluid in each of the regions is connected so as to form a single body of fluid. This does not mean that the fluids (or fluidic media) in the different regions are necessarily identical in composition. Rather, the fluids in different fluidically connected regions of a microfluidic device can have different compositions (e.g., different concentrations of solutes, such as proteins, carbohydrates, ions, or other molecules) which are in flux as solutes move down their respective concentration gradients and/or fluids flow through the device.

[00126] As used herein, a “flow path” refers to one or more fluidically connected circuit elements (e.g., channel(s), region(s), chamber(s) and the like) that define, and are subject to, the trajectory of a flow of medium. A flow path is thus an example of a swept region of a microfluidic device. Other circuit elements (e.g., unswept regions) may be fluidically connected with the circuit elements that comprise the flow path without being subject to the flow of medium in the flow path.

[00127] As used herein, “isolating a micro-object” confines a micro-object to a defined area within the microfluidic device. [00128] As used herein, “pen” or “penning” refers to disposing micro-objects within a chamber (e.g., a sequestration pen) within the microfluidic device. Forces used to pen a micro-object may be any suitable force as described herein such as dielectrophoresis (DEP), e.g., an optically actuated di electrophoretic force (OEP); gravity; magnetic forces; or tilting. In some embodiments, penning a plurality of microobjects may reposition substantially all the micro-objects. In some other embodiments, a selected number of the plurality of micro-objects may be penned, and the remainder of the plurality may not be penned. In some embodiments, when selected micro-objects are penned, a DEP force, e.g., an optically actuated DEP force or a magnetic force may be used to reposition the selected micro-objects. Typically, microobjects may be introduced to a flow region, e.g., a microfluidic channel, of the microfluidic device and introduced into a chamber by penning.

[00129] As used herein, “unpen” or “unpenning” refers to repositioning micro-objects from within a chamber, e.g., a sequestration pen, to a new location within a flow region, e.g., a microfluidic channel, of the microfluidic device. Forces used to unpen a micro-object may be any suitable force as described herein such as dielectrophoresis, e.g., an optically actuated di electrophoretic force; gravity; magnetic forces; or tilting. In some embodiments, unpenning a plurality of micro-objects may reposition substantially all the micro-objects. In some other embodiments, a selected number of the plurality of micro-objects may be unpenned, and the remainder of the plurality may not be unpenned. In some embodiments, when selected micro-objects are unpenned, a DEP force, e.g., an optically actuated DEP force or a magnetic force may be used to reposition the selected micro-objects.

[00130] As used herein, “export” or “exporting” refers to repositioning micro-objects from a location within a flow region, e.g., a microfluidic channel, of a microfluidic device to a location outside of the microfluidic device, such as a 96 well plate or other receiving vessel. The orientation of the chamber(s) having an opening to the microfluidic channel permits easy export of micro-objects that have been positioned or repositioned (e.g., unpenned from a chamber) to be disposed within the microfluidic channel. Micro-objects within the microfluidic channel may be exported without requiring disassembly (e.g., removal of the cover of the device) or insertion of a tool into the chamber(s) or microfluidic channel to remove micro-objects for further processing.

[00131] A microfluidic (or nanofluidic) device can comprise “swept” regions and “unswept” regions. As used herein, a “swept” region is comprised of one or more fluidically interconnected circuit elements of a microfluidic circuit, each of which experiences a flow of medium when fluid is flowing through the microfluidic circuit. The circuit elements of a swept region can include, for example, regions, channels, and all or parts of chambers. As used herein, an “unswept” region is comprised of one or more fluidically interconnected circuit element of a microfluidic circuit, each of which experiences substantially no flux of fluid when fluid is flowing through the microfluidic circuit. An unswept region can be fluidically connected to a swept region, provided the fluidic connections are structured to enable diffusion but substantially no flow of media between the swept region and the unswept region. The microfluidic device can thus be structured to substantially isolate an unswept region from a flow of medium in a swept region, while enabling substantially only diffusive fluidic communication between the swept region and the unswept region. For example, a flow channel of a micro-fluidic device is an example of a swept region while an isolation region (described in further detail below) of a microfluidic device is an example of an unswept region.

[00132] As used herein, a “non-sweeping” rate of fluidic medium flow means a rate of flow sufficient to permit components of a second fluidic medium in an isolation region of the sequestration pen to diffuse into the first fluidic medium in the flow region and/or components of the first fluidic medium to diffuse into the second fluidic medium in the isolation region; and further wherein the first medium does not substantially flow into the isolation region.

[00133] As used herein, a “prepolymer composition” refers to a composition comprising a flowable polymer (i.e., a prepolymer or a photoactivatable flowable polymer) and a crosslinker as described herein. The term “prepolymer composition” is interchangeable with the term “flowable polymer composition” or “polymer solution.” The prepolymer composition is configured to be introduced into a microfluidic device for forming a hydrogel barrier in situ at a selected area. In some embodiments, the prepolymer composition further comprises an initiator and/or an inhibitor as described herein.

[00134] In the bioproduction industry, one severe problem is the expense, time and difficulty in identifying clonal populations having desired levels of production and growth habits when employing the currently available instrumentation and workflows. The ability to screen and identify promising clones within a microfluidic device, very early in expanding populations, such as 3, 4, 5, 6, or 7 days after seeding individual founding cells, as described herein, can offer significant time and cost advantages. However, culturing, screening, and maintaining cells having desirable properties in a microfluidic environment imposes technical problems, especially when the cells are relatively small and/or highly mobile. Those cells can be difficult to be contained in a chamber during culturing. Detecting the desirable property, such as level of production of a biomolecule can be challenging when the cells have a lower secretion level, which impose uncertainties of whether the secreted products at early stage of culture are of sufficient concentration for detection and screening. Further, some screening techniques are destructive to the cells under investigation, and techniques for preserving a portion of a clonal population and/or not exposing potentially valuable cells are needed.

IN SITU-GENERATED HYDROGEL BARRIERS FOR MICROFLUIDIC WORKFLOWS

[00135] Microfluidic workflows permit growth and analysis of biological micro-object population not previously realizable at more macroscale environments. To more fully utilize the capability of the microfluidic environments, the flexibility and customizability of creating specialized structures to more optimally guide growth, assay, and retrieval of valuable cells, in situ-generated barriers of various kinds are invaluable additions to the arsenal of microfluidic techniques.

[00136] Generally, one of the functions of the in situ-generated barrier in the methods of the present disclosure is to contain a cell within a selected area of the microfluidic device. The term “in situ-generated barrier” refers to a barrier that is formed in a selected area while the microfluidic device is in operation. The barrier is generally not formed while manufacturing the microfluidic device or does not exist before the microfluidic device is used for experiments or research. The term “barrier” refers to a physical structure that is formed and fixed, at least for a certain period of time, in a selected area and is capable of impeding or blocking a particle from crossing through the barrier. As a result, the barrier formed in situ within the chamber can separate the inner space thereof into two areas on each side of the barrier. In some embodiments, the barrier defines an enclosed culture area within the chamber. In other embodiments, the barrier defines within the chamber an assay area and an enclosed culture area. In some other embodiments, the barrier preserves selected cells away from harmful or destructive assay reagents, such that undamaged cells may be retrieved for other purposes. In yet other embodiments, the barrier(s) define a transport region between two adjacent chambers. In further embodiments, the barrier(s) reduce the risk to clonality when unpenning cells from a chamber, and may also be used to reduce clonality risk when moving unpenned cells through the flow region, optionally out of the microfluidic device.

[00137] As used herein, “enclosed culture area” refers to an area pre-determined for maintaining or culturing a cell, but the methods of the present disclosure are not limited to maintain or culture the cell in the enclosed culture area. The term “enclosed” describes that the culture area is substantially closed so that the cell cultured cannot easily move or be moved out of the area. However, the term “enclosed” is not limited to require that the area is completely closed or sealed. Some substances can still move in and out of the area (for instance, the culture medium, containing nutrients and/or waste can diffuse in and out of the area). Furthermore, the cell cultured in the culture area can still move or be moved in and out of the area in some variations. In some embodiments, a specifically created opening exists to allow substance (including cells) to enter or leave the enclosed culture area. In certain embodiments, the “opening” is a space between the in situ-generated barrier and one or more surfaces of the chamber, wherein the space is at least 2. Ox, 2.5x, 3. Ox, 3.5x, 4. Ox, 4.5x, 5. Ox or greater, or any range defined by two of the foregoing endpoints, where x is the average diameter of the cell.

[00138] As used herein, “assay area” refers to an area pre-determined for performing an assay required by the methods of the present disclosure. However, it is not limited that an assay required by the method of the present disclosure can only be performed within that array. It is also not limited that any areas of the chamber other than the assay area cannot be used for performing an assay.

[00139] In some embodiments, the impediment or block produced by introduction of the barrier is sizedependent. A particle can be impeded, blocked, or allowed to cross through the barrier depending upon its size. In some embodiments, the in situ-generated barrier has a porosity that substantially prevents a cell from crossing through the in situ-generated barrier. In some embodiments, the in situ-generated barrier can comprise a gap having a width or diameter that allows a cell to pass through the barrier. Nevertheless, the movement of the cell through the barrier via the gap can still be impeded.

[00140] Hydrogel Barriers defining Assay Areas. To more effectively harness the potential for assay processes carried out within each of many individual chambers within the microfluidic device, barriers can be introduced into the chamber for a number of purposes that may improve the assay by enhancing the accuracy, sensitivity, or reproducibility, amongst other properties, of the assay. For example, a barrier may be introduced, e.g., generated in situ, in order to sequester cells away from the assay observation area (e.g., area of interest) so a rapidly secreting cell does not artificially enhance the detected signal for the entire chamber by being present within an area of interest as a point source. Barriers may also be introduced to prevent a secreted analyte that is bound to a reporter molecule (RMSA complex) from diffusing away from an area of interest. Barriers also can prevent molecules having a size (molecular weight) too large to pass through the barrier to reach an area of interest of an assay which might interfere with the assay mechanism.

[00141] In some embodiments, a hydrogel barrier can define a desired assay area, e.g., area of interest. In some embodiments, the hydrogel barrier provides a location to securely dispose a capture bead used in an assay. In other embodiments, the hydrogel barrier divides a chamber, e.g., a sequestration pen, into two areas, one more proximal to the pen opening which opens to a flow region and a second area distal to the barrier and to the pen opening. The distal area, in some embodiments may be used exclusively for a culturing area, and the proximal area is used for imaging/ob serving an assay. The assay is performed within an area of interest defined within the proximal area and may be any area of interest suitable for the assay. The in situ-generated hydrogel barrier permits the analyte to pass from the area where the cell is culturing and producing the analyte to the area of interest where the assay is observed, to determine the presence of the analyte.

[00142] In other embodiments, the distal area may be used for both a culturing area as well as containing the area of interest in which the assay is observed/imaged. In some embodiments, performing the assay in the distal area, e.g., the area in which culturing is taking place as well, is particularly useful to help concentrate, e.g., accumulate, a detectable signal from the assay, when the rate of production of the analyte by cell(s) within the culturing area is low. When the distal area is both a culturing area as well as containing the area of interest for detecting the signal from an assay, the area of interest is generally chosen to be in a portion of the distal area that contains no cells. For example, the area of interest may be selected to be in a portion of the distal area that is close to the distal side of the hydrogel barrier, but does not contain either cells or the hydrogel barrier.

[00143] In conducting an assay determining bioproductivity of the cell, the product of the cell may itself be detectable, e.g., is fluorescent, luminescent, visibly colored, uv- detectable, and the like. In other embodiments, one or more reagents may be introduced to the area of interest to interact with the analyte, e.g., bind or react with the analyte thereby creating a detectable signal. The hydrogel barrier may have selective permeability permitting one, some, or all of the one or more reagents to pass through the barrier to the distal area of the chamber. In some embodiments, the hydrogel barrier may have a selective permeability that decreases the permeability of the analyte when it is bound with one or more reagents in the assay.

[00144] Hydrogel Barriers defining a Preserving Region. In some embodiments of uses for hydrogel barriers, there can be assays where the assay reagent or conditions under which the reagents contact cells of interest, is inherently deleterious to the viability of the cells. Therefore, in some processes, a portion of a population of cells (e.g., clonal population) is divided into another region or chamber and protected by an in situ-generated hydrogel barrier that preserves the viability of that portion of cells, while the remainder of the population of cells is subjected to assay conditions determining the desirability of the population of cells, but also altering the viability or killing the remainder of cells outright. Further details are described in Experiment 4B, and shown in FIG. 21. [00145] Barriers defining a transport region. In other embodiments, of uses for in situ-generated hydrogel barriers, the barriers are used to define regions through which a cell population (e.g., clonal population) may be transported actively (DEP forces, centrifugation, and the like), or passively (the cells self propel) from an initial chamber to an adjacent “twinned” pen. Barriers used in this fashion can be supported with additional in situ-generated splitting elements. Examples of these are shown in FIGS. 22A-22B, and 23, and described in Experiment 4B.

[00146] Barriers reducing clonal risk during export. In another aspect, a hydrogel barrier may be additionally used to reduce clonality risk. As described below, a second hydrogel barrier may be introduced, for example, after completion of assay and identification of desired cells or clonal populations within a chamber. In order to prevent loss of clonality as the desired cells are exported from the chamber, and optionally from the microfluidic device, a uniform hydrogel barrier, as described herein, may be formed across the width of the openings of undesired/unselected chambers, reducing the risk that cells that do not belong to the selected clonal population will be unpenned and mix with the cells selected for export.

MATERIALS USEFUL FOR IN SITU-GENERATED BARRIERS

[00147] In certain embodiments, the in situ-generated barrier is a hydrogel. In certain embodiments, the in situ-generated barrier comprises a solidified polymer network. In some embodiments, the solidified polymer network comprises a synthetic polymer, a modified synthetic polymer, or a biological polymer. In certain embodiments, the solidified polymer network comprises at least one of a polyethylene glycol, modified polyethylene glycol, polyglycolic acid (PGA), modified polyglycolic acid, polyacrylamide (PAM), modified polyacrylamide, poly-N-isopropylacrylamide (PNIPAm), modified poly-N- isopropyl acrylamide, polyvinyl alcohol (PVA), modified polyvinyl alcohol, polyacrylic acid (PAA), modified polyacrylic acid, fibronectin, modified fibronectin, collagen, modified collagen, laminin, modified laminin, polysaccharide, modified polysaccharide, or a co-polymer in any combination. In some embodiments, the solidified polymer network does not include a silicone polymer.

[00148] Physical and chemical characteristics determining suitability of a polymer for use in the solidified polymer network may include molecular weight, hydrophobicity, solubility, rate of diffusion, viscosity (e.g., of the medium), excitation and/or emission range (e.g., of fluorescent reagents immobilized therein), known background fluorescence, characteristics influencing polymerization, and pore size of a solidified polymer network. The solidified polymer network is formed upon polymerization or thermal gelling of a flowable polymer solution containing at least one of a polyethylene glycol, modified polyethylene glycol, polyglycolic acid (PGA), modified polyglycolic acid, polyacrylamide (PAM), modified polyacrylamide, poly-N-isopropylacrylamide (PNIPAm), modified poly-N- isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinyl alcohol, polyacrylic acid (PAA), modified polyacrylic acid, fibronectin, modified fibronectin, collagen, modified collagen, laminin, modified laminin, polysaccharide, modified polysaccharide, or a co-polymer in any combination. Various co-polymer classes may be used, including but not limited to any of the above listed polymers, or biological polymers such as fibronectin, collagen or laminin. Polysaccharides such as dextran or modified collagens may be used. The flowable polymer may be referred alternatively here as a prepolymer, in the sense that the flowable polymer is crosslinked in situ. Biological polymers having photoactivatable functionalities for polymerization may also be used.

[00149] In some instances, a polymer may include a cleavage motif. A cleavage motif may include a peptide sequence inserted into the polymer that is a substrate for one or more proteases, including but not limited to a matrix metalloproteinase, a collagenase, or a serine proteinase such as Proteinase K. Another category of cleavage motif may include a photocleavable motif such as a nitrobenzyl photocleavable linker which may be inserted into selected locations of the prepolymer. In some embodiments, a nitrobenzyl photocleavable linker may include a l- methinyl, 2-nitrobenzyl moiety configured to be photocleavable. In other embodiments, the photocleavable linker may include a benzoin moiety, a 1, 3 nitrophenolyl moiety, a coumarin-4-ylmethyl moiety or a 1 -hydroxy 2- cinnamoyl moiety. A cleavage motif may be utilized to remove the solidified polymer network of an isolation structure. In other embodiments, the polymer may include cell recognition motifs including but not limited to a RGD peptide motif, which is recognized by integrins. Additional cleavable moieties useful for in situ-generated hydrogels are described below.

[00150] Polyethylene glycol moieties linked to acrylamide reactive moiety. One type of polymer, amongst the many polymers that may be used, is polyethylene glycol diacrylate (PEGDA) or polyethylene glycol acrylamide (di acrylamide, multi-armed acrylamide or substituted versions as described herein). In some embodiments, the PEGDA prepolymer can undergo gelation photoactivated by using a free radical initiator (e.g., Igracure® 2959 (BASF)). The mechanism of this gelation is shown in below equation. Other useful initiators can include but not limited to lithium acyl phosphinate salts, of which lithium phenyl 2, 4, 6, - trimethylbenzolylphosphinate has particular utility due to its more efficient absorption at longer wavelengths (e.g., 405nm) than that of the alpha hydroxy ketone class. Reaction

[00151] Polyethylene glycol molecules linked to a norbornene reactive moiety or to an alkyne moiety. In other embodiments, a polyethylene glycol polymer molecule including a polyethylene glycol moiety covalently linked to a norbomene reactive moiety or an alkyne reactive moiety may be used as part of a prepolymer, e.g., flowable polymer, composition to be solidified in situ within a microfluidic device. A first polyethylene glycol polymer molecule will be linked to a crosslinker, and the crosslinker may further be linked to a second polyethylene glycol molecule via a norbornene reactive moiety or an alkyne reactive moiety linked to a second polyethylene glycol moiety of the second polyethylene glycol molecule. The norbomene containing polyethylene glycol molecules and the alkyne containing polyethylene glycol molecules react with crosslinkers including thiols, upon photoinitiation in the presence of an initiator to produce thiol-ene crosslinked hydrogels in situ.

[00152] The composition may include: a first and a second polyethylene glycol polymer molecule, each comprising a respective polyethylene glycol moiety and a covalently linked reactive moiety R _x ; a crosslinker molecule comprising a first crosslinker moiety RXP disposed at a first end of a linker L moiety and a second crosslinker moiety RXP disposed at a second end of the linker L moiety, wherein each of the first and the second crosslinker moiety RXP is configured to be activatable to react with the respective reactive moiety R _x of the first and the second polyethylene polymer molecules, wherein the first and the second polyethylene polymer molecule comprises different polyethylene glycol moieties or each comprises a same polyethylene glycol moiety.

[00153] The first reactive moiety R _x and the second reactive moiety R _x of the first and the second polyethylene glycol molecule may be a norbomenyl moiety. In some embodiments, the norbomenyl moiety may be a substituted norbomenyl moiety. In some embodiments, the substituted norbomenyl moiety may have a hydrophilic substituent, such as but not limited to hydroxy or carboxylate. The substituent does not inhibit the reactivity of the norbomenyl moiety. The norbomenyl moiety is configured to react with a thiol moiety of a crosslinker molecule to form the hydrogel. In other embodiments, the first reactive moiety R _x and the second reactive moiety R _x of the first and the second polyethylene glycol molecule may be an alkyne moiety, which is configured to react with a thiol moiety of a crosslinker molecule to form the hydrogel.

[00154] The first polyethylene glycol moiety and/or the second polyethylene glycol moiety may include a 1-arm, 2- arm, 4- arm or 8- arm polyethylene glycol moiety. In some embodiments, each arm of the first polyethylene glycol moiety and/or the second polyethylene glycol moiety may include the covalently linked reactive moiety R _x . In other embodiments, when the first polyethylene glycol moiety and/or the second polyethylene glycol moiety has a 2-arm, 4- arm, or 8- arm polyethylene moiety structure, at least one arm of the first polyethylene glycol moiety and/or the second polyethylene glycol moiety may include the covalently linked reactive moiety R _x and at least one arm of the first polyethylene glycol moiety does not have a covalently linked reactive moiety R _x . For example, an 8-arm polyethylene glycol moiety may have seven arms including a reactive moiety R _x and one arm that does not include the reactive moiety Rx. In some embodiments, an 8-arm polyethylene glycol moiety may have 6-arms, 5-arms, 4-arms, 3 -arms, or 2-arms having the reactive moiety R _x while the other arms do not have the reactive moiety R _x .

[00155] Functionalizable moiety. In some embodiments, at least one arm of the first polyethylene glycol moiety and/or the second polyethylene glycol moiety, having no reactive moiety R _x , may include a functionalizable moiety configured to react with a type of moiety other than a thiol moiety or a norbomenyl moiety. For example, an 8-arm polyethylene glycol moiety may have 7-arms having the reactive moiety R _x , and the remaining arm may include the functionalizable moiety that does not react with thiol or norbomenyl moieties. In some embodiments, at least one end of the crosslinker molecule, having no crosslinker moiety Rxp, may include a functionalizable moiety configured to react with a type of moiety other than a thiol moiety or a norbomenyl moiety. In some embodiments, the functionalizable moiety may be a biotin. In other embodiments, functionalizable moiety may be an aldehyde. In yet other embodiments, the functionalizable moiety may be a succinimidyl moiety. In yet other embodiments, the functionalizable moiety may be an oligonucleotide. Functionalizing a hydrogel of the present disclosure will be described further below.

[00156] Molecular weight of the prepolymer. The first polyethylene glycol moiety may have a molecular weight from about 500 Da to about 40K Da; from about IK Da to about 25K Da; from about IK Da to about 25K Da; from about IK Da to about 5K Da; from about 5K Da to about 25K Da; from about 5K Da to about 20K Da; about 5K Da to about 15K Da; about 5K Da to about 10K Da, about 8K Da to about 12K Da (e.g., about 10K Da), or about 18K Da to about 22K Da (e.g., about 20K Da). The second polyethylene glycol moiety may have a molecular weight from about 500 Da to about 40K Da; from about IK Da to about 25K Da; from about IK Da to about 25K Da; from about IK Da to about 5K Da; from about 5K Da to about 25K Da; from about 5K Da to about 20K Da; about 5K Da to about 15K Da; about 5K Da to about 10K Da, about 8K Da to about 12K Da (e.g., about 10K Da), or about 18K Da to about 22K Da (e.g., about 20K Da).

[00157] In some embodiments, the first polyethylene glycol moiety may have a molecular weight of about 2K Da and the second polyethylene glycol moiety may have a molecular weight of about 2K Da. In some embodiments, the first polyethylene glycol moiety may have a molecular weight of about 10K Da and the second polyethylene glycol moiety may have a molecular weight of about 10K Da. In other embodiments, the first polyethylene glycol moiety may have a molecular weight of about 10K Da and the second polyethylene glycol moiety may have a molecular weight of about 20K Da. When the first polyethylene glycol moiety has a different molecular weight from that of the second polyethylene glycol moiety, the hydrogel once formed will include a mixture of molecules, e.g., some hydrogel molecules will have a 10K Da polyethylene glycol moiety linked through the crosslinker to a second 10K Da polyethylene glycol moiety; some hydrogel molecules will have a 10K Da polyethylene glycol moiety linked through the crosslinker to a 20K Da polyethylene glycol moiety; and some hydrogel molecules will have a 20K Da polyethylene glycol moiety linked through the crosslinker to a second 20K Da polyethylene glycol moiety.

[00158] In some embodiments, the first polyethylene glycol molecule and the second polyethylene molecule may be present in the composition in a ratio from about 1 : 100 to about 100: 1. In some further embodiments, the ratio of the first polyethylene glycol molecule and the second polyethylene molecule may be from about 1 :1; about 1 :2; about 1 :3; about 1 :4; about 1 :5; about 1 :6; about 1 :7; about 1 :8; about 1 :9; about 1 : 10; about 10: 1; about 9: 1; about 8: 1; about 7: 1; about 6: 1; about 5: 1; about 4: 1; about 3: 1; or about 2:1.

[00159] Crosslinker molecule. For the compositions described herein, the crosslinker molecule comprises a first thiol at a first end of the crosslinker molecule and a second thiol moiety at a second end of the molecule, where the first thiol moiety and the second thiol moiety are each configured to reach with a norbomenyl group of either the first or the second polyethylene glycol molecule including a reactive moiety, where the reactive moiety may be a norbornenyl moiety. [00160] In some embodiments, the crosslinker molecule and the prepolymer (e.g., the first polyethylene glycol moiety and/or the second polyethylene glycol moiety) have substantially the same rate of diffusion. Having substantially the same rate of diffusion can be particularly beneficial for efficiently and evenly distributing the crosslinker molecule and the prepolymer within a microfluidic device, especially between regions that the only fluidic communication is diffusion. As a result, hydrogels that are in situ-generated at different locations within the microfluidic device can have more consistent qualities.

[00161] While in the same environment (i.e., under same temperature and in the same fluidic medium), the rate of diffusion can mainly depend on, for example, the size of the crosslinker and the prepolymer and the configuration thereof (e.g., linear structure or branched structure). In certain embodiments, the crosslinker and the prepolymer have substantially the same molecular weight thereby having substantially the same rate of diffusion. In some embodiments, when the prepolymer has a branched structure, the crosslinker has a branched structure of similar molecular weight.

[00162] In some embodiments, the crosslinker molecule may have a molecular weight from about 500 Da to about 40K Da; from about IK Da to about 25K Da; from about IK Da to about 25K Da; from about IK Da to about 5K Da; from about 5K Da to about 25K Da; from about 5K Da to about 20K Da; about 5K Da to about 15K Da; about 5K Da to about 10K Da, about 8K Da to about 12K Da (e.g., about 10K Da), or about 18K Da to about 22K Da (e.g., about 20K Da).

[00163] In some embodiments, the crosslinker molecule may include a vicinal-diol moiety. A vicinal diol (vic-diol) moiety of the crosslinker provides an in situ-generated hydrogel that may be reversed, e.g, removed or dissolved at a later timepoint, by contact with a periodate (Sodium Periodate). This class of hydrogels are typically referred to herein as a reversible hydrogel.

[00164] Vicinal-diol (Vic-diol) containing crosslinker. In some embodiments of a crosslinker having a vic-diol moiety, the crosslinker has a structure having a molecular formula of Formula (2):

HS-LB-CH ₂ -C(H)(OH)-C(H)(OH)-CH ₂ -LB-SH Formula (2), where each instance of linker backbone LB is independently selected to comprise 0 to 200 nonhydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms. In some embodiments, the linker backbone LB may have a linear backbone having carbon atoms. In some embodiments, the linear backbone having carbon atoms does not include any silicon, nitrogen, oxygen, sulfur or phosphorus atoms. When referring to the linear backbone LB having carbon atoms, this does not exclude substitution of hydrogen atom substituents of the carbon backbone with other types of moieties containing alcohols, sulfonates, carboxylic acids and the like. In some embodiments, the crosslinker molecule is dithiothreitol.

[00165] Disulfide containing crosslinker. In other embodiments, the crosslinker molecule includes a disulfide moiety. A disulfide moiety of the crosslinker provides an in situ-generated hydrogel that may be reversed, e.g, removed or dissolved at a later timepoint, by contact with a disulfide reducing agent such as, but not limited to DTT (Dithiothreitol), BMMP (bis (mercaptomethyl)) pyrazine, BMS (Borane dimethylsulfide), DMH (1, 2 - Dimethyl hydrazine), or DTBA (Dithiobutylamine). The disulfide moiety is not disposed at the first end or the second end of the crosslinker molecule.

[00166] In some embodiments of a crosslinker having a disulfide moiety, the crosslinker has a structure having a molecular formula of Formula (3):

HS-LB2-CH ₂ -S-S-CH ₂ -LB2-SH Formula (3), where each instance of linker backbone LB2 is independently selected to comprise 0 to 200 nonhydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms. In some embodiments, the linker backbone LB2 may have a linear backbone having carbon atoms. In some embodiments, the linear backbone having carbon atoms does not include any silicon, nitrogen, oxygen, sulfur or phosphorus atoms. When referring to the linear backbone LB2 having carbon atoms, this does not exclude substitution of hydrogen atom substituents of the carbon backbone with other types of moieties containing alcohols, sulfonates, carboxylic acids and the like.

[00167] Peptide sequence containing crosslinker. In other embodiments, the crosslinker molecule includes a peptide sequence configured to be a substrate to a proteolytic enzyme. The crosslinker containing a peptide sequence that is configured to be cleaved upon contact with a proteolytic enzyme provides an in situ-generated hydrogel that may be reversed, e.g, removed or dissolved at a later timepoint, by contact with a proteolytic enzyme such as, but not limited to a tryptase such as TrypLE™.

[00168] In some embodiments of a crosslinker having a peptide moiety, the crosslinker has a structure having a molecular formula of Formula (4):

HS-PEPT-SH Formula (4), where PEPT is a peptidyl moiety comprising about 4 to about 16 amino acids, and additionally where the peptidyl moiety is susceptible to proteolytic enzymatic cleavage. In some embodiments, the peptide sequence may include GCRDLPRTGGDRCG (SEQ ID NO: 1).

[00169] Nonreversible crosslinker. In other embodiments, the crosslinker molecule has a structure of Formula (5):

HS-LB4-SH Formula (5), where linker backbone LB4 comprises 3 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms. In some embodiments, the linker backbone LB4 may have a linear backbone having carbon atoms. In some embodiments, the linear backbone having carbon atoms does not include any silicon, nitrogen, oxygen, sulfur or phosphorus atoms. When referring to the linear backbone LB4 having carbon atoms, this does not exclude substitution of hydrogen atom substituents of the carbon backbone with other types of moieties containing alcohols, sulfonates, carboxylic acids and the like. A crosslinker of Formula V provides a hydrogel that is non-reversible, e.g, not dissolvable. In some embodiments, the crosslinker is Sodium 2, 3 -dimercaptopropanesulfonate monohydrate. In some embodiments, the crosslinker is a PEG-dithiol.

[00170] Branched crosslinker. In some embodiments, a crosslinker having a branched structure is used. In certain embodiments, the branched structure can comprise multiple arms coupled to a branched core, and at least one of the arms has a structure having a molecular formula of Formula (6):

-LB5-SH Formula (6), wherein the linker backbone LB5 can comprise at least one PEG moiety. In some embodiments, the backbone LB5 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more PEG moieties, or a range defined by any two of the foregoing endpoints, e.g., between 1 to 15, between 2 to 15, between 3 to 15, between 4 to 15, between 5 to 15, between 6 to 15, between 10 to 15, between 1 to 12, between 1 to 10, between 1 to 8, between 1 to 5, between 2 to 12, between 2 to 10, between 2 to 8, or between 2 to 5.

[00171] In some embodiments, the linker backbone LB5 can further comprises a sulfide moiety, which can be derived from an interaction between a thiol moiety and a sulfhydryl-reactive moiety or a moiety comprising an unsaturated bond, such as a maleimide moiety. In certain embodiments, the linker backbone LB5 comprises a thiosuccinimide moiety, which can be derived from an interaction between a maleimide moiety and a thiol moiety.

[00172] In some embodiments, it is preferred to have a cleavable functionality in the backbone LB5. In such embodiments, the linker backbone LB5 can further comprise a vic-diol moiety. In one embodiment, the vic-diol moiety can be introduced to the linker backbone LB5 by reacting a DTT (Dithiothreitol) with a sulfhydryl-reactive moiety or a moiety comprising an unsaturated bond. In yet other embodiments, the backbone LB5 can comprises a disulfide moiety, a peptide moiety, and/or a LB4 moiety as described herein.

[00173] In some embodiments, the branched crosslinker may have a branched backbone having a plurality of arms (e.g., 2-arm, 4-arm, 6-arm, or 8-arm structure), each having a structure as depicted in Formula (6) In certain embodiments, the branched crosslinker can be 2-arm, 4-arm, 6-arm, or 8-arm PEG thiol. In some embodiments, the branched crosslinker can be 4-arm l-mercapto-2,4-diol terminated PEG.

[00174] In some embodiments, the branched crosslinker may have a molecular weight from about 500 Da to about 40K Da; from about IK Da to about 25K Da; from about IK Da to about 25K Da; from about IK Da to about 5K Da; from about 5K Da to about 25K Da; from about 5K Da to about 20K Da; about 5K Da to about 15K Da; about 5K Da to about 10K Da, about 8K Da to about 12K Da (e.g., about 10K Da), or about 18K Da to about 22K Da (e.g., about 20K Da).

[00175] In some embodiments, the compositions may further include an inhibitor configured to inhibit reaction of the crosslinker with the reactive moiety of the first and/or the second polyethylene glycol polymer molecule and/ or of the reactive moiety of the first and/or the second polyethylene glycol polymer molecules upon standing in solution. The inhibitor may be sodium ascorbate, MEHQ, or 4-hydroxy TEMPO. The composition including the first and the second polyethylene glycol polymer molecule, and the crosslinker molecule may further include an inhibitor. In some embodiments, the inhibitor is present within the composition at a concentration from about 0.5 millimolar to about 1.5 millimolar. In other embodiments, the composition may include another component having a photoinitiator and a second amount of the inhibitor. The inhibitor in the component having the photoinitiator may have the inhibitor present in a range from about 5 millimolar to about 20 millimolar. The composition component containing the photoinitiator and second amount of inhibitor may be separately packaged and not combined with the compositions including the first and the second polyethylene glycol polymer molecules until the compositions are introduced into the microfluidic device.

[00176] Thiol-Ene Hydrogels. The compositions described above provide a thiol-ene hydrogel once formed, and include a first polyethylene glycol polymer moiety covalently linked to a first end of a crosslinker moiety, wherein a second end of the crosslinker moiety is covalently linked to a second polyethylene glycol polymer moiety, wherein the first and the second polyethylene polymer molecule comprises different polyethylene glycol moieties or may each comprise a same polyethylene glycol moiety.

[00177] The hydrogel has a structure of Formula (1):

PEG1-CG1-L-CG1-PEG2 Formula (1),

[00178] where PEG1 is the first polyethylene glycol moiety and PEG2 is the second polyethylene glycol moiety; CGi is a coupled group formed from the reaction of the R _x moiety and the RXP moiety; and L is the crosslinker moiety. In some embodiments, L comprises a linear portion, wherein a backbone of the linear portion comprises 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms. The CGi coupled group can be the product of a reaction between a norbomenyl moiety and a thiol moiety, e.g., a thiol-ene coupled group. In some embodiments, CGi comprises a thioether group. In other embodiments, the CGi coupled group can be the product of a reaction between an alkynyl moiety and a thiol moiety. In some embodiments, CGi comprises an alkenyl sulfide group.

[00179] The first polyethylene glycol moiety and/or the second polyethylene glycol moiety may include a 1-arm, 2- arm, 4- arm or 8- arm polyethylene glycol moiety. In some embodiments, the first polyethylene glycol moiety and/or the second polyethylene glycol moiety may comprise a multi-arm polyethylene glycol moiety. In some embodiments, each arm of the first polyethylene glycol moiety and/or the second polyethylene glycol moiety may include the covalently linked reactive moiety R _x . In other embodiments, when the first polyethylene glycol moiety and/or the second polyethylene glycol moiety has a 2-arm, 4- arm, or 8- arm polyethylene moiety structure, at least one arm of the first polyethylene glycol moiety and/or the second polyethylene glycol moiety is covalently linked to the first end of the crosslinker moiety and at least one arm of the first polyethylene glycol moiety and/or the second polyethylene glycol moiety is not covalently linked to a second crosslinker moiety. For example, an 8- arm polyethylene glycol moiety may have seven arms that have been crosslinked to respective crosslinker moieties and one arm that was not crosslinked. In some embodiments, an 8-arm polyethylene glycol moiety may have 6-arms, 5-arms, 4-arms, 3-arms, or 2-arms that are crosslinked to respective crosslinker moieties while the other arms are not crosslinked.

[00180] In some embodiments, at least one arm of the first polyethylene glycol moiety and/or the second polyethylene glycol moiety of the hydrogel, may include a functionalizable moiety configured to react with a type of moiety other than a thiol moiety or a norbornenyl moiety. For example, a hydrogel incorporating an 8-arm polyethylene glycol moiety as PEG1 and/or PEG2, may have 7-arms that have been crosslinked, and the remaining arm may include the functionalizable moiety that does not react with thiol or norbornenyl moieties. In some embodiments, the crosslinker moiety may include a functionalizable moiety configured to react with a type of moiety other than a thiol moiety or a norbornenyl moiety. In some embodiments, the functionalizable moiety may be a biotin. In other embodiments, functionalizable moiety may be an aldehyde. In yet other embodiments, the functionalizable moiety may be a succinimidyl moiety. In yet other embodiments, the functionalizable moiety may be an oligonucleotide.

[00181] In some embodiments, the functionalizable moiety is coupled to a coupling partner. In some embodiments the couple partner is further covalently linked to a detectable label. The coupling partner may include, but not limited to a streptavidin moiety. In some embodiments, the detectable label is a visible label, luminescent label or a fluorescent label.

[00182] The first polyethylene glycol moiety may have a molecular weight from about 500 Da to about 40K Da; from about IK Da to about 25K Da; from about IK Da to about 20K Da; from about IK Da to about 5K Da; from about 5K Da to about 25K Da; from about 5K Da to about 20K Da; about 5K Da to about 15K Da; about 5K Da to about 10K Da, about 8K Da to about 12K Da (e.g., about 10K Da), or about 18K Da to about 22K Da (e.g., about 20K Da). The second polyethylene glycol moiety may have a molecular weight from about 500 Da to about 40K Da; from about IK Da to about 25K Da; from about IK Da to about 25K Da; from about IK Da to about 5K Da; from about 5K Da to about 25K Da; from about 5K Da to about 20K Da; about 5K Da to about 15K Da; about 5K Da to about 10K Da, about 8K Da to about 12K Da (e.g., about 10K Da), or about 18K Da to about 22K Da (e.g., about 20K Da).

[00183] In some embodiments, the first polyethylene glycol moiety may have a molecular weight of about 2K Da and the second polyethylene glycol moiety may have a molecular weight of about 2K Da. In some embodiments, the first polyethylene glycol moiety may have a molecular weight of about 10K Da and the second polyethylene glycol moiety may have a molecular weight of about 10K Da. In other embodiments, the first polyethylene glycol moiety may have a molecular weight of about 10K Da and the second polyethylene glycol moiety may have a molecular weight of about 20K Da. When the first polyethylene glycol moiety has a different molecular weight from that of the second polyethylene glycol moiety, the hydrogel once formed will include a mixture of molecules, e.g., some hydrogel molecules will have a 10K Da polyethylene glycol moiety linked through the crosslinker to a second 10K Da polyethylene glycol moiety; some hydrogel molecules will have a 10K Da polyethylene glycol moiety linked through the crosslinker to a 20K Da polyethylene glycol moiety; and some hydrogel molecules will have a 20K Da polyethylene glycol moiety linked through the crosslinker to a second 20K Da polyethylene glycol moiety. In some embodiments, the first polyethylene glycol moiety and the second polyethylene glycol moiety each has a molecular weight of about 8K Da to about 12K Da (e.g., about 10K Da), or about 18K Da to about 22K Da (e.g., about 20K Da).

[00184] In some embodiments, the first polyethylene glycol molecule and the second polyethylene molecule may be present in the composition in a ratio from about 1 : 100 to about 100: 1, about 1 :50 to about 50: 1, or about 1 :25 to about 25: 1. In some further embodiments, the ratio of the first polyethylene glycol molecule and the second polyethylene molecule may be from about 1 : 1; about 1 :2; about 1 :3; about 1 :4; about 1 :5; about 1 :6; about 1 :7; about 1 :8; about 1 :9; about 1 : 10; about 10: 1; about 9: 1; about 8: 1; about 7: 1; about 6: 1; about 5: 1; about 4: 1; about 3 : 1 ; or about 2: 1.

[00185] In some embodiments, the crosslinker moiety L is linked to PEG1 through a first thiol -ene coupled group at a first end of the crosslinker molecule and linked to PEG1 through a second thiol-ene coupled group at a second end of the moiety.

[00186] Vicinal-diol (Vic-diol) containing crosslinker moiety. In some embodiments, crosslinker moiety L comprises a vic-diol. A hydrogel having a vicinal diol (vic-diol) moiety within may be reversed, e.g, removed or dissolved at a later timepoint, by contact with a periodate (Sodium Periodate). This class of hydrogels are typically referred to herein as a reversible hydrogel.

[00187] In some embodiments, the crosslinker L moiety has a structure of Formula (7):

-LB-CH ₂ -C(H)(OH)-C(H)(OH)-CH ₂ -LB- Formula (7),

[00188] wherein each instance of linker backbone LB is independently selected to comprise 0 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms. In some embodiments, the linker backbone LB may have a linear backbone having carbon atoms. In some embodiments, the linear backbone having carbon atoms does not include any silicon, nitrogen, oxygen, sulfur or phosphorus atoms. When referring to the linear backbone LB having carbon atoms, this does not exclude substitution of hydrogen atom substituents of the carbon backbone with other types of moieties containing alcohols, sulfonates, carboxylic acids and the like. In some embodiments, the crosslinker molecule is derived from a threitol moiety.

[00189] Disulfide containing crosslinker. In other embodiments, the crosslinker moiety of the hydrogel includes a disulfide. The disulfide is not disposed at the first end or the second end of the crosslinker moiety. A hydrogel having a disulfide moiety within may be reversed, e.g, removed or dissolved at a later timepoint, by contact with disulfide reducing agents such as DTT, and the like. This class of hydrogels are typically referred to herein as a reversible hydrogel.

[00190] In some embodiments, the crosslinker moiety L has a structure of Formula (8):

-LB2-CH ₂ -S-S-CH ₂ -LB2- Formula (8), [00191] wherein each instance of linker backbone LB2 is independently selected to comprise 0 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms. In some embodiments, the linker backbone LB2 may have a linear backbone having carbon atoms. In some embodiments, the linear backbone having carbon atoms does not include any silicon, nitrogen, oxygen, sulfur or phosphorus atoms. When referring to the linear backbone LB2 having carbon atoms, this does not exclude substitution of hydrogen atom substituents of the carbon backbone with other types of moieties containing alcohols, sulfonates, carboxylic acids and the like.

[00192] Peptide sequence containing crosslinker moiety. In some embodiments, the crosslinker L includes a peptide sequence configured to be a substrate to a proteolytic enzyme. An in situ-generated hydrogel including the crosslinker containing the peptide sequence that is configured to be cleaved upon contact with a proteolytic enzyme may be reversed, e.g, removed or dissolved at a later timepoint, by contact with a proteolytic enzyme such as, but not limited to a tryptase such as TrypLE™.

[00193] In some other embodiments, the crosslinker L moiety has a structure of Formula (9):

-PEPT- Formula (9)

[00194] where PEPT is a peptidyl moiety comprising about 4 to about 12 amino acids, and further where the peptidyl moiety is susceptible to enzymatic cleavage. In some embodiments, the peptide sequence PEPT is GCRDLPRTGGDRCG (SEQ ID NO: 1).

[00195] Nonreversible crosslinker moiety. In yet other embodiments, the crosslinker L moiety of the hydrogel has a structure of Formula IX:

-LB4- Formula (10)

[00196] wherein linker backbone LB4 comprises 3 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms. A hydrogel having a crosslinker moiety of Formula(lO), is a non-reversible hydrogel. In some embodiments, the linker backbone LB4 may have a linear backbone having carbon atoms. In some embodiments, the linear backbone having carbon atoms does not include any silicon, nitrogen, oxygen, sulfur or phosphorus atoms. When referring to the linear backbone LB4 having carbon atoms, this does not exclude substitution of hydrogen atom substituents of the carbon backbone with other types of moieties containing alcohols, sulfonates, carboxylic acids and the like. In some embodiments, the crosslinker moiety is derived from Sodium 2, 3 -dimercaptopropanesulfonate monohydrate.

[00197] Branched crosslinker moiety. In yet other embodiments, the crosslinker L moiety of the hydrogel has a structure of Formula (11):

BC-(LB5) _n - Formula (11), wherein BC is a branched core, the linker backbone LB5 can comprise at least one PEG moiety, and n is an integer of at least 2. In some embodiments, n is 2, 4, 6, or 8. In some embodiments, the backbone LB5 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more PEG moieties, or a range defined by any two of the foregoing endpoints, e.g., between 1 to 15, between 2 to 15, between 3 to 15, between 4 to 15, between 5 to 15, between 6 to 15, between 10 to 15, between 1 to 12, between 1 to 10, between 1 to 8, between 1 to 5, between 2 to 12, between 2 to 10, between 2 to 8, or between 2 to 5.

[00198] In some embodiments, the linker backbone LB5 can further comprises a sulfide moiety, which can be derived from a sulfhydryl -reactive moiety, such as a maleimide moiety. In certain embodiments, the linker backbone LB5 comprises a thiosuccinimide moiety, which can be derived from an interaction between a maleimide moiety and a thiol moiety.

[00199] In some embodiments, it is preferred to have a cleavable functionality in the backbone LB5. In such embodiments, the linker backbone LB5 can further comprise a vic-diol moiety. In one embodiment, the vic-diol moiety can be introduced to the linker backbone LB5 by reacting a DTT (Dithiothreitol) with a sulfhydryl-reactive moiety. In yet other embodiments, the backbone LB5 can comprises a disulfide moiety, a peptide moiety, and/or a LB4 moiety as described herein.

[00200] Initiator. Photoactivated polymerization may be accomplished using a free radical initiator Igracure® 2959 (BASF), a highly efficient, non-yellowing radical, alpha hydroxy ketone photoinitiator, is typically used for initiation at wavelengths in the UV region (e.g., 365nm), but other initiators may be used. An example of another useful photoinitiator class for polymerization reactions is the group of lithium acyl phosphinate salts, of which lithium phenyl 2, 4, 6, - trimethylbenzolylphosphinate has particular utility due to its more efficient absorption at longer wavelengths (e.g., 405nm) than that of the alpha hydroxy ketone class. Another initiator that may be used are water soluble azo initiators, such as 2, 2-Azobis [2-methyl-N-(2-hydroxyethyl)propionamide], The initiator may be present within the flowable polymer solution at a concentration of about 5 millimolar, about 8 millimolar, about 10 millimolar, about 12 millimolar, about 15 millimolar, about 18 millimolar, about 20 millimolar, about 22 millimolar, about 25 millimolar, about 28 millimolar, about 30 millimolar, about 35 millimolar, or about 40 millimolar.

[00201] Crosslinking may be performed by photopatterning of linear or branched PEG polymers, free radical polymerization of PEG acrylates or PEG acrylamides, and specifically tailored chemical reactions such as Michael addition, condensation, Click chemistry, native chemical ligation and/or enzymatic reactions. In particular, photopatterning of crosslinking may be used to gain precise control of extent of the physical extent of the hydrogel barrier as well as the degree of crosslinking, as described in the following section and in the Examples.

[00202] Inhibitor. Inhibitors may be included within the flowable polymer solution to ensure precise control of photopatterning and to prevent extraneous or undesired polymerization. One useful inhibitor is hydroquinone monomethyl ether, MEHQ, but the compositions and methods are not so limited. Other suitable inhibitors include 4-hydroxy TEMPO (4-hydroxy-2,2,6,6-tetramethylpiperidin-l-oxyl) and sodium ascorbate, but any inhibitor known in the art may be used. The inhibitor may be present in the flowable polymer solution at a concentration of about 0.5 millimolar, about 0.6 millimolar, about 0.7 millimolar, about 0.8 millimolar, about 0.9 millmolar, about 1 millimolar, about 2 millimolar, about 5 millimolar, about 10 millimolar, about 15 millimolar, about 20 millimolar, about 25 millimolar, about 30 millimolar, about 35 millimolar, about 40 millimolar or more, as needed to provide the photopatterning control desired. The inhibitor may also be provided as part of a composition including the flowable polymer solutions to stabilize the flowable polymers prior to use.

[00203] Tuneable permeability. One aspect of performing assays using a hydrogel in a situ-generated isolation structure may include controlling the access of a molecule to an area of interest (e.g., completely exclude or control the rate of diffusion across the hydrogel). Selection of the chemical nature of the polymer (for example, molecular weight range, number of cross linkable moieties per polymer unit (linear, 2 arm, 4 arm, 8 arm, star or comb polymer), mixtures of polymers), the chemical nature of the crosslinker (for example, molecular weight range, number of cross linkable moieties per polymer unit (linear, 2 arm, 4 arm, 8 arm, star or comb polymer), mixtures of crosslinkers), the amount of initiator, and mode of polymerization are variables that may be modified to tune the hydrogel barrier formed. Generally, the initiator is a photoinitiator. Photopatterning provides precise control of the geometry of the polymerization as well as the extent of polymerization, and changes in exposure time and power of the illumination also can provide more control to arrive at a desired type of porosity and degree of robustness of the polymerized feature.

[00204] As described in Experiments 1, 5 and 6, non-limiting examples of controlled permeability are shown. Mixtures of two different flowable polymers having similar molecular weight were found to be advantageous in providing hydrogel barriers with differential permeability. Using polymers having similar molecular weights confers similar rates of diffusion, which simplifies delivery to the region within the chamber. Since chambers that are sequestration pens are unswept region of the microfluidic device, introduction of the polymer into the sequestration pen occurs substantially only by diffusion.

[00205] In many variations, polymer selection may depend upon the biocompatibility of the polymer species, and may be related to the specific application to which a hydrogel in situ-generated barrier may be used.

[00206] A wide range of molecular weights of the flowable polymer may be suitable, depending upon the structure of the polymer. In some embodiments, the flowable polymer may have a molecular weight of about 500 Da to about 20kDA, or about 500Da, about IkDa, about 3kDa, about 5kDa, about 10 kDa, about 12kDa, about 15kDa, about 20 kDa or any value therebetween. A useful star type polymer may have Mw (weight average molecular weight) in a range from about 500Da to about 20kDa (e.g., four arm polymer), or up to about 5kDa for each arm, or any value therebetween. In some embodiments, a polymer having a higher molecular weight range, may be used at lower concentrations in the flowable polymer, and still provide an in situ-generated barrier or isolation structure that may be used in the methods described herein.

[00207] Reversing/removing/minimizing the in situ-generated isolation structure. A number of mechanisms may be used to remove or reduce the in situ-generated hydrogel barrier when there is no further purpose for it. For example, once an assay is completed and desirable biological cells have been identified, it may be useful to remove the hydrogel barrier in order to export the cells and/or continue culturing and expanding the biological cell demonstrating desirable activities or properties.

[00208] Mechanical force. Increasing flow can be used if at least a portion of the hydrogel barrier is located within a flow region as opposed to an isolation region of a pen. For example, the at least one isolation structure may be located within an isolation region of a sequestration pen, and after the assay is complete, the sequestration pen or the isolation region therein may be modified to bring flow through the isolation region.

[00209] In other variations, such as is described herein, laser-initiated bubbles may provide forces that can deform or disrupt the hydrogel barrier, permitting export of the cells, as is more fully described in Experiment 1-5.

[00210] Hydrolytic susceptibility: Porogens, including polymers which are incapable of being chemically linked to the photoinitiated polymer(s), may be included when forming the hydrogel barrier. The degree/size of openings within the formed hydrogel can customize the hydrolysis rate via accessibility within the hydrogel barrier). In other embodiments, the pores formed may be employed to permit secreted materials or chemical reagents to pass through the hydrogel barrier but prevent a cell from moving into, out of, and/or through the isolation structure. In other embodiments, degradability of these polymers may be increased by introducing degradable segments such as polyester, acetal, fumarate, polypropylene fumarate) or polyhydroxyacids into polymers (e.g., PEG polymers).

[00211] Reducing agents: PEG may be formed with disulfide linkages at intervals along the macromere, which may be random or predetermined. The disulfide bonds may be broken by Dithiothreitol (DTT), mercaptoethanol, or TCEP.

[00212] Thermal: poly N-isopropyl acrylamide (PNIPAm) or other suitable LCST polymers may be used to introduce hydrogel barriers upon heating. They may be removed by decreasing the temperature of the formed polymer hydrogel barrier. The polymers may include ELPs or other motifs that also permit removal by other mechanisms such as hydrolysis or proteolysis. In particular, PNIPAm may be used to create a surface for adherent cells, but then switched to permit export.

[00213] Proteolytic susceptibility: Hydrogels may have any sort of peptide sequence engineered in, such that selective proteolysis upon a selected motif by a selected protease can remove/reverse/or minimize a hydrogel isolation structure. Some classes of modified PEG include PEG having elastin like peptide (ELP) motifs and/or having peptide motifs for susceptibility to a variety of proteases (enzyme sensitive peptide ESP). A large number of these motifs are known. One useful motif is RGD which may be constrained to be cyclic.

[00214] Osmotic susceptibility: Calcium concentration/other osmotic strategies can be employed to degrade and remove a hydrogel barrier. As above, changes of media flowed through the channel or flow region may dimensionally swell or de-swell hydrogel barriers. [00215] Photocleavage: As described above, if a polymer of the solidified polymer network includes a photocleavable moiety, directing illumination of an exciting wavelength to the solidified polymer network will cause cleavage within sections of the solidified polymer network. This cleavage may provide complete or partial disruption of the solidified polymer network, thereby removing or reducing the hydrogel barrier.

[00216] Chemical susceptibility. As described in the sections above directed to thiol -ene polymers, specific moieties of the hydrogel polymer may be susceptible to chemical cleavage, such as reducing agents or periodate cleavage.

[00217] In some applications, the hydrogel barrier may not be removed but may simply be swelled or de-swelled using light or media\solvent changes. Some types of hydrogels may incorporate moieties that respond reversibly to light (for example, change regiochemistry about a rigid bond; form reversible crosslinks within the polymer, or form/break ion pairs).

METHODS OF INTRODUCING HYDROGEL BARRIERS

[00218] In one aspect, a method of forming a hydrogel barrier within a microfluidic device is provided, wherein the microfluidic device has an enclosure including a base, a cover, and microfluidic circuit material defining a fluidic circuit therein. The fluidic circuit includes a flow region and chambers opening to the flow region. The process includes introducing a photoactivatable flowable polymer composition into the flow region of the microfluidic device; diffusing the composition from the flow region into the chambers; activating crosslinking of the composition in a selected area of the microfluidic device, thereby forming a hydrogel barrier within the flow region or one or more of the chambers of the microfluidic device. One or more biological micro-objects may be disposed within one or more of the chambers prior to introduction of the composition The photoactivatable flowable polymer composition may include modified polyethylene glycol moieties. In some instances, the photoactivatable polymer composition may be a composition described herein that produce a thiol-ene hydrogel, having any features described in any combination. In some embodiments, the composition may further include an inhibitor of crosslinking. The inhibitor may be MEHQ, 4-hydroxy TEMPO, or sodium ascorbate. The method may further include introducing a photoinitiator into the flow region, and permitting the photoinitiator to diffuse into the chambers. In some embodiments, the photoinitiator is LAP. The method may further include introducing a second portion of the inhibitor into the flow region and diffusing the second portion of the inhibitor into the chambers. Forming the hydrogel barrier may further include illuminating a selected area for a selected period of time with a wavelength of light configured to induce crosslinking.

[00219] In some embodiments, the one or more chambers are each a sequestration pen, and forming a hydrogel barrier in one or more of the sequestration pens may further include forming a hydrogel barrier within an isolation region of the one or more sequestration pens. The hydrogel barrier may form an assay region within the isolation region of the one or more sequestration pens. In some embodiments, the hydrogel barrier may form a culturing region within the isolation region of the one or more sequestration pens. In some instances, the culturing region is different from the assay region. [00220] In some embodiments, forming a hydrogel barrier in one or more of the chambers may further include forming a hydrogel barrier at an opening of the one or more chambers to the flow region. In other embodiments, forming a hydrogel barrier may include forming a barrier in the flow region.

[00221] In some embodiments, when the composition has a single type of crosslinker molecule comprising a vic-diol moiety or a peptide moiety, the method may further include removing the hydrogel barrier at a selected time by introducing a reversing reagent into the flow region and diffusing the reversing reagent into the sequestration pens, thereby removing the hydrogel barrier and providing one or more uncapped sequestration pens. The reversing reagent is a periodate reagent configured to cleave the vic-diol moiety or an enzyme configured to cleave the peptidyl moiety. In some embodiments, the enzyme configured to cleave the peptidyl moiety is a trypsin enzyme or analog thereof. The analog of the trypsin enzyme may be a TrypLE™ enzyme. The method may further include unpenning one or more biological micro-objects from at least one uncapped chamber.

[00222] In some embodiments, when the composition has a single type of crosslinker molecule having no cleavable moieties, forming the hydrogel barrier ( which is a non-reversible hydrogel barrier) further includes reducing a risk of loss of clonality for the one or more biological micro-objects or daughter micro-objects thereof disposed within at least one of the chambers. The method may further include forming the hydrogel barrier at an opening of a selected chamber. The method may further include unpenning biological micro-objects from the remaining chambers that comprise no hydrogel barrier at a respective opening of the remaining chambers.

[00223] Location of the hydrogel barrier. A hydrogel (e..g, a hydrogel barrier) can be disposed (e.g., in situ-generated) in various regions within a microfluidic device depending on the needs of an assay, for example, with the flow region (e.g., the microfluidic channel) or one or more of the plurality of chambers. In embodiments that the chamber is a sequestration pen as described herein, the hydrogel can be disposed in the connection region or the isolation region of the sequestration pen. In embodiments of disposing the hydrogel in a chamber, the hydrogel can be disposed distal to, at a mid-region of, or at the opening of the chamber.

[00224] In embodiments of disposing the hydrogel distal to the opening of the chamber, the hydrogel is disposed in a region within 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 or 1 micron (um) from the distal end of the chamber, or a range defined by two of the foregoing endpoints, e.g., between 1 um and 100 um, between 1 um to 50 um, between 1 um to 40 um, between 1 um to 30 um, between 1 um to 20 um, between 1 um to 10 um, between 1 um to 5 um, between 5 um to 50 um, between 5 um to 40 um, between 5 um to 30 um, between 5 um to 20 um, between 5 um to 10 um, between 10 um to 50 um, between 10 um to 40 um, between 10 um to 30 um, between 10 um to 20 um.

[00225] In embodiments of disposing the hydrogel at the opening of the chamber the hydrogel is disposed in a region within 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 or 1 micron (um) from the opening of the chamber, or a range defined by two of the foregoing endpoints, e.g., between 1 um and 100 um, between 1 um to 50 um, between 1 um to 40 um, between 1 um to 30 um, between 1 um to 20 um, between 1 um to 10 um, between 1 um to 5 um, between 5 um to 50 um, between 5 um to 40 um, between 5 um to 30 um, between 5 um to 20 um, between 5 um to 10 um, between 10 um to 50 um, between 10 um to 40 um, between 10 um to 30 um, between 10 um to 20 um. In some embodiments, the hydrogel is disposed in a region at the opening of the chamber and comprising part of the flow region and part of the chamber region.

[00226] In some embodiments, the hydrogel is disposed in a mid-region of the chamber. “Mid-region” used herein refers to a central portion of the chamber with respect to a long axis of the chamber, for example, with respect to an axis of the chamber that is substantially perpendicular to a direction of intended flow of medium within the flow region. In certain embodiments, disposing a hydrogel in the mid-region of the chamber bisects the chamber into a proximal region and a distal region of similar length along the long axis of the chamber.

[00227] In some embodiments, the hydrogel generated in situ can have a dimension (e.g., a thickness or a diameter) of 1, 3, 5, 6, 7, 8, 9 10, 11, 12, 13, 14 15, 16, 17, 18, 19, 20, 25, or 30 microns (um), or a range defined by two of the foregoing endpoints, e.g., between 1 um and 5 um, between 1 um and 10 um, between 1 um and 15 um, between 1 um and 20 um, between 1 um and 25 um, between 1 um and 30 um, between 3 um and 10 um, between 3 um and 15 um, between 3 um and 20 um, between 3 um and 25 um, between 3 um and 30 um, between 5 um and 10 um, between 5 um and 15 um, between 5 um and 20 um, between 5 um and 25 um, between 5 um and 30 um, between 3 um and 10 um, between 3 um and 15 um, between 3 um and 20 um, between 3 um and 25 um, between 3 um and 30 um, between 7 um and 10 um, between 7 um and 12 um, between 7 um and 15 um, between 7 um and 20 um, between 10 um and 12 um, between 10 um and 15 um, or between 10 um and 20 um. In some embodiments, the in situ-generated hydrogel encloses or caps a chamber. “Enclose” or “cap” used herein refers that the hydrogel restricts a micro-object from entering or leaving the chamber. In some embodiments, the hydrogel blocks a microobject from entering or leaving the chamber. Nevertheless, the hydrogel can have a certain permeability limit that allows a micro-object of a size to pass through the hydrogel while blocks another micro-object of a different (e.g. larger size) size from passing through.

[00228] In some embodiments, there can be a gap between the edge of an in situ-generated barrier and a wall of the chamber where the in situ-generated barrier is located. However, the gap can have a width that is smaller than a dimension of a micro-object so that the micro-object still is blocked/restricted by the hydrogel, or the gap can have a width that is about the same size as a dimension of a micro-object so that even if the micro-object is not completely blocked from passing through, its movement is restricted.

[00229] Introducing hydrogels with different physical properties. In situ-generated hydrogels provide additional physical structures for performing assays within an enclosure of a microfluidic device. In many circumstances, more than one hydrogel is formed within a microfluidic device. When the more than one hydrogel has different physical properties from one another, it allows higher flexibility to perform more complicated assays and further expands the capabilities of the microfluidic device. Therefore, in another aspect, a method of introducing hydrogel barriers having different physical properties to a microfluidic device is provided, where the microfluidic device has an enclosure comprising a base, a cover, and microfluidic circuit material defining a fluidic circuit therein. The fluidic circuit includes a flow region and a plurality of chambers opening to the flow region. The process includes introducing a first photoactivatable flowable polymer composition into the flow region of the microfluidic device, where the first composition has a first selected set of characteristics defining physical properties of a first hydrogel formed therefrom; diffusing the first composition from the flow region into the plurality of chambers; activating crosslinking of the first composition in a selected area of the microfluidic device, thereby forming a first hydrogel within the flow region or one or more of the plurality of chambers of the microfluidic device. The method further includes introducing a second photoactivatable flowable polymer composition into the flow region of the microfluidic device, where the second composition has a second selected set of characteristics defining physical properties of a second hydrogel formed therefrom; diffusing the second composition from the flow region into the chambers; and activating crosslinking of the second composition in a selected area of the microfluidic device, thereby forming a second hydrogel within the flow region or a sub-set of the plurality of chambers of the microfluidic device, where the first hydrogel differs from the second hydrogel based on at least one different physical property. In some embodiments, a plurality of biological micro-objects may be disposed in at least some of the plurality of chambers, prior to introduction of the first composition.

[00230] Hydrogels disposition arrangement in a microfluidic device. The first hydrogel and the second hydrogel can be disposed (e.g., in situ-generated) in various regions within a microfluidic device for facilitating assaying, for example, within the flow region (e.g., the microfluidic channel) or one or more of the plurality of chambers. Depending on the needs of the assays, a variety of arrangements can be made. In some embodiments, the first hydrogel and the second hydrogel can be independently disposed in flow region or a chamber. The first hydrogel and the second hydrogel can be disposed in the same chamber or in different chambers, where the different chambers can be adjacent chambers or distanced from each other.

[00231] In the embodiments where a first hydrogel and a second hydrogel are disposed in a chamber, the first hydrogel and the second hydrogel can be independently disposed distal to, at a mid-region of, or at the opening of the chamber, as described above. In some embodiments, the first hydrogel is generated in situ in a chamber thereby producing a capped chamber. When the microfluidic device comprises a plurality of chambers, a first subset of the plurality of chambers can be capped by the first hydrogel or by respective first hydrogels. In such embodiments, the second hydrogel can be generated in situ in a second subset of the plurality of chamber. In some embodiments, the second subset of the plurality of chambers can be (1) different from the first subset, (2) the same as the first subset, (3) selected to have one or more of the first subset, or (4) selected to comprise one or more of the first subset and one or more of the plurality of chambers that does not belong to the first subset.

[00232] Each of the first composition and the second composition may include an inhibitor of crosslinking. In some embodiments, the inhibitor is MEHQ, TEMPO, or sodium ascorbate The method may further include introducing a photoinitiator into the flow region, and permitting the photoinitiator to diffuse into the chambers after each introduction of the respective first and second composition. In some embodiments, the photoinitiator is LAP. The method may further include introducing a second portion of the inhibitor into the flow region and diffusing the second portion of the inhibitor into the chambers. Forming the hydrogel barrier may further include illuminating a selected area for a selected period of time with a wavelength of light configured to induce crosslinking.

[00233] In some embodiments, the first composition and the second composition each include modified polyethylene glycol moieties. In some embodiments, the first composition may be a composition described herein that produce a thiol-ene hydrogel, having any features described in any combination. In some embodiments, the second composition may be a composition described herein that produce a thiolene hydrogel, having any features described in any combination. In some embodiments, a viscosity of the first composition and a viscosity of the second composition are substantially the same. Using compositions having the same or similar viscosities can provide more uniform and predictable gelled polymer composition within a chamber.

[00234] Physical properties. In some embodiments, the physical property comprises reversibility, permeability, shrinkability, or a combination thereof. The difference in physical properties can be contributed by different orthogonality of the hydrogels formed in the microfluidic device. For example, a first hydrogel can be formed with a vic-diol crosslinker moiety (e.g., as described Formula (7) above) and can be reversible, while a second hydrogel can be formed with a nonreversible crosslinker moiety (e.g., as described Formula (10) above). In another example, a first hydrogel can be formed using prepolymer of PEG acrylamide, while a second hydrogel can be a thiol-ene hydrogel (e.g., using PEG norbomene) so that the first hydrogel is not shrinkable but the second hydrogel is.

[00235] In some embodiments, two or more hydrogels having different reversibility and permeability are generated in situ within a microfluidic device to cap respective chambers. This arrangement allows assaying the respective chambers with an assay reagent that is small enough to enter some of the hydrogels but is too large to enter the other. Furthermore, the difference in reversibility allows selectively removing only one or some of the hydrogels to uncap selected chambers for selectively exporting a micro-object from the uncapped chambers. Similarly, difference in shrinkability of hydrogels can be useful in controlling movement of a reagent/a micro-object within a microfluidic device (e.g., in or out of a chamber of the microfluidic device). Particularly in certain embodiments where the shrinking of a hydrogel can be initiated by light, the restriction of movement of a reagent/a micro-object can be controllably removed rapidly.

[00236] “Reversibility” used herein refers a mechanism of removing a formed hydrogel as to whether the hydrogel is reversible and/or how the hydrogel can be reversed or de-formed. In some embodiments, one of the first hydrogel or the second hydrogel is a reversible hydrogel and the respective other hydrogel is a non-reversible hydrogel. In some embodiments, the first hydrogel is reversible, and the second hydrogel is non-reversible. In some embodiments, both of the first hydrogel and the second hydrogel are reversible while the reversibility thereof is different in how the hydrogel can be reversed or de-formed. For example, the first hydrogel and the second hydrogel might be formed with different orthogonality, and therefore, different reversing reagents are required to reverse the first hydrogel and the second hydrogel. In yet some embodiments, the first hydrogel and the second hydrogel are formed with the same orthogonality, but they are formed with different amounts of the cleavable linkages. For example, the first hydrogel might have a higher amount of the cleavable vic-diol bonds than the second hydrogel so that the first hydrogel is more vulnerable to a reversing reagent than the second hydrogel.

[00237] In some embodiments, the first hydrogel is a reversible hydrogel. Forming the first reversible hydrogel barrier may include forming the first reversible hydrogel barrier within one or more chambers of the plurality of sequestration pens. Forming the first reversible hydrogel barrier may further include forming the first reversible hydrogel barrier distal to, at a mid-region of, or at the opening of the one or more chambers, thereby producing one or more reversibly capped chambers. In some embodiments, at least some of the one or more reversibly capped chambers contain one or more biological micro-objects, and the first reversible hydrogel barrier prevents the one or more biological micro-objects from exiting the one or more reversibly capped chambers. The first reversible hydrogel barrier of each of the one or more reversibly capped chamber may have a permeability limit, thereby preventing subsequently introduced reagents having a size greater than the permeability limit from entering the one or more reversibly capped chambers. In some embodiments, when the plurality of chambers is a plurality of sequestration pens, the first reversible hydrogel barrier may be formed within an isolation region of the one or more sequestration pens, forming an assay region or a biological micro-object maintenance region distal to the opening of the one or more reversibly capped sequestration pens. In some embodiments, the first reversible hydrogel barrier of each of the one or more reversibly capped chambers may prevent a biological micro-object from entering a region of each of the one or more reversibly capped chambers, thus preventing contamination of biological micro-objects that were disposed within the capped chambers, prior to introducing the first reversible hydrogel barrier.

[00238] In some embodiments, the method further includes forming the second hydrogel at the opening of a subset of the plurality of chambers, wherein the subset of the plurality of chambers: is different from the one or more reversibly capped chambers; is the same sequestration pens as the one or more reversibly capped chambers; selected to have some or all of the reversibly capped chambers; or selected to comprise some or all of the reversibly capped chambers and some of the remainder of the plurality of chambers, and the second hydrogel at each opening of the subset of the plurality of chambers prevents a biological micro-object from entering or exiting each chamber of the subset of chambers.

[00239] The method may further include introducing a reversing reagent configured to reverse the first reversible hydrogel barrier; contacting the first reversible hydrogel barriers of the one of more reversibly capped chambers; and reversing the first reversible hydrogel barriers, thereby providing a group of uncapped chambers while retaining the second hydrogel barriers in each of the subset of the plurality of chambers. The group of uncapped chambers are the chambers having only a first reversible hydrogel barrier and have no second hydrogel barrier. In some embodiments, the method further includes unpenning one or more biological micro-objects from at least one of the group of uncapped chambers.

[00240] In some other embodiments, the first hydrogel and the second hydrogel are each a reversible hydrogel and require orthogonal conditions for reversing. “Orthogonal conditions” as used herein refers to reversing conditions that will reverse the first hydrogel barriers and not the second reversible hydrogel barriers or will reverse the second hydrogel barriers and not the first reversible hydrogel barriers. Orthogonal conditions may be used in the presence of the non-susceptible set of hydrogel barriers without substantially reversing the non-susceptible barriers. Forming the first reversible hydrogel barrier may include forming the first reversible hydrogel barrier within one or more chambers of the plurality of chambers. Forming the first reversible hydrogel barrier may further include forming the first reversible hydrogel barrier distal to or at the opening of the one or more chambers, thereby producing one or more reversibly capped chambers. At least some of the one or more reversibly capped chambers may contain one or more biological micro-objects, and the first reversible hydrogel barrier may prevent the one or more biological micro-objects from exiting the one or more reversibly capped chambers. In some embodiments, the first reversible hydrogel barrier of each of the one or more reversibly capped chambers may further have a permeability limit, thereby preventing subsequently introduced reagents having a size greater than the permeability limit from entering the one or more reversibly capped chambers. In some embodiments, the first reversible hydrogel barrier of each of the one or more reversibly capped chambers prevents a biological micro-object from entering a region of each of the one or more reversibly capped chambers (where biological micro-objects has been disposed prior to introducing the first reversible hydrogel barrier, thereby preventing contamination of the cells of interest). In some embodiments, when the plurality of chambers is a plurality of sequestration pens, the first reversible hydrogel barrier may be formed within an isolation region of the one or more sequestration pens, forming an assay region or a biological micro-object maintenance region distal to the opening of the one or more reversibly capped sequestration pens.

[00241] The method may further include forming the second reversible hydrogel barrier at the opening of a subset of the plurality of chambers, wherein the subset of the plurality of chambers: is different from the one or more reversibly capped chambers; is the same sequestration pens as the one or more reversibly capped chambers; selected to have some or all of the reversibly capped chambers; or selected to comprise some or all of the reversibly capped chambers and some or all of the remainder of the plurality of chambers; thereby forming a first group of one or more capped chambers reversibly capped by a first reversible hydrogel barrier and a second group of chambers reversibly capped by the second reversible hydrogel barrier. The method may include introducing a first reversing reagent configured to reverse a hydrogel barrier having the composition of the first reversible hydrogel into the flow region of the microfluidic device; contacting the first hydrogel barriers of the first group of capped chambers with the first reversing reagent; and reversing the first hydrogel barriers, thereby removing the capping and providing a first group of uncapped chambers. The method may further include introducing a second reversing reagent configured to reverse a hydrogel barrier having the composition of the second hydrogel into the flow region of the microfluidic device; contacting the second hydrogel barriers of the second group of capped chambers with the second reversing reagent; and reversing the second hydrogel barriers, thereby providing a second group of uncapped chambers. The method may further include unpenning one or more biological micro-objects from at least one of the first group of uncapped chambers and/or the second group of uncapped chambers.

[00242] In some embodiments of the method, when the composition includes a crosslinker molecule comprising a vic-diol moiety or a peptide moiety, the reversing reagent is a periodate reagent configured to cleave the vic-diol moiety or an enzyme configured to cleave the peptidyl moiety. The enzyme configured to cleave the peptidyl moiety may be a trypsin enzyme or an analog thereof having similar enzymatic properties. The analog of the trypsin enzyme may be a TrypLE™ enzyme.

[00243] “Permeability” used herein refers describes how easily a molecule of interest passes through the in situ-generated structure. In some other embodiments, the first hydrogel has a different permeability from a permeability of the second hydrogel. Permeability can be tuned as described here including but not limited to adjusting the percentage of the prepolymer in a prepolymer composition, using prepolymers of smaller molecular weight, and/or using crosslinkers having branched structure. In some specific embodiments, a low porosity hydrogel, which provides low permeability, can be generated using 4-arm 2K PEG alkyne prepolymer crosslinking with 4 arm 2K PEG thiol crosslinker, compared with using 8- arm 10K/8-arm 20K PEG norbomene prepolymer mixture crosslinking with DTT crosslinker.

[00244] In some embodiments, the first hydrogel has a first permeability limit that is higher than a permeability limit of the second hydrogel. Forming the first permeable hydrogel barrier may include forming the first permeable hydrogel barrier within one or more chambers of the plurality of sequestration pens. Forming the first permeable hydrogel barrier may further include forming the first permeable hydrogel barrier distal to, at a mid-region of, or at the opening of the one or more chambers, thereby producing one or more reversibly capped chambers. At least some of the one or more capped chambers may contain one or more biological micro-objects, and the first permeable hydrogel barrier prevents the one or more biological micro-objects from exiting the one or more reversibly capped chambers, The first permeable hydrogel barrier of each of the one or more capped chambers may prevent subsequently introduced reagents having a size greater than the permeability limit from entering the one or more capped chambers but may permit subsequently introduced reagents having a size lower than the permeability limit to diffuse through the first permeable hydrogel barrier into an interior of the one or more capped chambers.

[00245] In some embodiments, when the plurality of chambers is a plurality of sequestration pens, the first permeable hydrogel barrier is formed within an isolation region of the one or more sequestration pens, forming an assay region or a biological micro-object maintenance region distal to the opening of the one or more capped sequestration pens. The first permeable hydrogel barrier of each of the one or more capped chambers may prevent a biological micro-object from entering a region of each of the one or more capped chambers wherein biological micro-objects were disposed prior to introducing the first permeable hydrogel barrier.

[00246] In some embodiments, the method may further include forming the second permeable hydrogel at the opening of a subset of the plurality of chambers, wherein the subset of the plurality of chambers: is different from the one or more capped chambers; is the same sequestration pens as the one or more capped chambers; is selected to have some or all of the one or more capped chambers; or is selected to comprise some or all of the one or more capped chambers and some of the remainder of the plurality of chambers, and the second permeable hydrogel at each opening of the subset of the plurality of chambers prevents a biological micro-object from entering or exiting each chamber of the subset of chambers. The second permeable hydrogel barrier of the subset of the plurality of chambers, may permit subsequently introduced reagents having a size lower than the permeability limit of the second permeable hydrogel barrier to diffuse through the second permeable hydrogel barrier and into the interior of the subset of chambers. A reagent configured to diffuse through the first permeable hydrogel barrier may be excluded from diffusing through the second permeable hydrogel barrier, when that reagent has a size greater than the permeability limit of the second permeable hydrogel barrier.

[00247] In some embodiments, when one of the first permeable hydrogel barrier and the second permeable hydrogel barrier is a reversible hydrogel barrier, the method may further include reversing the reversible hydrogel barrier, thereby uncapping selected chambers. The method may further include unpenning one or more biological micro-objects from at least one of the selected chambers.

[00248] “Shrinkability” used herein refers conformational changes of a hydrogel, which can include reducing of size. In some embodiments, one of the first hydrogel and the second hydrogel is a shrinkable hydrogel, and the respective other hydrogel is a non-shrinkable hydrogel. In some embodiments, the first hydrogel has a shrinkability different from a shrinkability of the second hydrogel. In some embodiments where the crosslinking between the prepolymer and crosslinker comprises a thiol-ene reaction, the shrinking of the hydrogel is thermoresponsive due to the hydrophobicity of the ene moieties. Without wishing being bound by theories, when the temperature of the hydrogel or the environment surrounding the hydrogel is increasing, the internal environment of the hydrogel becomes more and more unsuitable for water molecules to exist. As a result, water molecules are driven out of the polymer network of the hydrogel thereby shrinking the hydrogel.

[00249] In some embodiments, the more ene moieties existing in the polymer network, the more thermoresponsive the hydrogel is. In yet embodiments, the higher percentage of the ene moieties in the hydrogel structure, the more thermoresponsive the hydrogel is. In certain embodiment, the percentage of the ene moieties in a hydrogel structure can be adjusted by selecting the length of the PEG moieties of a prepolymer and/or a crosslinker. For example, the longer length of the PEG moieties, the lower percentage of the reactive moieties, which results in lower percentage of the ene moieties in the formed hydrogel. In some embodiments, the shrinking comprises reducing the size of the hydrogel by 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80%, or more of the original size before shrinking, or a range defined by two of the foregoing endpoints, e.g., between 5% to 80%, between 5% to 70%, between 5% to 60%, between 5% to 50%, between 5% to 40%, between 5% to 30%, between 5% to 20%, between 5% to 15%, between 10% to 80%, between 10% to 70%, between 10% to 60%, between 10% to 50%, between 10% to 40%, between 10% to 30%, between 10% to 20%, between 10% to 15%, between 20% to 80%, between 20% to 70%, between 20% to 60%, between 20% to 50%, between 20% to 40%, between 50% to 80%, between 50% to 70%, between 50% to 60%, between 60% to 80%, between 60% to 70%, or between 70% to 80%.

[00250] In some embodiments, forming the first hydrogel may include forming a first shrinkable hydrogel barrier within one or more chambers of the plurality of chambers. Forming the first shrinkage hydrogel barrier may further include forming the first shrinkage hydrogel barrier distal to, at a mid-region of, or at the opening of the one or more chambers, thereby producing one or more capped chambers. At least some of the one or more capped chambers may contain one or more biological micro-objects, and the first shrinkable hydrogel barrier prevents the one or more biological micro-objects from exiting the one or more capped chambers, The first shrinkable hydrogel barrier of each of the one or more capped chambers may prevent subsequently introduced reagents having a size greater than a permeability limit from entering the one or more capped chambers but may permit subsequently introduced reagents having a size lower than the permeability limit to diffuse through the first shrinkable hydrogel barrier into an interior of the one or more capped chambers.

[00251] In some embodiments, when the plurality of chambers is a plurality of sequestration pens, the first shrinkable hydrogel barrier is formed within an isolation region of the one or more sequestration pens, forming an assay region or a biological micro-object maintenance region distal to the opening of the one or more capped sequestration pens. The first shrinkable hydrogel barrier of each of the one or more capped chambers may prevent a biological micro-object from entering a region of each of the one or more capped chambers wherein biological micro-objects were disposed prior to introducing the first shrinkable hydrogel barrier.

[00252] In some embodiments, the method may further include forming the second shrinkable hydrogel at the opening of a subset of the plurality of chambers, wherein the subset of the plurality of chambers: is different from the one or more capped chambers; is the same chambers as the one or more capped chambers; is selected to have some or all of the one or more capped chambers; or is selected to comprise some or all of the one or more capped chambers and some of the remainder of the plurality of chambers, and the second shrinkable hydrogel at each opening of the subset of the plurality of chambers prevents a biological micro-object from entering or exiting each chamber of the subset of chambers. The second shrinkable hydrogel barrier of the subset of the plurality of chambers, may permit subsequently introduced reagents having a size lower than the permeability limit of the second shrinkable hydrogel barrier to diffuse through the second shrinkable hydrogel barrier and into the interior of the subset of chambers. A reagent configured to diffuse through the first shrinkabe hydrogel barrier may be excluded from diffusing through the second shrinkable hydrogel barrier, when that reagent has a size greater than the permeability limit of the second shrinkable hydrogel barrier.

[00253] In some embodiments, the method further comprises increasing a temperature of the microfluidic device whereby the first shrinkable hydrogel barrier experiences a higher degree of shrinking than a shrinking that the second shrinkable hydrogel barrier experiences. In some embodiments, the shrinking of the first shrinkable hydrogel barrier uncaps the one or more capped chambers, therefore, the method can further comprise unpenning a biological micro-object from the chambers.

[00254] Increasing the temperature can be achieved locally by projecting a light in a selected area, for example, an area comprising the first shrinkable hydrogel barrier. In some embodiments, the light is projected in an area proximal to the first shrinkable hydrogel barrier, and preferably avoiding an area comprising or proximal to the second shrinkable hydrogel barrier. In some embodiments, the light is not projected in an area proximal to the second shrinkable hydrogel barrier or is not projected on the second shrinkable hydrogel barrier. METHODS OF ASSAYING CELLS USING IN SITU-GENERATED HYDROGEL BARRIERS

[00255] Bioproductivity. As used herein, “bioproductivity” refers to productivity of a living cell in producing or secreting a molecule of interest. The term “bioproductivity” and “productivity” are interchangeable in this description. As used herein, “molecule of interest,” “biomolecule of interest,” “biomolecule,” “analyte,” “secreted analyte,” “secreted protein,” and alike are interchangeable and refer to a biomolecule or an organic molecule produced by a cell of which the bioproductivity is to be evaluated. In some embodiments, the analyte is an amino acid, a polypeptide, a protein, a nucleotide, a nucleic acid, a polysaccharide, or a combination.

[00256] In some embodiments, the biomolecule may be a small organic molecule having a molecular weight less than about 100, 90, 80, 60, 50, 40, 30, 20, 10 kDa. In some embodiments, the biomolecule may be a small organic molecule having a molecular weight less than about 2000, 1500, 1200, 1000 Da.

[00257] Secreted Analyte. In various embodiments, the analyte secreted by the cell, e.g., bioproduct, may include a protein, a saccharide, a nucleic acid, an organic molecule other than a protein, saccharide, or nucleic acid. A secreted analyte (e.g., analyte) can diffuse in the media, and can comprise a broad range of molecular weights. In various embodiments, the analyte secreted by the biological micro-object may be a protein. The secreted analyte can comprise a molecular weight, wherein said molecular weight is proportional to a diffusion rate and therefore correlated with how much (e.g., the concentration) of the secreted analyte that accumulates in the chamber under a steady state equilibrium.

[00258] A secreted analyte may be a naturally expressed analyte (e.g., natively expressed) or may be a bioengineered analyte (e.g., a product resulting from gene insertion, deletion, modification and the like).

[00259] Reporter molecule. Methods disclosed herein can comprise one or more reporter molecules (e.g., detection reagents, reagents, reporter, etc.). Reporter molecules can be configured to covalently or non-covalently bind to a secreted analyte of interest. In methods disclosed herein, the reporter molecule bound to the secreted analyte is configured to generate a signal that can be detected using imaging, such that the signal (raw or processed using one or more methods disclosed here in) provides direct or indirect measure of diffusion related properties such as concentration and diffusion rate constant which are proportional to the molecular weight of the reporter and/or reporter bound with secreted analyte (e.g., RMSA complexes). Signal is proportional to one or more of the amount of accumulated protein/complex resulting from one or more of: the secretion rate of a biological micro-object, the number of biological micro-objects, and/or the fraction bound of the analyte.

[00260] A reporter molecule may include a binding component designed to bind the secreted analyte and also may include one or more detectable label(s). The binding component may be any suitable binding partner configured to bind the secreted analyte (e.g., with a binding constant less than 10 micromolar). The binding component may be an amino acid, a polypeptide, a nucleotide, a nucleic acid, a small organic molecule, or a combination thereof. The binding component specifically binds to the secreted analytes, specific binding comprises a preference for the secreted analyte over one or more other components on or within the microfluidic device. In some embodiments, the reporter molecule may be multi-valent, comprising more than one binding component to bind more than one copy of the secreted analyte or to more than one member of a family of secreted analytes. The stoichiometry of the RMSA complex can therefore vary for example one or more reporter molecules can bind to one or more secreted molecules, and additionally or alternatively one or more secreted molecules can bind to one or more reporter molecules. The reporter molecule or secreted analyte must be soluble and can diffuse is solution or media disposed within the microfluidic device. In some embodiments, introducing the first fluidic medium comprising the reporter molecule comprises allowing the reporter molecule to diffuse into the chamber. In some embodiments, allowing the reporter molecule to diffuse into the chamber comprises allowing the reporter molecule to reach an equilibrium between the flow region and the chamber. The term “equilibrium” as defined herein refers to a state in which the average quantity of species (e.g., reporter, analyte, and reporter analyte or RMSA complex) don’t change as a function of time. In some instances, an equilibrium condition can comprise a closed system that attains equilibrium from nonequilibrium initial conditions.

[00261] Detectable label. The reporter molecule may also include a visible, luminescent, phosphorescent, or fluorescent detectable label. In some embodiments, the detectable label may be a fluorescent label. Any suitable fluorescent label may be used, including but not limited to fluorescein, rhodamine, cyanine, phenanthrene or any other class of fluorescent dye label. In some embodiments, the detectable label is covalently attached directly or indirectly to a reporter molecule.

[00262] In some other embodiments, a capture oligonucleotide may be a binding component of a reporter molecule and either an intrinsic or extrinsic fluorescent dye may be the detectable label, such that the detectable label of the reporter molecule may not be detectable until the capture oligonucleotide binds the analyte, for example, an intercalating dye. In some embodiments, a detectable label of a reporter molecule may not be detectable until after the RMSA complex has formed, as the detectable signal is shifted to a new wavelength not present prior to binding.

[00263] As used herein, “a signal associated with the detectable label” or similar phases refers to a signal that is directly or indirectly emitted by the detectable label within an area of interest. In some embodiments, the signal associated with the detectable label is detected after a steady state equilibrium is reached. In other embodiments, the signal associated with the detectable label is detected while perfusing another fluidic medium that does not comprise the reporter molecule into the flow region. The term “steady state” as defined herein refers to an equilibrium condition comprising an open system, which sustains equilibrium, wherein the net change of a species (e.g., reporter, analyte, and reporter analyte or RMSA complex) is zero. The term “kinetics or kinetic regime” as defined herein refers to any system or quantified metric associated therewith, that is not at equilibrium, wherein the reactants and products (e.g., reporter, analyte, and reporter analyte or RMSA complex (e.g., reporter molecule: secreted analyte complex)) change as a function of time.

[00264] Penning cells. In some embodiments, disposing a cell into a chamber of a microfluidic device comprises: obtaining a microfluidic device comprising a microfluidic circuit comprising a flow region and a chamber fluidically connected to the flow region; introducing a fluidic medium comprising the cell into the flow region; and disposing the cell into the chamber. In some embodiments, the cell is disposed into a chamber of a microfluidic device by gravity or by OEP as described herein. In some embodiments, positive OEP or negative OEP can be selected depending on the surface charge of the cell to be moved. For example, mammalian cells are generally negatively charged at physiological pH so that a negative OEP can be applied to move the cells by compelling them toward a direction. In other examples, cells such as yeast cells are typically positively charged so that a positive OEP will be suitable for moving the cells.

[00265] In some embodiments, when the cell to be penned is small (e.g., nonmammalian cells, which tend to be smaller than mammalian cells), for example, smaller than 10 microns in diameter, the OEP is performed at a higher voltage. In some embodiments, the voltage is higher than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15V. In some embodiments, the diameter of the cell is about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 microns.

[00266] The process of penning cells may be automated using image recognition software as described in U.S. Application Publication No. US2019/0384963, filed on May 31, 2019, and U.S. Application Serial No. 17/103414, filed on November 24, 2020, each of which disclosures is herein incorporated in its entirety by reference. In various embodiments, the cells are moved into separate chambers, e.g., NanoPens, to be cultured as individual colonies. By penning a single cell to an individual chamber, expansion to a population of cells provides a clonal population. The ability to observe, test, and export selectively a specific clonal population of cells that exhibit desired characteristics, provided by the methods described herein, is an important improvement over the macroscale techniques currently used for developing engineered cell lines that can produce a desired bioproduct.

[00267] Cell culture and induction. The cell to be evaluated by any of the methods of the present disclosure is not limited. In some embodiments, the cell can be a eukaryotic cell or a prokaryotic cell. In certain embodiments, the cell is an animal cell, a plant cell, fungal cell, or a bacteria cell. In some embodiments, the cell is a fungal cell. In some embodiments, the cell is a yeast cell, including but not limited to a Saccharomyces cell (e.g., Saccharomyces cerevisiae) or a Pichia cell (e.g., Pichia pastoris). In some other embodiments, the cell is a bacterial cell, which may be, but is not limited to Escherichia coli (E. coli), or any other bacterial cell that may be engineered to produce a desired bioproduct.

[00268] In some embodiments, the cell is maintained in the chamber. In some embodiments, the cell is cultured and expands (i.e., proliferates) in the chamber into a clonal population. In certain embodiments, the cell expands to a number of cells of which the secretion of a molecule of interest is of a level sufficient to be detected by the method of the present disclosure.

[00269] In some embodiments, culturing in the chamber of the microfluidic device may include culturing a cell or a clonal population thereof within a volume of medium less than 5 nanoliters. In some variations, the macroscale reactor of which the bioproductivity is predictive of may have a volume of 100 mL, I L, 10 L, 100 L or more. In some variations, culturing in the chamber of the microfluidic device may include culturing under substantially similar conditions to conditions of culturing in the macroscale reactor. [00270] In some embodiments, the cell is cultured in the presence of a selected level of a component of a fluidic medium. In some embodiments, the component may be a nutrient for the clonal population of the cells. In some embodiments, the selected level of the component may be a growth limiting level of the component.

[00271] In some embodiments, cell is induced to secrete the molecule of interest. The induction can be performed according to the general knowledge of the cell. In some embodiments that the cell is engineered to secrete the molecule of interest, the induction can be performed based on the nature of the promoter constructed for the expression of the molecule of interest in the cell. In some embodiments that the cell is a yeast cell engineered with an AOX1 promoter, a BMMY medium or a BM1M medium is introduced into the flow region and allows to diffuse into the chamber to induce the secretion. Preferably, a BM1M medium is used to induce the secretion. In certain embodiments, the medium used for induction is oxygenated with at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of oxygen. In other embodiments, no induction is necessary for the cell to secrete the molecule of interest.

[00272] Assays. In light of the foregoing, the methods of the present disclosure provide a variety of different approaches to evaluate bioproductivity of a cell. Three of those variations are shown in FIGS. 7A 7C. FIG.7A shows a bead assay. FIG. 7B shows a diffusion gradient assay, and FIG. 7C shows an accumulation assay herein. FIGS. 7A-7C are top views of the chambers 805, and the chambers normally are disposed horizontally, similarly to the sequestration pens 124, 126, 128, 130 shown in FIG. 1A. The chambers 705 open laterally from the flow region, e.g., channel 780, where flow 770 is contained. As shown in FIGS. 7A-7C, each chamber 705 has an in situ-generated hydrogel barrier 740 or 750 formed within the chamber, dividing the chamber into two areas. The hydrogel barrier is generally introduced after cell(s) have already been introduced into the chamber. The first area is a culturing area 710, which is disposed distal from the hydrogel barrier, in relationship to the opening 715 of the chamber to the channel 780. This culturing area 710 is an unswept region of chamber, and no flow 770 from the channel 780 flows directly into that part of the chamber, even if the hydrogel barrier were not there. The second area 720 created by installation of the hydrogel barrier, is an assaying area and is located to the proximal side (in relation to the opening 715 of the chamber to the channel) of the hydrogel barrier 740 or 750.

[00273] As shown in FIG. 7A, an assay is provided (“bead assay”), where detection within the assay area 720 is performed by capturing secreted analyte to a bead 730. The secreted analyte diffuses either through the hydrogel barrier 740 or through a small gap between segments of the hydrogel barrier that is shown in FIG. 7A. The methods for the bead assay includes: disposing the cell into a chamber of a microfluidic device, the microfluidic device having a microfluidic circuit comprising a flow region and the chamber, wherein the chamber comprising an opening to the flow region; forming an in situ-generated barrier within the chamber, wherein the in situ-generated barrier defines within the chamber an assay area and an enclosed culture area for culturing the cell; disposing a micro-object in the assay area of the chamber, wherein the micro-object comprises a capture moiety configured to bind an analyte secreted by the cell; allowing the cell to secreting the analyte; introducing a first fluidic medium comprising a reporter molecule into the flow region, wherein the reporter molecule comprises a first detectable label and a binding component configured to bind to the analyte; and detecting a first signal associated with the first detectable label within an area of interest within the microfluidic circuit, thereby evaluating the bioproductivity of the cell.

[00274] In some embodiments, the in situ-generated barrier has a porosity that allows diffusion of the analyte through the in situ-generated barrier so that the analyte can bind the micro-object within the assay area. In some embodiments, the in situ-generated barrier used in the diffusion gradient assay comprises a gap (e.g., a bowtie barrier as described above) through which the analyte can diffuse (e.g., and thereby cross the in situ-generated barrier) while the micro-object cannot pass through the gap.

[00275] In some embodiments, the micro-object is a surface coated with the capture moiety. In certain embodiments, the micro-object is a bead coated with the capture moiety. The bead may be made of any suitable material, such as polymer, metal, ceramic, glass, or any combination thereof. The bead may be magnetic or may not be magnetic. As used herein, “capture moiety” is a chemical or biological species, functionality, or motif that provides a recognition site for the analyte. Further details of the bead assay may be found in International Application No. PCT/US2022/023598, entitled “Methods of Microfluidic Assay and Bioproduction from Non-Mammalian Cells and Kits Therefor” and filed on April 6, 2022.

[00276] FIG. 9A and FIG. 9B shows images of an exemplary bead assay of the present disclosure. FIG. 9A is a fluorescent image, and FIG. 9B is a brightfield image. In this experiment, four strains (Strain 8, Strain 9, Strain 10, Strain 11) of yeast were used and their bioproductivities had been pre-determined. Their bioproductivities were consistent with the numbering of the strains with Strain 11 being the best producer among the four, and Strain 8 the least productive. An in situ-generated barrier 940 having a v- shaped cap shape, like 640 of FIG. 6A was formed at mid-pen to contain the cells within the enclosed culture area 920. The bead 930 was positioned in the assay area and disposed between the v-shape of the barrier. As shown in the fluorescent FIG. 9A, the bead of Strain 11 exhibited the stronger intensity compared to the others, e.g., had the most reporter molecule labeling captured analyte. The bead of Strain 8 had the lowest intensity, which is consistent with the pre-determined bioproductivities. Additionally, it is noticeable that the bead of Strain 8 was brighter at the side close to the enclosed culture area in the fluorescence image FIG. 9A, indicating the RMS A was diffusing in the direction from the hydrogel barrier and did not saturate the side of the bead 930 that faces the opening of the chamber to the channel, only slightly shown in these figures.

[00277] As shown in FIG. 7B, an assay is provided (“diffusion gradient assay”) where detection of the secreted analyte occurs in the same region as that of the bead assay, but no bead is provided. Alternatively, an area of interest is selected within the assay area 720, and a labelling reagent is permitted to diffuse into the assay area and bind to the analyte of interest. A gradient profile of the labelled analyte, as it diffuses towards the opening of the chamber and into the channel can be obtained, providing an amount of fluorescence detected that is proportional to the amount of analyte. The detected amount of fluorescence may be used as a raw total, raw ranking or a raw score. The detected signal may also be further processed to provide a normalized (for biomass), corrected (for background unbound labelling reagent and the like), and/or normalized against other detected signals in other chambers on the chip to produce a total or score. Further details of how to conduct a diffusion assay are described in International Application Serial No. PCT/2017/027795, entitled “Methods, Systems, and Kits for In-Pen Assays”, filed on April 14, 2017, published as International Application Publication WO2017/1811135; International Application Serial No. PCT/US2018/055918, entitled “Methods, Systems, and Kits for In-Pen Assays”, filed on October 15, 2018, published as International Application Publication WO2019/075476; and International Application Serial No. PCT/US2021/021417, entitled “Methods, Systems, and Kits for In-Pen Assays”, filed on March 09, 2020, published as International Application Publication WO2021/184458, the entirety of each of which disclosures are herein incorporated by reference for any purpose.

[00278] The in situ-generated barrier has a first permeability with respect to the analyte and a second permeability with respect to the reporter molecule. In some embodiments, the first permeability is lower than the second permeability. Preferably, the in situ-generated barrier has a porosity that allows diffusion of the RMSA complex through the in situ-generated barrier. In some embodiments, the in situ-generated barrier used in the diffusion gradient assay comprises a gap (e.g., a bowtie barrier as described above) through which the RMSA complex can diffuse (e.g., and thereby cross the in situ-generated barrier). Because the analyte is secreted by the cell in the chamber and diffuse from the distal end of the chamber toward the proximal end thereof, a gradient of signal associated with the detectable label can be observed within the assay area.

[00279] In FIG. 7C, a third type of assay is shown. In this assay (“accumulation assay”), a hydrogel barrier 850 that spans the width of the chamber without interruption is introduced into the chamber. The chamber is divided into two areas, a culturing area 710 and second area 725 that is separated from cells. The secreted analyte is permitted to accumulate within the culturing area 720 and a culturing area 720 is selected within the culturing area, but in a portion of the culturing area where cells are not disposed. This permits a cell/colony of cells to secrete an analyte and accumulate within area 720. The hydrogel barrier in this case does not readily permit diffusion of the secreted analyte across the hydrogel, so the secreted analyte will accumulate. This is particularly useful if the rate of secretion of the analyte is low. The labelling reagent is introduced from the channel into the chamber by diffusion. Because the labelling reagent is selected to have a smaller molecular weight (size), the labelling reagent can diffuse from area 725 through the hydrogel barrier 750, and into the culturing area 710/Assay area 720 and bind to the secreted analyte.

[00280] The in situ-generated barrier has a first permeability with respect to the analyte and a second permeability with respect to the reporter molecule. In some embodiments, the first permeability is lower than the second permeability. In other words, a diffusion rate of the analyte diffusing out of the barrier is slower than a diffusion rate of the reporter molecule diffusing. Given that the RMSA complex is a larger than the secreted analyte, its diffusion rate would be even slower, resulting in the RMSA complex accumulating with the enclosed culture area. In some embodiments, to offer a better accumulation of the RMSA complex, the in situ-generated barrier has a porosity that impedes diffusion of the RMSA complex through the in situ-generated barrier. In certain embodiments, the porosity of the in situ-generated barrier substantially prevents diffusion of the RMSA complex through the in situ-generated barrier. In some embodiments, the area of interest is within the enclosed culture area. In some embodiments, the area of interest does not include a portion of the in situ-generated barrier. [00281] An image can be taken after a steady state equilibrium is reached as described above for detecting a signal associated with the detectable label of the reporter molecule. As shown in FIGS. 8 A and 8B, fluorescence images of the chambers 805, which open to channel 880, where hydrogel barriers 850 have been introduced in each chamber at the same point distal from the opening of the chamber to the channel. FIG 8A is taken at a timepoint when labelled reporter molecule is being flowed through channel 880 and has diffused into the pens 805. Fluorescent signal is observed in both the channel and chambers, and additionally in the culturing area 820. Here, the free reporter molecule (e.g.., unbound) in the channel and the chamber and the RMSA both emit fluorescent signals. A range of intensities of signal can be observed at the distal end of the chambers, representing different productivities of the cells cultured in each chamber. Among them, better producers can be identified, two of which 872 are annotated for both FIG. 8A and 8B, and the cells can be exported.

[00282] A fluorescence image can also be taken at a later timepoint, while flushing a fluidic medium that does not comprise the reporter molecule into the microfluidic device as described above, as shown in FIG. 8B. The channel in this image is dark because the fluidic medium that is perfused at this moment does not comprise the reporter molecule. The in situ-generated barrier 850 is not clearly seen in this image but it is at the same position as indicated in FIG. 8A. A range of intensities of signal can also be observed at the distal end of the chambers in the culturing area 820, showing differential signal still accumulated within the culturing areas below the barrier 850. These show different productivities of the cells cultured in each chamber. It can be seen that the signal in positive secretor chambers 872 is brighter than other chambers, correlating with the observation from FIG. 8A. Alternatively, two poorly performing colonies are identified at chambers 874, where the image is slightly less fluorescent in FIG. 8A and very dim in FIG. 8B. This demonstrates that while substantial amounts of reporter molecule have diffused in the respective culturing areas 820, once flushing with medium carrying no labelling molecule has been performed, unbound reporter molecule has diffused out and little to no bound RMSA is found.

[00283] Without intending to be bound by theory, the bead assay may be used for evaluating a predicted secretion level of about 0.01 mg/liter to about 0.25 mg/liter; a diffusion gradient assay may be used for a predicted secretion level of from about 20 micrograms/ml (about 1 picogram/cell/day or about 7 attomoles/cell/day) to about 2500 micrograms/ml ( about 17 picograms/cell/day) ; and an accumulation assay may be used for a predicted secretion level of about 0.1 mg/liter to more than about 2.5 mg/liter.

[00284] Many different variations of assays may be used to assess bioproductivity of a cell or a clonal population derived therefrom. Alternative assays may be adapted as suitable for the particular use case, and a variety configurations of hydrogel barriers may be used. For example, in another type of assay, a hydrogel barrier may be generated in situ, more distal to the opening of the chamber to the flow region, creating a culture region proximal to the barrier and an assay region distal to the barrier. FIG. 7D shows this variation, where sequestration pen 715 has a hydrogel barrier 750 generated at a point distal relative to the mid point of the length of the pen. Cells were imported after generation of the hydrogel barrier 750 and are maintained and expanded in culture region 710, proximal to the hydrogel barrier but still within the isolation region of the pen 715. The area 720 was then used as an assay region, where analytes produced by the cells permeate through hydrogel barrier 750. The permeability of the barrier may be tuned to choose the molecular weight limit that the hydrogel barrier will permit through to the culture area 720.

HYDROGEL FUNCTIONALIZATION AND CAPTURE HYDROGELS

[00285] A hydrogel barrier formed within a selected area with a microfluidic device can serve not only as a physical barrier but also a surface for capturing a molecule of interest so that the molecule of interest can be retained and concentrated within a selected area for detection or assays. In some embodiments, in order to capture a molecule of interest, the hydrogel barrier might need to be functionalized with a capture moiety.

[00286] Therefore, in another aspect, a method of functionalizing an in situ-generated hydrogel within a microfluidic device is provided, where the microfluidic device has an enclosure comprising a base, a cover, and microfluidic circuit material defining a fluidic circuit therein. The fluidic circuit includes a flow region and a plurality of chambers opening to the flow region. The process includes: introducing at least one hydrogel barrier in at least some chambers of a plurality of chambers, wherein the at least one hydrogel barrier is any hydrogel barrier described herein, and is introduced by any method described herein, wherein at least one of the first and the second polyethylene glycol polymer moieties of the hydrogel further includes a functionalizable moiety configured to react with a moiety other than a thiol moiety or a norborenyl moiety. In some embodiments, the functionalizable moiety includes a biotin, an aldehyde, a succinimidyl moiety, or an oligonucleotide.

[00287] Introducing the functionalizable moiety before in situ hydrogel formation. In some embodiments, the functionalizable moiety is introduced to a prepolymer or a crosslinker before a prepolymer composition comprising the prepolymer and the crosslinker is introduced into a microfluidic device. In some embodiments that the prepolymer is a polyethylene glycol polymer as described above, a reactive moiety R _x of the polyethylene glycol polymer can be replaced with a functionalizable moiety. For example, an 8-arms PEG norbomene can be modified to replace the norbornene moiety of one of the eight arms with a functionalizable moiety, such as a biotin. In some other embodiments, a crosslinker can be modified to replace a crosslinker moiety RXP with a functionalizable moiety. For example, a thiol moiety of DTT or a thiol moiety of a 4-arm PEG thiol crosslinker can be coupled with a biotin. Therefore, after the gelation of the hydrogel in situ within the microfluidic device, the first and/or the second polyethylene glycol polymer moieties of the hydrogel can include the functionalizable moiety configured to react with a moiety other than a thiol moiety or a norborenyl moiety. In some embodiments, the functionalizable moiety includes a biotin, an aldehyde, or a succinimidyl moiety.

[00288] Introducing the functionalizable moiety after in situ hydrogel gelation (post modification). In some embodiments, the functionalizable moiety is introduced to the hydrogel after the hydrogel is formed in situ within the microfluidic device. In such embodiments, the process further comprises, after the hydrogel is formed at a selected area, introducing a molecule having a functionalizable moiety and a reactive moiety, wherein the reactive moiety is configured to react with a thiol group on the formed hydrogel (e.g., a free thiol group of the crosslinker) thereby functionalizing the hydrogel via a thiol-ene reaction. In some embodiments, the reactive moiety can be a sulfhydryl-reactive moiety or a moiety comprising an unsaturated bond, including but not limited to a maleimide moiety, an alkyne moiety, or an olefin (alkene) moiety. In some examples, the molecule having the functionalizable moiety and the reactive moiety can be a maleimide PEG biotin reagent with the biotin being the functionalizable moiety and the maleimide being the reactive moiety.

[00289] Introducing the functionalizable moiety during the in situ hydrogel gelation. In other embodiments, the functionalizable moiety is introduced to the hydrogel during in situ gelation of the hydrogel within the microfluidic device. In such embodiments, a molecule having a functionalizable moiety and a reactive moiety can be doped in the prepolymer composition. The reactive moiety is configured to react with the reactive moiety R _x of the first polyethylene glycol polymer and/or the second polyethylene glycol polymer upon photoactivation. For example, the reactive moiety of the molecule can comprise a dithiol moiety, which can be reduced into free thiol groups. The process can comprise treating the molecule with a reducing reagent (e.g., Zrz (2-carboxyethyl)phosphine; TCEP) to reduce the dithiol moiety, doping the treated molecule into the prepolymer composition, introducing the prepolymer composition into the microfluidic device and activating the solidification of the prepolymers at a selected region within the microfluidic device. The free thiol moieties of the molecule will react with the reactive moiety R _x of the first polyethylene glycol polymer and/or the second polyethylene glycol polymer thereby introducing the functionalizable moiety to the hydrogel.

[00290] In some embodiments, the molecule having the functionalizable moiety and the reactive moiety is a dithiol-PEG-biotin reagent such as lipoic acid-PEG-biotin, where the biotin is the functionalizable moiety and the dithiol moiety is the reactive moiety. In some specific embodiments, the dithiol-PEG- biotin reagent is lipoic acid-PEG3 -biotin or lipoic acid-PEGl 1 -biotin. In another specific embodiment, the molecule having the functionalizeable moiety and the reactive moiety is an oligonucleotide (being the functionalizable moiety) conjugated with a reactive moiety, wherein the reactive moiety can be but not limited to dithiol phosphorami di te (DTP A) or S-Trityl-6-mercaptohexyl-l-[(2-cyanoethyl)-(N,N- diisopropyl)]-phosphoramidite (5'-Thiol-Modifier C6).

[00291] Hydrogel comprising a functionalizable moiety. A hydrogel comprising a functionalizable moiety is provided. In some embodiments, the hydrogel comprising a functionalizable moiety can be provided by method described above.

[00292] In one aspect, the hydrogel comprises a first polyethylene glycol polymer moiety covalently linked to a first end of a first crosslinker moiety, wherein a second end of the first crosslinker moiety is covalently linked to a second polyethylene glycol polymer moiety, wherein the first and the second polyethylene polymer molecule comprises different polyethylene glycol moieties or each comprises a same polyethylene glycol moiety, and wherein at least one of the first and the second polyethylene glycol polymer moieties further comprises a first functionalizable moiety configured to react with a moiety other than a thiol moiety or a norborenyl moiety; and/or wherein the crosslinker moiety further comprises a second functionalizable moiety configured to react with a moiety other than a thiol moiety or a norborenyl moiety. [00293] In another aspect, a hydrogel comprising a functionalizable moiety is provided. The hydrogel comprises a polymer network comprising a plurality of the first polyethylene glycol polymer moieties, a plurality of the second polyethylene glycol polymer moieties, and a plurality of crosslinker moieties, wherein a first end of a first crosslinker moiety of the plurality of crosslinker moieties is covalently linked to one of the plurality of the first polyethylene glycol polymer moieties and a second end of the first crosslinker moiety moieties is covalently linked to one of the plurality of the second polyethylene glycol polymer moieties, and wherein a first end of a second crosslinker moiety of the plurality of crosslinker moieties is covalently linked to one of the plurality of the first polyethylene glycol polymer moieties or one of the plurality of the second polyethylene glycol polymer moieties, wherein at least one of the first and the second polyethylene glycol polymer moieties further comprises a functionalizable moiety, or a second end of the second crosslinker moiety is linked to a functionalizable moiety.

[00294] For example, a hydrogel incorporating an 8-arm polyethylene glycol moiety as PEG1 and/or PEG2, may have 7-arms that have been crosslinked, and the remaining arm may include the functionalizable moiety that does not react with thiol or norbomenyl moieties. In some embodiments, the functionalizable moiety may be a biotin. In other embodiments, functionalizable moiety may be an aldehyde. In yet other embodiments, the functionalizable moiety may be a succinimidyl moiety.

[00295] For example, a crosslinker comprising a 4-arm polyethylene glycol moiety may have 3-arm that have been crosslinked, and the remaining arm may include the functionalizable moiety that does not react with thiol or norbomenyl moieties. For example, a crosslinker comprising two thiol moieties may have one of the thiol moieties crosslinked with a norbornene moiety of PEG1 or PEG 2, and the remaining thiol moiety linked to the functionalizable moiety. In some embodiments, the functionalizable moiety may be a biotin. In other embodiments, functionalizable moiety may be an aldehyde. In yet other embodiments, the functionalizable moiety may be a succinimidyl moiety.

[00296] Functionalization reagent. The process can further include introducing a functionalization reagent comprising a functionalizing reaction pair moiety (i.e. a coupling partner) configured to react with the functionalizable moiety of the hydrogel barrier; contacting the functionalization reagent with the hydrogel barrier; and coupling the functionalization reagent to the hydrogel barrier. In some embodiments, the functionalization reagent can comprise the functionalizing reaction pair moiety and a capture moiety. The capture moiety is configured to bind to a molecule of interest present, being secreted/produced (e.g., by a cell), being introduced in a microfluidic device In some embodiments, the functionalizable moiety is coupled to a coupling partner covalently linked to a detectable label. In some embodiments, the functionalization reagent may include a streptavidin functionalizing reaction pair moiety configured to couple with the biotin moiety of the hydrogel. In some embodiments, the functionalization reagent may further include a detectable label, which can be a visible label, a fluorescent label, or a luminescent label. In some embodiments, the functionalizing reaction pair moiety and the capture moiety can be independently a protein, a peptide, an oligonucleotide, an organic molecule, or a saccharide.

[00297] Kit. In another aspect, a kit for providing a hydrogel comprising a functionalizable moiety is provided. The kit can comprise a prepolymer composition as described above and, in some embodiments of post modification, a reagent comprising a molecule having a molecule having a functionalizable moiety and a reactive moiety, wherein the reactive moiety is configured to react with a thiol group on the formed hydrogel. In some embodiments, the reactive moiety can be a sulfhydryl -reactive moiety or a moiety comprising an unsaturated bond, including but not limited to a maleimide moiety, an alkyne moiety, or an olefin (alkene) moiety. In some examples, the molecule having the functionalizable moiety and the reactive moiety can be a maleimide PEG biotin reagent with the biotin being the functionalizable moiety and the maleimide being the reactive moiety.

[00298] In some other embodiments, the prepolymer composition is doped with a molecule having a functionalizable moiety and a reactive moiety, wherein the reactive moiety of the molecule can comprise a dithiol moiety. In some specific embodiments, the dithiol-PEG-biotin reagent is lipoic acid-PEG3 -biotin or lipoic acid-PEGl l -biotin. In another specific embodiment, the molecule having the functionalizeable moiety and the reactive moiety is an oligonucleotide (being the functionalizable moiety) conjugated with a reactive moiety, wherein the reactive moiety can be but not limited to dithiol phosphoramidite (DTP A) or S-Trityl-6-mercaptohexyl-l-[(2-cyanoethyl)-(N,N-diisopropyl) ]-phosphoramidite (5'-Thiol -Modifier C6). In some embodiments, the dithiol moiety of the reactive moiety is reduced. In some embodiments, the kit can further comprise a reducing reagent for reducing the dithiol moiety of the reactive moiety. The reducing reagent can be but not limited to TCEP.

[00299] Assaying a micro-object in a microfluidic device using capture hydrogel. In another aspect, a method for assaying a micro-object in a microfluidic device is provided. The microfluidic device can be as described herein. In some embodiments, the microfluidic device comprises an enclosure comprising a base, a cover, and microfluidic circuit material defining a fluidic circuit therein, and further wherein the fluidic circuit of the microfluidic device comprises a flow region and a chamber opening to the flow region. The method can comprise disposing a micro-object in the microfluidic device, introducing a first in situ-generated hydrogel into the microfluidic device, wherein the first in situ-generated hydrogel is functionalized with a first capture moiety configured to bind a first molecule of interest, allowing a biomolecule produced by the micro-object to interact with the first capture moiety within the microfluidic device; and detecting a first interaction between the first capture moiety and the biomolecule.

[00300] The first in situ-generated hydrogel can be a hydrogel according to the present disclosure. In some embodiment, the first in situ-generated hydrogel is introduced according to the methods of introducing hydrogel barriers of the present disclosure. Furthermore, the first in situ-generated hydrogel can have functionalizable moieties and can be functionalized with the first capture moiety.

[00301] In some embodiments, the first in situ-generated hydrogel comprises a reversible moiety. In certain embodiments, the first in situ-generated hydrogel comprises a structure of Formula (1) or Formula (7) as described above. In some embodiments, allowing a biomolecule produced by the micro-object to interact with the first capture moiety comprises binding the biomolecule with the first capture moiety. In further embodiments, the method further comprises introducing a reversing reagent configured to react on the vic-diol moiety or the peptide moiety thereby reversing the hydrogel barrier and releasing the bound biomolecule from the first capture moiety. In this way, the biomolecule produced by the microobject can be retained within the chamber and controllably released at a subsequent time point. [00302] In some embodiments, the method further comprises introducing a second in situ-generated hydrogel into the microfluidic device, wherein the second in situ-generated hydrogel is functionalized with a second capture moiety configured to bind a second molecule of interest, allowing a biomolecule produced by the micro-object to interact with the first capture moiety and the second capture moiety within the microfluidic device, and detecting a first interaction between the first capture moiety and the biomolecule and/or a second interaction between the second capture moiety and the biomolecule.

[00303] The second in situ-generated hydrogel can be introduced according to the methods of introducing hydrogel barriers of the present disclosure. Furthermore, the second in situ-generated hydrogel can have functionalizable moieties and can be functionalized with the second capture moiety respectively as described above according to the present disclosure.

[00304] In some embodiments, the first in situ-generated hydrogel is functionalized with the first capture moiety by introducing a first functionalization reagent comprising a first functionalizing reaction pair moiety and the first capture moiety; and contacting the first functionalization reagent with the first in situ- generated hydrogel thereby coupling the first functionalization reagent to the first in situ-generated hydrogel. Similarly, the second in situ-generated hydrogel is functionalized with the second capture moiety by introducing a second functionalization reagent comprising a second functionalizing reaction pair moiety and the second capture moiety; and contacting the second functionalization reagent with the second in situ-generated hydrogel thereby coupling the second functionalization reagent to the second in situ -generated hydrogel. In some embodiments, wherein the first capture moiety and the second capture moiety are independently a protein, a nucleic acid, an organic molecule, a saccharide, a combination thereof.

[00305] The first in situ-generated hydrogel can comprise a first functionalizable moiety configured to couple with the first functionalizing reaction pair moiety, and the second in situ-generated hydrogel can comprise a second functionalizable moiety configured to couple with the second functionalizing reaction pair moiety. It is favorable that the first functionalizable moiety and the second functionalizable moiety are different so that the first functionalizable moiety does not couple with the second functionalizing reaction pair moiety, and the second functionalizable moiety does not couple with the first functionalizing reaction pair moiety. In this way, after the first in situ-generated hydrogel comprising the first functionalizable moiety and the second in situ-generated hydrogel comprising the second functionalizable moiety are introduced within the microfluidic device, a cocktail reagent comprising the first functionalization reagent and the second functionalization reagent can be introduced to functionalize the first in situ-generated hydrogel and the second in situ-generated hydrogel respectively in one experimental step.

[00306] In certain embodiments, the first functionalizable moiety comprises a first oligonucleotide comprising a first sequence, and the first functionalizing reaction pair moiety comprises a first complementary oligonucleotide comprises a sequence complementary to the first sequence; the second functionalizable moiety comprises a second oligonucleotide comprising a second sequence, and the second functionalizing reaction pair moiety comprises a second complementary oligonucleotide comprises a sequence complementary to the second sequence. [00307] In such embodiments, the first functionalization reagent can be a first oligonucleotide-antibody conjugate comprising the first oligonucleotide and a first antibody, and the second functionalization reagent can be a second oligonucleotide-antibody conjugate comprising the second oligonucleotide and a second antibody. The first antibody and the second antibody can be configured to recognize different antigens, which can be a first biomolecule and a second biomolecule produced by the biological microobject.

[00308] For example, in certain embodiments, the micro-object can be a cell. In certain embodiments, the micro-object can be a T cell, and the biomolecule can be a cytokine comprising but not limited to CCL-11, GM-CSF, Gran B, IFN-g, IL-10, IL-12, IL-13, IL-15, IL-17A,IL-17F, IL-lb, IL-2, IL-21, IL- 22, IL-4, IL-5, IL-6, IL-7, IL-8, IL-19, IP-10, MCP-1, MCP-4, MIP-lalpha, MIP-lbeta, perforin, RANTES, TGFbetal, TNF-alpha, TNF-beta, sCD137, and sCD4. In other embodiments, the microobject can be a virus-producing cell, and the biomolecule can be a viral particle comprising but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. In yet other embodiments where the micro-object is a virus-producing cell, the biomolecule can comprise a full viral particle comprising a payload of interest and an empty viral particle without the payload of interest. In some other embodiments, the micro-obj ect can be a bead configured to express a protein of interest within a chamber of the microfluidic device. In certain embodiments, the bead is conjugated with a nucleic acid configured to encode a protein of interest.

[00309] In some embodiment, the first in situ-generated hydrogel is disposed at a first location and the second in situ-generated hydrogel is disposed at a second location, and the first location and the second location are physically distinguishable. In such embodiments, signals associated with the first interaction and the section interaction can be determined respectively based on the locations where the signals are detected. In other words, it is not necessary to use different labels (e.g., fluorescent labels) to determine/distinguish the first interaction and the second interaction.

[00310] Detecting the first interaction and/or detecting the second interaction can comprise detecting a first signal from the first location and/or a second signal from the second location. In some embodiments, detecting the first interaction comprises introducing a first detection reagent comprising a first detectable label to a region adjacent to the first in situ-generated hydrogel; and detecting the second interaction comprises introducing a second detection reagent comprising a second detectable label to a region adjacent to the second in situ-generated hydrogel. The first detectable label and the second detectable label can be independently a fluorescent label, a colorimetric label, or a luminescent label. Preferably, the first detectable label and the second detectable label are spectrally distinct.

[00311] In some embodiments, the first location and the second location are both within a chamber of the microfluidic device where the biological micro-object is disposed. In certain embodiments, the first in situ-generated hydrogel can be formed on a wall of the chamber, and the second in situ-generated hydrogel can be formed on another wall of the chamber. In yet other embodiments, the first in situ- generated hydrogel and the second in situ-generated hydrogel are formed on the same wall of the chamber. In some embodiments, a plurality of the first in situ-generated hydrogels and a plurality of the second in situ-generated hydrogel can be formed. CELL EXPORTATION AND GEOMETRY OF THE IN SITU-GENERATED HYDROGEL BARRIER

[00312] The in situ-generated barrier of the methods of the present disclosure provides advantages of containing a cell within the chamber and accumulation effects for assays. In some embodiments, the methods of the present disclosure further comprise exporting a cell from the chamber, e.g., unpenning the cell. In some embodiments, the cell is further exported out of the microfluidic device. In some embodiments, exporting the cell comprises directing a laser illumination upon a selected area of the chamber, as mentioned above, to create a bubble pushing the cell toward the opening of the chamber. In other embodiments, exporting the cell comprises directing laser illumination upon a selected area of the chamber to create a bubble dislodging the cell and di electrophoretic forces may be used to move the cell once it is dislodged from the surface of the culturing area.

[00313] Without intending to be limited by theory, the laser illumination is set to project on a thermal target on a surface of the chamber to generate heating in the fluidic medium surrounding the thermal target may nucleate and propagate bubbles. The bubble, upon collapse, creates a cavitating force. In other embodiments, the bubble may be grown by continued illumination to create a shear flow of fluid directed towards nearby substances. The force and/or the flow can push the in situ-generated barrier, fluidic medium, and the cell away, typically in a direction towards the opening of the chamber. As a result, once the in situ-generated barrier no long holds its original position containing the cell, the cell can be moved out of the chamber. In some embodiments, the cell is moved by the bubble to a location close to an opening of the chamber to the microfluidic channel, and a OEP force is applied to further move the cell into the microfluidic channel where later the cell is flushed out of the microfluidic device by a flow introduced therein. In some other embodiments, once the laser pulse has been stopped the induced bubble collapses resultingly drawing fluid back towards the distal end of the chamber.

[00314] The site of illumination may be selected to be any discrete selected region of the microfluidic device, as may be useful. In some embodiments, the discrete selected region of illumination may be a location within a chamber (e.g., a sequestration pen) of a microfluidic device. In various embodiments, the discrete selected region of illumination is located within an isolation region of a sequestration pen, which may be configured like any sequestration pen described herein. In some embodiments, the laser illumination is projected at a cell-free area of the chamber. In some embodiments, the laser illumination is projected at an area near the distal end of the chamber. In certain embodiments, an OEP force is applied first to move the cell cultured in the chamber away from the distal end thereof to create a cell-free area, and then a laser illumination can be applied at the cell-free area to create a bubble. One method of exporting cells using laser illumination in the presence of a hydrogel barrier is further discussed below and is shown in FIGS. I6A-I6F.

[00315] The optical illumination, power of the laser, and other information regarding bubble dislodgement have been described, for example, in U.S. Patent No. 10,829,728 (issued on November 10, 2020) and U.S. Publication No. 20220033758 (published on February 3, 2022), each of which disclosures is incorporated herein by reference in its entirety. [00316] The geometry of the in situ-generated barrier can be selected to facilitate the cell exportation. Without intending to be bound by any theory, in some embodiments, the in situ-generated barrier is designed to have a structurally vulnerable portion so that, upon application of a threshold pressure (for instance, a force generated by the bubble created by the laser illumination), the in situ-generated barrier can be deformed, flipped or slipped from its original position, or changed of position resulting in an open space for a cell to be moved through. In other words, in those embodiments, the in situ-generated barrier will no longer impede or block the cell after the laser illumination.

[00317] In some embodiments, the in situ-generated barrier comprises one or more discrete sections, each of which is moveably connected to one or more surfaces of the chamber. As used here, “moveably connected” describes that the discrete section can be moved upon a threshold pressure. Therefore, application of a threshold pressure to the one or more discrete sections of the in situ-generated barrier moves at least one of the one or more discrete sections with respect to the one or more surfaces of the chamber and thereby creates an opening in the enclosed culture area. The opening can facilitate the exportation of the cell out of the chamber. It may be desired to select a hydrogel barrier design that provides non-uniformity in a center point of the width of the chamber, decreasing the width, thickness or height of the hydrogel gels/segments comprising the barrier as the forces created by the laser illumination are directed more forcefully there.

[00318] In some embodiments, the in situ-generated barrier comprises two or more discrete sections, wherein adjacent sections are separated from one another by a gap. In some embodiments, the in situ- generated barrier consists of (or consists essentially of) two discrete sections which are separated from one another by a gap. In some embodiments, the gap can be designed to cross the in situ-generated barrier and have an axis aligning with an axis of the chamber at an angle of 0°, 5°, 10°, 15°, 20°, 30°, 40°, 50°, 60°, 70°, 80°, or 90°, or any number between any two of listed numbers. In some embodiments, the in situ-generated barrier can have one, two, or more gaps, each of which may be on the order of the diameter of a single cell size, for example, O. lx to 3. Ox, 0.2x to 2.5x, 0.3x to 2. Ox, 0.4x to 1.5x, 0.5x to l.Ox, or any range defined by two of the foregoing endpoints, where x is the average diameter of the cell.

[00319] In some embodiments, the structurally vulnerable portion can be of a suitable thickness. For instance, in some embodiments, a portion of the in situ-generated barrier has a thickness that is smaller than the height of the chamber.

[00320] In some embodiments, the in situ-generated barrier comprises a non-uniform thickness with respect to an axis of the chamber such that a portion of the in situ-generated barrier is less thick than other portions of the in situ-generated barrier. In some embodiments, the less thick portion of the in situ- generated barrier has a thickness that is smaller than the height of the chamber.

[00321] Accordingly, various exemplary shapes of in situ-generated barrier can be formed within the chamber, as shown in FIG 6A. These include, but are not limited to a rectangular fully capping barrier (i.e. a fully sealed cap, 605, a center bar 610, a rectangular half bar 615, a side bars (two discrete side rectangular bars separated by a gap and extended from both sides of the wall, 620, a “bowtie” (two discrete side triangular bars separated by a gap and extended from both sides of the wall, 625, a single triangular bar 630, a V-shaped bar 635, and a V-cap (a rectangular bar having a v shaped depression on the side of the barrier facing the opening to the channel, 640. The center bar has two gaps (arrowed) respectively on each side between the walls of the chamber. The side bars, bowtie and single triangle bar both have a single gap (arrowed). The size of the gap can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 microns or any range defined by two of the foregoing endpoints.

[00322] Further the size of the components of the hydrogel barrier may vary considerably. As shown in the photographs of FIGS. 6B-6C), where two embodiments of the “bowtie” barrier are shown. These examples of nonuniform barriers may have a variety of dimensions that are nonuniform. Non-uniformity may be located in the width (pen wall to pen wall dimension), in the “thickness” dimension (from distal- to-pen opening to proximal -to-pen opening dimension), and/or the “height” dimension (from inner surface of the substrate to the inner surface of the cover of the microfluidic device, e.g., z-dimension). The non-uniformity in the “thickness” dimension, e.g., the center of the “bowtie”, provides a convenient and standard location for bead disposition for the assay. The “bowtie” barrier may have a “thickness” at its largest dimension of the triangular shaped individual gel form of about 15 microns. This dimension is not limiting, and the “thickness” of the bowtie may be less than about 15 microns, less than about 12 microns, less than about 10 microns, less than about 8 microns, or less than about 5 microns. In other embodiments, the “thickness” of a triangular segment of the “bowtie” barrier may be more than about 10 microns, more than about 15 microns, or more than about 18 microns. As shown in FIG. 6B-6C, two different embodiments of hydrogel barriers having a “bowtie” non-uniform shape are shown, where the dimension 760 of the triangular segment forming the barrier (“thickness” dimension) is larger in the barrier of FIG. 6C than the dimension of the segment 750 of the barrier of FIG.6B. Since the pens are of the same width, it can be seen that the barrier formed in FIG. 6B has a distinct gap between the two barriers, while the barrier of FIG.6C does not have an obvious gap. While in the embodiment of FIG. 6B, a few cells have escaped the culture area below the barrier, the barrier of FIG. 6C does not permit cells to move past the barrier.

[00323] However, the use of hydrogel barriers does not limit the ability to selectively export cells from chambers having hydrogel barriers. As discussed in detail in Example 1-5, and shown in FIGS. 16A-16F, export of desired cells, at a user-defined timepoint may be performed.

[00324] Methods of Reducing Clonal Risk. In another aspect, a method of reducing a risk of loss of clonality within a microfluidic device is provided, wherein the microfluidic device has an enclosure comprising a base, a cover, and microfluidic circuit material defining a fluidic circuit therein. The fluidic circuit includes a flow region and a plurality of chambers opening to the flow region. The process includes: introducing a biological micro-object into one of the plurality of chambers; assaying or culturing the biological micro-object or a daughter micro-object thereof, thereby identifying a biological microobject or daughter micro-object thereof of interest; identifying the chamber containing the biological micro-object or daughter micro-object of interest; introducing a photoactivatable flowable polymer composition into the flow region of the microfluidic device; diffusing the composition from the flow region into the plurality of chambers; activating crosslinking of the composition in a selected area of the microfluidic device, thereby forming a hydrogel barrier at the opening of chambers other than the identified chamber, thereby producing a subset of capped chambers of the plurality of chambers; and unpenning and exporting the biological micro-object or daughter micro-object thereof of interest, thereby preserving clonality of the biological micro-object or daughter micro-object thereof of interest. The composition may include modified polyethylene glycol moieties. In some embodiments, the composition may be a composition described herein that produce a thiol-ene hydrogel, having any features described in any combination.

[00325] In yet another aspect, a method of reducing a risk of loss of clonality within a microfluidic device, is provided wherein the microfluidic device has an enclosure comprising a base, a cover, and microfluidic circuit material defining a fluidic circuit therein. The fluidic circuit includes a flow region and a first chamber and a second chamber each opening to the flow region. The process includes: introducing a plurality of biological micro-objects into the first chamber; actively separating a second portion of the plurality of biological micro-objects from a first portion remaining in a first chamber and disposing the second portion into the second chamber; introducing a photoactivatable flowable polymer composition having a reversible crosslinker into the flow region of the microfluidic device; diffusing the composition from the flow region into the plurality of chambers; activating crosslinking of the composition in a selected area of the microfluidic device, thereby forming a hydrogel barrier at the opening of the second chamber and preserving the second portion of the biological micro-objects;

[00326] assaying the first portion of the plurality of biological micro-obj ects in the first chamber, thereby identifying one or more biological micro-objects or daughter micro-objects thereof of interest;; removing biological micro-objects or assay materials from the first pen; reversing the reversible hydrogel barrier at the second chamber; and unpenning and exporting the one or more biological micro-objects or daughter micro-objects thereof of interest from the second chamber, thereby preserving clonality of the biological micro-object or daughter micro-object thereof of interest.

[00327] In some other aspects, a method of reducing a risk of loss of clonality within a microfluidic device, is provided wherein the microfluidic device has an enclosure comprising a base, a cover, and microfluidic circuit material defining a fluidic circuit therein. The fluidic circuit includes a flow region and a plurality of chambers opening to the flow region. The process includes: introducing a biological micro-object into one of the plurality of chambers; introducing a first photoactivatable flowable polymer composition into the flow region of the microfluidic device; diffusing the first composition from the flow region into the plurality of chambers; activating crosslinking of the first composition in a selected area of the microfluidic device, thereby forming a first hydrogel barrier within a first sub-set of the plurality of chambers of the microfluidic device; assaying or culturing the biological micro-object or a daughter micro-object thereof, thereby identifying one or more biological micro-objects or daughter micro-objects thereof of interest; identifying one or more chambers containing one or more biological micro-objects or daughter micro-objects of interest; introducing a second photoactivatable flowable polymer composition into the flow region of the microfluidic device; diffusing the second composition from the flow region into the plurality of chambers; activating crosslinking of the second composition in a selected area of the microfluidic device, thereby forming a second hydrogel barrier at the opening of chambers other than the identified chambers, thereby producing a second capped subset of chambers of the plurality of chambers; and unpenning and exporting the one or more biological micro-objects or daughter micro-objects thereof of interest from the identified chambers, thereby preserving clonality of the biological micro-object or daughter micro-object thereof of interest.

[00328] The first and the second composition may include modified polyethylene glycol moieties. In some embodiments, the first and the second composition may be a composition described herein that produce a thiol-ene hydrogel, having any features described in any combination.

[00329] In some embodiments, the first composition may include a crosslinker providing a non- reversible hydrogel once formed. In some embodiments, the crosslinker has a structure of Formula (5):

HS-LB4-SH Formula (5)

[00330] wherein linker backbone LB4 comprises 3 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms. In some embodiments, the linker backbone LB4 has a linear backbone having carbon atoms. In some embodiments, the linear backbone of LB4 has no silicon, nitrogen, oxygen, sulfur or phosphorus atoms.

[00331] In some embodiments, the first hydrogel barrier creates a first area proximal to the opening of the chamber to the flow region and a second area distal to the opening, where the second area contains the biological micro-obj ect or the daughter micro-obj ect thereof. In some embodiments, the first hydrogel barrier comprises one or more discrete sections, each of which is moveably connected to one or more surfaces of the chamber, that substantially prevents biological micro-object or the daughter micro-object thereof from crossing through the first hydrogel barrier. In some embodiments, application of a threshold pressure to the one or more discrete sections of the first hydrogel barrier moves at least one of the one or more discrete sections with respect to the one or more surfaces of the chamber and thereby creates an opening in the second distal area.

[00332] Unpenning may further include applying the threshold pressure to the one or more discrete sections of the first hydrogel barrier of the identified chambers, thereby unpenning the one or more biological micro-objects or daughter micro-objects thereof of interest. Applying the threshold pressure may include illuminating a distal region of each of the identified chambers with laser illumination, thereby generating a dislodging force to move the one or more biological micro-objects or daughter micro-objects past the one or more discreet sections of the first hydrogel barrier to the proximal region of the chambers.

[00333] In other embodiments, the first composition may include a crosslinker providing a reversible first hydrogel barrier once formed, which may be any crosslinker described herein that provides a reversible hydrogel barrier once formed. Unpenning the one or more biological micro-objects or daughter micro-objects thereof of interest may then include introducing a reversing reagent configured to reverse the first hydrogel barrier comprising the composition of the first reversible hydrogel into the flow region of the microfluidic device; contacting the first hydrogel barriers of the first group of capped chambers with the reversing reagent; and reversing the first hydrogel barriers, thereby providing a first group of uncapped chambers suitable for unpenning. [00334] In yet other aspects, a method of reducing a risk of loss of clonality within a microfluidic device, is provided wherein the microfluidic device has an enclosure comprising a base, a cover, and microfluidic circuit material defining a fluidic circuit therein. The fluidic circuit includes a flow region and a plurality of chambers opening to the flow region. The process includes: introducing a plurality of biological microobjects into alternating chambers of the plurality of chambers; introducing a first photoactivatable flowable polymer composition having a reversible crosslinker into the flow region of the microfluidic device; activating crosslinking of the first composition in a selected area of the microfluidic device, thereby forming a first reversible hydrogel barrier within the flow region of the microfluidic device, wherein the first hydrogel barrier created a transport region between each of two adjacent chambers, thereby forming twinned chambers including a first chamber including the plurality of biological microobject and a second chamber having no biological micro-objects;

[00335] Separating the plurality of biological micro-objects between the twinned chambers, thereby separating a first portion of the plurality of biological micro-objects into the first chamber and a second portion of the plurality of biological micro-objects into the second chamber; reversing the first reversible hydrogel; introducing a second photoactivatable flowable polymer composition having a reversible crosslinker into the flow region of the microfluidic device; diffusing the second composition from the flow region into the plurality of chambers; activating crosslinking of the second composition in a selected area of the microfluidic device, thereby forming a second reversible hydrogel barrier at the opening of one of the first or second chambers; assaying or culturing the respective portion of the plurality of biological micro-objects in an uncapped first or second chamber, thereby identifying one or more biological micro-objects or daughter micro-objects thereof of interest; identifying one or more chambers containing one or more biological micro-objects or daughter micro-objects of interest; introducing a third photoactivatable non-reversible flowable polymer composition into the flow region of the microfluidic device; diffusing the third composition from the flow region into the plurality of chambers; activating crosslinking of the second composition in a selected area of the microfluidic device, thereby forming a non-reversible hydrogel barrier at the opening of chambers other than the identified chambers, thereby producing a non-reversible capped subset of chambers of the plurality of chambers; reversing the second reversible hydrogel barrier at the identified chambers; and unpenning and exporting the one or more biological micro-objects or daughter micro-objects thereof of interest from the identified chambers, thereby preserving clonality of the biological micro-object or daughter micro-object thereof of interest.

[00336] The first and the second composition may include modified polyethylene glycol moieties. In some embodiments, the first and the second composition may be a composition described herein that produce a thiol-ene hydrogel, having any features described in any combination.

METHOD OF CONTROLLING PERMEABILITY OF A HYDROGEL

[00337] An in situ-generated structure (e.g. a hydrogel barrier) can have a porosity that restricts the passage of a molecule so that the diffusion of the molecule passing through the in situ-generated structure is impeded while not blocked. In this way, the diffusion rate of the molecule can be adjusted by tuning the porosity of the in situ-generated structure. As used herein, “diffusion rate” has the same definition as “rate of diffusion” described herein. [00338] As used herein, “porosity” describes an average pore size of an in situ-generated structure, and “permeability” describes how easily a molecule of interest passes through the in situ-generated structure. A molecule of interest can pass through the in situ-generated structure easier or faster if the in situ- generated structure has a high permeability to it. In contrast, the molecule of interest can pass through the in situ-generated structure more slowly if the permeability is lower. The permeability of the in situ- generated structure can be tuned to affect the diffusion of a molecule passing through.

[00339] Controlling diffusion of a molecule of interest within a microfluidic device. A method of controlling diffusion of a molecule of interest within a microfluidic device, wherein the microfluidic device comprises an enclosure comprising a base, a cover, and microfluidic circuit material defining a fluidic circuit therein, and further wherein the fluidic circuit of the microfluidic device comprises a flow region and a chamber opening to the flow region, the method comprising: introducing a prepolymer composition for forming a hydrogel into the flow region of the fluidic circuit; introducing a molecule of interest into the flow region of the fluidic circuit; activating crosslinking of the prepolymer composition in a selected area within the fluidic circuit of the microfluidic device thereby forming a hydrogel barrier between the flow region and the chamber; and diffusing the molecule of interest through the hydrogel barrier.

[00340] In some embodiments, the hydrogel barrier is formed after introducing the molecule of interest. The molecule of interest can be introduced before introducing the prepolymer composition for forming a hydrogel. For example, the method can comprise introducing the molecule of interest into the flow region of the microfluidic device and diffusing the molecule of interest into the chamber. Then, other remaining molecules of interest still present within the flow region can be flushed out of the microfluidic device while the diffused molecule of interest within the chamber is retained therewithin. Subsequently, the method can comprise introducing the prepolymer composition into the flow region and activating crosslinking of the prepolymer composition in a selected area within the fluidic circuit of the microfluidic device, thereby forming a hydrogel barrier in the selected area.

[00341 ] In such embodiments, the molecule of interest is retained within the chamber while the hydrogel barrier is formed, and diffusing the molecule of interest through the hydrogel barrier comprises diffusing the molecule of interest from the chamber to the flow region.

[00342] In other embodiments, the prepolymer composition may be introduced simultaneously with the molecule of interest, followed by activating crosslinking to form the hydrogel barrier in the selected area. Subsequent to barrier formation, media flow through the flow region may be effected to flush any molecules of interest remaining in the flow region as well as flushing un-crosslinked prepolymer composition molecules from the flow region.

[00343] In some embodiments, the hydrogel barrier is formed before introducing the molecule of interest. The prepolymer composition for forming a hydrogel can be introduced before introducing the molecule of interest. For example, the method can comprise introducing the prepolymer composition into the flow region of the microfluidic device and diffusing the prepolymer composition into chamber. Then, the remaining prepolymer composition within the flow region can be flushed out of the microfluidic device while the prepolymer composition diffused into the chamber is retained therewithin. Activating crosslinking of the prepolymer composition in a selected area can be conducted to form the hydrogel barrier. After that, the method can comprises introducing the molecule of interest into the flow region and diffusing the molecule of interest passing through the hydrogel into the chamber. In some embodiments, the activating crosslinking of the composition for forming a hydrogel in a selected area can be conducted after the molecule of interest is introduced into the flow region.

[00344] In some embodiments, introducing a molecule of interest into the fluidic circuit comprises introducing a fluidic medium comprising the molecule of interest, wherein the molecule of interest is soluble in the fluidic medium.

[00345] The chamber of the microfluidic device can comprise an opening to the flow region. In some embodiments, the hydrogel barrier can be formed at the opening. For example, the hydrogel barrier can be formed at a location within the chamber and close to the opening. In certain embodiments, the hydrogel barrier can seal the opening of the chamber so that the molecule of interest must pass through the hydrogel barrier while entering the chamber from the flow region or leaving the chamber into the flow region.

[00346] As used herein, “seal” refers to that the in situ-generated structure fully enclose the opening of the chamber to the flow region. In some embodiments, the in situ-generated structure is an in situ- generated cap having a dimension across a width of the connection region or a width of the opening of the chamber. However, sealing does not necessarily mean that all kinds of molecules will be blocked from passing through the in situ-generated structure. Whether a molecule is restricted, blocked, or allowed to pass the in situ-generated cap is dependent on the porosity (i.e., pore size) of the in situ-generated structure, that is, in a size-dependent manner.

[00347] FIG. 26 illustrates an example of controlling diffusion of a molecule of interest within a microfluidic device. A microfluidic channel 2601 and three chambers 2611, 2612, 2613 are depicted in the figure. Each of the three chambers 2611, 2612, 2613 comprises a proximal opening to the microfluidic channel 2601. FIG. 26 illustrates a moment that molecules of interest 2620 retained in the chambers 2611, 2612, 2613 are diffusing toward the microfluidic channel 2601.

[00348] Among those three chambers, an in situ-generated structure 2631 is formed at mid-chamber of chamber 2612, and an in situ-generated structure 2632 is formed in an area proximal to the opening of chamber 2613. The in situ-generated structure 2631 and the in situ-generated structure 1232 both have a porosity that impedes but does not block passage of the molecules of interest 2620. As a result, the molecule of interest 2620 is diffusing more slowly out from a region of chamber 2612 distal to in situ- generated structure 2631 and out from a region of chamber 2613 distal to in situ-generated structure 2632 compared to the rate of diffusion of molecule of interest 2620 in chamber 2611.

[00349] Adjusting permeability of a hydrogel to a molecule of interest. A method of adjusting permeability of a hydrogel to a molecule of interest is provided. The method comprises preparing a prepolymer composition for forming the hydrogel, wherein the prepolymer composition can be a composition as described herein comprising a first polyethylene glycol polymer molecule, a second polyethylene glycol polymer molecule, and a crosslinker; and adjusting a working concentration of the first polyethylene glycol polymer molecule to be different from a pre-determined standard concentration thereof, and/or adjusting a working concentration of the second polyethylene glycol polymer molecule to be different from a pre-determined standard concentration thereof thereby adjusting permeability of the hydrogel to a molecule of interest.

[00350] A standard concentration of a certain component of the composition for forming the hydrogel can be determined based on a need of an assay and is not limited. The present disclosure teaches, once a standard concentration is determined, the permeability of the hydrogel can be tuned according to the disclosure herein.

[00351] In some embodiments, the method does not comprise adjusting a working concentration of the crosslinker to be different from a pre-determined standard concentration thereof for purposes of controlling permeability of the hydrogel to the molecule of interest. In certain embodiments, the composition for forming the hydrogel can further comprises an inhibitor and/or an initiator (e.g., a photoinitiator, and the method does not comprise adjusting a working concentration of the inhibitor and/or adjusting a working concentration of the initiator to be different from a pre-determined standard concentration thereof for purposes of controlling permeability of the hydrogel to the molecule of interest.

[00352] In some embodiments, wherein adjusting a working concentration of the first polyethylene glycol polymer molecule to be different from a pre-determined standard concentration thereof comprises adjusting the working concentration of the first polyethylene glycol polymer molecule to be higher than the pre-determined standard concentration. For example, the working concentration of the first polyethylene glycol polymer molecule can be +5%, +10%, +20%, +30%, +40%, +50%, +60%, +70%, +80%, +90%, +100%, +150%, +175%, +200%, +225%, +250%, +275%, +300%, +350%, +400%, or higher, or any value therebetween of the pre-determined standard concentration.

[00353] In other embodiments, wherein adjusting a working concentration of the first polyethylene glycol polymer molecule to be different from a pre-determined standard concentration thereof comprises adjusting the working concentration of the first polyethylene glycol polymer molecule to be lower than the pre-determined standard concentration. For example, the working concentration of the first polyethylene glycol polymer molecule can be -95%, -90%, -80%, -70%, -60%, -50%, -40%, -30%, -20%, -10%, -5%, or any value therebetween of the pre-determined standard concentration.

[00354] In some embodiments, wherein adjusting a working concentration of the second polyethylene glycol polymer molecule to be different from a pre-determined standard concentration thereof comprises adjusting the working concentration of the second polyethylene glycol polymer molecule to be higher than the pre-determined standard concentration. For example, the working concentration of the second polyethylene glycol polymer molecule can be +5%, +10%, +20%, +30%, +40%, +50%, +60%, +70%, +80%, +90%, +100%, +150%, +175%, +200%, +225%, +250%, +275%, +300%, +350%, +400%, or higher, or any value therebetween of the pre-determined standard concentration. [00355] In other embodiments, wherein adjusting a working concentration of the second polyethylene glycol polymer molecule to be different from a pre-determined standard concentration thereof comprises adjusting the working concentration of the second polyethylene glycol polymer molecule to be lower than the pre-determined standard concentration. For example, the working concentration of the second polyethylene glycol polymer molecule can be -95%, -90%, -80%, -70%, -60%, -50%, -40%, -30%, -20%, -10%, -5%, or any value therebetween of the pre-determined standard concentration.

[00356] In some embodiments, increasing the amount of a component of the prepolymer composition decreases the pore size of the hydrogel formed thereby slowing down diffusion of a molecule of interest. For example, increasing the amount of a prepolymer can result in a denser solidified polymer network that slows down the diffusion. In some embodiments, increasing the amount of a component increases the pore size of the hydrogel formed thereby accelerating diffusion of a molecule of interest. For example, increasing the amount of an inhibitor can result in a less dense solidified polymer network whereby an assay reagent can diffuse faster (e.g., diffusion is slowed to a lesser degree by the barrier). In yet some other embodiments, the porosity of a hydrogel formed at a standard amount does not restrict passage of a molecule, therefore adjusting the amount of the components to get a less dense solidified polymer network would not affect the diffusion of the molecule.

[00357] In some embodiments, an in situ-generated structure comprises a porosity that can restrict passage of a molecule no smaller than a critical size (e.g. a cut-off value or threshold of the size of the molecule), and the porosity of the in situ-generated structure can be adjusted as described herein so that the diffusion rate of the molecule passing through the in situ-generated structure can be tuned. The critical size can be 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, 600 kDa, 650 kDa, 700 kDa, 750 kDa, 800 kDa, 850 kDa, 900 kDa, 950 kDa, 1000 kDa, or higher, or any value therebetween.

[00358] In a specific example, permeability of two reagents passing through an in situ-generated structure (a hydrogel barrier) was tested in a microfluidic device as described herein. In this example, a permeability test mixture comprising a mixture of AF647 labeled ovalbumin (AF647-OVA; Invitrogen™, #034784) and AF488Plus labeled IgG (AF488P-IgG; Invitrogen™, # A32223) was prepared. The ovalbumin and the IgG in those reagents had a molecular weight of 45 kDa and 150 kDa respectively, and their final concentration in the permeability test mixture was 0.02 mg/mL and 0.05 mg/mL respectively.

[00359] The microfluidic device was first calibrated and flushed with PBS. Then, the permeability test mixture was introduced while there was no hydrogel structure formed within the device. The perfusion of the permeability test mixture was continued for one hour to let the reagents diffuse into the chambers of the microfluidic device. Time-lapsed brightfield images and fluorescent images were taken. See FIG. 27A, showing Time 0 when the permeability test mixture was just introduced into the channel 2701. The channel 2701 had a bright fluorescent image due to the presence of the fluorescent label of the AF647- OVA, while the chambers 2702 were still dark because the permeability test mixture had not yet diffused into the chambers 2702. The images of AF488P-IgG were also taken but not shown in this figure. After 60 minutes, the permeability test mixture had diffused and reached equilibration across the channel 2701 and the chambers 2702 so that the channel 2701 and the chambers 2702 were about equally bright in the image (FIG. 27B).

[00360] Then, the microfluidic device was flushed with PBS to remove the permeability test mixture remaining therewithin. A prepolymer composition comprising a 8 Al OK PEG norbornene (prepolymer component A in FIG. 27), a 8A20K PEG norbornene (prepolymer B in FIG. 27), a crosslinker (dithiothreitol), an initiator (LAP), and an inhibitor (sodium ascorbate) at a standard concentration for in situ-generation of a hydrogel barrier was introduced into the microfluidic device. Solidification of the prepolymer was activated by illuminating UV light at a selected area within each chamber 2702 close to the opening 2703. The in situ-generated hydrogel barrier 2710 formed and sealed the opening 2703 as shown in FIG. 27C. Then, the permeability test mixture was introduced again into the channel 2701 and allowed to diffuse into the chambers 2702 for 60 minutes. With the in situ-generated hydrogel barrier 2710 in place, the permeability test mixture must pass through the barrier to reach the chambers 2702, thus slowing diffusion.

[00361] FIG. 27D shows an image at Time 0 wherein the permeability test mixture was just introduced. The channel 2701 had a bright fluorescent image due to the presence of the fluorescent label of the AF647- OVA, while the chambers 2702 were still dark because the permeability test mixture had not yet diffused into the chambers 2702. Compared with FIG. 27 A, the chambers 2702 in FIG. 27D were darker showing that the in situ-generated hydrogel barrier 2710 impeded the diffusion of the permeability mixture. In contrast, the permeability test mixture can quickly diffuse into the chambers 2702 in FIG. 27A even just upon introducing the mixture into the flow region.

[00362] After diffusion for 60 minutes, most of the permeability test mixture still remained in the channel 2701. As the chambers 2702 were brighter compared with that in FIG. 27D, indicating that the in situ-generated hydrogel barrier 2710 did not block diffusion but slowed down the diffusion of the permeability mixture, which can also be seen clearly from the comparison between FIG. 27E and FIG. 27B.

[00363] Alternatively, the final concentration of each component of the hydrogel solution was adjusted to tune the permeability of the permeability mixture. The intensities of the fluorescent signals of the permeability test mixture (AF647-OVA and AF488P-IgG respectively) detected from regions of interest in channel and in chambers were detected to calculate channel-to-chamber ratios. At time 0 when there was no permeability test mixture existing within the microfluidic device, the ratio should be close to 0. At full equilibration (the substantial difference in concentration of the permeability test mixture across the channel and the chamber), the ratio should be close to 1. The ratios were used in the following exponential equation to calculate a diffusion coefficient representing diffusion behavior in various conditions. The Ichamber/Ichannei represents the channel-to-chamber ratio as described above; t is time; D is the diffusion coefficient.

Ichamber/Ichannei ^— 1 ^— e ^A (-D*t) [00364] The concentration of each component (prepolymer A, prepolymer B, crosslinker, initiator, and inhibitor) of the hydrogel solution varied independently from -50% to 200% of the standard concentration. FIG. 28 shows that the diffusion coefficient varied along with variation of the concentration of each component. A strong relationship between hydrogel permeability and polymer concentration was observed. In both cases, as the concentration of polymer went up, the permeability of the hydrogel went down. In contrast, the concentrations of the crosslinker, the initiator, and the inhibitor only had a modest impact on the permeability. The data verifies that the permeability of hydrogel to a molecule of interest can be tuned by adjusting the concentration of the component of the hydrogel solution resulting in changes of the porosity of the hydrogel.

[00365] Since the crosslinker is designed to be incorporated into the formed hydrogel structure, it is surprising that the concentration of the crosslinker is not the primary factor in determining the porosity of a hydrogel. Hydrogels of different porosities can be formed from prepolymer compositions comprising a same or similar density of crosslinkers. This is particularly beneficial for forming reversible hydrogel structures that can be dissolved by cleaving the crosslinkers in the hydrogel structures. For example, a first reversible hydrogel of high permeability and a second reversible hydrogel of low permeability may comprise a same cleavable crosslinker moiety. Even though the first reversible hydrogel and the second reversible hydrogel have different permeabilities, they can be dissolved with similar efficiencies using the same dissolution reagent.

[00366] In some embodiments where a microfluidic device comprising a microfluidic circuit material defining a flow region, a first chamber and a second chamber is used, the first chamber and the second chamber can have a first permeable hydrogel barrier and a second permeable hydrogel barrier formed therewithin respectively. The first permeable hydrogel barrier and the second permeable hydrogel barrier can have different physical properties, for example, different porosities or different permeabilities to a reagent. The first permeable hydrogel barrier and the second permeable hydrogel barrier can be both reversible hydrogel barrier, non-reversible hydrogel barrier, or one of them is a reversible hydrogel barrier and the other is a non-reversible hydrogel barrier.

[00367] In some embodiments, the first permeable hydrogel barrier has a different permeability from a permeability of the second permeable hydrogel barrier. In some embodiments, the first permeable hydrogel barrier has a permeability limit that is higher than a permeability limit of the second permeable hydrogel barrier. A reagent configured to diffuse through the first permeable hydrogel barrier may be excluded from diffusing through the second permeable hydrogel barrier, when that reagent has a size greater than the permeability limit of the second permeable hydrogel barrier. In some other embodiments, a reagent configured to diffuse through the first permeable hydrogel barrier and the second permeable hydrogel barrier might diffuse at a different rate passing through the first permeable hydrogel barrier and the second permeable hydrogel barrier. For example, a first rate of the reagent to pass through the first permeable hydrogel barrier might be higher than a second rate of the reagent to pass through the second permeable hydrogel barrier.

[00368] Forming the first permeable hydrogel barrier may include forming the first permeable hydrogel barrier within one or more chambers of the plurality of sequestration pens. Forming the first permeable hydrogel barrier may further include forming the first permeable hydrogel barrier distal to or at the opening of the one or more chambers, thereby producing one or more reversibly capped chambers.

[00369] A process for forming the first permeable hydrogel barrier and the second permeable hydrogel barrier can comprise: introducing a first prepolymer composition into the flow region of the microfluidic device, where the first prepolymer composition has a first selected set of characteristics defining physical properties of a first permeable hydrogel barrier formed therefrom; diffusing the first prepolymer composition from the flow region into the plurality of chambers; activating crosslinking of the first prepolymer composition in a selected area of the microfluidic device, thereby forming a first permeable hydrogel barrier within the flow region or one or more of the plurality of chambers of the microfluidic device.

[00370] The process can further comprise introducing a second prepolymer composition into the flow region of the microfluidic device, where the second prepolymer composition has a second selected set of characteristics defining physical properties of a second permeable hydrogel barrier formed therefrom; diffusing the second prepolymer composition from the flow region into the chambers; and activating crosslinking of the second prepolymer composition in a selected area of the microfluidic device, thereby forming a second permeable hydrogel barrier within the flow region or a sub-set of the plurality of chambers of the microfluidic device.

[00371] In a specific example, a reversible hydrogel barrier 2910 was formed at the mid-pen location (mid-area) of each chamber, a first non -reversible hydrogel barrier 2921 was formed in chamber 2901 and chamber 2904 respectively, a second non-reversible hydrogel barrier 2922 was formed in chamber 2902 and chamber 2905 respectively (FIG. 29A).

[00372] The reversible hydrogel barrier 2910 was formed by using a prepolymer composition comprising a 8 Al OK PEG norbomene, a 8A20K PEG norbomene, a crosslinker (a peptide: GCRDLPRTGGDRCG; SEQ ID NO: 1), an initiator (LAP), and an inhibitor (sodium ascorbate).

[00373] Both the first non-reversible hydrogel barrier 2921 and second non-reversible hydrogel barrier 2922 were formed by using a first prepolymer composition and a second prepolymer composition respectively. The first prepolymer composition and the second prepolymer composition comprises the same components: a 8A10K PEG norbomene, a 8A20K PEG norbomene, a crosslinker (sodium 2,3- dimercaptopropanesulfonate), an initiator (LAP), and an inhibitor (sodium ascorbate), but the second prepolymer composition comprises the 8 Al OK PEG norbomene and the 8A20K PEG norbomene at a concentration 2-fold the concentration of that in the first prepolymer composition.

[00374] A dissolution reagent (TrypLE™; 24 kDa) was introduced into the channel of the microfluidic device and allowed to diffuse into those chambers. The dissociation of the reversible hydrogels barrier 2910 was recorded. Six images were extracted from the recording including an image at Time 0 (FIG. 29 A) and images of other five different timepoints (FIGS. 29B to 29F).

[00375] It was observed that the reversible hydrogel barrier 2910 within chamber 2903 and chamber

1506 started to be dissolved while the reversible hydrogels barrier 2910 within other chambers remained substantially intact (See FIG. 29B. the arrows indicate the dissociation of the hydrogels). Along time, the reversible hydrogels barrier 2910 in chamber 2901 and chamber 2904 started to be dissolved (See FIG. 29C. the arrows indicate the dissociation of the hydrogels). The reversible hydrogels barrier 2910 in chamber 2903 and chamber 2906 were almost gone at this moment while no substantial change was observed in the reversible hydrogel barriers 2910 in chamber 2902 and chamber 2905. Later in FIG. 29D, the reversible hydrogels barrier 2910 in chamber 2903 and chamber 2906 were dissolved, and so were most of the reversible hydrogel barriers 2910 in chamber 2901 and chamber 2904. Then in FIG. 29E, the reversible hydrogel barriers 2910 in chamber 2903 and chamber 2906 started to be dissolved and eventually dissolved completely as shown in FIG. 29F.

[00376] According to the images, the second non-reversible hydrogel barrier 2922 having higher concentration of the prepolymer had a porosity impeding passage of the dissolution reagent greater than the first non-reversible hydrogel barrier 2921. In other words, the permeability of the second non- reversible hydrogel barrier 2922 to the dissolution reagent was lower than that of the first non-reversible hydrogel barrier 2921 whereby the dissolution reagent diffused more slowly in chamber 2902 and chamber 2905. The result verifies that, by adjusting the concentration of the prepolymer used for forming the in situ-generated hydrogel barrier, the porosity of the hydrogel barrier formed can be tuned resulting different in permeability to a molecule of interest.

KITS

[00377] A kit for introducing a hydrogel barrier within a microfluidic device is provided. The kit comprises a composition comprising a first and a second polyethylene glycol polymer molecule, each comprising a respective polyethylene glycol moiety and a covalently linked reactive moiety R _x ; and a crosslinker molecule comprising a first reactive moiety RXP disposed at a first end of a linker L moiety and a second reactive moiety RXP disposed at a second end of the linker L moiety, wherein each of the first and the second crosslinker moiety RXP is configured to be activatable to react with the respective reactive moiety R _x of the first and the second polyethylene polymer molecules, wherein the first and the second polyethylene polymer molecule comprises different polyethylene glycol moieties or each comprises a same polyethylene glycol moiety.

[00378] The first reactive moiety Rxp and the second reactive moiety Rxp of the first and the second polyethylene glycol molecule may be a norbomenyl moiety. In some embodiments, the norbomenyl moiety may be a substituted norbomenyl moiety. In some embodiments, the substituted norbomenyl moiety may have a hydrophilic substituent, such as but not limited to hydroxy or carboxylate.

[00379] The first polyethylene glycol moiety and/or the second polyethylene glycol moiety may include a 1-arm, 2- arm, 4- arm or 8- arm polyethylene glycol moiety. In some embodiments, each arm of the first polyethylene glycol moiety and/or the second polyethylene glycol moiety may include the covalently linked reactive moiety R _x . In other embodiments, when the first polyethylene glycol moiety and/or the second polyethylene glycol moiety has a 2-arm, 4- arm, or 8- arm polyethylene moiety structure, at least one arm of the first polyethylene glycol moiety and/or the second polyethylene glycol moiety may include the covalently linked reactive moiety R _x and at least one arm of the first polyethylene glycol moiety does not have a covalently linked reactive moiety R _x .

[00380] The first polyethylene glycol moiety and/or the second polyethylene glycol moiety may have a molecular weight from about 500 Da to about 40K Da; from about IkDa to about 25KDa; from about 5KDa to about 25KDa; from about 5KDa to about 20KDa; about 5KDA to about 15KDa; or about 5KDA to about lOKDa. The second polyethylene glycol moiety may have a molecular weight from about 500 Da to about 40K Da; from about IkDa to about 25KDa; from about 5KDa to about 25KDa; from about 5KDa to about 20KDa; about 5KDA to about 15KDa; or about 5KDA to about lOKDa.

[00381] In some embodiments, the first polyethylene glycol moiety may have a molecular weight of about 10K Da and the second polyethylene glycol moiety may have a molecular weight of about 10K Da. In other embodiments, the first polyethylene glycol moiety may have a molecular weight of about 10K Da and the second polyethylene glycol moiety may have a molecular weight of about 20K.

[00382] In some embodiments, the crosslinker of the composition has a structure having a molecular formula of Formula II:

HS-LB-CH ₂ -C(H)(OH)-C(H)(OH)-CH ₂ -LB-SH Formula (2)

[00383] where each instance of linker backbone LB is independently selected to comprise 0 to 200 nonhydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms. In some embodiments, the crosslinker molecule is dithiothreitol.

[00384] In some embodiments of the kit, the crosslinker of the composition has a structure having a molecular formula of Formula (4):

HS-PEPT-SH Formula (4)

[00385] where PEPT is a peptidyl moiety comprising about 4 to about 12 amino acids, and additionally where the peptidyl moiety is susceptible to proteolytic enzymatic cleavage. In some embodiments, the peptide sequence may include GCRDLPRTGGDRCG (SED ID NO: 1).

[00386] In other embodiments, the crosslinker molecule has a structure of Formula (5):

HS-LB4-SH Formula (5)

[00387] where linker backbone LB4 comprises 3 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms. In some embodiments, the crosslinker is Sodium 2,3-dimercaptopropanesulfonate monohydrate.

[00388] In some embodiments, the composition may further include an inhibitor configured to inhibit reaction of the crosslinker with the reactive moiety of the first and/or the second polyethylene glycol polymer molecule and/ or of the reactive moiety of the first and/or the second polyethylene glycol polymer molecules upon standing in solution. The inhibitor may be sodium ascorbate, MEHQ, or 4-hydroxy TEMPO. In some embodiments, the inhibitor is present within the composition at a concentration from about 0.5 millimolar to about 1.5 millimolar.

[00389] In other embodiments, the kit may include a second component having a photoinitiator and a second amount of the inhibitor. The inhibitor in the second component having the photoinitiator may have the inhibitor present in a range from about 5 millimolar to about 20 millimolar. The composition component containing the photoinitiator and second amount of inhibitor may be separately packaged from the composition including the first and the second polyethylene glycol polymer molecules. In some embodiments, the kit further comprises a microfluidic device comprising a microfluidic circuit comprising a flow region and a chamber, wherein the chamber comprises an opening to the flow region. The microfluidic device can be any microfluidic device as described herein.

MICROFLUIDIC DEVICE/SYSTEM FEATURE CROSS- APPLICABILITY

[00390] It should be appreciated that various features of microfluidic devices, systems, and motive technologies described herein may be combinable or interchangeable. For example, features described herein with reference to the microfluidic device 100, 175, 200, 300, 320, 400, 450, 520 and system attributes as described in FIGS. 1 A-5B may be combinable or interchangeable.

[00391] Microfluidic devices. FIG. 1A illustrates an example of a microfluidic device 100. A perspective view of the microfluidic device 100 is shown having a partial cut-away of its cover 110 to provide a partial view into the microfluidic device 100. The microfluidic device 100 generally comprises a microfluidic circuit 120 comprising a flow path 106 through which a fluidic medium 180 can flow, optionally carrying one or more micro-objects (not shown) into and/or through the microfluidic circuit 120.

[00392] As generally illustrated in FIG. 1 A, the microfluidic circuit 120 is defined by an enclosure 102. Although the enclosure 102 can be physically structured in different configurations, in the example shown in FIG. 1A the enclosure 102 is depicted as comprising a support structure 104 (e.g., a base), a microfluidic circuit structure 108, and a cover 110. The support structure 104, microfluidic circuit structure 108, and cover 110 can be attached to each other. For example, the microfluidic circuit structure 108 can be disposed on an inner surface 109 of the support structure 104, and the cover 110 can be disposed over the microfluidic circuit structure 108. Together with the support structure 104 and cover 110, the microfluidic circuit structure 108 can define the elements of the microfluidic circuit 120, forming a three-layer structure.

[00393] The support structure 104 can be at the bottom and the cover 110 at the top of the microfluidic circuit 120 as illustrated in FIG. 1A. Alternatively, the support structure 104 and the cover 110 can be configured in other orientations. For example, the support structure 104 can be at the top and the cover 110 at the bottom of the microfluidic circuit 120. Regardless, there can be one or more ports 107 each comprising a passage into or out of the enclosure 102. Examples of a passage include a valve, a gate, a pass-through hole, or the like. As illustrated, port 107 is a pass-through hole created by a gap in the microfluidic circuit structure 108. However, the port 107 can be situated in other components of the enclosure 102, such as the cover 110. Only one port 107 is illustrated in FIG. 1A but the microfluidic circuit 120 can have two or more ports 107. For example, there can be a first port 107 that functions as an inlet for fluid entering the microfluidic circuit 120, and there can be a second port 107 that functions as an outlet for fluid exiting the microfluidic circuit 120. Whether a port 107 function as an inlet or an outlet can depend upon the direction that fluid flows through flow path 106.

[00394] The support structure 104 can comprise one or more electrodes (not shown) and a substrate or a plurality of interconnected substrates. For example, the support structure 104 can comprise one or more semiconductor substrates, each of which is electrically connected to an electrode (e.g., all or a subset of the semiconductor substrates can be electrically connected to a single electrode). The support structure 104 can further comprise a printed circuit board assembly (“PCBA”). For example, the semiconductor substrate(s) can be mounted on a PCBA.

[00395] The microfluidic circuit structure 108 can define circuit elements of the microfluidic circuit 120. Such circuit elements can comprise spaces or regions that can be fluidly interconnected when microfluidic circuit 120 is filled with fluid, such as flow regions (which may include or be one or more flow channels), chambers (which class of circuit elements may also include sub-classes including sequestration pens), traps, and the like. Circuit elements can also include barriers, and the like. In the microfluidic circuit 120 illustrated in Figure 1A, the microfluidic circuit structure 108 comprises a frame 114 and a microfluidic circuit material 116. The frame 114 can partially or completely enclose the microfluidic circuit material 116. The frame 114 can be, for example, a relatively rigid structure substantially surrounding the microfluidic circuit material 116. For example, the frame 114 can comprise a metal material. However, the microfluidic circuit structure need not include a frame 114. For example, the microfluidic circuit structure can consist of (or consist essentially of) the microfluidic circuit material 116.

[00396] The microfluidic circuit material 116 can be patterned with cavities or the like to define the circuit elements and interconnections of the microfluidic circuit 120, such as chambers, pens and microfluidic channels. The microfluidic circuit material 116 can comprise a flexible material, such as a flexible polymer (e.g., rubber, plastic, elastomer, silicone, polydimethylsiloxane (“PDMS”), or the like), which can be gas permeable. Other examples of materials that can form the microfluidic circuit material 116 include molded glass, an etchable material such as silicone (e.g., photo-pattemable silicone or “PPS”), photoresist (e.g., SU8), or the like. In some embodiments, such materials — and thus the microfluidic circuit material 116 — can be rigid and/or substantially impermeable to gas. Regardless, microfluidic circuit material 116 can be disposed on the support structure 104 and inside the frame 114.

[00397] The microfluidic circuit 120 can include a flow region in which one or more chambers can be disposed and/or fluidically connected thereto. A chamber can have one or more openings fluidically connecting the chamber with one or more flow regions. In some embodiments, a flow region comprises or corresponds to a microfluidic channel 122. Although a single microfluidic circuit 120 is illustrated in FIG. 1 A, suitable microfluidic devices can include a plurality (e.g., 2 or 3) of such microfluidic circuits. In some embodiments, the microfluidic device 100 can be configured to be a nanofluidic device. As illustrated in FIG. 1 A, the microfluidic circuit 120 may include a plurality of microfluidic sequestration pens 124, 126, 128, and 130, where each sequestration pens may have one or more openings. In some embodiments of sequestration pens, a sequestration pen may have only a single opening in fluidic communication with the flow path 106. In some other embodiments, a sequestration pen may have more than one opening in fluidic communication with the flow path 106, e.g., n number of openings, but with n-1 openings that are valved, such that all but one opening is closable. When all the valved openings are closed, the sequestration pen limits exchange of materials from the flow region into the sequestration pen to occur only by diffusion. In some embodiments, the sequestration pens comprise various features and structures (e.g., isolation regions) that have been optimized for retaining micro-objects within the sequestration pen (and therefore within a microfluidic device such as microfluidic device 100) even when a medium 180 is flowing through the flow path 106.

[00398] The cover 110 can be an integral part of the frame 114 and/or the microfluidic circuit material 116. Alternatively, the cover 110 can be a structurally distinct element, as illustrated in Figure 1 A. The cover 110 can comprise the same or different materials than the frame 114 and/or the microfluidic circuit material 116. In some embodiments, the cover 110 can be an integral part of the microfluidic circuit material 116. Similarly, the support structure 104 can be a separate structure from the frame 114 or microfluidic circuit material 116 as illustrated, or an integral part of the frame 114 or microfluidic circuit material 116. Likewise, the frame 114 and microfluidic circuit material 116 can be separate structures as shown in FIG. 1A or integral portions of the same structure. Regardless of the various possible integrations, the microfluidic device can retain a three-layer structure that includes a base layer and a cover layer that sandwich a middle layer in which the microfluidic circuit 120 is located.

[00399] In some embodiments, the cover 110 can comprise a rigid material. The rigid material may be glass or a material with similar properties. In some embodiments, the cover 110 can comprise a deformable material. The deformable material can be a polymer, such as PDMS. In some embodiments, the cover 110 can comprise both rigid and deformable materials. For example, one or more portions of cover 110 (e.g., one or more portions positioned over sequestration pens 124, 126, 128, 130) can comprise a deformable material that interfaces with rigid materials of the cover 110. Microfluidic devices having covers that include both rigid and deformable materials have been described, for example, in U.S. Patent No. 10,058,865 (Breinlinger et al.), the contents of which are incorporated herein by reference. In some embodiments, the cover 110 can further include one or more electrodes. The one or more electrodes can comprise a conductive oxide, such as indium-tin-oxide (ITO), which may be coated on glass or a similarly insulating material. Alternatively, the one or more electrodes can be flexible electrodes, such as singlewalled nanotubes, multi-walled nanotubes, nanowires, clusters of electrically conductive nanoparticles, or combinations thereof, embedded in a deformable material, such as a polymer (e.g., PDMS). Flexible electrodes that can be used in microfluidic devices have been described, for example, in U.S. Patent No. 9,227,200 (Chiou et al.), the contents of which are incorporated herein by reference. In some embodiments, the cover 110 and/or the support structure 104 can be transparent to light. The cover 110 may also include at least one material that is gas permeable (e.g., PDMS or PPS).

[00400] In the example shown in FIG. 1A, the microfluidic circuit 120 is illustrated as comprising a microfluidic channel 122 and sequestration pens 124, 126, 128, 130. Each pen comprises an opening to channel 122, but otherwise is enclosed such that the pens can substantially isolate micro-objects inside the pen from fluidic medium 180 and/or micro-objects in the flow path 106 of channel 122 or in other pens. The walls of the sequestration pen extend from the inner surface 109 of the base to the inside surface of the cover 110 to provide enclosure. The opening of the sequestration pen to the microfluidic channel 122 is oriented at an angle to the flow 106 of fluidic medium 180 such that flow 106 is not directed into the pens. The vector of bulk fluid flow in channel 122 may be tangential or parallel to the plane of the opening of the sequestration pen, and is not directed into the opening of the pen. In some instances, pens 124, 126, 128, 130 are configured to physically isolate one or more micro-objects within the microfluidic circuit 120. Sequestration pens in accordance with the present disclosure can comprise various shapes, surfaces and features that are optimized for use with DEP, OET, OEW, fluid flow, magnetic forces, centripetal, and/or gravitational forces, as will be discussed and shown in detail below.

[00401] The microfluidic circuit 120 may comprise any number of microfluidic sequestration pens. Although five sequestration pens are shown, microfluidic circuit 120 may have fewer or more sequestration pens. As shown, microfluidic sequestration pens 124, 126, 128, and 130 of microfluidic circuit 120 each comprise differing features and shapes which may provide one or more benefits useful for maintaining, isolating, assaying or culturing biological micro-objects. In some embodiments, the microfluidic circuit 120 comprises a plurality of identical microfluidic sequestration pens.

[00402] In the embodiment illustrated in FIG. 1A, a single flow path 106 containing a single channel 122 is shown. However, other embodiments may contain multiple channels 122 within a single flow path 106, as shown in FIG. IB. The microfluidic circuit 120 further comprises an inlet valve or port 107 in fluid communication with the flow path 106, whereby fluidic medium 180 can access the flow path 106 (and channel 122). In some instances, the flow path 106 comprises a substantially straight path. In other instances, the flow path 106 is arranged in a non-linear or winding manner, such as a zigzag pattern, whereby the flow path 106 travels across the microfluidic device 100 two or more times, e.g., in alternating directions. The flow in the flow path 106 may proceed from inlet to outlet or may be reversed and proceed from outlet to inlet.

[00403] One example of a multi-channel device, microfluidic device 175, is shown in FIG. IB, which may be like microfluidic device 100 in other respects. Microfluidic device 175 and its constituent circuit elements (e.g., channels 122 and sequestration pens 128) may have any of the dimensions discussed herein. The microfluidic circuit illustrated in FIG. IB has two inlet/outlet ports 107 and a flow path 106 containing four distinct channels 122. The number of channels into which the microfluidic circuit is subdivided may be chosen to reduce fluidic resistance. For example, the microfluidic circuit may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more channels to provide a selected range of fluidic resistance. Microfluidic device 175 further comprises a plurality of sequestration pens opening off of each channel 122, where each of the sequestration pens is similar to sequestration pen 128 of FIG. 1A, and may have any of the dimensions or functions of any sequestration pen as described herein. However, the sequestration pens of microfluidic device 175 can have different shapes, such as any of the shapes of sequestration pens 124, 126, or 130 of FIG. 1A or as described anywhere else herein. Moreover, microfluidic device 175 can include sequestration pens having a mixture of different shapes. In some instances, a plurality of sequestration pens is configured (e.g., relative to a channel 122) such that the sequestration pens can be loaded with target micro-objects in parallel.

[00404] Returning to FIG. 1 A, microfluidic circuit 120 further may include one or more optional microobject traps 132. The optional traps 132 may be formed in a wall forming the boundary of a channel 122, and may be positioned opposite an opening of one or more of the microfluidic sequestration pens 124, 126, 128, 130. The optional traps 132 may be configured to receive or capture a single micro-object from the flow path 106, or may be configured to receive or capture a plurality of micro-objects from the flow path 106. In some instances, the optional traps 132 comprise a volume approximately equal to the volume of a single target micro-object. In some instances, the trap 132 comprises a side passage 134 that is smaller than the target micro-object in order to facilitate flow through the trap 132.

[00405] Sequestration pens. The microfluidic devices described herein may include one or more sequestration pens, where each sequestration pen is suitable for holding one or more micro-objects (e.g., biological cells, or groups of cells that are associated together). The sequestration pens may be disposed within and open to a flow region, which in some embodiments is a microfluidic channel. Each of the sequestration pens can have one or more openings for fluidic communication to one or more microfluidic channels. In some embodiments, a sequestration pen may have only one opening to a microfluidic channel.

[00406] FIGS. 2A-2C show sequestration pens 224, 226, and 228 of a microfluidic device 200, which may be like sequestration pen 128 of FIG. 1 A. Each sequestration pen 224, 226, and 228 can comprise an isolation region 240 and a connection region 236 fluidically connecting the isolation region 240 to a flow region, which may, in some embodiments include a microfluidic channel, such as channel 122. The connection region 236 can comprise a proximal opening 234 to the flow region (e.g., microfluidic channel 122) and a distal opening 238 to the isolation region 240. The connection region 236 can be configured so that the maximum penetration depth of a flow of a fluidic medium (not shown) flowing in the microfluidic channel 122 past the sequestration pen 224, 226, and 228 does not extend into the isolation region 240, as discussed below for FIG. 2C. In some embodiments, streamlines from the flow in the microfluidic channel do not enter the isolation region. Thus, due to the connection region 236, a microobject (not shown) or other material (not shown) disposed in the isolation region 240 of a sequestration pen 224, 226, and 228 can be isolated from, and not substantially affected by, a flow of fluidic medium 180 in the microfluidic channel 122.

[00407] The sequestration pens 224, 226, and 228 of FIGS.2A-2C each have a single opening which opens directly to the microfluidic channel 122. The opening of the sequestration pen may open laterally from the microfluidic channel 122, as shown in FIG. 2 A, which depicts a vertical cross-section of microfluidic device 200. FIG. 2B shows a horizontal cross-section of microfluidic device 200. An electrode activation substrate 206 can underlie both the microfluidic channel 122 and the sequestration pens 224, 226, and 228. The upper surface of the electrode activation substrate 206 within an enclosure of a sequestration pen, forming the floor of the sequestration pen, can be disposed at the same level or substantially the same level of the upper surface the of electrode activation substrate 206 within the microfluidic channel 122 (or flow region if a channel is not present), forming the floor of the flow channel (or flow region, respectively) of the microfluidic device. The electrode activation substrate 206 may be featureless or may have an irregular or patterned surface that varies from its highest elevation to its lowest depression by less than about 3 micrometers (microns), 2.5 microns, 2 microns, 1.5 microns, 1 micron, 0.9 microns, 0.5 microns, 0.4 microns, 0.2 microns, 0.1 microns or less. The variation of elevation in the upper surface of the substrate across both the microfluidic channel 122 (or flow region) and sequestration pens may be equal to or less than about 10%, 7%, 5%, 3%, 2%, 1%, 0.9%, 0.8%, 0.5%, 0.3% or 0.1% of the height of the walls of the sequestration pen. Alternatively, the variation of elevation in the upper surface of the substrate across both the microfluidic channel 122 (or flow region) and sequestration pens may be equal to or less than about 2%, 1%. 0.9%, 0.8%, 0.5%, 0.3%, 0.2%, or 0.1% of the height of the substrate. While described in detail for the microfluidic device 200, this may also apply to any of the microfluidic devices described herein.

[00408] The microfluidic channel 122 and connection region 236 can be examples of swept regions, and the isolation regions 240 of the sequestration pens 224, 226, and 228 can be examples of unswept regions. Sequestration pens like 224, 226, 228 have isolation regions wherein each isolation region has only one opening, which opens to the connection region of the sequestration pen. Fluidic media exchange in and out of the isolation region so configured can be limited to occurring substantially only by diffusion. As noted, the microfluidic channel 122 and sequestration pens 224, 226, and 228 can be configured to contain one or more fluidic media 180. In the example shown in Figures 2A-2B, ports 222 are connected to the microfluidic channel 122 and allow the fluidic medium 180 to be introduced into or removed from the microfluidic device 200. Prior to introduction of the fluidic medium 180, the microfluidic device may be primed with a gas such as carbon dioxide gas. Once the microfluidic device 200 contains the fluidic medium 180, the flow 242 (see FIG. 2C) of fluidic medium 180 in the microfluidic channel 122 can be selectively generated and stopped. For example, as shown, the ports 222 can be disposed at different locations (e.g., opposite ends) of the flow region (microfluidic channel 122), and a flow 242 of the fluidic medium can be created from one port 222 functioning as an inlet to another port 222 functioning as an outlet.

[00409] FIG. 2C illustrates a detailed view of an example of a sequestration pen 224, which may contain one or more micro-objects 246, according to some embodiments. The flow 242 of fluidic medium 180 in the microfluidic channel 122 past the proximal opening 234 of the connection region 236 of sequestration pen 224 can cause a secondary flow 244 of the fluidic medium 180 into and out of the sequestration pen 224. To sequester the micro-obj ects 246 in the isolation region 240 of the sequestration pen 224 from the secondary flow 244, the length L _CO n of the connection region 236 of the sequestration pen 224 (i.e., from the proximal opening 234 to the distal opening 238) should be greater than the penetration depth D _p of the secondary flow 244 into the connection region 236. The penetration depth D _p depends upon a number of factors, including the shape of the microfluidic channel 122, which may be defined by a width W _CO n of the connection region 236 at the proximal opening 234; a width W _C h of the microfluidic channel 122 at the proximal opening 234; a height H _C h of the channel 122 at the proximal opening 234; and the width of the distal opening 238 of the connection region 236. Of these factors, the width Wcon of the connection region 236 at the proximal opening 234 and the height H _C h of the channel 122 at the proximal opening 234 tend to be the most significant. In addition, the penetration depth D _p can be influenced by the velocity of the fluidic medium 180 in the channel 122 and the viscosity of fluidic medium 180. However, these factors (i.e., velocity and viscosity) can vary widely without dramatic changes in penetration depth D _p . For example, for a microfluidic chip 200 having a width W _CO n of the connection region 236 at the proximal opening 234 of about 50 microns, a height H _C h of the channel 122 at the proximal opening 122 of about 40 microns, and a width W _C h of the microfluidic channel 122 at the proximal opening 122 of about 100 microns to about 150 microns, the penetration depth D _p of the secondary flow 244 ranges from less than 1.0 times W _CO n (i.e., less than 50 microns) at a flow rate of 0.1 microliters/sec to about 2.0 times W _CO n (i.e., about 100 microns) at a flow rate of 20 microliters/sec, which represents an increase in D _p of only about 2.5-fold over a 200-fold increase in the velocity of the fluidic medium 180.

[00410] In some embodiments, the walls of the microfluidic channel 122 and sequestration pen 224, 226, or 228 can be oriented as follows with respect to the vector of the flow 242 of fluidic medium 180 in the microfluidic channel 122: the microfluidic channel width W _C h (or cross-sectional area of the microfluidic channel 122) can be substantially perpendicular to the flow 242 of medium 180; the width Wcon (or cross-sectional area) of the connection region 236 at opening 234 can be substantially parallel to the flow 242 of medium 180 in the microfluidic channel 122; and/or the length L _CO n of the connection region can be substantially perpendicular to the flow 242 of medium 180 in the microfluidic channel 122. The foregoing are examples only, and the relative position of the microfluidic channel 122 and sequestration pens 224, 226 and 228 can be in other orientations with respect to each other.

[00411] In some embodiments, for a given microfluidic device, the configurations of the microfluidic channel 122 and the opening 234 may be fixed, whereas the rate of flow 242 of fluidic medium 180 in the microfluidic channel 122 may be variable. Accordingly, for each sequestration pen 224, a maximal velocity Vmaxfor the flow 242 of fluidic medium 180 in channel 122 may be identified that ensures that the penetration depth D _p of the secondary flow 244 does not exceed the length L _CO n of the connection region 236. When Vmax is not exceeded, the resulting secondary flow 244 can be wholly contained within the connection region 236 and does not enter the isolation region 240. Thus, the flow 242 of fluidic medium 180 in the microfluidic channel 122 (swept region) is prevented from drawing micro-objects 246 out of the isolation region 240, which is an unswept region of the microfluidic circuit, resulting in the micro-objects 246 being retained within the isolation region 240. Accordingly, selection of microfluidic circuit element dimensions and further selection of the operating parameters (e.g., velocity of fluidic medium 180) can prevent contamination of the isolation region 240 of sequestration pen 224 by materials from the microfluidic channel 122 or another sequestration pen 226 or 228. It should be noted, however, that for many microfluidic chip configurations, there is no need to worry about Vmax per se, because the chip will break from the pressure associated with flowing fluidic medium 180 at high velocity through the chip before V max can be achieved.

[00412] Components (not shown) in the first fluidic medium 180 in the microfluidic channel 122 can mix with the second fluidic medium 248 in the isolation region 240 substantially only by diffusion of components of the first medium 180 from the microfluidic channel 122 through the connection region 236 and into the second fluidic medium 248 in the isolation region 240. Similarly, components (not shown) of the second medium 248 in the isolation region 240 can mix with the first medium 180 in the microfluidic channel 122 substantially only by diffusion of components of the second medium 248 from the isolation region 240 through the connection region 236 and into the first medium 180 in the microfluidic channel 122. In some embodiments, the extent of fluidic medium exchange between the isolation region of a sequestration pen and the flow region by diffusion is greater than about 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or greater than about 99% of fluidic exchange.

[00413] In some embodiments, the first medium 180 can be the same medium or a different medium than the second medium 248. In some embodiments, the first medium 180 and the second medium 248 can start out being the same, then become different (e.g., through conditioning of the second medium 248 by one or more cells in the isolation region 240, or by changing the medium 180 flowing through the microfluidic channel 122).

[00414] As illustrated in FIG. 2C, the width W _CO n of the connection region 236 can be uniform from the proximal opening 234 to the distal opening 238. The width W _CO n of the connection region 236 at the distal opening 238 can be any of the values identified herein for the width W _CO n of the connection region 236 at the proximal opening 234. In some embodiments, the width of the isolation region 240 at the distal opening 238 can be substantially the same as the width W _CO n of the connection region 236 at the proximal opening 234. Alternatively, the width W _CO n of the connection region 236 at the distal opening 238 can be different (e.g., larger or smaller) than the width W _CO n of the connection region 236 at the proximal opening 234. In some embodiments, the width W _CO n of the connection region 236 may be narrowed or widened between the proximal opening 234 and distal opening 238. For example, the connection region 236 may be narrowed or widened between the proximal opening and the distal opening, using a variety of different geometries (e.g., chamfering the connection region, beveling the connection region). Further, any part or subpart of the connection region 236 may be narrowed or widened (e.g., a portion of the connection region adjacent to the proximal opening 234).

[00415] FIG. 3 depicts another exemplary embodiment of a microfluidic device 300 containing microfluidic circuit structure 308, which includes a channel 322 and sequestration pen 324, which has features and properties like any of the sequestration pens described herein for microfluidic devices 100, 175, 200, 400, 520 and any other microfluidic devices described herein.

[00416] The exemplary microfluidic devices of FIG. 3 include a microfluidic channel 322, having a width Wch, as described herein, and containing a flow 310 of first fluidic medium 302 and one or more sequestration pens 324 (only one illustrated in FIG. 3). The sequestration pens 324 each have a length L _s , a connection region 336, and an isolation region 340, where the isolation region 340 contains a second fluidic medium 304. The connection region 336 has a proximal opening 334, having a width W _CO ni, which opens to the microfluidic channel 322, and a distal opening 338, having a width W _CO n2, which opens to the isolation region 340. The width W _CO ni may or may not be the same as W _CO n2, as described herein. The walls of each sequestration pen 324 may be formed of microfluidic circuit material 316, which may further form the connection region walls 330. A connection region wall 330 can correspond to a structure that is laterally positioned with respect to the proximal opening 334 and at least partially extends into the enclosed portion of the sequestration pen 324. In some embodiments, the length L _CO n of the connection region 336 is at least partially defined by length L _wa ii of the connection region wall 330. The connection region wall 330 may have a length L _wa ii, selected to be more than the penetration depth D _p of the secondary flow 344. Thus, the secondary flow 344 can be wholly contained within the connection region without extending into the isolation region 340.

[00417] The connection region wall 330 may define a hook region 352, which is a sub-region of the isolation region 340 of the sequestration pen 324. Since the connection region wall 330 extends into the inner cavity of the sequestration pen, the connection region wall 330 can act as a physical barrier to shield hook region 352 from secondary flow 344, with selection of the length of L _wa ii, contributing to the extent of the hook region. In some embodiments, the longer the length L _wa ii of the connection region wall 330, the more sheltered the hook region 352.

[00418] In sequestration pens configured like those of FIGS. 2A-2C and 3, the isolation region may have a shape and size of any type, and may be selected to regulate diffusion of nutrients, reagents, and/or media into the sequestration pen to reach to a far wall of the sequestration pen, e.g., opposite the proximal opening of the connection region to the flow region (or microfluidic channel). The size and shape of the isolation region may further be selected to regulate diffusion of waste products and/or secreted products of a biological micro-object out from the isolation region to the flow region via the proximal opening of the connection region of the sequestration pen. In general, the shape of the isolation region is not critical to the ability of the sequestration pen to isolate micro-objects from direct flow in the flow region.

[00419] In some other embodiments of sequestration pens, the isolation region may have more than one opening fluidically connecting the isolation region with the flow region of the microfluidic device. However, for an isolation region having a number of n openings fluidically connecting the isolation region to the flow region (or two or more flow regions), n-1 openings can be valved. When the n-1 valved openings are closed, the isolation region has only one effective opening, and exchange of materials into/out of the isolation region occurs only by diffusion.

[00420] Examples of microfluidic devices having pens in which biological micro-objects can be placed, cultured, and/or monitored have been described, for example, in U.S. Patent No. 9,857,333 (Chapman, et al.), U.S. Patent No. 10,010,882 (White, et al.), and U.S. Patent No. 9,889,445 (Chapman, et al.), each of which is incorporated herein by reference in its entirety.

[00421] Microfluidic circuit element dimensions. Various dimensions and/or features of the sequestration pens and the microfluidic channels to which the sequestration pens open, as described herein, may be selected to limit introduction of contaminants or unwanted micro-objects into the isolation region of a sequestration pen from the flow region/microfluidic channel; limit the exchange of components in the fluidic medium from the channel or from the isolation region to substantially only diffusive exchange; facilitate the transfer of micro-objects into and/or out of the sequestration pens; and/or facilitate growth or expansion of the biological cells. Microfluidic channels and sequestration pens, for any of the embodiments described herein, may have any suitable combination of dimensions, may be selected by one of skill from the teachings of this disclosure. [00422] For any of the microfluidic devices described herein, a microfluidic channel may have a uniform cross sectional height along its length that is a substantially uniform cross sectional height, and may be any cross sectional height as described herein. At any point along the microfluidic channel, the substantially uniform cross sectional height of the channel, the upper surface of which is defined by the inner surface of the cover and the lower surface of which is defined by the inner surface of the base, may be substantially the same as the cross sectional height at any other point along the channel, e.g., having a cross sectional height that is no more than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2% or about 1% or less, different from the cross-sectional height of any other location within the channel.

[00423] Additionally, the chamber(s), e.g., sequestration pen(s), of the microfluidic devices described herein, may be disposed substantially in a coplanar orientation relative to the microfluidic channel into which the chamber(s) open. That is, the enclosed volume of the chamber(s) is formed by an upper surface that is defined by the inner surface of the cover, a lower surface defined by the inner surface of the base, and walls defined by the microfluidic circuit material. Therefore, the lower surface of the chamber(s) may be coplanar to the lower surface of the microfluidic channel, e.g., substantially coplanar. The upper surface of the chamber may be coplanar to the upper surface of the microfluidic channel, e.g., substantially coplanar. Accordingly, the chamber(s) may have a cross-sectional height, which may have any values as described herein, that is the same as the channel, e.g., substantially the same, and the chamber(s) and microfluidic channel(s) within the microfluidic device may have a substantially uniform cross sectional height throughout the flow region of the microfluidic device, and may be substantially coplanar throughout the microfluidic device.

[00424] Coplanarity of the lower surfaces of the chamber(s) and the microfluidic channel(s) can offer distinct advantage with repositioning micro-objects within the microfluidic device using DEP or magnetic force. Penning and unpenning of micro-objects, and in particular selective penning/ selective unpenning, can be greatly facilitated when the lower surfaces of the chamber(s) and the microfluidic channel to which the chamber(s) open have a coplanar orientation.

[00425] The proximal opening of the connection region of a sequestration pen may have a width (e.g., Wcon or Wconi) that is at least as large as the largest dimension of a micro-object (e.g., a biological cell, which may be a plant cell, such as a plant protoplast) for which the sequestration pen is intended. In some embodiments, the proximal opening has a width (e.g., W _CO n or W _CO ni) of about 20 microns, about 40 microns, about 50 microns, about 60 microns, about 75 microns, about 100 microns, about 150 microns, about 200 microns, or about 300 microns. The foregoing are examples only, and the width (e.g., W _CO n or Wconi) of a proximal opening can be selected to be a value between any of the values listed above (e.g., about 20-200 microns, about 20-150 microns, about 20-100 microns, about 20-75 microns, about 20-60 microns, about 50-300 microns, about 50-200 microns, about 50-150 microns, about 50-100 microns, about 50-75 microns, about 75-150 microns, about 75-100 microns, about 100-300 microns, about 100- 200 microns, or about 200-300 microns).

[00426] In some embodiments, the connection region of the sequestration pen may have a length (e.g., Leon) from the proximal opening to the distal opening to the isolation region of the sequestration pen that is at least 0.5 times, at least 0.6 times, at least 0.7 times, at least 0.8 times, at least 0.9 times, at least 1.0 times, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.75 times, at least 2.0 times, at least 2.25 times, at least 2.5 times, at least 2.75 times, at least 3.0 times, at least 3.5 times, at least 4.0 times, at least 4.5 times, at least 5.0 times, at least 6.0 times, at least 7.0 times, at least 8.0 times, at least 9.0 times, or at least 10.0 times the width (e.g., W _CO n or W _CO ni) of the proximal opening. Thus, for example, the proximal opening of the connection region of a sequestration pen may have a width (e.g., W _C0 n Or W _CO ni) from about 20 microns to about 200 microns (e.g., about 50 microns to about 150 microns), and the connection region may have a length L _CO n that is at least 1.0 times (e.g., at least 1.5 times, or at least 2.0 times) the width of the proximal opening. As another example, the proximal opening of the connection region of a sequestration pen may have a width (e.g., W _CO n or W _CO ni) from about 20 microns to about 100 microns (e.g., about 20 microns to about 60 microns), and the connection region may have a length L _CO n that is at least 1.0 times (e.g., at least 1.5 times, or at least 2.0 times) the width of the proximal opening.

[00427] The microfluidic channel of a microfluidic device to which a sequestration pen opens may have specified size (e.g., width or height). In some embodiments, the height (e.g., H _C h) of the microfluidic channel at a proximal opening to the connection region of a sequestration pen can be within any of the following ranges: 20-100 microns, 20-90 microns, 20-80 microns, 20-70 microns, 20-60 microns, 20-50 microns, 30-100 microns, 30-90 microns, 30-80 microns, 30-70 microns, 30-60 microns, 30-50 microns, 40-100 microns, 40-90 microns, 40-80 microns, 40-70 microns, 40-60 microns, or 40-50 microns. The foregoing are examples only, and the height (e.g., H _C h) of the microfluidic channel (e.g., 122) can be selected to be between any of the values listed above. Moreover, the height (e.g., H _C h) of the microfluidic channel 122 can be selected to be any of these heights in regions of the microfluidic channel other than at a proximal opening of a sequestration pen.

[00428] The width (e.g., W _C h) of the microfluidic channel at the proximal opening to the connection region of a sequestration pen can be within any of the following ranges: about 20-500 microns, 20-400 microns, 20-300 microns, 20-200 microns, 20-150 microns, 20-100 microns, 20-80 microns, 20-60 microns, 30-400 microns, 30-300 microns, 30-200 microns, 30-150 microns, 30-100 microns, 30-80 microns, 30-60 microns, 40-300 microns, 40-200 microns, 40-150 microns, 40-100 microns, 40-80 microns, 40-60 microns, 50-1000 microns, 50-500 microns, 50-400 microns, 50-300 microns, 50-250 microns, 50-200 microns, 50-150 microns, 50-100 microns, 50-80 microns, 60-300 microns, 60-200 microns, 60-150 microns, 60-100 microns, 60-80 microns, 70-500 microns, 70-400 microns, 70-300 microns, 70-250 microns, 70-200 microns, 70-150 microns, 70-100 microns, 80-100 microns, 90-400 microns, 90-300 microns, 90-250 microns, 90-200 microns, 90-150 microns, 100-300 microns, 100-250 microns, 100-200 microns, 100-150 microns, 100-120 microns, 200-800 microns, 200-700 microns, or 200-600 microns. The foregoing are examples only, and the width (e.g., W _C h) of the microfluidic channel can be a value selected to be between any of the values listed above. Moreover, the width (e.g., W _C h) of the microfluidic channel can be selected to be in any of these widths in regions of the microfluidic channel other than at a proximal opening of a sequestration pen. In some embodiments, the width W _C h of the microfluidic channel at the proximal opening to the connection region of the sequestration pen (e.g., taken transverse to the direction of bulk flow of fluid through the channel) can be substantially perpendicular to a width (e.g., W _CO n or W _CO ni) of the proximal opening.

[00429] A cross-sectional area of the microfluidic channel at a proximal opening to the connection region of a sequestration pen can be about 500-50,000 square microns, 500-40,000 square microns, SOO- SO, 000 square microns, 500-25,000 square microns, 500-20,000 square microns, 500-15,000 square microns, 500-10,000 square microns, 500-7,500 square microns, 500-5,000 square microns, 1,000-25,000 square microns, 1,000-20,000 square microns, 1,000-15,000 square microns, 1,000-10,000 square microns, 1,000-7,500 square microns, 1,000-5,000 square microns, 2,000-20,000 square microns, 2, GOO- 15, 000 square microns, 2,000-10,000 square microns, 2,000-7,500 square microns, 2,000-6,000 square microns, 3,000-20,000 square microns, 3,000-15,000 square microns, 3,000-10,000 square microns, 3,000-7,500 square microns, or 3,000 to 6,000 square microns. The foregoing are examples only, and the cross-sectional area of the microfluidic channel at the proximal opening can be selected to be between any of the values listed above. In various embodiments, and the cross-sectional area of the microfluidic channel at regions of the microfluidic channel other than at the proximal opening can also be selected to be between any of the values listed above. In some embodiments, the cross-sectional area is selected to be a substantially uniform value for the entire length of the microfluidic channel.

[00430] In some embodiments, the microfluidic chip is configured such that the proximal opening (e.g., 234 or 334) of the connection region of a sequestration pen may have a width (e.g., W _C0 nOr W _CO ni) from about 20 microns to about 200 microns (e.g., about 50 microns to about 150 microns), the connection region may have a length L _CO n (e.g., 236 or 336) that is at least 1.0 times (e.g., at least 1.5 times, or at least 2.0 times) the width of the proximal opening, and the microfluidic channel may have a height (e.g., H _C h) at the proximal opening of about 30 microns to about 60 microns. As another example, the proximal opening (e.g., 234 or 334) of the connection region of a sequestration pen may have a width (e.g., W _CO n or Wconi) from about 20 microns to about 100 microns (e.g., about 20 microns to about 60 microns), the connection region may have a length L _CO n (e.g., 236 or 336) that is at least 1.0 times (e.g., at least 1.5 times, or at least 2.0 times) the width of the proximal opening, and the microfluidic channel may have a height (e.g., H _C h) at the proximal opening of about 30 microns to about 60 microns. The foregoing are examples only, and the width (e.g., W _C0 nOr W _CO ni) of the proximal opening (e.g., 234 or 274), the length (e.g., Leon) of the connection region, and/or the width (e.g., W _C h) of the microfluidic channel (e.g., 122 or 322), can be a value selected to be between any of the values listed above. Generally, however, the width (Wcon or Wconi) of the proximal opening of the connection region of a sequestration pen is less than the width (Wch) of the microfluidic channel. In some embodiments, the width (W _CO n or W _CO ni) of the proximal opening is about 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 22%, 24%, 25%, or 30% of the width (W _C h) of the microfluidic channel. That is, the width (W _C h) of the microfluidic channel may be at least 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 6.0 times, 7.0 times, 8.0 times, 9.0 times or at least 10.0 times the width (W _CO n or W _CO ni) of the proximal opening of the connection region of the sequestration pen.

[00431] In some embodiments, the size Wc (e.g., cross-sectional width W _C h, diameter, area, or the like) of the channel 122, 322, 618, 718 can be about one and a quarter (1.25), about one and a half (1.5), about two, about two and a half (2.5), about three (3), or more times the size Wo (e.g., cross-sectional width Wcon, diameter, area, or the like) of a chamber opening, e.g., sequestration pen opening 234, 334, and the like. This can reduce the extent of secondary flow and the rate of diffusion (or diffusion flux) through the opening 234, 334 for materials diffusing from a selected chamber (e.g., like sequestration pens 224, 226 of FIG. 2B) into channel 122, 322, 618, 718 and subsequently re-entering a downstream or adjacent chamber (e.g., like sequestration pen 228). The rate of diffusion of a molecule (e.g., an analyte of interest, such as an antibody) is dependent on a number of factors, including (without limitation) temperature, viscosity of the medium, and the coefficient of diffusion Do of the molecule. For example, the Do for an IgG antibody in aqueous solution at about 20°C is about 4.4xl0' ⁷ cm ² /sec, while the kinematic viscosity of cell culture medium is about 9xl0' ⁴ m ² /sec. Thus, an antibody in cell culture medium at about 20°C can have a rate of diffusion of about 0.5 microns/sec. Accordingly, in some embodiments, a time period for diffusion from a biological micro-object located within a sequestration pen such as 224, 226, 228, 324 into the channel 122, 322, 618, 718 can be about 10 minutes or less (e.g., about 9, 8, 7, 6, 5 minutes, or less). The time period for diffusion can be manipulated by changing parameters that influence the rate of diffusion. For example, the temperature of the media can be increased (e.g., to a physiological temperature such as about 37°C) or decreased (e.g., to about 15°C, 10°C, or 4°C) thereby increasing or decreasing the rate of diffusion, respectively. Alternatively, or in addition, the concentrations of solutes in the medium can be increased or decreased as discussed herein to isolate a selected pen from solutes from other upstream pens.

[00432] Accordingly, in some variations, the width (e.g., W _C h) of the microfluidic channel at the proximal opening to the connection region of a sequestration pen may be about 50 to 500 microns, about 50 to 300 microns, about 50 to 200 microns, about 70 to 500 microns, about to 70-300 microns, about 70 to 250 microns, about 70 to 200 microns, about 70 to 150 microns, about 70 to 100 microns, about 80 to 500 microns, about 80 to 300 microns, about 80 to 250 microns, about 80 to 200 microns, about 80 to 150 microns, about 90 to 500 microns, about 90 to 300 microns, about 90 to 250 microns, about 90 to 200 microns, about 90 to 150 microns, about 100 to 500 microns, about 100 to 300 microns, about 100 to 250 microns, about 100 to 200 microns, or about 100 to 150 microns. In some embodiments, the width W _C h of the microfluidic channel at the proximal opening to the connection region of a sequestration pen may be about 70 to 250 microns, about 80 to 200 microns, or about 90 to 150 microns. The width W _CO n of the opening of the chamber (e.g., sequestration pen) may be about 20 to 100 microns; about 30 to 90 microns; or about 20 to 60 microns. In some embodiments, W _C h is about 70-250 microns and W _CO n is about 20 to 100 microns; W _C h is about 80 to 200 microns and W _CO n is about 30 to 90 microns; W _C h is about 90 to 150 microns, and W _CO n is about 20 to 60 microns; or any combination of the widths of W _C h and W _CO n thereof.

[00433] In some embodiments, the proximal opening (e.g., 234 or 334) of the connection region of a sequestration pen has a width (e.g., Wcon or Wconi) that is 2.0 times or less (e.g., 2.0, 1.9, 1.8, 1.5, 1.3, 1.0, 0.8, 0.5, or 0.1 times) the height (e.g., H _C h) of the flow region/ microfluidic channel at the proximal opening, or has a value that lies within a range defined by any two of the foregoing values.

[00434] In some embodiments, the width W _CO ni of a proximal opening (e.g., 234 or 334) of a connection region of a sequestration pen may be the same as a width W _CO n2 of the distal opening (e.g., 238 or 338) to the isolation region thereof. In some embodiments, the width W _CO ni of the proximal opening may be different than a width W _CO n2 of the distal opening, and W _CO ni and/or W _CO n2 may be selected from any of the values described for W _CO n or W _CO ni. In some embodiments, the walls (including a connection region wall) that define the proximal opening and distal opening may be substantially parallel with respect to each other. In some embodiments, the walls that define the proximal opening and distal opening may be selected to not be parallel with respect to each other.

[00435] The length (e.g., L _CO n) of the connection region can be about 1-600 microns, 5-550 microns, 10- 500 microns, 15-400 microns, 20-300 microns, 20-500 microns, 40-400 microns, 60-300 microns, 80- 200 microns, about 100-150 microns, about 20-300 microns, about 20 -250 microns, about 20-200 microns, about 20-150 microns, about 20-100 microns, about 30-250 microns, about 30-200 microns, about 30- 150 microns, about 30-100 microns, about 30-80 microns, about 30-50 microns, about 45-250 microns, about 45-200 microns, about 45-100 microns, about 45- 80 microns, about 45-60 microns, about 60-200 microns, about 60-150 microns, about 60-100 microns or about 60-80 microns. The foregoing are examples only, and length (e.g., L _CO n) of a connection region can be selected to be a value that is between any of the values listed above.

[00436] The connection region wall of a sequestration pen may have a length (e.g., L _wa ii) that is at least 0.5 times, at least 0.6 times, at least 0.7 times, at least 0.8 times, at least 0.9 times, at least 1.0 times, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.75 times, at least 2.0 times, at least 2.25 times, at least 2.5 times, at least 2.75 times, at least 3.0 times, or at least 3.5 times the width (e.g., W _CO n or W _CO ni) of the proximal opening of the connection region of the sequestration pen. In some embodiments, the connection region wall may have a length L _wa ii of about 20-200 microns, about 20-150 microns, about 20-100 microns, about 20-80 microns, or about 20-50 microns. The foregoing are examples only, and a connection region wall may have a length L _wa ii selected to be between any of the values listed above.

[00437] A sequestration pen may have a length L _s of about 40-600 microns, about 40-500 microns, about 40-400 microns, about 40-300 microns, about 40-200 microns, about 40-100 microns or about 40-80 microns. The foregoing are examples only, and a sequestration pen may have a length L _s selected to be between any of the values listed above.

[00438] According to some embodiments, a sequestration pen may have a specified height (e.g., H _s ). In some embodiments, a sequestration pen has a height H _s of about 20 microns to about 200 microns (e.g., about 20 microns to about 150 microns, about 20 microns to about 100 microns, about 20 microns to about 60 microns, about 30 microns to about 150 microns, about 30 microns to about 100 microns, about 30 microns to about 60 microns, about 40 microns to about 150 microns, about 40 microns to about 100 microns, or about 40 microns to about 60 microns). The foregoing are examples only, and a sequestration pen can have a height H _s selected to be between any of the values listed above.

[00439] The height H _CO n of a connection region at a proximal opening of a sequestration pen can be a height within any of the following heights: 20-100 microns, 20-90 microns, 20-80 microns, 20-70 microns, 20-60 microns, 20-50 microns, 30-100 microns, 30-90 microns, 30-80 microns, 30-70 microns, 30-60 microns, 30-50 microns, 40-100 microns, 40-90 microns, 40-80 microns, 40-70 microns, 40-60 microns, or 40-50 microns. The foregoing are examples only, and the height H _CO n of the connection region can be selected to be between any of the values listed above. Typically, the height H _CO n of the connection region is selected to be the same as the height H _C h of the microfluidic channel at the proximal opening of the connection region. Additionally, the height H _s of the sequestration pen is typically selected to be the same as the height H _CO n of a connection region and/or the height H _C h of the microfluidic channel. In some embodiments, H _s , H _CO n, and H _C h may be selected to be the same value of any of the values listed above for a selected microfluidic device.

[00440] The isolation region can be configured to contain only one, two, three, four, five, or a similar relatively small number of micro-objects. In other embodiments, the isolation region may contain more than 10, more than 50 or more than 100 micro-objects. Accordingly, the volume of an isolation region can be, for example, at least IxlO ⁴ , IxlO ⁵ , 5xl0 ⁵ , 8xl0 ⁵ , IxlO ⁶ , 2xl0 ⁶ , 4xl0 ⁶ , 6xl0 ⁶ , IxlO ⁷ , 3xl0 ⁷ , 5xl0 ⁷ IxlO ⁸ , 5xl0 ⁸ , or 8xl0 ⁸ cubic microns, or more. The foregoing are examples only, and the isolation region can be configured to contain numbers of micro-objects and volumes selected to be between any of the values listed above (e.g., a volume between IxlO ⁵ cubic microns and 5xl0 ⁵ cubic microns, between 5xl0 ⁵ cubic microns and IxlO ⁶ cubic microns, between IxlO ⁶ cubic microns and 2xl0 ⁶ cubic microns, or between 2xl0 ⁶ cubic microns and IxlO ⁷ cubic microns).

[00441] According to some embodiments, a sequestration pen of a microfluidic device may have a specified volume. The specified volume of the sequestration pen (or the isolation region of the sequestration pen) may be selected such that a single cell or a small number of cells (e.g., 2-10 or 2-5) can rapidly condition the medium and thereby attain favorable (or optimal) growth conditions. In some embodiments, the sequestration pen has a volume of about 5xl0 ⁵ , 6xl0 ⁵ , 8xl0 ⁵ , IxlO ⁶ , 2xl0 ⁶ , 4xl0 ⁶ , 8xl0 ⁶ , IxlO ⁷ , 3xl0 ⁷ , 5xl0 ⁷ , or about 8xl0 ⁷ cubic microns, or more. In some embodiments, the sequestration pen has a volume of about 1 nanoliter to about 50 nanoliters, 2 nanoliters to about 25 nanoliters, 2 nanoliters to about 20 nanoliters, about 2 nanoliters to about 15 nanoliters, or about 2 nanoliters to about 10 nanoliters. The foregoing are examples only, and a sequestration pen can have a volume selected to be any value that is between any of the values listed above.

[00442] According to some embodiments, the flow of fluidic medium within the microfluidic channel (e.g., 122 or 322) may have a specified maximum velocity (e.g., Vmax). In some embodiments, the maximum velocity (e.g., Vmax) may be set at around 0.2, 0.5, 0.7, 1.0, 1.3, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.7, 7.0, 7.5, 8.0, 8.5, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, or 25 microliters/sec. The foregoing are examples only, and the flow of fluidic medium within the microfluidic channel can have a maximum velocity (e.g., Vmax) selected to be a value between any of the values listed above. The flow of fluidic medium within the microfluidic channel typically may be flowed at a rate less than the Vmax. While the Vmax may vary depending on the specific size and numbers of channel and sequestration pens opening thereto, a fluidic medium may be flowed at about 0.1 microliters/sec to about 20 microliters/sec; about 0.1 microliters/sec to about 15 microliters/sec; about 0.1 microliters/sec to about 12 microliters/sec, about 0.1 microliters/sec to about 10 microliters/sec; about 0.1 microliter/sec to about 7 microliters/sec without exceeding the Vmax. In some portions of a typical workflow, a flow rate of a fluidic medium may be about 0.1 microliters/sec; about 0.5 microliters/sec; about 1.0 microliters/sec; about 2.0 microliters/sec; about 3.0 microliters/sec; about 4.0 microliters/sec; about 5.0 microliters/sec; about 6.0 microliters/sec; about 7.0 microliters/sec; about 8.0 microliters/sec; about 9.0 microliters/sec; about 10.0 microliters/sec; about 11.0 microliters/sec; or any range defined by two of the foregoing values, e.g., 1-5 microliters/sec or 5-10 microliters/sec. The flow rate of a fluidic medium in the microfluidic channel may be equal to or less than about 12 microliters/sec; about 10 microliters/sec; about 8 microliters/sec, or about 6 microliters/sec.

[00443] In various embodiment, the microfluidic device has sequestration pens configured as in any of the embodiments discussed herein where the microfluidic device has about 5 to about 10 sequestration pens, about 10 to about 50 sequestration pens, about 25 to about 200 sequestration pens, about 100 to about 500 sequestration pens, about 200 to about 1000 sequestration pens, about 500 to about 1500 sequestration pens, about 1000 to about 2500 sequestration pens, about 2000 to about 5000 sequestration pens, about 3500 to about 7000 sequestration pens, about 5000 to about 10,000 sequestration pens, about 7,500 to about 15,000 sequestration pens, about 12,500 to about 20,000 sequestration pens, about 15,000 to about 25,000 sequestration pens, about 20,000 to about 30,000 sequestration pens, about 25,000 to about 35,000 sequestration pens, about 30,000 to about 40,000 sequestration pens, about 35,000 to about 45,000 sequestration pens, or about 40,000 to about 50,000 sequestration pens. The sequestration pens need not all be the same size and may include a variety of configurations (e.g., different widths, different features within the sequestration pen).

[00444] Coating solutions and coating agents. In some embodiments, at least one inner surface of the microfluidic device includes a coating material that provides a layer of organic and/or hydrophilic molecules suitable for maintenance, expansion and/or movement of biological micro-object(s) (i.e., the biological micro-object exhibits increased viability, greater expansion and/or greater portability within the microfluidic device). The conditioned surface may reduce surface fouling, participate in providing a layer of hydration, and/or otherwise shield the biological micro-objects from contact with the non-organic materials of the microfluidic device interior.

[00445] In some embodiments, substantially all the inner surfaces of the microfluidic device include the coating material. The coated inner surface(s) may include the surface of a flow region (e.g., channel), chamber, or sequestration pen, or a combination thereof. In some embodiments, each of a plurality of sequestration pens has at least one inner surface coated with coating materials. In other embodiments, each of a plurality of flow regions or channels has at least one inner surface coated with coating materials. In some embodiments, at least one inner surface of each of a plurality of sequestration pens and each of a plurality of channels is coated with coating materials. The coating may be applied before or after introduction of biological micro-object(s), or may be introduced concurrently with the biological microobjects). In some embodiments, the biological micro-object(s) may be imported into the microfluidic device in a fluidic medium that includes one or more coating agents. In other embodiments, the inner surface(s) of the microfluidic device (e.g., a microfluidic device having an electrode activation substrate such as, but not limited to, a device including dielectrophoresis (DEP) electrodes) may be treated or “primed” with a coating solution comprising a coating agent prior to introduction of the biological micro- object(s) into the microfluidic device. Any convenient coating agent/coating solution can be used, including but not limited to: serum or serum factors, bovine serum albumin (BSA), polymers, detergents, enzymes, and any combination thereof.

[00446] Synthetic polymer-based coating materials. The at least one inner surface may include a coating material that comprises a polymer. The polymer may be non-covalently bound (e.g., it may be non-specifically adhered) to the at least one surface. The polymer may have a variety of structural motifs, such as found in block polymers (and copolymers), star polymers (star copolymers), and graft or comb polymers (graft copolymers), all of which may be suitable for the methods disclosed herein. A wide variety of alkylene ether containing polymers may be suitable for use in the microfluidic devices described herein, including but not limited to Pluronic® polymers such as Pluronic® L44, L64, P85, and F127 (including Fl 27NF). Other examples of suitable coating materials are described in US2016/0312165, the contents of which are herein incorporated by reference in their entirety.

[00447] Covalently linked coating materials. In some embodiments, the at least one inner surface includes covalently linked molecules that provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) within the microfluidic device, providing a conditioned surface for such cells. The covalently linked molecules include a linking group, wherein the linking group is covalently linked to one or more surfaces of the microfluidic device, as described below. The linking group is also covalently linked to a surface modifying moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/ expansion/ movement of biological micro-object(s).

[00448] In some embodiments, the covalently linked moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) may include alkyl or fluoroalkyl (which includes perfluoroalkyl) moieties; mono- or polysaccharides (which may include but is not limited to dextran); alcohols (including but not limited to propargyl alcohol); polyalcohols, including but not limited to polyvinyl alcohol; alkylene ethers, including but not limited to polyethylene glycol; polyelectrolytes ( including but not limited to polyacrylic acid or polyvinyl phosphonic acid); amino groups (including derivatives thereof, such as, but not limited to alkylated amines, hydroxyalkylated amino group, guanidinium, and heterocylic groups containing an unaromatized nitrogen ring atom, such as, but not limited to morpholinyl or piperazinyl); carboxylic acids including but not limited to propiolic acid (which may provide a carboxylate anionic surface); phosphonic acids, including but not limited to ethynyl phosphonic acid (which may provide a phosphonate anionic surface); sulfonate anions; carboxybetaines; sulfobetaines; sulfamic acids; or amino acids.

[00449] In various embodiments, the covalently linked moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device may include non-polymeric moieties such as an alkyl moiety, amino acid moiety, alcohol moiety, amino moiety, carboxylic acid moiety, phosphonic acid moiety, sulfonic acid moiety, sulfamic acid moiety, or saccharide moiety. Alternatively, the covalently linked moiety may include polymeric moieties, which may include any of these moieties. [00450] In some embodiments, a microfluidic device may have a hydrophobic layer upon the inner surface of the base which includes a covalently linked alkyl moiety. The covalently linked alkyl moiety may comprise carbon atoms forming a linear chain (e.g., a linear chain of at least 10 carbons, or at least 14, 16, 18, 20, 22, or more carbons) and may be an unbranched alkyl moiety. In some embodiments, the alkyl group may include a substituted alkyl group (e.g., some of the carbons in the alkyl group can be fluorinated or perfluorinated). In some embodiments, the alkyl group may include a first segment, which may include a perfluoroalkyl group, joined to a second segment, which may include a non- substituted alkyl group, where the first and second segments may be joined directly or indirectly (e.g., by means of an ether linkage). The first segment of the alkyl group may be located distal to the linking group, and the second segment of the alkyl group may be located proximal to the linking group.

[00451] In other embodiments, the covalently linked moiety may include at least one amino acid, which may include more than one type of amino acid. Thus, the covalently linked moiety may include a peptide or a protein. In some embodiments, the covalently linked moiety may include an amino acid which may provide a zwitterionic surface to support cell growth, viability, portability, or any combination thereof.

[00452] In other embodiments, the covalently linked moiety may further include a streptavidin or biotin moiety. In some embodiments, a modified biological moiety such as, for example, a biotinylated protein or peptide may be introduced to the inner surface of a microfluidic device bearing covalently linked streptavidin, and couple via the covalently linked streptavidin to the surface, thereby providing a modified surface presenting the protein or peptide.

[00453] In other embodiments, the covalently linked moiety may include at least one alkylene oxide moiety and may include any alkylene oxide polymer as described above. One useful class of alkylene ether containing polymers is polyethylene glycol (PEG M _w <100,000Da) or alternatively polyethylene oxide (PEO, M _w >100,000). In some embodiments, a PEG may have an M _w of about lOOODa, 5000Da, 10,000Da or 20,000Da. In some embodiments, the PEG polymer may further be substituted with a hydrophilic or charged moiety, such as but not limited to an alcohol functionality or a carboxylic acid moiety.

[00454] The covalently linked moiety may include one or more saccharides. The covalently linked saccharides may be mono-, di-, or polysaccharides. The covalently linked saccharides may be modified to introduce a reactive pairing moiety which permits coupling or elaboration for attachment to the surface. One exemplary covalently linked moiety may include a dextran polysaccharide, which may be coupled indirectly to a surface via an unbranched linker.

[00455] The coating material providing a conditioned surface may comprise only one kind of covalently linked moiety or may include more than one different kind of covalently linked moiety. For example, a polyethylene glycol conditioned surface may have covalently linked alkylene oxide moieties having a specified number of alkylene oxide units which are all the same, e.g., having the same linking group and covalent attachment to the surface, the same overall length, and the same number of alkylene oxide units. Alternatively, the coating material may have more than one kind of covalently linked moiety attached to the surface. For example, the coating material may include the molecules having covalently linked alkylene oxide moieties having a first specified number of alkylene oxide units and may further include a further set of molecules having bulky moieties such as a protein or peptide connected to a covalently attached alkylene oxide linking moiety having a greater number of alkylene oxide units. The different types of molecules may be varied in any suitable ratio to obtain the surface characteristics desired. For example, the conditioned surface having a mixture of first molecules having a chemical structure having a first specified number of alkylene oxide units and second molecules including peptide or protein moieties, which may be coupled via a biotin/ streptavidin binding pair to the covalently attached alkylene linking moiety, may have a ratio of first molecules: second molecules of about 99: 1; about 90: 10; about 75:25; about 50:50; about 30:70; about 20:80; about 10:90; or any ratio selected to be between these values. In this instance, the first set of molecules having different, less sterically demanding termini and fewer backbone atoms can help to functionalize the entire substrate surface and thereby prevent undesired adhesion or contact with the silicon/silicon oxide, hafnium oxide or alumina making up the substrate itself. The selection of the ratio of mixture of first molecules to second molecules may also modulate the surface modification introduced by the second molecules bearing peptide or protein moieties.

[00456] Conditioned surface properties. Various factors can alter the physical thickness of the conditioned surface, such as the manner in which the conditioned surface is formed on the substrate (e.g., vapor deposition, liquid phase deposition, spin coating, flooding, and electrostatic coating). In some embodiments, the conditioned surface may have a thickness of about Inm to about lOnm. In some embodiments, the covalently linked moieties of the conditioned surface may form a monolayer when covalently linked to the surface of the microfluidic device (which may include an electrode activation substrate having dielectrophoresis (DEP) or electrowetting (EW) electrodes) and may have a thickness of less than 10 nm (e.g., less than 5 nm, or about 1.5 to 3.0 nm). These values are in contrast to that of a surface prepared by spin coating, for example, which may typically have a thickness of about 30nm. In some embodiments, the conditioned surface does not require a perfectly formed monolayer to be suitably functional for operation within a DEP-configured microfluidic device. In other embodiments, the conditioned surface formed by the covalently linked moieties may have a thickness of about 10 nm to about 50 nm.

[00457] Unitary or Multi-part conditioned surface. The covalently linked coating material may be formed by reaction of a molecule which already contains the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device, and may have a structure of Formula A, as shown below. Alternatively, the covalently linked coating material may be formed in a two-part sequence, having a structure of Formula B, by coupling the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance and/or expansion of biological micro-object(s) to a surface modifying ligand that itself has been covalently linked to the surface. In some embodiments, the surface may be formed in a two-part or three-part sequence, including a streptavidin/biotin binding pair, to introduce a protein, peptide, or mixed modified surface.

DEP substrate DEP substrate or

Formula A Formula B

[00458] The coating material may be linked covalently to oxides of the surface of a DEP-configured or EW- configured substrate. The coating material may be attached to the oxides via a linking group (“LG”), which may be a siloxy or phosphonate ester group formed from the reaction of a siloxane or phosphonic acid group with the oxides. The moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device can be any of the moieties described herein. The linking group LG may be directly or indirectly connected to the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device. When the linking group LG is directly connected to the moiety, optional linker (“L”) is not present and n is 0. When the linking group LG is indirectly connected to the moiety, linker L is present and n is 1. The linker L may have a linear portion where a backbone of the linear portion may include 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and/or phosphorus atoms, subject to chemical bonding limitations as is known in the art. It may be interrupted with any combination of one or more moieties, which may be chosen from ether, amino, carbonyl, amido, and/or phosphonate groups, arylene, heteroarylene, or heterocyclic groups. In some embodiments, the coupling group CG represents the resultant group from reaction of a reactive moiety R _x and a reactive pairing moiety R _px (i. e., a moiety configured to react with the reactive moiety R _x ). CG may be a carboxamidyl group, a triazolylene group, substituted triazolylene group, a carboxamidyl, thioamidyl, an oxime, a mercaptyl, a disulfide, an ether, or alkenyl group, or any other suitable group that may be formed upon reaction of a reactive moiety with its respective reactive pairing moiety. In some embodiments, CG may further represent a streptavidin/biotin binding pair.

[00459] Further details of suitable coating treatments and modifications, as well as methods of preparation, may be found at U.S. Patent Application Publication No. US2016/0312165 (Lowe, Jr., et al.), U.S. Patent Application Publication No US2017/0173580 (Lowe, Jr., et al), International Patent Application Publication W02017/205830 (Lowe, Jr., et al.), and International Patent Application Publication W02019/01880 (Beemiller et al.), each of which disclosures is herein incorporated by reference in its entirety.

[00460] Microfluidic device motive technologies. The microfluidic devices described herein can be used with any type of motive technology. As described herein, the control and monitoring equipment of the system can comprise a motive module for selecting and moving objects, such as micro-objects or droplets, in the microfluidic circuit of a microfluidic device. The motive technology(ies) may include, for example, dielectrophoresis (DEP), electrowetting (EW), and/or other motive technologies. The microfluidic device can have a variety of motive configurations, depending upon the type of object being moved and other considerations. Returning to FIG. 1A, for example, the support structure 104 and/or cover 110 of the microfluidic device 100 can comprise DEP electrode activation substrates for selectively inducing motive forces on micro-objects in the fluidic medium 180 in the microfluidic circuit 120 and thereby select, capture, and/or move individual micro-objects or groups of micro-objects.

[00461] In some embodiments, motive forces are applied across the fluidic medium 180 (e.g., in the flow path and/or in the sequestration pens) via one or more electrodes (not shown) to manipulate, transport, separate and sort micro-objects located therein. For example, in some embodiments, motive forces are applied to one or more portions of microfluidic circuit 120 in order to transfer a single microobject from the flow path 106 into a desired microfluidic sequestration pen. In some embodiments, motive forces are used to prevent a micro-object within a sequestration pen from being displaced therefrom. Further, in some embodiments, motive forces are used to selectively remove a micro-object from a sequestration pen that was previously collected in accordance with the embodiments of the current disclosure.

[00462] In some embodiments, the microfluidic device is configured as an optically-actuated electrokinetic device, such as in optoelectronic tweezer (OET) and/or optoelectrowetting (OEW) configured device. Examples of suitable OET configured devices (e.g., containing optically actuated dielectrophoresis electrode activation substrates) can include those illustrated in U.S. Patent No. RE 44,711 (Wu, et al.) (originally issued as U.S. Patent No. 7,612,355), U.S. Patent No. 7,956,339 (Ohta, et al.), U.S. Patent No. 9,908,115 (Hobbs et al.), and U.S. Patent No. 9,403,172 (Short et al), each of which is incorporated herein by reference in its entirety. Examples of suitable OEW configured devices can include those illustrated in U.S. Patent No. 6,958,132 (Chiou, et al.), and U.S. Patent Application No. 9,533,306 (Chiou, et al.), each of which is incorporated herein by reference in its entirety. Examples of suitable optically-actuated electrokinetic devices that include combined OET/OEW configured devices can include those illustrated in U.S. Patent Application Publication No. 2015/0306598 (Khandros, et al.), U.S. Patent Application Publication No 2015/0306599 (Khandros, et al.), and U.S. Patent Application Publication No. 2017/0173580 (Lowe, et al.), each of which is incorporated herein by reference in its entirety.

[00463] It should be understood that, for purposes of simplicity, the various examples of FIGS. 1-5B may illustrate portions of microfluidic devices while not depicting other portions. Further, Figures 1-5B may be part of, and implemented as, one or more microfluidic systems. In one non-limiting example, FIGS. 4 A and 4B show a side cross-sectional view and a top cross-sectional view, respectively, of a portion of an enclosure 102 of the microfluidic device 400 having a region/chamber 402, which may be part of a fluidic circuit element having a more detailed structure, such as a growth chamber, a sequestration pen (which may be like any sequestration pen described herein), a flow region, or a flow channel. For instance, microfluidic device 400 may be similar to microfluidic devices 100, 175, 200, 300, 520 or any other microfluidic device as described herein. Furthermore, the microfluidic device 400 may include other fluidic circuit elements and may be part of a system including control and monitoring equipment 152, described above, having one or more of the media module 160, motive module 162, imaging module 164, optional tilting module 166, and other modules 168. Microfluidic devices 175, 200, 300, 520 and any other microfluidic devices described herein may similarly have any of the features described in detail for FIGS. 1 A-1B and 4A-4B.

[00464] As shown in the example of FIG. 4A, the microfluidic device 400 includes a support structure 104 having a bottom electrode 404 and an electrode activation substrate 406 overlying the bottom electrode 404, and a cover 110 having a top electrode 410, with the top electrode 410 spaced apart from the bottom electrode 404. The top electrode 410 and the electrode activation substrate 406 define opposing surfaces of the region/ chamber 402. A fluidic medium 180 contained in the region/chamber 402 thus provides a resistive connection between the top electrode 410 and the electrode activation substrate 406. A power source 412 configured to be connected to the bottom electrode 404 and the top electrode 410 and create a biasing voltage between the electrodes, as required for the generation of DEP forces in the region/chamber 402, is also shown. The power source 412 can be, for example, an alternating current (AC) power source.

[00465] In certain embodiments, the microfluidic device 400 illustrated in FIGS. 4A and 4B can have an optically-actuated DEP electrode activation substrate. Accordingly, changing patterns of light 418 from the light source 416, which may be controlled by the motive module 162, can selectively activate and deactivate changing patterns of DEP electrodes at regions 414 of the inner surface 408 of the electrode activation substrate 406. (Hereinafter the regions 414 of a microfluidic device having a DEP electrode activation substrate are referred to as “DEP electrode regions.”) As illustrated in Figure 4B, a light pattern 418 directed onto the inner surface 408 of the electrode activation substrate 406 can illuminate select DEP electrode regions 414a (shown in white) in a pattern, such as a square. The non-illuminated DEP electrode regions 414 (cross-hatched) are hereinafter referred to as “dark” DEP electrode regions 414. The relative electrical impedance through the DEP electrode activation substrate 406 (i.e., from the bottom electrode 404 up to the inner surface 408 of the electrode activation substrate 406 which interfaces with the fluidic medium 180 in the flow region 106) is greater than the relative electrical impedance through the fluidic medium 180 in the region/chamber 402 (i.e., from the inner surface 408 of the electrode activation substrate 406 to the top electrode 410 of the cover 110) at each dark DEP electrode region 414. An illuminated DEP electrode region 414a, however, exhibits a reduced relative impedance through the electrode activation substrate 406 that is less than the relative impedance through the fluidic medium 180 in the region/chamber 402 at each illuminated DEP electrode region 414a.

[00466] With the power source 412 activated, the foregoing DEP configuration creates an electric field gradient in the fluidic medium 180 between illuminated DEP electrode regions 414a and adjacent dark DEP electrode regions 414, which in turn creates local DEP forces that attract or repel nearby microobjects (not shown) in the fluidic medium 180. DEP electrodes that attract or repel micro-objects in the fluidic medium 180 can thus be selectively activated and deactivated at many different such DEP electrode regions 414 at the inner surface 408 of the region/chamber 402 by changing light patterns 418 projected from a light source 416 into the microfluidic device 400. Whether the DEP forces attract or repel nearby micro-objects can depend on such parameters as the frequency of the power source 412 and the dielectric properties of the fluidic medium 180 and/or micro-objects (not shown). Depending on the frequency of the power applied to the DEP configuration and selection of fluidic media (e.g., a highly conductive media such as PBS or other media appropriate for maintaining biological cells), negative DEP forces may be produced. Negative DEP forces may repel the micro-objects away from the location of the induced non-uniform electrical field. In some embodiments, a microfluidic device incorporating DEP technology may generate negative DEP forces.

[00467] The square pattern 420 of illuminated DEP electrode regions 414a illustrated in FIG. 4B is an example only. Any pattern of the DEP electrode regions 414 can be illuminated (and thereby activated) by the pattern of light 418 projected into the microfluidic device 400, and the pattern of illuminated/activated DEP electrode regions 414 can be repeatedly changed by changing or moving the light pattern 418.

[00468] In some embodiments, the electrode activation substrate 406 can comprise or consist of a photoconductive material. In such embodiments, the inner surface 408 of the electrode activation substrate 406 can be featureless. For example, the electrode activation substrate 406 can comprise or consist of a layer of hydrogenated amorphous silicon (a-Si:H). The a-Si:H can comprise, for example, about 8% to 40% hydrogen (calculated as 100 * the number of hydrogen atoms / the total number of hydrogen and silicon atoms). The layer of a-Si:H can have a thickness of about 500 nm to about 2.0 pm. In such embodiments, the DEP electrode regions 414 can be created anywhere and in any pattern on the inner surface 408 of the electrode activation substrate 406, in accordance with the light pattern 418. The number and pattern of the DEP electrode regions 414 thus need not be fixed, but can correspond to the light pattern 418. Examples of microfluidic devices having a DEP configuration comprising a photoconductive layer such as discussed above have been described, for example, in U.S. Patent No. RE 44,711 (Wu, et al.) (originally issued as U.S. Patent No. 7,612,355), each of which is incorporated herein by reference in its entirety.

[00469] In other embodiments, the electrode activation substrate 406 can comprise a substrate comprising a plurality of doped layers, electrically insulating layers (or regions), and electrically conductive layers that form semiconductor integrated circuits, such as is known in semiconductor fields. For example, the electrode activation substrate 406 can comprise a plurality of phototransistors, including, for example, lateral bipolar phototransistors, with each phototransistor corresponding to a DEP electrode region 414. Alternatively, the electrode activation substrate 406 can comprise electrodes (e.g., conductive metal electrodes) controlled by phototransistor switches, with each such electrode corresponding to a DEP electrode region 414. The electrode activation substrate 406 can include a pattern of such phototransistors or phototransistor-controlled electrodes. The pattern, for example, can be an array of substantially square phototransistors or phototransistor-controlled electrodes arranged in rows and columns. Alternatively, the pattern can be an array of substantially hexagonal phototransistors or phototransistor-controlled electrodes that form a hexagonal lattice. Regardless of the pattern, electric circuit elements can form electrical connections between the DEP electrode regions 414 at the inner surface 408 of the electrode activation substrate 406 and the bottom electrode 404, and those electrical connections (i.e., phototransistors or electrodes) can be selectively activated and deactivated by the light pattern 418, as described above.

[00470] Examples of microfluidic devices having electrode activation substrates that comprise phototransistors have been described, for example, in U.S. Patent No. 7,956,339 (Ohta et al.) and U.S. Patent No. 9,908,115 (Hobbs et al.), the entire contents of each of which are incorporated herein by reference. Examples of microfluidic devices having electrode activation substrates that comprise electrodes controlled by phototransistor switches have been described, for example, in U.S. Patent No. 9,403,172 (Short et al.), which is incorporated herein by reference in its entirety.

[00471] In some embodiments of a DEP configured microfluidic device, the top electrode 410 is part of a first wall (or cover 110) of the enclosure 402, and the electrode activation substrate 406 and bottom electrode 404 are part of a second wall (or support structure 104) of the enclosure 102. The region/chamber 402 can be between the first wall and the second wall. In other embodiments, the electrode 410 is part of the second wall (or support structure 104) and one or both of the electrode activation substrate 406 and/or the electrode 410 are part of the first wall (or cover 110). Moreover, the light source 416 can alternatively be used to illuminate the enclosure 102 from below.

[00472] With the microfluidic device 400 of FIGS. 4A-4B having a DEP electrode activation substrate, the motive module 162 of control and monitoring equipment 152, as described for FIG. 1A herein, can select a micro-object (not shown) in the fluidic medium 180 in the region/chamber 402 by projecting a light pattern 418 into the microfluidic device 400 to activate a first set of one or more DEP electrodes at DEP electrode regions 414a of the inner surface 408 of the electrode activation substrate 406 in a pattern (e.g., square pattern 420) that surrounds and captures the micro-object. The motive module 162 can then move the in situ -generated captured micro-object by moving the light pattern 418 relative to the microfluidic device 400 to activate a second set of one or more DEP electrodes at DEP electrode regions 414. Alternatively, the microfluidic device 400 can be moved relative to the light pattern 418.

[00473] In other embodiments, the microfluidic device 400 may be a DEP configured device that does not rely upon light activation of DEP electrodes at the inner surface 408 of the electrode activation substrate 406. For example, the electrode activation substrate 406 can comprise selectively addressable and energizable electrodes positioned opposite to a surface including at least one electrode (e.g., cover 110). Switches (e.g., transistor switches in a semiconductor substrate) may be selectively opened and closed to activate or inactivate DEP electrodes at DEP electrode regions 414, thereby creating a net DEP force on a micro-object (not shown) in region/chamber 402 in the vicinity of the activated DEP electrodes. Depending on such characteristics as the frequency of the power source 412 and the dielectric properties of the medium (not shown) and/or micro-objects in the region/chamber 402, the DEP force can attract or repel a nearby micro-object. By selectively activating and deactivating a set of DEP electrodes (e.g., at a set of DEP electrodes regions 414 that forms a square pattern 420), one or more micro-objects in region/chamber 402 can be selected and moved within the region/chamber 402. The motive module 162 in FIG. 1A can control such switches and thus activate and deactivate individual ones of the DEP electrodes to select, and move particular micro-objects (not shown) around the region/chamber 402. Microfluidic devices having a DEP electrode activation substrate that includes selectively addressable and energizable electrodes are known in the art and have been described, for example, in U.S. Patent No. 6,294,063 (Becker, et al.) and U.S. Patent No. 6,942,776 (Medoro), each of which is incorporated herein by reference in its entirety.

[00474] Regardless of whether the microfluidic device 400 has a di electrophoretic electrode activation substrate, an electrowetting electrode activation substrate or a combination of both a di electrophoretic and an electrowetting activation substrate, a power source 412 can be used to provide a potential (e.g., an AC voltage potential) that powers the electrical circuits of the microfluidic device 400. The power source 412 can be the same as, or a component of, the power source 192 referenced in Fig. 1A. Power source 412 can be configured to provide an AC voltage and/or current to the top electrode 410 and the bottom electrode 404. For an AC voltage, the power source 412 can provide a frequency range and an average or peak power (e.g., voltage or current) range sufficient to generate net DEP forces (or electrowetting forces) strong enough to select and move individual micro-objects (not shown) in the region/chamber 402, as discussed above, and/or to change the wetting properties of the inner surface 408 of the support structure 104 in the region/chamber 202, as also discussed above. Such frequency ranges and average or peak power ranges are known in the art. See, e.g., U.S. Patent No. 6,958,132 (Chiou, et al.), US Patent No. RE44,711 (Wu, et al.) (originally issued as US Patent No. 7,612,355), and U.S. Patent Application Publication Nos. 2014/0124370 (Short, et al.), 2015/0306598 (Khandros, et al.), 2015/0306599 (Khandros, et al.), and 2017/0173580 (Lowe, Jr. et al.), each of which disclosures are herein incorporated by reference in its entirety.

[00475] Other forces may be utilized within the microfluidic devices, alone or in combination, to move selected micro-objects. Bulk fluidic flow within the microfluidic channel may move micro-objects within the flow region. Localized fluidic flow, which may be operated within the microfluidic channel, within a sequestration pen, or within another kind of chamber (e.g., a reservoir) can also be used to move selected micro-objects. Localized fluidic flow can be used to move selected micro-objects out of the flow region into a non-flow region such as a sequestration pen or the reverse, from a non-flow region into a flow region. The localized flow can be actuated by deforming a deformable wall of the microfluidic device, as described in U.S. Patent No. 10,058,865 (Breinlinger, et al.), which is incorporated herein by reference in its entirety.

[00476] Gravity may be used to move micro-obj ects within the microfluidic channel, into a sequestration pen, and/or out of a sequestration pen or other chamber, as described in U.S. Patent No. 9,744,533 (Breinlinger, et al.), which is incorporated herein by reference in its entirety. Use of gravity (e.g., by tilting the microfluidic device and/or the support to which the microfluidic device is attached) may be useful for bulk movement of cells into or out of the sequestration pens from/to the flow region. Magnetic forces may be employed to move micro-objects including paramagnetic materials, which can include magnetic micro-objects attached to or associated with a biological micro-object. Alternatively, or in additional, centripetal forces may be used to move micro-objects within the microfluidic channel, as well as into or out of sequestration pens or other chambers in the microfluidic device.

[00477] In another alternative mode of moving micro-objects, laser-generated dislodging forces may be used to export micro-objects or assist in exporting micro-objects from a sequestration pen or any other chamber in the microfluidic device, as described in International Patent Publication No. WO2017/117408 (Kurz, et al.), which is incorporated herein by reference in its entirety.

[00478] In some embodiments, DEP forces are combined with other forces, such as fluidic flow (e.g., bulk fluidic flow in a channel or localized fluidic flow actuated by deformation of a deformable surface of the microfluidic device, laser generated dislodging forces, and/or gravitational force), so as to manipulate, transport, separate and sort micro-objects and/or droplets within the microfluidic circuit 120. In some embodiments, the DEP forces can be applied prior to the other forces. In other embodiments, the DEP forces can be applied after the other forces. In still other instances, the DEP forces can be applied in an alternating manner with the other forces. For the microfluidic devices described herein, repositioning of micro-objects may not generally rely upon gravity or hydrodynamic forces to position or trap microobjects at a selected position. Gravity may be chosen as one form of repositioning force, but the ability to reposition of micro-objects within the microfluidic device does not rely solely upon the use of gravity. While fluid flow in the microfluidic channels may be used to introduce micro-objects into the microfluidic channels (e.g., flow region), such regional flow is not relied upon to pen or unpen micro-objects, while localized flow (e.g., force derived from actuating a deformable surface) may, in some embodiments, be selected from amongst the other types of repositioning forces described herein to pen or unpen microobjects or to export them from the microfluidic device.

[00479] When DEP is used to reposition micro-objects, bulk fluidic flow in a channel is generally stopped prior to applying DEP to micro-objects to reposition the micro-objects within the microfluidic circuit of the device, whether the micro-objects are being repositioned from the channel into a sequestration pen or from a sequestration pen into the channel. Bulk fluidic flow may be resumed thereafter.

[00480] System. Returning to FIG. 1 A, a system 150 for operating and controlling microfluidic devices is shown, such as for controlling the microfluidic device 100. The electrical power source 192 can provide electric power to the microfluidic device 100, providing biasing voltages or currents as needed. The electrical power source 192 can, for example, comprise one or more alternating current (AC) and/or direct current (DC) voltage or current sources.

[00481] System 150 can further include a media source 178. The media source 178 (e.g., a container, reservoir, or the like) can comprise multiple sections or containers, each for holding a different fluidic medium 180. Thus, the media source 178 can be a device that is outside of and separate from the microfluidic device 100, as illustrated in FIG. 1A. Alternatively, the media source 178 can be located in whole or in part inside the enclosure 102 of the microfluidic device 100. For example, the media source 178 can comprise reservoirs that are part of the microfluidic device 100.

[00482] FIG. 1A also illustrates simplified block diagram depictions of examples of control and monitoring equipment 152 that constitute part of system 150 and can be utilized in conjunction with a microfluidic device 100. As shown, examples of such control and monitoring equipment 152 can include a master controller 154 comprising a media module 160 for controlling the media source 178, a motive module 162 for controlling movement and/or selection of micro-objects (not shown) and/or medium (e.g., droplets of medium) in the microfluidic circuit 120, an imaging module 164 for controlling an imaging device (e.g., a camera, microscope, light source or any combination thereof) for capturing images (e.g., digital images), and an optional tilting module 166 for controlling the tilting of the microfluidic device 100. The control equipment 152 can also include other modules 168 for controlling, monitoring, or performing other functions with respect to the microfluidic device 100. As shown, the monitoring equipment 152 can further include a display device 170 and an input/output device 172.

[00483] The master controller 154 can comprise a control module 156 and a digital memory 158. The control module 156 can comprise, for example, a digital processor configured to operate in accordance with machine executable instructions (e.g., software, firmware, source code, or the like) stored as non- transitory data or signals in the memory 158. Alternatively, or in addition, the control module 156 can comprise hardwired digital circuitry and/or analog circuitry. The media module 160, motive module 162, imaging module 164, optional tilting module 166, and/or other modules 168 can be similarly configured. Thus, functions, processes acts, actions, or steps of a process discussed herein as being performed with respect to the microfluidic device 100 or any other microfluidic apparatus can be performed by any one or more of the master controller 154, media module 160, motive module 162, imaging module 164, optional tilting module 166, and/or other modules 168 configured as discussed above. Similarly, the master controller 154, media module 160, motive module 162, imaging module 164, optional tilting module 166, and/or other modules 168 may be communicatively coupled to transmit and receive data used in any function, process, act, action or step discussed herein.

[00484] The media module 160 controls the media source 178. For example, the media module 160 can control the media source 178 to input a selected fluidic medium 180 into the enclosure 102 (e.g., through an inlet port 107). The media module 160 can also control removal of media from the enclosure 102 (e.g., through an outlet port (not shown)). One or more media can thus be selectively input into and removed from the microfluidic circuit 120. The media module 160 can also control the flow of fluidic medium 180 in the flow path 106 inside the microfluidic circuit 120. The media module 160 may also provide conditioning gaseous conditions to the media source 178, for example, providing an environment containing 5% CO2 (or higher). The media module 160 may also control the temperature of an enclosure of the media source, for example, to provide feeder cells in the media source with proper temperature control.

[00485] Motive module. The motive module 162 can be configured to control selection and movement of micro-objects (not shown) in the microfluidic circuit 120. The enclosure 102 of the microfluidic device 100 can comprise one or more electrokinetic mechanisms including a dielectrophoresis (DEP) electrode activation substrate, optoelectronic tweezers (OET) electrode activation substrate, electrowetting (EW) electrode activation substrate, and/or an opto-electrowetting (OEW) electrode activation substrate, where the motive module 162 can control the activation of electrodes and/or transistors (e.g., phototransistors) to select and move micro-objects and/or droplets in the flow path 106 and/or within sequestration pens 124, 126, 128, and 130. The electrokinetic mechanism(s) may be any suitable single or combined mechanism as described within the paragraphs describing motive technologies for use within the microfluidic device. A DEP configured device may include one or more electrodes that apply a non- uniform electric field in the microfluidic circuit 120 sufficient to exert a di electrophoretic force on microobjects in the microfluidic circuit 120. An OET configured device may include photo-activatable electrodes to provide selective control of movement of micro-objects in the microfluidic circuit 120 via light-induced dielectrophoresis.

[00486] The imaging module 164 can control the imaging device. For example, the imaging module 164 can receive and process image data from the imaging device. Image data from the imaging device can comprise any type of information captured by the imaging device (e.g., the presence or absence of micro-objects, droplets of medium, accumulation of label, such as fluorescent label, etc.). Using the information captured by the imaging device, the imaging module 164 can further calculate the position of objects (e.g., micro-objects, droplets of medium) and/or the rate of motion of such objects within the microfluidic device 100.

[00487] The imaging device (part of imaging module 164, discussed below) can comprise a device, such as a digital camera, for capturing images inside microfluidic circuit 120. In some instances, the imaging device further comprises a detector having a fast frame rate and/or high sensitivity (e.g., for low light applications). The imaging device can also include a mechanism for directing stimulating radiation and/or light beams into the microfluidic circuit 120 and collecting radiation and/or light beams reflected or emitted from the microfluidic circuit 120 (or micro-objects contained therein). The emitted light beams may be in the visible spectrum and may, e.g., include fluorescent emissions. The reflected light beams may include reflected emissions originating from an LED or a wide spectrum lamp, such as a mercury lamp (e.g., a high-pressure mercury lamp) or a Xenon arc lamp. The imaging device may further include a microscope (or an optical train), which may or may not include an eyepiece.

[00488] Support Structure. System 150 may further comprise a support structure 190 configured to support and/or hold the enclosure 102 comprising the microfluidic circuit 120. In some embodiments, the optional tilting module 166 can be configured to activate the support structure 190 to rotate the microfluidic device 100 about one or more axes of rotation. The optional tilting module 166 can be configured to support and/or hold the microfluidic device 100 in a level orientation (i.e., at 0° relative to x- and y-axes), a vertical orientation (i.e., at 90° relative to the x-axis and/or the y-axis), or any orientation therebetween. The orientation of the microfluidic device 100 (and the microfluidic circuit 120) relative to an axis is referred to herein as the “tilt” of the microfluidic device 100 (and the microfluidic circuit 120). For example, support structure 190 can optionally be used to tilt the microfluidic device 100 (e.g., as controlled by optional tilting module 166) to 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 90° relative to the x- axis or any degree therebetween. When the microfluidic device is tilted at angles greater than about 15, tilting may be performed to create bulk movement of micro-objects into/out of sequestration pens from/into the flow region (e.g., microfluidic channel). In some embodiments, the support structure 190 can hold the microfluidic device 100 at a fixed angle of 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, or 10° relative to the x-axis (horizontal), so long as DEP is an effective force to move micro-objects out of the sequestration pens into the microfluidic channel. Since the surface of the electrode activation substrate is substantially flat, DEP forces may be used even when the far end of the sequestration pen, opposite its opening to the microfluidic channel, is disposed at a position lower in a vertical direction than the microfluidic channel.

[00489] In some embodiments where the microfluidic device is tilted or held at a fixed angle relative to horizontal, the microfluidic device 100 may be disposed in an orientation such that the inner surface of the base of the flow path 106 is positioned at an angle above or below the inner surface of the base of the one or more sequestration pens opening laterally to the flow path. The term “above” as used herein denotes that the flow path 106 is positioned higher than the one or more sequestration pens on a vertical axis defined by the force of gravity (i.e., an object in a sequestration pen above a flow path 106 would have a higher gravitational potential energy than an object in the flow path), and inversely, for positioning of the flow path 106 below one or more sequestration pens. In some embodiments, the support structure 190 may be held at a fixed angle of less than about 5°, about 4°, about 3° or less than about 2 ° relative to the x-axis (horizontal), thereby placing the sequestration pens at a lower potential energy relative to the flow path. In some other embodiments, when long term culturing (e.g., for more than about 2, 3, 4, 5, 6, 7 or more days) is performed within the microfluidic device, the device may be supported on a culturing support and may be tilted at a greater angle of about 10°, 15°, 20°, 25°, 30°, or any angle therebetween to retain biological micro-objects within the sequestration pens during the long-term culturing period. At the end of the culturing period, the microfluidic device containing the cultured biological micro-objects may be returned to the support 190 within system 150, where the angle of tilting is decreased to values as described above, affording the use of DEP to move the biological micro-objects out of the sequestration pens. Further examples of the use of gravitational forces induced by tilting are described in U.S. Patent No. 9,744,533 (Breinlinger et al.), the contents of which are herein incorporated by reference in its entirety.

[00490] Nest. Turning now to FIG. 5 A, the system 150 can include a structure (also referred to as a “nest”) 500 configured to hold a microfluidic device 520, which may be like microfluidic device 100, 200, or any other microfluidic device described herein. The nest 500 can include a socket 502 capable of interfacing with the microfluidic device 520 (e.g., an optically actuated electrokinetic device 100, 200, etc.) and providing electrical connections from power source 192 to microfluidic device 520. The nest 500 can further include an integrated electrical signal generation subsystem 504. The electrical signal generation subsystem 504 can be configured to supply a biasing voltage to socket 502 such that the biasing voltage is applied across a pair of electrodes in the microfluidic device 520 when it is being held by socket 502. Thus, the electrical signal generation subsystem 504 can be part of power source 192. The ability to apply a biasing voltage to microfluidic device 520 does not mean that a biasing voltage will be applied at all times when the microfluidic device 520 is held by the socket 502. Rather, in most cases, the biasing voltage will be applied intermittently, e.g., only as needed to facilitate the generation of electrokinetic forces, such as dielectrophoresis or electro-wetting, in the microfluidic device 520.

[00491] As illustrated in FIG. 5 A, the nest 500 can include a printed circuit board assembly (PCBA) 522. The electrical signal generation subsystem 504 can be mounted on and electrically integrated into the PCBA 522. The exemplary support includes socket 502 mounted on PCBA 522, as well. [00492] In some embodiments, the nest 500 can comprise an electrical signal generation subsystem 504 configured to measure the amplified voltage at the microfluidic device 520 and then adjust its own output voltage as needed such that the measured voltage at the microfluidic device 520 is the desired value. In some embodiments, the waveform amplification circuit can have a +6.5V to -6.5V power supply generated by a pair of DC-DC converters mounted on the PCBA 522, resulting in a signal of up to 13 Vpp at the microfluidic device 520.

[00493] In certain embodiments, the nest 500 further comprises a controller 508, such as a microprocessor used to sense and/or control the electrical signal generation subsystem 504. Examples of suitable microprocessors include the Arduino™ microprocessors, such as the Arduino Nano™. The controller 508 may be used to perform functions and analysis or may communicate with an external master controller 154 (shown in Figure 1A) to perform functions and analysis. In the embodiment illustrated in Figure 5 A the controller 508 communicates with the master controller 154 (of Figure 1A) through an interface (e.g., a plug or connector).

[00494] As illustrated in FIG. 5A, the support structure 500 (e.g., nest) can further include a thermal control subsystem 506. The thermal control subsystem 506 can be configured to regulate the temperature of microfluidic device 520 held by the support structure 500. For example, the thermal control subsystem 506 can include a Peltier thermoelectric device (not shown) and a cooling unit (not shown). In the embodiment illustrated in Figure 5 A, the support structure 500 comprises an inlet 516 and an outlet 518 to receive cooled fluid from an external reservoir (not shown) of the cooling unit, introduce the cooled fluid into the fluidic path 514 and through the cooling block, and then return the cooled fluid to the external reservoir. In some embodiments, the Peltier thermoelectric device, the cooling unit, and/or the fluidic path 514 can be mounted on a casing 512 of the support structure 500. In some embodiments, the thermal control subsystem 506 is configured to regulate the temperature of the Peltier thermoelectric device so as to achieve a target temperature for the microfluidic device 520. Temperature regulation of the Peltier thermoelectric device can be achieved, for example, by a thermoelectric power supply, such as a Pololu™ thermoelectric power supply (Pololu Robotics and Electronics Corp.). The thermal control subsystem 506 can include a feedback circuit, such as a temperature value provided by an analog circuit. Alternatively, the feedback circuit can be provided by a digital circuit.

[00495] The nest 500 can include a serial port 524 which allows the microprocessor of the controller 508 to communicate with an external master controller 154 via the interface. In addition, the microprocessor of the controller 508 can communicate (e.g., via a Plink tool (not shown)) with the electrical signal generation subsystem 504 and thermal control subsystem 506. Thus, via the combination of the controller 508, the interface, and the serial port 524, the electrical signal generation subsystem 504 and the thermal control subsystem 506 can communicate with the external master controller 154. In this manner, the master controller 154 can, among other things, assist the electrical signal generation subsystem 504 by performing scaling calculations for output voltage adjustments. A Graphical User Interface (GUI) (not shown) provided via a display device 170 coupled to the external master controller 154, can be configured to plot temperature and waveform data obtained from the thermal control subsystem 506 and the electrical signal generation subsystem 504, respectively. Alternatively, or in addition, the GUI can allow for updates to the controller 508, the thermal control subsystem 506, and the electrical signal generation subsystem 504.

[00496] Optical sub-system. FIG. 5B is a schematic of an optical sub-system 550 having an optical apparatus 510 for imaging and manipulating micro-objects in a microfluidic device 520, which can be any microfluidic device described herein. The optical apparatus 510 can be configured to perform imaging, analysis and manipulation of one or more micro-objects within the enclosure of the microfluidic device 520.

[00497] The optical apparatus 510 may have a first light source 552, a second light source 554, and a third light source 556. The first light source 552 can transmit light to a structured light modulator 560, which can include a digital mirror device (DMD) or a microshutter array system (MSA), either of which can be configured to receive light from the first light source 552 and selectively transmit a subset of the received light into the optical apparatus 510. Alternatively, the structured light modulator 560 can include a device that produces its own light (and thus dispenses with the need for a light source 552), such as an organic light emitting diode display (OLED), a liquid crystal on silicon (LCOS) device, a ferroelectric liquid crystal on silicon device (FLCOS), or a transmissive liquid crystal display (LCD). The structured light modulator 560 can be, for example, a projector. Thus, the structured light modulator 560 can be capable of emitting both structured and unstructured light. In certain embodiments, an imaging module and/or motive module of the system can control the structured light modulator 560.

[00498] In embodiments when the structured light modulator 560 includes a mirror, the modulator can have a plurality of mirrors. Each mirror of the plurality of mirrors can have a size of about 5 microns x 5 microns to about 10 microns xlO microns, or any values therebetween. The structured light modulator 560 can include an array of mirrors (or pixels) that is 2000 x 1000, 2580 x 1600, 3000 x 2000, or any values therebetween. In some embodiments, only a portion of an illumination area of the structured light modulator 560 is used. The structured light modulator 560 can transmit the selected subset of light to a first dichroic beam splitter 558, which can reflect this light to a first tube lens 562.

[00499] The first tube lens 562 can have a large clear aperture, for example, a diameter larger than about 40 mm to about 50 mm, or more, providing a large field of view. Thus, the first tube lens 562 can have an aperture that is large enough to capture all (or substantially all) of the light beams emanating from the structured light modulator 560.

[00500] The structured light 515 having a wavelength of about 400 nm to about 710 nm, may alternatively or in addition, provide fluorescent excitation illumination to the microfluidic device.

[00501] The second light source 554 may provide unstructured brightfield illumination. The brightfield illumination light 525 may have any suitable wavelength, and in some embodiments, may have a wavelength of about 400 nm to about 760 nm. The second light source 554 can transmit light to a second dichroic beam splitter 564 (which also may receive illumination light 535 from the third light source 556), and the second light, brightfield illumination light 525, may be transmitted therefrom to the first dichroic beam splitter 558. The second light, brightfield illumination light 525, may then be transmitted from the first dichroic beam splitter 558 to the first tube lens 562.

[00502] The third light source 556 can transmit light through a matched pair relay lens (not shown) to a mirror 566. The third illumination light 535 may therefrom be reflected to the second dichroic beam splitter 564 and be transmitted therefrom to the first beam splitter 558, and onward to the first tube lens 562. The third illumination light 535 may be a laser and may have any suitable wavelength. In some embodiments, the laser illumination 535 may have a wavelength of about 350 nm to about 900 nm. The laser illumination 535 may be configured to heat portions of one or more sequestration pens within the microfluidic device. The laser illumination 535 may be configured to heat fluidic medium, a microobject, a wall or a portion of a wall of a sequestration pen, a metal target disposed within a microfluidic channel or sequestration pen of the microfluidic channel, or a photoreversible physical barrier within the microfluidic device, and described in more detail in U. S. Application Publication Nos. 2017/0165667 (Beaumont, et al.) and 2018/0298318 (Kurz, et al.), each of which disclosure is herein incorporated by reference in its entirety. In other embodiments, the laser illumination 535 may be configured to initiate photocleavage of surface modifying moieties of a modified surface of the microfluidic device or photocleavage of moieties providing adherent functionalities for micro-objects within a sequestration pen within the microfluidic device. Further details of photocleavage using a laser may be found in International Application Publication No. W02017/205830 (Lowe, Jr. et al.), which disclosure is herein incorporated by reference in its entirety.

[00503] The light from the first, second, and third light sources (552, 554, 556) passes through the first tube lens 562 and is transmitted to a third dichroic beam splitter 568 and filter changer 572. The third dichroic beam splitter 568 can reflect a portion of the light and transmit the light through one or more filters in the filter changer 572 and to the objective 570, which may be an objective changer with a plurality of different objectives that can be switched on demand. Some of the light (515, 525, and/or 535) may pass through the third dichroic beam splitter 568 and be terminated or absorbed by a beam block (not shown). The light reflected from the third dichroic beam splitter 568 passes through the objective 570 to illuminate the sample plane 574, which can be a portion of a microfluidic device 520 such as the sequestration pens described herein.

[00504] The nest 500, as described in FIG. 5A, can be integrated with the optical apparatus 510 and be a part of the apparatus 510. The nest 500 can provide electrical connection to the enclosure and be further configured to provide fluidic connections to the enclosure. Users may load the microfluidic apparatus 520 into the nest 500. In some other embodiments, the nest 500 can be a separate component independent of the optical apparatus 510.

[00505] Light can be reflected off and/or emitted from the sample plane 574 to pass back through the objective 570, through the filter changer 572, and through the third dichroic beam splitter 568 to a second tube lens 576. The light can pass through the second tube lens 576 (or imaging tube lens 576) and be reflected from a mirror 578 to an imaging sensor 580. Stray light baffles (not shown) can be placed between the first tube lens 562 and the third dichroic beam splitter 568, between the third dichroic beam splitter 568 and the second tube lens 576, and between the second tube lens 576 and the imaging sensor 580.

[00506] Objective. The optical apparatus can comprise the objective lens 570 that is specifically designed and configured for viewing and manipulating of micro-objects in the microfluidic device 520. For example, conventional microscope objective lenses are designed to view micro-objects on a slide or through 5mm of aqueous fluid, while micro-objects in the microfluidic device 520 are inside the plurality of sequestration pens within the viewing plane 574 which have a depth of 20, 30, 40, 50, 6070, 80 microns or any values therebetween. In some embodiments, a transparent cover 520a, for example, glass or ITO cover with a thickness of about 750 microns, can be placed on top of the plurality of sequestration pens, which are disposed above a microfluidic substrate 520c. Thus, the images of the micro-objects obtained by using the conventional microscope objective lenses may have large aberrations such as spherical and chromatic aberrations, which can degrade the quality of the images. The objective lens 570 of the optical apparatus 510 can be configured to correct the spherical and chromatic aberrations in the optical apparatus 510. The objective lens 570 can have one or more magnification levels available such as, 4X, 10X, 20X.

[00507] Modes of illumination. In some embodiments, the structured light modulator 560 can be configured to modulate light beams received from the first light source 552 and transmits a plurality of illumination light beams 515, which are structured light beams, into the enclosure of the microfluidic device, e.g., the region containing the sequestration pens. The structured light beams can comprise the plurality of illumination light beams. The plurality of illumination light beams can be selectively activated to generate a plurality of illuminations patterns. In some embodiments, the structured light modulator 560 can be configured to generate an illumination pattern, similarly as described for FIGS. 4A-4B, which can be moved and adjusted. The optical apparatus 510 can further comprise a control unit (not shown) which is configured to adjust the illumination pattern to selectively activate the one or more of the plurality of DEP electrodes of a substrate 520c and generate DEP forces to move the one or more micro-objects inside the plurality of sequestration pens within the microfluidic device 520. For example, the plurality of illuminations patterns can be adjusted over time in a controlled manner to manipulate the micro-objects in the microfluidic device 520. Each of the plurality of illumination patterns can be shifted to shift the location of the DEP force generated and to move the structured light for one position to another in order to move the micro-objects within the enclosure of the microfluidic apparatus 520.

[00508] In some embodiments, the optical apparatus 510 may be configured such that each of the plurality of sequestration pens in the sample plane 574 within the field of view is simultaneously in focus at the image sensor 580 and at the structured light modulator 560. In some embodiments, the structured light modulator 560 can be disposed at a conjugate plane of the image sensor 580. In various embodiments, the optical apparatus 510 can have a confocal configuration or confocal property. The optical apparatus 510 can be further configured such that only each interior area of the flow region and/or each of the plurality of sequestration pens in the sample plane 574 within the field of view is imaged onto the image sensor 580 in order to reduce overall noise to thereby increase the contrast and resolution of the image. [00509] In some embodiments, the first tube lens 562 can be configured to generate collimated light beams and transmit the collimated light beams to the objective lens 570. The objective 570 can receive the collimated light beams from the first tube lens 562 and focus the collimated light beams into each interior area of the flow region and each of the plurality of sequestration pens in the sample plane 574 within the field of view of the image sensor 580 or the optical apparatus 510. In some embodiments, the first tube lens 562 can be configured to generate a plurality of collimated light beams and transmit the plurality of collimated light beams to the objective lens 570. The objective 570 can receive the plurality of collimated light beams from the first tube lens 562 and converge the plurality of collimated light beams into each of the plurality of sequestration pens in the sample plane 574 within the field of view of the image sensor 580 or the optical apparatus 510.

[00510] In some embodiments, the optical apparatus 510 can be configured to illuminate the at least a portion of sequestration pens with a plurality of illumination spots. The objective 570 can receive the plurality of collimated light beams from the first tube lens 562 and project the plurality of illumination spots, which may form an illumination pattern, into each of the plurality of sequestration pens in the sample plane 574 within the field of view. For example, each of the plurality of illumination spots can have a size of about 5 microns X 5 microns; 10 microns X 10 microns; 10 microns X 30 microns, 30 microns X 60 microns, 40 microns X 40 microns, 40 microns X 60 microns, 60 microns X 120 microns, 80 microns X 100 microns, 100 microns X 140 microns and any values there between. The illumination spots may individually have a shape that is circular, square, or rectangular. Alternatively, the illumination spots may be grouped within a plurality of illumination spots (e.g., an illumination pattern) to form a larger polygonal shape such as a rectangle, square, or wedge shape. The illumination pattern may enclose (e.g., surround) an unilluminated space that may be square, rectangular or polygonal. For example, each of the plurality of illumination spots can have an area of about 150 to about 3000, about 4000 to about 10000, or 5000 to about 15000 square microns. An illumination pattern may have an area of about 1000 to about 8000, about 4000 to about 10000, 7000 to about 20000, 8000 to about 22000, 10000 to about 25000 square microns and any values there between.

[00511] The optical system 510 may be used to determine how to reposition micro-objects and into and out of the sequestration pens of the microfluidic device, as well as to count the number of micro-objects present within the microfluidic circuit of the device. Further details of repositioning and counting microobjects are found in U. S. Application Publication No. 2016/0160259 (Du); U. S. Patent No. 9,996,920 (Du et al.); and International Application Publication No. WO2017/102748 (Kim, et al.). The optical system 510 may also be employed in assay methods to determine concentrations of reagents/assay products, and further details are found in U. S. Patent Nos. 8,921,055 (Chapman), 10,010,882 (White et al.), and 9,889,445 (Chapman et al.); International Application Publication No. WO2017/181135 (Lionberger, et al.); and International Application Serial No. PCT/US2018/055918 (Lionberger, et al.). Further details of the features of optical apparatuses suitable for use within a system for observing and manipulating micro-objects within a microfluidic device, as described herein, may be found in WO2018/102747 (Lundquist, et al), the disclosure of which is herein incorporated by reference in its entirety. [00512] Additional system components for maintenance of viability of cells within the sequestration pens of the microfluidic device. In order to promote growth and/or expansion of cell populations, environmental conditions conducive to maintaining functional cells may be provided by additional components of the system. For example, such additional components can provide nutrients, cell growth signaling species, pH modulation, gas exchange, temperature control, and removal of waste products from cells.

EXPERIMENTAL

[00513] System and device: An OptoSelect™ device, a nanofluidic device controlled by an optical instrument, Beacon® were employed (Both are manufactured by Berkeley Lights, Inc.) The instrument includes: a mounting stage for the chip coupled to a temperature controller; a pump and fluid medium conditioning component; and an optical train including a camera and a structured light source suitable for activating phototransistors within the chip. The OptoSelect device includes a substrate configured with OptoElectroPositioning (OEP™) technology, which provides a phototransistor-activated OET force. The chip also included a plurality of microfluidic channels, each having a plurality of NanoPen™ chambers (or chambers) fluidically connected thereto. The volume of each chamber is around IxlO ⁶ cubic microns.

[00514] Device priming. 250 microliters of 100% carbon dioxide was flowed in to the OptoSelect device at a rate of 12 microliters/sec, followed by 250 microliters of PBS containing 0.1% Pluronic® F27 (Life Technologies® Cat# P6866) flowed in at 12 microliters/sec, and finally 250 microliters of PBS flowed in at 12 microliters/sec. Introduction of a Wetting Solution follows, which introduces a conditioned surface to the surfaces within the microfluidic device. The details of the surface and its introduction are described in US Application Publication US 2016/0312165, filed on April 22, 2016 and U.S. Application Publication No. US2019/0275516, filed on November 20, 2018, each of which disclosures are herein incorporated by reference in its entirety.

[00515] Media perfusion during culture. Medium is perfused through the OptoSelect™ device according to either of the following two methods: (1) Perfuse at 0.01 microliters/sec for 2h; perfuse at 2 microliters/sec for 64 sec; and repeat. (2) Perfuse at 0.02 microliters/sec for 100 sec; stop flow 500 sec; perfuse at 2 microliters/sec for 64 sec; and repeat.

[00516] Experiment 1: Biomass measurement of nonmammalian cells

[00517] Yeast culture and standard curve. Cells from a Pichia cell population engineered to secrete a non-endogenous product, e.g., protein, organic molecule, and the like, were suspended in PBS and introduced into an OptoSelect™ microfluidic device, e.g., chip, in a Beacon® Optofluidic system for biomass measurement. In Examples 1-1 to 1-5, the chip is a sealed chip, which prevents exchange of gaseous components through the edges of the chip construction. The sealed chip is described in further detail in International Application Serial No. PCT/US2021/048196, entitled “Apparatuses, Methods and Kits for Microfluidic Assays”, filed on August 30, 2021, and published as International Application Publication W02022/047290, the entire disclosure of which is herein incorporated by reference. Single cells were disposed into respective sequestration pens and cultured on-chip at 27 °C with constant perfusion of fresh medium BMMY (Buffered Methanol-Complex Medium) 1% methanol and 80% air fraction. Time-lapse bright-field images were taken every 30 minutes to keep track of cell growth for each colony. After 48 hours, the bright-field biomass measurements were analyzed using the Cell Analysis Suite 2.1 Software from Berkeley Lights (See International Application No. PCT/US2020/060784, entitled “Systems and Methods for Analysis of Biological Samples, filed on November 16, 2020 and published as International Application Publication WO20210987449 Al, the entire disclosure of which is incorporated by reference for any purpose).

[00518] Example 1-1: Hydrogels forming assay barriers. In Examples 1-2 through 1-4, variations on an 8 arm 20K PEG polymer were used to form the hydrogel barriers. The extent of crosslinking with resultant effects on permeability/impermeability was controlled by varying the length of photopatterning (about 1000 msec to about 5 sec, or repeated exposures of about 1000 msec); the concentration of inhibitor present in the hydrogel solution introduced into the microfluidic device; and the proportion of crosslinkable moieties on the 8 armed modified PEG polymer.

[00519] Variation A. The effect of hydrogel composition and extent of crosslinking was examined. Two polymer compositions were examined: the first as an 8 arm, 8 acrylamide terminated 20K PEG polymer. The second composition was a 90: 10 mixture of the 8 arm, 8 acrylamide terminated 20K PEG polymer: linear (larm) acrylamide 20kDa PEG polymer. Each was introduced to a microfluidic chip and polymerized in different sections using different lengths of exposure and power, ranging from 40% power to 100% power, and 3 sec to 20 sec. Some of these conditions did not create fully polymerized hydrogel barriers ( in this example, barriers like FIG. 6A, 605 type having a thickness of about 100 microns were introduced). However, 50% power, 20 sec ensures fully sealing barriers and was used for diffusion experiments.

[00520] A mixture of FITC-labelled IgG (150kDA) and Alexa-647 labeled streptavidin (66kDa) was perfused through the device after hydrogel formation. Images were obtained Ihr after initial introduction of the labelled materials in the respective color cube. As seen in FIG. 10A, the hydrogel barrier permitted a significant amount of fluorescently labeled streptavidin to diffuse into the culturing area distal to the hydrogel barrier. In contrast, in FIG. 10B, the same pen (annotated as 336) did not permit diffusion of the FITC -IgG across the hydrogel barrier. Results (data not shown) were the same with the hydrogel barrier formed from the 90: 10 mixture of 8arm: linear PEG polymers.

[00521] Variation B. Permeability control was examined by creating hydrogel barriers fully spanning the width of the sequestration pen.

[00522] The behavior of two different formulations was examined (formulation Fl equals 100% 8 arm 20K PEG having 8 acrylamide termini; formulation F2 equals 25% 8 arm 20K PEG having 8 acrylamide termini: 75% 8 arm 20K PEG having 1 acrylamide terminus, 7 non-crosslinkable termini (e.g., hydroxyl termini). The initiator and inhibitor ratios were the same: initiator (Lithium phenyl- 2, 4, 6 trimethylbenzoylphosphinate, LAP) and inhibitor (hydroquinone monomethyl ether, MEHQ). Different sections of sequestration pens on the same microfluidic chip had two different types of hydrogel barriers introduced. The first type was a mid-pen barrier having a thickness of about 15 microns, leaving a culturing region distal to the barrier. The second type of barrier was a hydrogel plug that extended to the distal end of the sequestration pen. Fl formulation barriers were introduced using a 1 to 1.5 second exposure, 10X objective, 50% power for both types of barriers. F2 formulation barriers were introduced using a 3.5 sec to 5 sec exposure, 10X objective, 50% power.

[00523] Three different reagent flows were successively introduced. For each reagent flow, images were obtained after a 90min period of equilibration.

[00524] Flow 1 : Fluorescent anti-Spot nanobody (MW = 30kDa), shown in FIG. 11 A.

[00525] Flow 2: Fluorescent anti-SPOT nanobody plus Protein (MW= 50-55 kDa), shown in FIG. 1 IB.

[00526] Flow 3: Fluorescent anti-SPOT nanobody plus Protein (MW= 50-55 kDa) plus anti -FLAG antibody (MW= 150 kDa), shown in FIG. 11C.

[00527] As is shown in FIGS. 11 A-l 1C, the low molecular weight labelled nanobody can easily diffuse through the F2 mid-pen 15 micron barrier, as shown in each of pens 1110, 1130, 1150, equilibrating to essentially the same concentration as that of the channel and the area of the pen proximal to the barrier. In FIG. 11 A, the hydrogel plug in pen 1115 showed significant amounts of the small labelled reagent, demonstrating that diffusion occurs through the hydrogel and is not due to diffusion around the barrier. Additionally, the barrier having the denser Fl formulation, as shown in pen 1120 also permits diffusion through the barrier, but at a reduced concentration. In pen 1125, the denser Fl formulation hydrogel plus shows quite limited diffusion of the low molecular weight reagent.

[00528] In FIG. 1 IB, where the larger protein is present and which binds to the fluorescently labelled reagent, forming a higher weight complex, affecting diffusion of the bound pair. While not easily seen in pen 1130, the effect is more evident in pen 1135, having the hydrogel plug. The larger bound complex cannot as easily diffuse into the hydrogel, even with the F2 formulation. The denser hydrogels of pens 1140 and 1145 show increasingly decreased amounts of fluorescent reagent, and the large reagent: analyte complex is cannot easily diffuse through.

[00529] FIG. 11C shows the effect of adding an anti -FLAG antibody to the reagent mixture, which can also bind to the protein, thus making an even larger tri-component complex. The levels of diffusion were slightly more inhibited in pens 1150, 1155, 1160, 1165.

[00530] Further variations: The ratios of 8 arm, 8 acrylamide termini polymer: 8 arm, 1 acrylamide termini polymer may be varied to be in any suitable ratio, e.g., about 10:90; 20:80; 30:70; 40:60: 50:50; 60:40; 70:30; 80:20; 90: 10 w/w% or any value therebetween. The molecular weights of the PEG polymers may be varied and do not need to be 20K polymers, but may have a MW of about 5kDA, lOkDa, 15kDa, 20kDa or more. A consideration in combining polymers of different molecular weights that the rate of diffusion into the pen depends upon the molecular weight. Therefore, the actual ratio of polymers in the pen available for polymerization will therefore reflect the difference in diffusion rates.

[00531] Example 1-2: Hydrogel geometry for assaying soluble bioproduct. [00532] In this example, various hydrogel shapes were formed in the sequestration pen. They were tested for their performance for retaining cells within the distal culturing region of the sequestration pen, as well as assaying productivity, for example, but not limited to using a diffusion gradient assay. Protocols for typical diffusion gradient assays are more fully described in International Application Serial No. PCT/2017/027795, entitled “Methods, Systems, and Kits for In-Pen Assays”, filed on April 14, 2017, published as International Application Publication WO2017/1811135; International Application Serial No. PCT/US2018/055918, entitled “Methods, Systems, and Kits for In-Pen Assays”, filed on October 15, 2018, published as International Application Publication WO2019/075476; and International Application Serial No. PCT/US2021/021417, entitled “Methods, Systems, and Kits for In-Pen Assays”, filed on March 09, 2020, published as International Application Publication WO2021/184458; and International Application Serial No. PCT/US2022/23598, entitled “Methods of Microfluidic Assay and Bioproduction from Non-Mammalian Cells and Kits Therefor”, filed on April 6, 2022, the entirety of each of which disclosures are herein incorporated by reference for any purpose.).

[00533] Pichia cells engineered to secrete a non-endogenous protein were suspended in BMGY (Buffered Glycerol-complex Medium) medium before loading. The engineered non-endogenous protein sequence also included Spot-tag, an inert, unstructured 12 amino acid sequence added to the genetic insert. The additional 12 amino acid sequence is added to a region of the engineered insertion sequence located outside (to the N-terminal end or the C-terminal end) of the desired non-endogenous protein sequence, which can be detected by anti-SPOT nanobodies (Chromotek™). The cells were introduced and disposed into respective sequestration pens by gravity. Hydrogel mixture was introduced into the flow region and allowed to diffuse into the sequestration pens. Then, gel polymerization was initiated by photoactivation at selected area to form various shapes, including full cap (15 pm band fully spanning pen, FIG. 6A, barrier 605, center bar (15 microns, as measured in the distal-to-pen opening to proximal-to-pen opening dimension “thickness” x 20 microns, “width”, as measured from pen wall to pen wall, forming a pillar centered in the pen leaving a 10 micron gap to the pen walls on each side, FIG. 6A, barrier 610, half bar (15 micron thick band extending from the left wall to the center of the pen, FIG. 6A, barrier 615, and side bars (Two 15 micron thick bands extending from left and right wall leaving an approximately 20 micron gap at the center of the pen, FIG. 6A, barrier 620. The hydrogel barrier(s) were formed in mid-pen and separated the sequestration pen into two areas. The area distal from the opening of the sequestration pen was used as a culture area for cells to live and expand.

[00534] Cells were cultured on-chip with constant perfusion of fresh BMGY medium at 30 °C and 80% air fraction (cycle duration was 10 minutes and the flow rate was 0.1 microliters/s) for 17 hours. Then, BM1M medium with 10% MeOH was introduced to induce the secretion for 4 hours (27 °C, 5 microliters/s flow rate). After that, BM1M medium with anti-SPOT nanobodies (labeled with ATTO594, Chromotek™) was introduced. The perfusion was continued at 0.014 microliters/s for 45 minutes for equilibration, and then BM1M medium without anti-SPOT nanobodies was introduced and flushed at 0.1 microliters/s for 30 minutes. Images were taken to observe the gradient of soluble fluorescently labeled product at indicated time points.

Ill [00535] FIGS. 12A-12C shows cell expansion in the sequestration pens and that the hydrogel barrier succeeded in containing the cells within the culturing area, for a variety of hydrogel barrier shapes. In some circumstances, a small number of cells might breach the hydrogel barrier; nevertheless, substantially most, e.g., more than about 80%, more than about 90%, more than about 95% or more than about 99% of all cells were still contained inside the culture area. The results (FIGS. 12D-12F) of a diffusion gradient assay for each respective barrier type, performed as described within Example 1-3, showed that the free anti-SPOT nanobodies rapidly equilibrated in pens with partial hydrogels (center bar, half bar, and side bars) and different intensities can be observed between pens.

[00536] Example 1-3: Bioproductivity assessment assays

[00537] In this example, four different Pichia strains (Strains 1-4) engineered to secrete a first protein (Protein 1), three different Pichia strains (Strains 5-7) engineered to secrete a second protein (Protein 2), and four further different strains (Strains 8-11) engineered to secrete a third protein (Protein 3), were tested in this assay, but already had known productivities. Each of Proteins 1, 2, and 3 were labeled with Spot-tag, which can be detected by anti-SPOT nanobodies (Chromotek™) as well as the FLAG- tag. Cells were resuspended in PBS before loading and introduced into the flow region. For each stain, single cells were disposed into respective individual sequestration pens using di electrophoretic forces, which in this example, are optically actuated dielectrophoretic forces. In this example, a single cell was disposed into an individual sequestration pen using positive light actuated dielectrophoretic forces. Details of positive dielectrophoretic transport of cells are described in International Application Serial No. PCT/US2020/066229, entitled “Methods of Penning Micro-Objects Using Positive Dielectrophoresis”, filed on December 18, 2020, and published as International Patent Application Publication WO2021/127576, the entire disclosure of which is herein incorporated by reference in its entirety for any purpose. However, the disclosure is not so limited, for example, when other cell types such as bacterial cells, negative dielectrophoretic forces may be used to selectively place a single cell (or more than one cell) into each individual sequestration pen.

[00538] Hydrogel formation. In assays where hydrogel barriers were used in the productivity assay, flowable hydrogel polymer was introduced in solution, and allowed to diffuse into the sequestration pens. Photoinitiator was also included within the solution containing the flowable hydrogel polymer. The hydrogel barriers were formed by photopatterning, e.g., photoactivation of polymerization to form the solidified hydrogel barriers. The formed hydrogel defined the sequestration pen into two regions (e.g., areas). The region that was furthest away from the opening and contained cells therewithin was a culture area. In some instances, the other region that was closest to the opening was an assay area (See FIG. 7A), for example using a bead assay and a hydrogel barrier.

[00539] Variation 1. Culturing, Induction and Assay using Capture Bead. Strains 1-1 Iwere used in this experiment. The hydrogel formulation included 8 arm 20K PEG having 8 acrylamide termini, included initiator (Lithium phenyl-2, 4, 6 trimethylbenzoylphosphinate, LAP) and inhibitor (hydroquinone monomethyl ether, MEHQ). A nitrogen gas purge followed the introduction of the flowable polymer mixture. Gel polymerization was initiated by photoactivation in the DAPI filter cube, using 10X objective, for Is at 50% power. Gel polymerization was initiated by photoactivation at mid-pen to form a cap formed from two individual triangular gel barriers, like the barrier shown in FIG. 6 A, barrier 625. The nominal size of the illumination used to form the barrier produced a “bowtie” shape with about a gap in the center) in each pen. Depending on the exposure time, exact amount of initiator, inhibitor and/or polymer composition, the triangular gel barriers may meet in the center rather than leaving a discernable gap. In other instances, the triangular barriers may leave a gap that is less than about 1, 2, 3, 4, 5, 6, 7, 8, 9 or about 10 microns wide. “Bowtie” barriers can function equally well in either case. What these barriers share is a non-uniform thickness. That non-uniformity may be in the width (pen wall to pen wall dimension), in the “thickness” dimension (from distal-to-pen opening to proximal-to-pen opening dimension), and/or the “height” dimension (from inner surface of the substrate to the inner surface of the cover of the microfluidic device, e.g., z-dimension). The non-uniformity in the “thickness” dimension, e.g., the center of the “bowtie”, provides a convenient and standard location for bead disposition for the assay. The “bowtie” barrier had a “thickness” at its largest dimension of the triangular shaped individual gel form of about 15 microns. This dimension is not limiting and the “thickness” of the bowtie may be less than about 15 microns, less than about 12 microns, less than about 10 microns, less than about 8 microns, or less than about 5 microns. In other embodiments, the “thickness” of a triangular segment of the “bowtie” barrier may be more than about 10 microns, more than about 15 microns, or more than about 18 microns. As shown in FIG. 6B-6C, two different embodiments of hydrogel barriers having a “bowtie” non-uniform shape are shown, where the dimension 660 of the triangular segment forming the barrier (“thickness” dimension) is larger in the barrier of FIG. 6C than the dimension of the segment 650 of the barrier of FIG.6B. Since the pens are of the same width, it can be seen that the barrier formed in FIG. 6B has a distinct gap between the two barriers, while the barrier of FIG.6C does not have an obvious gap. While in the embodiment of FIG. 6B, a few cells have escaped the culture area below the barrier, the barrier of FIG. 6C does not permit cells to move past the barrier.

[00540] Further variations. While the “bowtie” non-uniform barrier was used in this experiment, other non-uniform barriers may be suitable barriers for productivity assays using beads to capture secreted biomolecules, and may include some of the non-uniform barriers shown in FIG. 6A. Depending on culture period duration and the specific type of cells being assayed, many different configurations of barriers will work. The specific type of cell may determine what type of barrier may be used, as cells that tend to grow in close association within the culturing region of the divided pen, may not even require a barrier that blocks more than half the width of the pen.

[00541] Cell culture, Bead Load, and Induction. Cells were then cultured on chip with constant perfusion of fresh BMGY medium at 30 °C and 80% air fraction (cycle duration was 10 minutes and the flow rate was 0.1 pL/s) for 14 hours. The period of time for culturing may be selected as desired, and may be less than about 14 h, 12 h, 10 h, 8 h, or less or may be more than about lOh, 12 h, 14h, 16 h, 18h, or about 20h. Overgrowth of the culturing area may be a determining factor in selecting the culturing period.

[00542] Assay beads coated with anti-FLAG antibodies (which bind to the FLAG® peptide sequence) were suspended in loading buffer and then introduced into the flow region before the induction. Single beads were disposed into each pen and located in the assay region. In some further variations, a second period of culturing may be added after bead introduction. This period of culture may be for about 1 h, 2 h, 3 h, 4 h, or any value therebetween, and assures that the cells are in a state such that induction will be more uniformly successful. Flow of liquid media (BMGY) is alternated with air perfusion, in a ratio of 20% liquid: 80% gaseous (air) in a 10 min cycle, at 1 microliter/sec.

[00543] After introduction of the bead (and in some variations, after the second period of culturing), BM1M medium with 10% MeOH was introduced to induce secretion of the molecule of interest (the analyte). The induction was performed for 5 hours with the perfusion of the BM1M medium continued (1 microliter/s, 27 °C). In some variations, the induction period is varied from a 5 h period, and may be selected to be about 1 h, 2 h, 3 h, 4 h, 6 h, 7 h or more.

[00544] Assaying. BM1M medium with anti-SPOT nanobodies (labeled with ATTO594, Chromotek™) was introduced. The perfusion was continued at 0.011 pL/s for 30 minutes for equilibration, and then BM1M medium without anti-SPOT nanobodies was introduced and flushed at 5 pL/s for 60 minutes. Brightfield images were taken before assaying for biomass measurement, which was used to normalize the results of the measurement.

[00545] Mean bead fluorescent signal was detected and normalized with biomass (OD score). The results were shown in the histograms of FIG. 13 showing the production results for Protein 1, Protein 2, and Protein 3 (from left column to right column) for respective Strains 1-11. The x-axis shows the OD normalized score (AU) (marked at IK, 2K, 3K, and the y-axis shows count (0 to 100). The histograms match the expectation of productivities as Strain 1, Strain 5, and Strain 8 were expected to be the worst producers of the respective proteins of the tested groups. Furthermore, it was observed that there were different levels of productivities between sequestration pens containing cells from the same strain. The locations of the highest scoring pens were stored by the analysis software of the Beacon system and can be used to export the cells exhibiting high productivities in this assay. Therefore, the better producers from each strain may be exported for expansion and further development.

[00546] Further variation. In another variation, the “bowtie” or other non-uniform hydrogel barrier may be introduced, where the hydrogel is permeable to the secreted analyte or where there is a gap between portions of the hydrogel barrier. The secreted analyte, being a soluble product may diffuse out of the culturing area, through the hydrogel and/or through a gap in portions of the hydrogel barrier. A diffusion gradient assay may then be performed in an area of interest that is selected to be located between the opening of the pen into the channel and the surface of the hydrogel facing the opening of the pen into the channel (“assay area” as shown in FIG. 7B). BM1M medium with anti-SPOT nanobodies (labeled with ATTO594, Chromotek™) is introduced, and the soluble analyte is labelled with the anti-SPOT nanobodies. Many variations of diffusion gradient assay imaging may be used, and details of these variations are described in International Application Serial No. PCT/2017/027795, entitled “Methods, Systems, and Kits for In-Pen Assays”, filed on April 14, 2017, published as International Application Publication WO2017/1811135; International Application Serial No. PCT/US2018/055918, entitled “Methods, Systems, and Kits for In-Pen Assays”, filed on October 15, 2018, published as International Application Publication WO2019/075476; and International Application Serial No. PCT/US2021/021417, entitled “Methods, Systems, and Kits for In-Pen Assays”, filed on March 09, 2020, published as International Application Publication WO2021/184458, the entirety of each of which disclosures are herein incorporated by reference for any purpose.

[00547] Variation 2. Accumulation Assay. In another variation, secreted product was accumulated within the same region as the cells, below a hydrogel barrier which substantially spans the width of the sequestration pen, as described herein (e.g., located within a pen to divide the pen into two regions, distal and proximal to the opening, forming a full cap as shown in FIG. 6A, barrier 605. Cells were cultured within the distal, culturing region. An area of interest for monitoring the amount of detectable product was located within the culturing region, but in a portion of the region that preferably does not contain any cells. For example, a portion of the culturing region closest to the distal side of the hydrogel barrier was defined as the area of interest for the assay. The area of interest typically may not include any portion of the gel itself, when performing this assay. An accumulation-type assay may be useful where the culturing cells produce the bioproduct at a low rate, which may be difficult to detect using a bead to capture secreted bioproduct.

[00548] Cell culture and Induction. After introduction of individual cells into respective sequestration pens, as described above for Strains 1-11, cells were then cultured on chip with constant perfusion of fresh BMGY medium at 30 °C and 80% air fraction (cycle duration was 10 minutes and the flow rate was 0.1 pL/s) for 14 hours. The period of time for culturing may be selected as desired, and may be less than about 14 h, 12 h, 10 h, 8 h, or less or may be more than about lOh, 12 h, 14h, 16 h, 18h, or about 20h. Overgrowth of the culturing area may be a determining factor in selecting the culturing period.

[00549] BM1M medium with 10% MeOH was introduced to induce secretion of the molecule of interest (the analyte). The induction was performed for 5 hours with the perfusion of the BM1M medium continued (1 microliter/s, 27 °C). In some variations, the induction period is varied from a 5 h period, and may be selected to be about 1 h, 2 h, 3 h, 4 h, 6 h, 7 h or more.

[00550] Accumulation Assay. BM1M medium with anti-SPOT nanobodies (labeled with ATTO594, Chromotek™) was introduced. The perfusion was continued at 0.011 microliters/s for 30 minutes for equilibration, and then BM1M medium without anti-SPOT nanobodies was introduced and flushed at 5 microliters/s for 60 minutes. Brightfield images were taken before assaying for biomass measurement, which was used to normalize the results of the measurement. Fluorescent Images were taken during flushing to determine signal intensities of the anti-SPOT nanobodies within an area of interest that was selected to be within the culture area of each pen.

[00551] The results, shown in FIG. 14, showed that the presence of a hydrogel fully sealing the pen results in concentration of secreted analytes within the culture area that can then be detected by using the anti-SPOT nanobodies. The x-axis shows biomass, as measured by OD, and the y-axis shows signal intensity in the area of interest below the hydrogel barrier. A linear correlation with colony size demonstrates that this method of quantifying secretion is highly predictive of productivity . FIG. 15 shows histograms of the signals normalized to the OD, where the y-axis shows the score (e.g., detectable signal intensity) normalized for biomass and the x-axis shows the number of pens having each score. The results were consistent with expectation, and the distribution of scores provides additional information for selecting a better producer. For example, Strain 3 and Strain 4 were overall better producers than Strain 2. Nevertheless, some pens of Strain 2 exhibited signals stronger than most of the pens of Strain 3 and Strain 4. Accordingly, the diversity of secretion productivity within a given strain can be captured and selection of individual clonal populations from a strain can yield superior candidates for further development.

[00552] Example 1-4. Export. After culturing and assay of cells as described in Example 1-3, Variation 1, selected pens were prepared in order to export the cells within the culture area, and a sequence of photographs illustrating the export process is shown in FIGS. 16A-16F. In FIG. 16A, the non-uniform hydrogel barrier used for the bead assay (“bowtie”) is annotated for clarity as seen in the figures, for example barrier 1605, as well as the uniform barrier 1610. The central pen was the only pen in this view for which export was desired. At this first time point, for all pens for which export is not desired, additional uniform hydrogel barriers 1610 were already introduced, which prevents cells from exiting those pens. This was performed in case any cells in non-desired pens were located in the area proximal to the opening of the pen, thus reducing risk of clonality loss for the cells being exported. Therefore, the non-selected pens have two hydrogel barriers, and the selected pens have a single barrier. Starting export of cells 1615, a light actuated di electrophoretic bar was programmed to move towards the opening of the pen to the channel. The dielectric force in this instance repels the cells ahead of the light 1620, which is the visible light activating transistors in the substrate, inducing the negative dielectrophoretic force. At a second, later timepoint, as shown in FIG.16B, the light bar 1620 is shown at a location closer to the opening of the pens, having activated the transistors within the substrate, thus moving the cells closer to the hydrogel barrier 1605. The non-uniform hydrogel barrier, however, prevented cells from moving past the barrier.

[00553] At a third still later timepoint, as shown in FIG. 16C, a laser pulse had been directed towards an area of the substrate most distal to the opening of the pen, and the location of the effect 1630 is shown. The dielectrophoretic force had already cleared cells from that portion of the culturing area, so direct impact of laser illumination on cells was prevented. The z-focus was changed for this individual photo, and the focus was resultingly affected. However, it is clear that the laser pulse induced heating and/or a bubble in the media that deformed two triangular segments forming the non-uniform “bowtie” barrier. The two segments 1625 and 1625’ have flipped within the pen from their original position, and now provided a passage for cells to move through. The heating (including expansion of the media) or bubble exerts a force towards the proximal end of the pen, e.g., towards the opening of the pen, and therefore exerts a force upon the center of the hydrogel barrier. The “bowtie” non-uniform hydrogel barrier permitted deformation in the center, and more easily deformed to permit passage of cells.

[00554] At a fourth time point, as shown in FIG. 16D, (z focus is resumed once the laser is not operating) where the sweep of the pen with the light bar 1620 was resumed, moving cells past the deformed barrier segments 1625 and 1625’ (also annotated for clarity) that no longer prevent cell transport. Another light bar 1622 preceded the central group of cells, the dielectrophoretic forces inducing clearance of the path for the cells. At a fifth subsequent time point, as shown in FIG. 16E, the light bar 1620 continues moving the cells towards the opening of the pen, which opens into the channel at the top of the figure. Finally, at a sixth timepoint, as shown in FIG. 16F, the light bar 1620 has initiated dielectrophoretic forces that transported the cells 1615 into the channel. A third light bar 1624 swept through the pen, following the first group of cells 1615, to initiate di electrophoretic forces to capture any remaining cells into the channel to be joined with the first group of cells. After this timepoint, flow was started in the channel to carry the selected cells to an external vessel such as a well in a wellplate. The cells may be further expanded or processed in another manner.

[00555] Experiment 2. Formation and reversal of a reversible hydrogel barrier.

[00556] 2A. Reversible hydrogel barrier including a vicinal diol moiety. A solution of 8-Arm PEG- Norbornene (MW 20 kDa, 8.75 micromolar); 8- Arm PEG-Norbomene (MW lOkDA, 17.5 micromolar) in 4 ml ultrapure distilled water was combined with 450 microliters of a 1.0M solution of dithiothreitol (crosslinker); 250 microliters of a 100 millimolar solution of LAP (Lithium phenyl -2,4,6- trimethylbenzoylphosphinate (initiator); and 250 microliters of a 15 millimolar solution of sodium ascorbate (inhibitor), and sterile filtered. A diluent solution containing 5.05 micromolar initiator and 0.758 micromolar inhibitor in deionized water was also made and sterile filtered. A diluent solution containing 5.05 micromolar initiator and 0.758 micromolar inhibitor was also made and sterile filtered.

[00557] The solution containing the polymer resin components (50 microliters) was combined with 350 microliters of the diluent solution, diluted with 44 microliters of lOx DPBS solution (to arrive at a final IX DPBS concentration), and introduced into the flow region of a microfluidic device (as in the general materials above, having 3500 pens) at a rate between 0.25 microliters/sec to about 0.50 microliters/second, for about 10 to 20 minutes. This permitted full equilibration within the enclosure of the microfluidic device.

[00558] Hydrogels were then generated in situ, using a 5000ms exposure, 50% power, with a DAPI filter cube, permitting efficient photoinitiation. The hydrogels were formed as a barrier within the sequestration pen, and the pattern performed throughout each field of view as desired. The patterning was performed once using a 4X objective. Typically, polymerization was conducted at the temperature of the subsequent culturing or assaying. FIG. 17A shows a brightfield image of a set of reversible hydrogel barriers generated in situ within a row of sequestration pens. The specific hydrogel barrier introduces here was a full-width (50 microns) barrier and had a 100 micron dimension in the distal to proximal direction within the pen. After gelation, perfusion was continued for 20 minutes to effectively flush remaining uncrosslinked polymer solution from the microfluidic device. The uncrosslinked materials present within the sequestration pen typically diffused through the gel caps and entered the microfluidic channel to be flushed out of the microfluidic device.

[00559] The hydrogel barriers were then dissolved, using a reagent, sodium periodate that was introduced as a solution (5.0 millimolar in DPBS), at reduced temperature (11 degrees C), and diffused into and through the hydrogel barriers, as sodium periodate has a very low molecular weight and can permeate through the formed hydrogel. FIG. 17B shows the same sequestration pens after a 3 minute treatment period, and showed the complete removal of all the hydrogel barriers. [00560] In FIGS. 18A and 18B, an experiment having CHO cells present within the sequestration pens during the process of introducing the hydrogel barriers and subsequent cleavage by sodium periodate at low temperature, 10 millimolar concentration, and 3 minute exposure showed that viability of the cells was not damaged. The image in FIG. 18B was taken 24h post periodate treatment (i.e., hydrogel barrier dissolution) and cell counting showed no significant impact at this concentration, exposure time, and temperature.

[00561] 2B. Reversible hydrogel barrier including a cleavable peptide sequence. A Prepolymer composition was made similar to that of Experiment 2, except that the crosslinker was a peptide having the sequence GCRDLPRTGGDRCG (SEQ ID NO: 1). Additionally, for the Prepolymer composition and the Diluent, the inhibitor was 4-hydroxy TEMPO instead of sodium ascorbate. Gellation was performed as for the reversible hydrogel of Experiment 2A. A solution of TrypLe was introduced to reverse the gelation on chip.

[00562] Experiment 3.

[00563] Formation of a non-reversible hydrogel barrier. A solution of 8-Arm PEG-Norbomene (MW 20 kDa, 8.75 micromolar); 8- Arm PEG-Norbomene (MW lOkDA, 17.5 micromolar) in 4 ml ultrapure distilled water was combined with 450 microliters of a 1 ,0M solution of 2, 3 -dimercapto propane sulfonate (crosslinker); 250 microliters of a 100 millimolar solution of LAP (Lithium phenyl -2,4, 6- trimethylbenzoylphosphinate (initiator); and 250 microliters of a 15 millimolar solution of sodium ascorbate (inhibitor), and sterile filtered. A diluent solution containing 5.05 micromolar initiator and 0.758 micromolar inhibitor in deionized water was also made and sterile filtered.

[00564] The solution containing the polymer resin components (50 microliters) was combined with 350 microliters of the diluent solution, and diluted with 44 microliters of lOx DPBS solution (to arrive at a final IX DPS concentration) and introduced into the flow region of a microfluidic device (as in the general materials above, having 3500 pens) at a rate between 0.25 microliters/sec to about 0.50 microliters/second, for about 10 to 20 minutes. This permitted full equilibration within the sequestration pens. Hydrogels were then generated in situ, using a 5000ms exposure, repeated three times, 50% power, 4X objective with a DAPI filter cube, permitting efficient photoinitiation. The hydrogels were formed as a barrier within the sequestration pen, and the pattern performed throughout each field of view as desired. Typically, polymerization was conducted at the temperature of the subsequent culturing or assaying. In FIGS. 18A and 18B, an experiment having CHO cells present within the sequestration pens during the process of introducing the hydrogel barriers and subsequent cleavage by sodium periodate at low temperature, 0.5 millimolar concentration, and 20 minute exposure showed that viability of the cells was not damaged. The image in FIG. 18B was taken 24h post periodate treatment (i.e., hydrogel barrier dissolution) and cell counting showed no significant impact at this concentration, exposure time, and temperature.

[00565] Experiment 4. Formation of multiple hydrogel barriers having different physical properties. [00566] 4A. Use of both reversible and non-reversible hydrogel barriers. Introduction of a reversible hydrogel barrier, mid pen. Prepolymer composition and Diluent solution were made similarly to that of Experiment 2, except that the inhibitor used was 4-hydroxy TEMPO instead of sodium ascorbate. Mid pen barriers were introduced to every sequestration pen as described in Experiment 2. Introduction of non-reversible hydrogel barriers followed, using a Prepolymer composition and Diluent as described in Experiment 2, except that the inhibitor was 4-hydroxy-TEMPO. Gellation was induced within the sequestration pen but proximal to the opening to the channel for every other pen. The final patterned hydrogel barriers are shown in FIG. 19A. After a IX PBS flush, 50 microliters of 5 millimolar sodium periodate was introduced at 1 microliter/sec. FIG. 19B shows a brightfield image of the same pens after 20 min, showing that all of the mid-pen hydrogel barriers have been removed (dissolved by cleavage with periodate).

[00567] 4B. Peptide reversible hydrogel barrier plus non reversible hydrogel cap. A Prepolymer composition having a peptidyl crosslinker susceptible to TrypLE was made as in Experiment 2B and Diluent solution (similar to that of Experiment 2B but both solutions include 4-hydroxy TEMPO as an inhibitor). Mid-pen hydrogel barriers were introduced in every sequestration pen. Introduction of non- reversible hydrogel barriers followed, using a Prepolymer composition and Diluent as described in Experiment 2, except that the inhibitor was 4-hydroxy-TEMPO. Gellation was induced within the sequestration pen but proximal to the opening to the channel for every other pen, in a specific pattern. For every group of three pens, a first pen has a doubly gelled non-reversible hydrogel barrier (cap), a second pen a singly gelled non-reversible hydrogel barrier (cap), and the third pen had no non-reversible barrier introduced. The patterned pens are shown in FIG. 20A. After a IX PBS flush, 85 microliters of lOx TrypLE was introduced into the flow region, and images were taken over time. FIG. 20B shows an image taken nearly three minutes after introduction. The pen having no cap, e.g., the third pen of each sequence, showed nearly complete dissolution of the mid-pen reversible peptidyl hydrogel barrier. At time = 5 min, FIG. 20C shows complete dissolution of the mid-pen hydrogel barrier within the pen having a singly gelled non-reversible cap. At time = 13 minutes, the reversible mid-pen gel is completely dissolved, even with the slowed diffusion of TrypLE through the doubly gelled top cap, as shown in FIG. 20D.

[00568] Other Hydrogel Barrier combinations. Hydrogel barriers are not limited to simple caps or barriers across the width of a chamber in service to assaying, preserving or maintaining cells. Many other configurations are possible, and the following description is not limiting as to the variety of hydrogel barriers that may be useful for microfluidic workflows.

[00569] 4C. Hydrogels for splitting clonal populations. Hydrogels were used to split a portion of a clonal population from the parent sequestration pen and protect the separated portion from assay conditions that could destroy the separated clonal cells. A microfluidic device (Berkeley Lights, Inc.) having a plurality of sequestration pens was prepared for use as above in the general conditions. To aid identification of stable producer cell clones useful for viral vector manufacturing, including but not limited to adeno-associated virus (AAV) and lentivirus (LV) vectors, fluidic medium comprising a plurality of cells, which may be gene-edited or otherwise genetically manipulated was introduced in the microfluidic device. Single cells were disposed into each selected sequestration pen of the plurality of the sequestration pens, and each selected sequestration pen was spaced with an adjacent empty sequestration pen (i.e., a sequestration pen without any cells present). The disposed cells were cultured on chip and expanded into a clonal population in each sequestration pen.

[00570] The sequestration pen having a cell disposed therein was designated as an assay chamber, and the empty sequestration pen next to the assay chamber was designated as a preserving chamber. Taken together the two pens function as a culture unit. A subset of each clonal population in a sequestration pen was moved to the preserving chamber by DEP force. In this instance, the DEP force was optically actuated but optical actuation is not required to successfully transfer cells. The remaining cells of that clonal population were kept in the assay chamber of the culture unit. Then, the preserving chamber was sealed with an in situ-generated hydrogel barrier (“capped”) to preserve the cells therewithin from being affected by the assay to be performed in the assay chamber. The in situ-generated hydrogel cap was formed from the reversible hydrogel composition and gelled as described in Experiment 2A. The hydrogel barrier cap was permeable to nutrients and permitted waste products to diffuse into/out of the sequestration pen, but does not permit the destructive assay reagents (in this instance a viral particle) to pass through the hydrogel barrier cap. As shown in FIG. 21 the paired pens (2110, 2112) show a point in time where the destructive assay was in progress (in pen 2110), while the separated cell in pen 2112 are not affected and remained viable. After evaluation of each clonal population, the assay pens were either cleared of cells and viral particles by DEP, or the assay pens may be capped with a non-reversible in situ-generated hydrogel barrier (non-reversible cap). In either case, the reversible hydrogel caps may then be removed as described in Experiment 2A, and the remaining viable cells that did not undergo destructive testing may be unpenned using DEP forces. The use of these protective hydrogel barriers is not limited to experiments using viral particles but may be more broadly applied whenever the assaying process may damage the cells under examination.

[00571] The initial splitting of the clonal populations was also performed in an alternative manner. Each pair of pens were linked together with a transport barrier, disposed within the flow region, as shown schematically in FIGS. 22A and photographically in FIGS. 22B to 22D. The transport barrier was an in situ-generated barrier formed from the reversible hydrogel compositions of Experiment 2A. Cells that are mobile may propel themselves from the assay chamber to the preserving chamber, thereby splitting the population. This movement from assay chamber to preserving chamber was also performed, subsequent to hydrogel transport barrier introduction, by centrifuging the entire microfluidic chip in a first direction to propel cells from the chambers into the hydrogel transport barrier, then reversing the direction of spin, to propel the cells from the hydrogel transport barrier spanning the assay chamber and the preserving chamber. The cell population was divided by the dual centrifugation to provide cells of the clonal population in each of the two twinned chambers. Further, the hydrogel barriers reduce clonality risk, as cells originating outside of each group of two twinned pens were excluded. Once the cell population was divided, the hydrogel transport barrier was removed as described in Experiment 2A, and a hydrogel barrier (“cap”) across the proximal opening of the preserving chamber was then introduced as described above, permitting destructive assaying in the assay chamber, and eventual removal or displacement of the hydrogel barrier, and unpenning of desired cells from the preserving chamber. [00572] As shown in FIG. 22A, the transport barriers may be of various shapes. For the twinned chambers 2211, hydrogel barriers 2221a, 2221b spanning the microfluidic channel 2230 can provide rectangular transport region 2241. For twinned chambers 2212, athree sided hydrogel feature 2222, which does not extend across the entire channel, provides a smaller rectangular transport region. For the twinned chambers 2213, a triangular transport region 2243 is provided by angled hydrogel barrier 2223. For the twinned chambers 2214, a semi-circular transport area 2244 is formed by the arched hydrogel barrier 2224. FIG, 22B is a photographic image of a series of twinned chambers having semicircular transport regions defined by transport hydrogel barrier 2224 which spans across many sets of twinned chambers. FIG. 22C is a photographic image of a series of twinned chambers having rectangular transport regions, which do not extend all the way across the microfluidic channel, defined by transport hydrogel barrier 2222 which spans across many sets of twinned chambers. FIG. 22D is a photographic image of a series of twinned chambers having triangular transport regions defined by transport hydrogel barrier 2223 which spans across many sets of twinned chambers. Additional refinements to the use of transport hydrogel barriers, is shown in FIG. 23, where the hydrogel barrier 2324, spanning many twinned chambers, providing semicircular transport regions within the channel 2330, are further assisted by including reversible hydrogel splitting elements to support directing cells to each of the twinned pens. These are gelled into a variety of shapes. Hydrogel splitting elements 2351, 2352, 2353, 2354, 2355, 2356 may all be used to help direct (2362, 2362) cells into one of the two twinned pens. The splitting element is generated in situ, shown for 2351, to be centered on the chamber wall 2312 that divides the twinned chambers 2311.

[00573] Experiment 5. Permeability.

[00574] 5A. The permeability of the hydrogels was tuned by changing the amount of resin and diluent used. A specific ratio of Prepolymer composition (as described in Experiment 2A. and Diluent solution (as described in Experiment 2) is combined along with lOx PBS to arrive at a final target of lx PBS. For example, 100 microliters of a 1 :3 Prepolymer /Diluent sample, as shown below, 22.5 microliters Prepolymer composition, 67.5 microliters Diluent Solution, and 10 microliters lx PBS were combined to prepare the working solution. Shown below are the different hydrogel formulations (patterned with the 4x objective, -180 um offset, 3 x 5s) where each individual labelled species was flowed through the flow region and images were examined for extent of permeability for each formulation.

[00575] 5B. The permeability of the hydrogels was tuned by changing the type of resin and crosslinkers. Prepolymer compositions were prepared generally following the procedures of Experiment 2 A, but three prepolymer compositions with different resin and crosslinkers were prepared for this experiment. The three prepolymer compositions include: Prepolymer composition #1, which was according to the Experiment 2 A (Hydrogel 1), Prepolymer composition #2 using 4A2K PEG- Alkyne as the resin and 4A2K PEG-thiol as the crosslinker (Hydrogel 2), and Prepolymer composition #3 using 4A2K PEG- Norbornene as the resin and 4A2K PEG-thiol as the crosslinker (Hydrogel 3). The three prepolymer compositions were mixed with a dilution solution according to Experiment 2A at a ratio of 1 : 1 and then introduced into an OptoSelect® 3500 chip respectively. The chip was photopatterened to form respective hydrogels at mid-region of every other three sequestration pens (FIG. 30A).

[00576] After the hydrogels were formed, a testing solution having a reporter molecule of around 14k Da was perfused at 0.01 uL/s into the flow region of the chip. Images were taken to observe whether the hydrogels block the reporter molecules from crossing. The reporter molecule was attached with a fluorescent label. Therefore, if the hydrogel successfully blocks the entry of the reporter molecule, the sequestration pen should be dark in the image. On the other hand, if the hydrogel cannot block or the sequestration pen does not have any hydrogel, then the sequestration pen should be bright in the image.

[00577] FIG. 30B shows an image taken right after the testing solution was introduced. The image shows that all hydrogels blocked the reporter molecules from entering the sequestration pen. There were not too many report molecules entering the sequestration pen without a hydrogel barrier either, because the perfusion was just started, and the reporter molecules had not yet fully diffused into the sequestration pen. After the perfusion of the testing solution, FIG. 30C shows that the reporter molecules diffused into the sequestration pen without a hydrogel barrier. The hydrogel #1 can partially block the entry of the reporter molecules while hydrogel #2 and hydrogel #3 both efficiently blocked the reporter molecules. The intensities of the fluorescent signal associated with the label were also detected. According to the detection (data not shown), hydrogel #2 had the best performance among the three hydrogels in blocking the reporter molecules.

[00578] Then, IxPBS was perfused at 5 uL/s into the flow region of the chip. Images were taken to observe the residual reporter molecules within the sequestration pen. FIG. 30D shows an image taken after the remaining testing solution in the flow region was flushed away by the PBS. The perfusion of the PBS also facilitated removing the reporter molecules within the sequestration pens. While there was no reporter molecule in the flow region and most part of the chip, the image was able to more clearer shows that some reporter molecules were able to enter the sequestration pen with the hydrogen #1. In contrast, the sequestration pens of hydrogen #2 and hydrogen #3 were obviously dark.

[00579] The results shows that hydrogels formed from 4A2K PEG-Alkyne or 4A2K PEG-Norbornene using 4A2K PEG-thiol as the crosslinker had lower porosities than the hydrogel formed from 8 Al OK and 8A20K PEG Norbornenes using Dithiothreitol as the crosslinker (the formula in Experiment 2 A). Without wishing to be bound by theories, the resins of lower molecule weight, meaning shorter arms, together with crosslinkers of higher molecule weight were able to form more compact network, hence hydrogels with denser structure.

[00580] Experiment 6. Functionalization and permeability of a hydrogel barrier.

[00581] 6A. Functionalization of a PEG acrylamide hydrogel. A control (no functionalization) hydrogel mixture (Control) was prepared including: 270 microliters 20% w/v 8-arm acrylamide-PEG 20 kDA in 2 millimolar aqueous hydroquinone monomethyl ether (MEHQ, inhibitor); 270 microliters Initiator solution (40 millimolar LAP); and 60 microliters 10X PBS.

[00582] A biotin-hydrogel mixture (Biotin) was prepared including 81 microliters 20% w/v 8-arm acrylamide-PEG 20 kDa in 2 millimolar aqueous MEHQ; 1 microliter 20% w/v 8-arm biotin(l) acrylamide(7)-PEG 20 kDa in 2 millimolar aqueous MEHQ; 90 microliters aqueous Initiator Solution (40 mM LAP); and 20 microliters 10X PBS.

[00583] 20 microliters of the Biotin mixture were added to 180 microliters of the Control mixture to prepare a "1% Biotin" solution.

[00584] 20 microliters of the 1% biotin solution were added to 180 microliters of the Control mixture to prepare a "0.1% Biotin" solution.

[00585] 20 microliters of the 0.1% biotin solution were added to 180 microliters of the Control mixture to prepare a "0.01% Biotin" solution.

[00586] A microfluidic chip having 1750 sequestration pens (PhenomeX Inc), and prepared as described in the general conditions above, was maintained at 25° C, and flushed 3X with PBS/0.05% Tween 20.

[00587] 100 microliters of the Control Prepolymer composition were imported at 20 microliters /s.

Following the pattern "01020304", hydrogels were formed in the channel above pens in the " 1" position by illuminating the assay area, shifted by -150 in y axis with the DAPI cube. 3 s exposures at 50% power were used to ensure polymerization for this test. The chip was flushed 3X with PBS/Tween. This process was repeated with the 0.01% Biotin Prepolymer composition in the "2" position, 0.1% Biotin Prepolymer composition in the "3" position, and 1% Biotin Prepolymer composition in the "4" position. A final flush 3X with PBS/Tween was performed to remove unpolymerized materials. Each successive introduction of the increasingly biotinylated gel is shown in the brightfield images of FIGS. 24A to 24D, showing that equivalent sized hydrogel features were formed. [00588] 60 microliters of a 0.1 micrograms/mL Alexa 350 labelled streptavidin (Sav) solution were slowly introduced to the flow region of the microfluidic device at 0.032 microliters /s. (-1800 s). The chip was flushed, then imaged using a fluorescence channel appropriate for the Alexa 350 dye. FIG. 24E shows increasing concentration of Alexa 350 signal in each increasingly biotinylated hydrogel, which correlated to the increases in biotinylation. The Alexa 350 Sav labelling of the biotin functional sites on the hydrogel did not penetrate very far into the hydrogel feature, due to the particular concentration of the formulation chosen and the extent of gelation induced. In other variations, more accessible, i.e. permeable features can be produced by changing the polymer ratios and the power/exposure time for gelation.

[00589] 6B. Functionalization of a PEG norbornene-based hydrogel.

[00590] A 1 :3 Prepolymer composition: Diluent solution was made as described in Experiment 5, (using the Prepolymer composition and Diluent solution as described in Experiment 2A.

[00591] A 200 mg/mL solution of 8 Arm 20K (l)biotin(7)norbomene was prepared in ultrapure water, and the biotinylated norbornene prepolymer composition was spiked into the 1 :3 Prepolymer composition for use as the biotinylated prepolymer.

[00592] The 1:3 PrePolymer preparation, 50 microliters (without biotinylated prepolymer present), was imported onto the chip. The chip was then allowed to equilibrate for 10 minutes, then photopatterning was performed using the using 3x5s, 50% power DAPI irradiation. This was followed by introducing 1 :3 PrePolymer preparation including biotinylated prepolymer, 50 microliters) onto the chip, equilibrated for 10 min, and photopattemed using 3x5s, 50% power DAPI irradiation. The chip was flushed with 250 microliters IX PBS, after each round of photopatteming. After photopatteming, a solution of 0.1 mg/mL AF488-SAv was then imported onto the chip. 80 uL of solution was imported at 1 uL/s and 60 uL was then perfused over 30 minutes. The mixture was then allowed to incubate for another 5 hours.

[00593] The chip was then washed with 2 x 5 mL lx PBS at 1 uL/s, followed by 5 mL of lx PBS + 2%Tween20 at 1 microliter/s. The chip was then imaged in brightfield (FIG. 25 A for the non-biotinylated hydrogel, and FIG, 25C for the biotinylated hydrogel) and in fluorescence channel using 5 ms FITC (exceedingly bright).

[00594] In the fluorescence image, Streptavidin non-specifically binds to the non-biotinylated hydrogel of FIG. 25B, and appears to be evenly distributed within the hydrogel. Streptavidin does bind in greater concentration on the biotinylated hydrogel of FIG. 25D. However, here the Streptavidin accumulated strongly to the region closest to the channel (upper 40% of the gel in this representation), and has induced a morphological change within the hydrogel that may be due to further crosslinking.

[00595] Experiment 7. Functionalization of in situ-generated hydrogel

[00596] 7A. An OptoSelect™ device was prepared and primed as described above while the wetting solution used in this experiment was doped with anchor reagent (DBCO-PEG-alkyne reagent). A prepolymer composition comprising 8A20K PEG-Norbomene, 8 Al OK PEG-Norbornene, dithiothreitol (crosslinker), LAP (initiator), sodium ascorbate (inhibitor) was prepared and sterile filtered as described in Experiment 2A. In this experiment, the prepolymer composition is doped with oligonucleotide conjugates to functionalize the hydrogel barrier. The oligonucleotide conjugates comprise oligonucleotides conjugated with DTPA (dithiol phosphoramidite) as reactive moieties. Moreover, the oligonucleotide conjugates were treated with a reducing reagent (in this experiment: tris(2- carboxyethyljphosphine; TCEP)) Four prepolymer compositions doped respectively with four different oligonucleotide conjugates, each having different nucleic acid sequences (herein “Oligo A,” “Oligo B,” “Oligo C,” and “Oligo D”), were prepared.

[00597] Each of the four prepolymer compositions was mixed a dilution solution according to the Experiment 2A at a ratio of 1 : 1. Then, each mixture of the prepolymer composition/dilution solution was introduced into the flow region of a microfluidic device and photopatterned to generate hydrogels in situ in respective chambers sequentially. As shown in FIG. 31. A control prepolymer composition without oligonucleotide conjugates was also introduced and hydrogels thereof formed in respective chambers.

[00598] After the hydrogel formation, a cocktail reagent comprising four kinds of probes (herein “Probe A,” “Probe B,” “Probe C,” and “Probe D”), each has a nucleic acid sequence complementary to one of the Oligo A, Oligo B, Oligo C, and Oligo D, was introduced into the flow region. Each kind of probes was attached with respective fluorescent labels for observation purposes. Then, the microfluidic device was flushed with PBS to remove residual probes within the device. FIG. 32 shows images detecting the fluorescent signals of each probe respectively. According to the respective position of each type of hydrogels formed in the microfluidic device (FIG. 32(a)), each probe specifically bound to the respective hydrogels as shown in the images. Probe B showed a slight non-specific binding to Oligo D hydrogel (FIG. 32(c)) while the non-specific binding can be further optimized by adjusting the nucleic acid sequences of Oligo B or Oligo D. On the other hands, the signal associates with the binding of Probe D to Oligo D hydrogel seems to be weak in the image (FIG. 32 (e)), but it was because the signal was interfered by the auto-fluorescence of the materials of the chamber wall.

[00599] 7B. The experiment was performed according to 7A except that only two hydrogel compositions respectively doped with a kind of oligonucleotide conjugates were prepared (herein “Oligo A” and “Oligo B”) and the multiple hydrogels were formed in situ within a same chamber of the microfluidic device. Specifically, the hydrogels were formed on the side walls of the chambers (FIG. 33A), and every other hydrogel was the oligo A hydrogel while every other hydrogel was the oligo B hydrogel. The size of the hydrogels was about 10 um (±2) in diameter, which is smaller than those in 7A. FIG. 33B shows fluorescence image indicating specific binding of each probe to respective hydrogels in the chambers. The arrangement of the hydrogels allows better fluidic communication between the chamber and the flow region and allows multiplexed assay for capturing multiple kinds of molecules of interested within the chamber.

[00600] In sum, the Experiments 7A and 7B show that the hydrogel generated in situ within the microfluidic device can be efficiently functionalized with oligonucleotides as functionalizable moieties, which in turn can be used to couple with a functionalization reagent. For example, an antibody- oligonucleotide conjugate having a complementary nucleic acid sequence can be anchored on the hydrogel so that the anchored antibody can capture a molecule of interest within the chamber of the microfluidic device.

[00601] 7C. A prepolymer composition was prepared according to the Prepolymer composition#2 of Experiment 5B having 4R2K PEG alkyne and 4R2K PEG thiol. A solution of lipoic acid-PEG3 -biotin was prepared, treated with TCEP, and then doped into the prepolymer composition. The doped prepolymer composition was mixed with a dilution solution and introduced into a microfluidic device as described herein. In this experiment, three prepolymer compositions doped with serially diluted lipoic acid-PEG3 -biotin reagent (10X, 100X, and 1000X dilution) were prepared. Photoactivation was initiated to generate four hydrogels, including three biotinylated hydrogels and one control hydrogel without doping, in the microfluidic channel. After gelation, a solution of Alexa 647-labeled streptavidin was introduced. Fluorescence images were taken to observe the binding of the Alexa 647-labeled streptavidin to the biotinylated hydrogels.

[00602] The results confirms that the hydrogel can be functionalized with biotin functionalities for anchoring streptavidin moieties. The streptavidin moieties can then be used to anchor a biotinylated reagent, for example, biotinylated antibody, for capturing a molecule of interest within the microfluidic device.

PARTIAL LISTING OF EMBODIMENTS

[00603] Embodiment 1. A composition for forming a hydrogel, comprising: a first and a second polyethylene glycol polymer molecule, each comprising a respective polyethylene glycol moiety and a covalently linked reactive moiety Rx; and a crosslinker molecule comprising a first crosslinker moiety RxP disposed at a first end of a linker L moiety and a second crosslinker moiety RxP disposed at a second end of the linker moiety, wherein each of the first and the second crosslinker moiety RxP is configured to be activatable to react with the respective reactive moiety Rx of the first or the second polyethylene polymer molecules.

[00604] Embodiment 2. The composition of embodiment 1, wherein the polyethylene glycol moiety of the first polyethylene glycol polymer is the same as the polyethylene glycol moiety of the second polyethylene glycol polymer.

[00605] Embodiment 3. The composition of embodiment 1, wherein the polyethylene glycol moiety of the first polyethylene glycol polymer is different from the polyethylene glycol moiety of the second polyethylene glycol polymer.

[00606] Embodiment 4. The composition of any one of embodiments 1 to 3, wherein the hydrogel once formed, includes a structure of Formula (1): PEG1-CG1-L-CG1-PEG2 Formula (1), wherein PEG1 is the first polyethylene glycol moiety and PEG2 is the second polyethylene glycol moiety; CGI is a coupled group formed from the reaction of the Rx moiety and the RxP moiety; and L is the linker moiety comprising a linear portion, wherein a backbone of the linear portion comprises 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms.

[00607] Embodiment 5. The composition of embodiment 4, wherein the CGI coupled group is the product of a reaction between a norbomenyl moiety and a thiol moiety.

[00608] Embodiment 6. The composition of embodiment 4, wherein CGI comprises a thioether group. [00609] Embodiment 7. The composition of embodiment 4, wherein the CGI coupled group is the product of a reaction between an alkynyl moiety and a thiol moiety.

[00610] Embodiment 8. The composition of embodiment 4, wherein CGI comprises an alkenyl sulfide group.

[00611] Embodiment 9. The composition of any one of claims 1 to 8, wherein the first polyethylene glycol moiety comprises a multi-arm (e.g., 2-arm, 4-arm, or 8-arm) polyethylene glycol moiety.

[00612] Embodiment 10. The composition of embodiment 9, wherein each arm of the first polyethylene glycol moiety comprises a covalently linked reactive moiety Rx.

[00613] Embodiment 11. The composition of embodiment 9, wherein at least one arm of the first polyethylene glycol moiety comprises a covalently linked reactive moiety Rx, and at least one arm of the first polyethylene glycol moiety does not have a covalently linked reactive moiety Rx.

[00614] Embodiment 12. The composition of any one of embodiments 1 to 11, wherein the first polyethylene glycol moiety has a molecular weight from about 500 Da to about 25K Da (e.g., about IK Da to about 20K Da, about 5K Da to about 15K Da, or about 8K Da to about 12K Da).

[00615] Embodiment 13. The composition of any one of embodiments 1 to 12, wherein the second polyethylene glycol moiety comprises a multi-arm (e.g., 2-arm, 4-arm, or 8-arm) polyethylene glycol moiety. [00616] Embodiment 14. The composition of embodiment 13, wherein each arm of the second polyethylene glycol moiety comprises a covalently linked reactive moiety Rx.

[00617] Embodiment 15. The composition of embodiment 13, wherein at least one arm of the second polyethylene glycol moiety comprises a covalently linked reactive moiety Rx, and at least one arm of the second polyethylene glycol moiety does not have a covalently linked reactive moiety Rx.

[00618] Embodiment 16. The composition of any one of embodiments 1 to 15, wherein the second polyethylene glycol moiety has a molecular weight from about 500 Da to about 25K Da (e.g., about IK Da to about 20K Da, about 5K Da to about 15K Da, or about 8K Da to about 12K Da).

[00619] Embodiment 17. The composition of any one of embodiments 1 to 16, wherein the first polyethylene glycol moiety has a molecular weight of about 8K Da to about 12K Da (e.g., about 10K Da), and the second polyethylene glycol moiety has a molecular weight of about 18K Da to about 22K Da (e.g., about 20K Da).

[00620] Embodiment 18. The composition of any one of embodiments 4 to 17, wherein the hydrogel, once formed, comprises a mixture of hydrogel sub-structures, each having a structure of Formula (1).

[00621] Embodiment 19. The composition of any one of embodiments 1 to 18, wherein the first polyethylene glycol molecule and the second polyethylene glycol molecule each comprise a polyethylene glycol moiety covalently linked to a norbomene reactive moiety, and wherein each polyethylene glycol moiety has a molecular weight of about 8K Da to about 12K Da (e.g., about 10K Da).

[00622] Embodiment 20. The composition of any one of embodiments 1 to 19, wherein the first polyethylene glycol polymer molecule and the second polyethylene polymer molecule are present in the composition in a ratio from about 1 : 100 to about 100: 1, or about 1 :50 to about 50: 1, or about 1 :25 to about 25: 1.

[00623] Embodiment 21. The composition of embodiment 20, wherein the ratio of the first polyethylene glycol polymer molecule and the second polyethylene polymer molecule is about 1: 1; about 1:2; about 1:3; about 1:4; about 1:5; about 1:6; about 1:7; about 1:8; about 1:9; about 1: 10; about 10: 1; about 9: 1; about 8: 1; about 7: 1; about 6: 1; about 5: 1; about 4: 1; about 3: 1; or about 2: 1.

[00624] Embodiment 22. The composition of any one of embodiments 1 to 21, wherein the crosslinker molecule comprises a vic-diol (vicinal diol). [00625] Embodiment 23. The composition of embodiment 22, wherein the crosslinker molecule further comprises a first thiol moiety at a first end of the crosslinker, and a second thiol moiety at a second end of the crosslinker, each configured to react with a norbomenyl moiety or an alkynyl moiety.

[00626] Embodiment 24. The composition of embodiment 22 or 23, wherein the crosslinker molecule has a molecular formula: HS-LB-CH2-C(H)(OH)-C(H)(OH)-CH2-LB-SH Formula (2), wherein each instance of linker backbone LB is independently selected to comprise 0 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms.

[00627] Embodiment 25. The composition of embodiment 24, wherein the linker backbone LB has a linear backbone having carbon atoms.

[00628] Embodiment 26. The composition of embodiment 25, wherein the linear backbone has no silicon, nitrogen, oxygen, sulfur or phosphorus atoms.

[00629] Embodiment 27. The composition of any one of embodiments 1 to 26, wherein the crosslinker molecule is dithiothreitol.

[00630] Embodiment 28. The composition of any one of embodiments 1 to 26, wherein the crosslinker molecule comprises a disulfide.

[00631] Embodiment 29. The composition of embodiment 28, wherein the disulfide is not disposed at the first end or the second end of the crosslinker molecule.

[00632] Embodiment 30. The composition of embodiment 28, wherein the crosslinker has a molecular formula: HS-LB2-CH2-S-S-CH2-LB2-SH Formula (3), wherein each instance of linker backbone LB2 is independently selected to comprise 0 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms.

[00633] Embodiment 31. The composition of embodiment 30, wherein the linker backbone LB2 has a linear backbone having carbon atoms.

[00634] Embodiment 32. The composition of embodiment 31, wherein the linear backbone of LB2 has no silicon, nitrogen, oxygen, sulfur, or phosphorus atoms.

[00635] Embodiment 33. The composition of any one of embodiments 1 to 21, wherein the crosslinker molecule comprises a peptide sequence configured to be a substrate to a protease.

[00636] Embodiment 34. The composition of embodiment 33, wherein the crosslinker molecule has a formula HS-PEPT-SH Formula (4), wherein PEPT is a peptidyl moiety comprising about 4 to about 16 amino acids, wherein the peptidyl moiety is susceptible to enzymatic cleavage.

[00637] Embodiment 35. The composition of embodiment 33 or 34, wherein the peptide sequence comprises GCRDLPRTGGDRCG (SEQ ID NO: 1).

[00638] Embodiment 36. The composition of any one of embodiments 33 to 35, wherein the crosslinker molecule comprises a peptide sequence configured to be a tryptase substrate.

[00639] Embodiment 371. The composition of any one of embodiments 1 to 26, wherein the crosslinker molecule has a formula: HS-LB4-SH Formula (5), wherein linker backbone LB4 comprises 3 to 200 nonhydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms.

[00640] Embodiment 38. The composition of embodiment 37, wherein the linker backbone LB4 has a linear backbone having carbon atoms.

[00641] Embodiment 39. The composition of embodiment 38, wherein the linear backbone of LB4 has no silicon, nitrogen, oxygen, sulfur or phosphorus atoms. [00642] Embodiment 40. The composition of any one of embodiments 37 to 39, wherein the crosslinker is Sodium 2,3-dimercaptopropanesulfonate monohydrate.

[00643] Embodiment 41. The composition of any one of embodiments 1 to 40, wherein the crosslinker molecule comprises a branched moiety having multiple arms coupled to a branched core, at least one of the arms having a structure of Formula (6): -LB5-SH Formula (6), wherein linker backbone LB5 comprises at least one PEG moiety.

[00644] Embodiment 42. The composition of embodiment 41, wherein the linker backbone LB5 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 PEG moieties.

[00645] Embodiment 43. The composition of embodiment 41 or claim 42, wherein the linker backbone LB5 further comprises a sulfide moiety.

[00646] Embodiment 44. The composition of embodiment 43, wherein the sulfide moiety is derived from an interaction between a thiol moiety and a sulfhydryl-reactive moiety or a moiety comprising an unsaturated bond. [00647] Embodiment 45. The composition of embodiment 42 or embodiment 44, wherein the linker backbone LB5 comprises a thiosuccinimide moiety comprising the sulfide moiety.

[00648] Embodiment 46. The composition of any one of embodiments 41 to 45, wherein the linker backbone LB5 further comprises a vic-diol moiety, a disulfide moiety, or a peptide moiety.

[00649] Embodiment 47. The composition of any one of embodiments 41 to 46, wherein the linker backbone LB5 comprises 3 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms.

[00650] Embodiment 48. The composition of any one of embodiments 41 to 47, wherein the branched moiety has a plurality of arms (e.g., 2 arms, 4 arms, 6 arms, or 8 arms) each having the structure of Formula (6).

[00651] Embodiment 49. The composition of any one of embodiments 41 to 48, wherein the crosslinker molecule has a molecular weight from about 500 Da to about 25K Da (e.g., about IK Da to about 20K Da, about 5K Da to about 15K Da, or about 8K Da to about 12K Da).

[00652] Embodiment 50. The composition of any one of embodiments 1 to 49, wherein the crosslinker molecule and the first polyethylene glycol polymer molecule and/or the second polyethylene glycol polymer molecule have substantially the same rate of diffusion.

[00653] Embodiment 51. The composition of any one of embodiments 1 to 50, further comprising an inhibitor configured to inhibit reaction of the crosslinker with the reactive moiety of the first and/or the second polyethylene glycol polymer molecule.

[00654] Embodiment 52. The composition of embodiment 51, wherein the inhibitor is sodium ascorbate, MEHQ, or 4-hydroxy TEMPO.

[00655] Embodiment 53. The composition of embodiment 51 or 52, wherein the inhibitor is present within the composition at a concentration from about 5 millimolar to about 20 millimolar.

[00656] Embodiment 54. The composition of any one of embodiments 1 to 53, wherein at least one of the first and the second polyethylene glycol polymer molecules further comprises a first functionalizable moiety configured to react with a moiety other than a thiol moiety or a norborenyl moiety.

[00657] Embodiment 55. The composition of embodiment 54, wherein the functionalizable moiety comprises a biotin, an aldehyde, a succinimidyl moiety, or an oligonucleotide.

[00658] Embodiment 56. The composition of any one of embodiments 1 to 55, further comprising a first molecule having a functionalizable moiety and a reactive moiety, wherein the functionalizable moiety is configured to react with a moiety other than a thiol moiety or a norbomenyl moiety, and the reactive moiety of the first molecule is configured to react with the reactive moiety Rx of the first polyethylene glycol polymer and/or the second polyethylene glycol polymer.

[00659] Embodiment 57. The composition of embodiment 56, wherein the reactive moiety of the first molecule comprises a thiol moiety, which is optionally derived from a reduced dithiol moiety.

[00660] Embodiment 58. The composition of embodiment 56 or 57, wherein the first molecule having the functionalizable moiety and the reactive moiety is a reduced dithiol-PEG-biotin or an oligonucleotide conjugated with a reduced dithiol phosphoramidite or a S-Trityl-6-mercaptohexyl-l-[(2-cyanoethyl)-(N,N-diisopropyl) ]- phosphoramidite.

[00661] Embodiment 59. The composition of embodiment 57 or 58, wherein the first molecule having the functionalizable moiety and the reactive moiety is a reduced lipoic acid-PEG-biotin.

[00662] Embodiment 60. The composition of any one of embodiments 1 to 59, wherein the crosslinker molecule further comprises a second functionalizable moiety configured to react with a moiety other than a thiol moiety or a norbomenyl moiety.

[00663] Embodiment 61. The composition of embodiment 60, wherein the second functionalizable moiety comprises a biotin, an aldehyde, a succinimidyl moiety, or an oligonucleotide.

[00664] Embodiment 62. A hydrogel comprising: a first polyethylene glycol polymer moiety covalently linked to a first end of a first crosslinker moiety, wherein a second end of the first crosslinker moiety is covalently linked to a second polyethylene glycol polymer moiety.

[00665] Embodiment 63. The hydrogel of embodiment 62, wherein the polyethylene glycol moiety of the first polyethylene glycol polymer moiety is the same as the polyethylene glycol moiety of the second polyethylene glycol polymer moiety.

[00666] Embodiment 64. The hydrogel of embodiment 62, wherein the polyethylene glycol moiety of the first polyethylene glycol polymer moiety is different from the polyethylene glycol moiety of the second polyethylene glycol polymer moiety.

[00667] Embodiment 65. The hydrogel of any one of esmbodiment 62 to 64, wherein the hydrogel comprises a structure of Formula (1): PEG1-CG1-L-CG1-PEG2 Formula (1), wherein PEG1 is the first polyethylene glycol moiety and PEG2 is the second polyethylene glycol moiety; CGI is a coupled group covalently linking each moiety; and L is the crosslinker moiety, comprising a linear portion wherein a backbone of the linear portion comprises 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms.

[00668] Embodiment 66. The hydrogel of embodiment 65, wherein the CG coupled group is the product of a reaction between a norbomenyl moiety and a thiol moiety or the produce of a reaction between an alkynyl moiety and a thiol moiety.

[00669] Embodiment 67. The hydrogel of embodiment 66, wherein the CGI comprises a thioether group or an alkenyl sulfide group.

[00670] Embodiment 68. The hydrogel of any one of embodiments 62 to 67, wherein the first polyethylene glycol moiety comprises a multi-arm (e.g., 2-arm, 4-arm, or 8-arm) polyethylene glycol moiety.

[00671] Embodiment 69. The hydrogel of embodiment 68, wherein when the first polyethylene glycol moiety comprises the 2-arm, 4-arm, or 8-arm polyethylene moiety, at least one arm of the first polyethylene glycol moiety is covalently linked to the first end of the crosslinker moiety and at least one arm of the first polyethylene glycol moiety is not covalently linked to a second crosslinker moiety. [00672] Embodiment 70. The hydrogel of any one of embodiments 62 to 69, wherein the first polyethylene glycol moiety has a molecular weight from about 500 Da to about 25K Da (e.g., about IK Da to about 20K Da, about 5K Da to about 15K Da, or about 8K Da to about 12K Da).

[00673] Embodiment 71. The hydrogel of any one of embodiments 62 to 70, wherein the second polyethylene glycol moiety comprises a multi-arm (e.g., 2-arm, 4-arm, or 8-arm) polyethylene glycol moiety.

[00674] Embodiment 72. The hydrogel of embodiment 71, wherein when the second polyethylene glycol moiety comprises the 2-arm, 4-arm, or 8-arm polyethylene moiety, is covalently linked to the first end of the crosslinker moiety and at least one arm of the first polyethylene glycol moiety is not covalently linked to a second crosslinker moiety.

[00675] Embodiment 73. The hydrogel of any one of embodiments 62 to 72, wherein the second polyethylene glycol moiety has a molecular weight from about 500 Da to about 25K Da (e.g., about IK Da to about 20K Da, about 5K Da to about 15K Da, or about 8K Da to about 12K Da).

[00676] Embodiment 74. The hydrogel of any one of embodiments 62 to 73, wherein the first polyethylene glycol moiety has a molecular weight of about 10K Da and the second polyethylene glycol moiety has a molecular weight of about 10K Da.

[00677] Embodiment 75. The hydrogel of any one of embodiments 62 to 74, wherein the hydrogel comprises a mixture of hydrogels, each having a structure of Formula (1).

[00678] Embodiment 76. The hydrogel of any one of embodiments 62 to 75, wherein the first polyethylene glycol moiety and the second polyethylene moiety are present in the hydrogel in a ratio from about 1 : 100 to about 100: 1, or about 1:50 to about 50: 1, or about 1:25 to about 25: 1.

[00679] Embodiment 77. The hydrogel of embodiment 76, wherein the ratio of the first polyethylene glycol moiety and the second polyethylene moiety is about 1: 1; about 1:2; about 1:3; about 1:4; about 1:5; about 1:6; about 1:7; about 1:8; about 1:9; about 1: 10; about 10: 1; about 9: 1; about 8: 1; about 7: 1; about 6: 1; about 5: 1; about 4: 1; about 3: l; or about 2: 1.

[00680] Embodiment 78. The hydrogel of any one of embodiments 62 to 77, wherein the crosslinker moiety comprises a vic -diol.

[00681] Embodiment 79. The hydrogel of embodiment 78, wherein the crosslinker moiety has a molecular formula: -LB-CH2-C(H)(OH)-C(H)(OH)-CH2-LB- Formula (7), wherein each instance of linker backbone LB is independently selected to comprise 0 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms.

[00682] Embodiment 80. The hydrogel of embodiment 79, wherein the linker backbone LB has a linear backbone having carbon atoms.

[00683] Embodiment 81. The hydrogel of embodiment 79 or 80, wherein the linear backbone has no silicon, nitrogen, oxygen, sulfur, or phosphorus atoms.

[00684] Embodiment 82. The hydrogel of any one of embodiments 62 to 81, wherein the crosslinker moiety is derived from a threitol moiety.

[00685] Embodiment 83. The hydrogel of any one of embodiments 62 to 82, wherein the crosslinker moiety comprises a disulfide.

[00686] Embodiment 84. The hydrogel of embodiment 83, wherein the disulfide is not disposed at the first end or the second end of the crosslinker moiety.

[00687] Embodiment 85. The hydrogel of embodiment 83 or claim 84, wherein the crosslinker moiety has a molecular formula: -LB2-CH2-S-S-CH2-LB2- Formula (8), wherein each instance of linker backbone LB2 is independently selected to comprise 0 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms.

[00688] Embodiment 86. The hydrogel of embodiment 85, wherein the linker backbone LB2 has a linear backbone having carbon atoms.

[00689] Embodiment 87. The hydrogel of embodiment 86, wherein the linear backbone of LB2 has no silicon, nitrogen, oxygen, sulfur, or phosphorus atoms.

[00690] Embodiment 88. The hydrogel of any one of embodiments 62 to 87, wherein the crosslinker moiety comprises a peptide sequence configured to be a substrate to a protease.

[00691] Embodiment 89. The hydrogel of embodiment 88, wherein the crosslinker moiety has a formula: -PEPT- Formula (9), wherein PEPT is a peptidyl moiety comprising about 4 to about 12 amino acids, wherein the peptidyl moiety is susceptible to enzymatic cleavage.

[00692] Embodiment 90. The hydrogel of embodiment 88 or 89, wherein the peptide sequence comprises GCRDLPRTGGDRCG (SEQ ID NO: 1).

[00693] Embodiment 91. The hydrogel of any one of embodiments 88 to 90, wherein the crosslinker moiety comprises a peptide sequence configured to be a tryptase substrate.

[00694] Embodiment 92. The hydrogel of any one of embodiments 62 to 91, wherein the crosslinker moiety has a formula: -LB4- Formula (10), wherein linker backbone LB4 comprises 3 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms

[00695] Embodiment 93. The hydrogel of embodiment 92, wherein the linker backbone LB4 has a linear backbone having carbon atoms.

[00696] Embodiment 94. The hydrogel of embodiment 93, wherein the linear backbone of LB4 has no silicon, nitrogen, oxygen, sulfur, or phosphorus atoms.

[00697] Embodiment 95. The hydrogel of any one of embodiments 92 to 94, wherein the crosslinker moiety is derived from Sodium 2,3-dimercaptopropanesulfonate monohydrate.

[00698] Embodiment 96. The hydrogel of any one of embodiments 62 to 95, wherein the crosslinker moiety L has a formula: BC-(LB5)n- Formula (11), wherein BC is a branched core, the linker backbone LB5 comprises at least one PEG moiety, and n is an integer of at least 2.

[00699] Embodiment 97. The hydrogel of embodiment 96, wherein the linker backbone LB5 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 PEG moieties.

[00700] Embodiment 98. The hydrogel of embodiment 96 or 97, wherein the linker backbone LB5 further comprises a sulfide moiety.

[00701] Embodiment 99. The hydrogel of embodiment 98, wherein the sulfide moiety is derived from an interaction between a thiol moiety and a sulfhydryl-reactive moiety or a moiety comprising an unsaturated bond. [00702] Embodiment 100. The hydrogel of embodiment 98 or 99, wherein the linker backbone LB5 comprises a thiosuccinimide moiety comprising the sulfide moiety.

[00703] Embodiment 101. The hydrogel of any one of embodiments 96 to 100, wherein the linker backbone LB5 further comprises a vic -diol moiety, a disulfide moiety, or a peptide moiety.

[00704] Embodiment 102. The hydrogel of any one of embodiments 96 to 101, wherein the linker backbone LB5 comprises 3 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms.

[00705] Embodiment 103. The hydrogel of any one of embodiments 96 to 102, wherein n is 4, 6, or 8. [00706] Embodiment 104. The hydrogel of any one of embodiments 62 to 103, wherein at least one of the first and the second polyethylene glycol polymer moieties further comprises a first functionalizable moiety configured to react with a moiety other than a thiol moiety or a norbomenyl moiety; and/or wherein the crosslinker moiety further comprises a second functionalizable moiety configured to react with a moiety other than a thiol moiety or a norbomenyl moiety.

[00707] Embodiment 105. The hydrogel of embodiment 104, wherein the functionalizable moiety comprises a biotin, an aldehyde, a succinimidyl moiety, or an oligonucleotide.

[00708] Embodiment 106. The hydrogel of embodiment 104 or claim 105, wherein the first functionalizable moiety and/or the second functionalizable moiety is coupled to a coupling partner covalently linked to a detectable label.

[00709] Embodiment 107. The hydrogel of embodiment 106, wherein the detectable label is a visible label, a fluorescent label, or a luminescent label.

[00710] Embodiment 108. A hydrogel comprising: a plurality of polyethylene glycol polymer moieties having covalent linkages to crosslinker moieties, wherein: the covalent linkages comprise at least one of a thioether linkage and an alkenyl sulfide linkage; and the plurality of polyethylene glycol polymer moieties and crosslinker moieties form a matrix that restricts passage of 20nm -diameter nanoparticles through the hydrogel.

[00711] Embodiment 109. The hydrogel of embodiment 108, wherein the plurality of polyethylene glycol polymer moieties comprises branched polyethylene glycol polymer moieties.

[00712] Embodiment 110. The hydrogel of embodiment 109, wherein the branched polyethylene glycol polymer moieties comprise a plurality of polyethylene glycol arms (e.g., 2 arms, 4 arms, 8 arms, etc.) linked via a branched core, and, optionally, wherein the polyethylene glycol arms having similar molecular weights.

[00713] Embodiment 111. The hydrogel of any one of embodiments 108 to 110, wherein the polyethylene glycol polymer moieties have a molecular weight of about 500 Da to about 25K Da (e.g., about IK Da to about 5K Da, about 1.5K Da to about 3K Da, about 5 K Da to about 15K Da, or about 8K Da to about 12K Da, about 15K Da to about 25K Da, or about 18K Da to about 22K Da).

[00714] Embodiment 112. The hydrogel of any one of embodiment 108 to 111, wherein each crosslinker moiety comprises a polyethylene glycol polymer moiety.

[00715] Embodiment 113. The hydrogel of embodiment 112, wherein the polyethylene glycol polymer moieties of the crosslinker moieties are branched polyethylene glycol polymer moieties.

[00716] Embodiment 114. The hydrogel of embodiment 113, wherein the branched polyethylene glycol polymer moieties of the crosslinker moieties comprise a plurality of polyethylene glycol arms (e.g., 2 arms, 4 arms, 8 arms, etc.) linked via a branched core, and, optionally, wherein the polyethylene glycol arms having similar molecular weights.

[00717] Embodiment 115. The hydrogel of any one of embodiments 112 to 114, wherein the polyethylene glycol polymer moieties of the crosslinker moieties have a molecular weight of about 500 Da to about 25K Da (e.g., about IK Da to about 5K Da, about 1.5K Da to about 3K Da, about 5K Da to about 15K Da, or about 8K Da to about 12K Da, about 15K Da to about 25K Da, or about 18K Da to about 22K Da).

[00718] Embodiment 116. The hydrogel of any one of embodiments 108 to 115, wherein the polyethylene glycol polymer moieties of the plurality of polyethylene glycol polymer moieties have an average molecular weight that is similar to an average molecular weight of the crosslinker moieties.

[00719] Embodiment 117. The hydrogel of any one of embodiments 108 to 116, wherein the polyethylene glycol polymer moieties of the plurality of polyethylene glycol polymer moieties diffuse through aqueous medium at a rate that is similar to the rate of diffusion of the crosslinker moieties through an identical aqueous medium.

[00720] Embodiment 118. The hydrogel of any one of embodiments 109 to 117, wherein the polyethylene glycol polymer moieties of the plurality of polyethylene glycol polymer moieties have a molecular weight of about IK Da to about 3K Da, and wherein the crosslinker moieties have a molecular weight of about IK Da to about 3K Da.

[00721] Embodiment 119. The hydrogel of embodiment 118, wherein the polyethylene glycol polymer moieties of the plurality of polyethylene glycol polymer moieties are branched and have four arms.

[00722] Embodiment 120. The hydrogel of embodiment 118 or 119, wherein each crosslinker moieties comprises a branched polyethylene glycol polymer moiety having four arms.

[00723] Embodiment 121. The hydrogel of any one of embodiments 118 to 120, wherein the matrix formed by the plurality of polyethylene glycol polymer moieties and crosslinker moieties restricts passage of molecules having a molecular weight of greater than 140K Da, greater than 100K Da, or greater than 60K Da through the hydrogel.

[00724] Embodiment 122. The hydrogel of any one of embodiments 108 to 117, wherein the polyethylene glycol polymer moieties of the plurality of polyethylene glycol polymer moieties have a molecular weight of about 8K Da to about 12K Da, and wherein the crosslinker moieties have a molecular weight of about 8K Da to about 12K Da.

[00725] Embodiment 123. The hydrogel of embodiment 122, wherein the polyethylene glycol polymer moieties of the plurality of polyethylene glycol polymer moieties are branched and have four arms.

[00726] Embodiment 124. The hydrogel of embodiment 122, wherein the polyethylene glycol polymer moieties of the plurality of polyethylene glycol polymer moieties are branched and have eight arms.

[00727] Embodiment 125. The hydrogel of any one of embodiments 122 to 124, wherein each crosslinker moieties comprises a branched polyethylene glycol polymer moiety having four arms.

[00728] Embodiment 126. The hydrogel of any one of embodiments 122 to 124, wherein each crosslinker moieties comprises a branched polyethylene glycol polymer moiety having eight arms.

[00729] Embodiment 127. The hydrogel of any one of embodiments 108 to 117, wherein the polyethylene glycol polymer moieties of the plurality of polyethylene glycol polymer moieties have a molecular weight of about IK Da to about 3K Da, and wherein the crosslinker moieties have a molecular weight of about IK Da to about 3K Da.

[00730] Embodiment 128. The hydrogel of embodiment 127, wherein the polyethylene glycol polymer moieties of the plurality of polyethylene glycol polymer moieties are branched and have four arms.

[00731] Embodiment 129. The hydrogel of embodiment 127, wherein the polyethylene glycol polymer moieties of the plurality of polyethylene glycol polymer moieties are branched and have eight arms.

[00732] Embodiment 130. The hydrogel of any one of embodiments 127 to 129, wherein each crosslinker moieties comprises a branched polyethylene glycol polymer moiety having four arms.

[00733] Embodiment 131. The hydrogel of any one of embodiments 127 to 129, wherein each crosslinker moieties comprises a branched polyethylene glycol polymer moiety having eight arms.

[00734] Embodiment 132. A method of forming a hydrogel within a microfluidic device, the method comprising: introducing a photoactivatable flowable polymer composition into a flow region of the microfluidic device; and activating crosslinking of the composition in a selected area of the microfluidic device, thereby forming a hydrogel. [00735] Embodiment 133. The method of embodiment 132, wherein the photoactivatable flowable polymer composition comprises modified polyethylene glycol moieties.

[00736] Embodiment 134. The method of embodiment 132 or embodiment 133, wherein the photoactivable flowable polymer composition comprises the composition of any one of embodiments 1 to 59.

[00737] Embodiment 135. The method of any one of embodiments 132 to 134, further comprising introducing a photoinitiator into the flow region, and permitting the photoinitiator to diffuse into the chambers.

[00738] Embodiment 136. The method of embodiment 135, wherein the photoinitiator is Lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP) .

[00739] Embodiment 137. The method of any one of embodiments 132 to 136, wherein forming the hydrogel further comprises illuminating the selected area for a selected period of time with a wavelength of light configured to induce crosslinking.

[00740] Embodiment 138. The method of any one of embodiments 132 to 137, wherein the photoactivatable flowable polymer composition further comprises an inhibitor of crosslinking.

[00741] Embodiment 139. The method of embodiment 138, wherein the inhibitor is MEHQ, 4-hydroxy TEMPO, or sodium ascorbate.

[00742] Embodiment 140. The method of embodiment 138 or embodiment 139, further comprising introducing a second portion of the inhibitor into the flow region of the microfluidic device.

[00743] Embodiment 141. The method of any one of embodiments 132 to 140, wherein the microfluidic device comprises an enclosure comprising a base, a cover, and microfluidic circuit material defining a fluidic circuit therein, and further wherein the fluidic circuit comprises the flow region and a chamber opening to the flow region.

[00744] Embodiment 142. The method of embodiment 141, wherein a biological micro-object is disposed within the chamber prior to introduction of the photoactivatable flowable polymer composition.

[00745] Embodiment 143. The method of embodiment 142, wherein forming the hydrogel comprises capping the chamber with the formed hydrogel thereby retaining the biological micro-object within the chamber.

[00746] Embodiment 144. The method of embodiment 143, further comprising removing the hydrogel and exporting the biological micro-object from the chamber.

[00747] Embodiment 145. The method of any one of embodiments 141 to 144, wherein introducing a photoactivatable flowable polymer composition comprises diffusing the photoactivatable flowable polymer composition from the flow region into the chamber.

[00748] Embodiment 146. The method of any one of embodiments 141 to 145, wherein the chamber is a sequestration pen, and the selected area is in the sequestration pen,

[00749] Embodiment 147. The method of embodiment 146, wherein the selection area is within an isolation region of the sequestration pen.

[00750] Embodiment 148. The method of embodiment 146 or embodiment 147, wherein the hydrogel forms an assay region within the sequestration pen.

[00751] Embodiment 149. The method of any one of embodiments 146 to 148, wherein the hydrogel forms a culturing region within the sequestration pen.

[00752] Embodiment 150. The method of any one of embodiments 141 to 149, wherein forming the hydrogel comprises forming the hydrogel at an opening of the chamber to the flow region

[00753] Embodiment 151. The method of any one of embodiments 141 to 150, wherein the selected area is in the flow region. [00754] Embodiment 152. The method of any one of embodiments 132 to 151, wherein when the composition comprises a single type of crosslinker molecule comprising a vic -diol moiety or a peptide moiety, the method further comprises removing the hydrogel at the selected time by introducing a reversing reagent into the flow region configured to react on the vic-diol moiety or the peptide moiety thereby reversing the hydrogel.

[00755] Embodiment 153. The method of embodiment 152, wherein the reversing reagent is a periodate reagent or an enzyme configured to cleave the peptidyl moiety.

[00756] Embodiment 154. The method of embodiment 153, wherein the enzyme configured to cleave the peptidyl moiety is a trypsin enzyme or analog thereof.

[00757] Embodiment 155. The method of embodiment 154, wherein the analog of the trypsin enzyme is a TrypLETM enzyme.

[00758] Embodiment 156. The method of any one of embodiments 132 to 155, further comprising introducing a second molecule having a functionalizable moiety and a reactive moiety, wherein the functionalizable moiety is configured to react with a moiety other than a thiol moiety or a norbomenyl moiety, and the reactive moiety comprises an unsaturated bond.

[00759] Embodiment 157. The method of embodiment 156, wherein the reactive moiety of the second molecule comprises a maleimide moiety, an alkyne moiety, or an olefin moiety.

[00760] Embodiment 158. The method of embodiment 156 or embodiment 157, wherein the functionalizable moiety of the second molecule comprises a biotin, an aldehyde, a succinimidyl moiety, or an oligonucleotide. [00761] Embodiment 159. A method of introducing hydrogel having different physical properties to a microfluidic device, wherein the microfluidic device comprises an enclosure comprising a base, a cover, and microfluidic circuit material defining a fluidic circuit therein, and further wherein the fluidic circuit comprises a flow region and a plurality of chambers opening to the flow region, the method comprising: introducing a first photoactivatable flowable polymer composition into the flow region of the microfluidic device, wherein the first photoactivatable flowable polymer composition has a first selected set of characteristics defining physical properties of a first hydrogel formed therefrom; activating crosslinking of the first photoactivatable flowable polymer composition in a first selected area of the microfluidic device, thereby forming the first hydrogel; introducing a second photoactivatable flowable polymer composition into the flow region of the microfluidic device, wherein the second photoactivatable flowable polymer composition has a second selected set of characteristics defining physical properties of a second hydrogel formed therefrom; and activating crosslinking of the second photoactivatable flowable polymer composition in a second selected area of the microfluidic device, thereby forming a second hydrogel barrier, wherein the first hydrogel differs from the second hydrogel, based on at least one different physical property.

[00762] Embodiment 160. The method of embodiment 159, wherein the first photoactivatable flowable polymer composition and the second photoactivatable flowable polymer composition each comprise modified polyethylene glycol moieties.

[00763] Embodiment 161. The method of embodiment 159 or 160, wherein the first photoactivatable flowable polymer composition is the composition of any one of embodiments 1 to 61.

[00764] Embodiment 162. The method of any one of embodiments 159 to 161, wherein the second photoactivatable flowable polymer composition is the composition of any one of embodiments 1 to 61. [00765] Embodiment 163. The method of any one of embodiments 159 to 162, wherein a viscosity of the first photoactivatable flowable polymer composition and a viscosity of the second photoactivatable flowable polymer composition are substantially the same. [00766] Embodiment 164. The method of any one of embodiments 159 to 163, wherein the at least one different physical property comprises reversibility, permeability, shrinkability, or a combination thereof. [00767] Embodiment 165. The method of embodiment 164, wherein one of the first hydrogel or the second hydrogel is a reversible hydrogel and the respective other hydrogel is a non-reversible hydrogel.

[00768] Embodiment 166. The method of embodiment 165, wherein the first hydrogel is a reversible hydrogel.

[00769] Embodiment 167. The method of embodiment 166, wherein forming the first hydrogel comprises forming a first reversible hydrogel barrier within one or more chambers of the plurality of chambers.

[00770] Embodiment 168. The method of embodiment 167, wherein forming the first reversible hydrogel barrier further comprises forming the first reversible hydrogel barrier distal to, at a mid-region of, or at the opening of the one or more chambers, thereby producing one or more reversibly capped chambers.

[00771] Embodiment 169. The method of embodiment 168, wherein the first reversible hydrogel barrier of the reversibly capped chamber comprises a permeability limit, thereby preventing subsequently introduced reagents having a size greater than the permeability limit from entering the one or more reversibly capped chambers. [00772] Embodiment 170. The method of embodiment 168 or embodiment 169, wherein at least some of the one or more reversibly capped chambers contain one or more biological micro-objects, and the first reversible hydrogel barrier prevents the one or more biological micro-objects from exiting the one or more reversibly capped chambers.

[00773] Embodiment 171. The method of any one of embodiments 167 to 170, wherein when the plurality of chambers is a plurality of sequestration pens, the first reversible hydrogel barrier is formed within an isolation region of the plurality of sequestration pens, forming an assay region or a biological micro-object maintenance region distal to the opening of the one or more reversibly capped .

[00774] Embodiment 172. The method of any one of embodiments 167 to 171, wherein the first reversible hydrogel barrier of each of the one or more reversibly capped chambers prevents a biological micro-object from entering a region of each of the one or more reversibly capped chambers wherein biological micro-objects were disposed prior to forming the first reversible hydrogel barrier.

[00775] Embodiment 173. The method of any one of embodiments 168 to 172, wherein the method further comprises forming the second hydrogel at the opening of a subset of the plurality of chambers, wherein the subset of the plurality of chambers: is different from the one or more reversibly capped chambers; is the same chambers as the one or more reversibly capped chamber; is selected to have some or all of the reversibly capped chambers; or is selected to comprise some or all of the reversibly capped chambers and some of the remainder of the plurality of chambers, and further wherein: the second hydrogel at each opening of the subset of the plurality of chambers prevents a biological micro-object from entering or exiting each chamber of the subset of chambers. [00776] Embodiment 174. The method of embodiment 173, further comprising introducing a reversing reagent configured to reverse the first reversible hydrogel barrier; contacting the reversing reagent with the first reversible hydrogel barriers of the one of more reversibly capped chambers; and reversing the first reversible hydrogel barriers, thereby providing a group of uncapped chambers while retaining the second hydrogel barriers in each of the subset of the plurality of chambers.

[00777] Embodiment 175. The method of embodiment 174, wherein the group of uncapped chambers are the chambers having only a first reversible hydrogel barrier and have no second hydrogel barrier.

[00778] Embodiment 176. The method of embodiment 174 or 175, further comprising unpenning one or more biological micro-objects from at least one of the group of uncapped chambers. [00779] Embodiment 177. The method of embodiment 164, wherein the wherein first hydrogel and the second hydrogel are each a reversible hydrogel and require orthogonal conditions for reversing.

[00780] Embodiment 178. The method of embodiment 177, wherein forming the first hydrogel comprises forming a first reversible hydrogel barrier within one or more chambers of the plurality of chambers.

[00781] Embodiment 179. The method of embodiment 178, wherein forming the first reversible hydrogel barrier further comprises forming the first reversible hydrogel barrier distal to, at a mid-region of, or at the opening of the one or more chambers, thereby producing one or more reversibly capped chambers.

[00782] Embodiment 180. The method of embodiment 179, wherein the first reversible hydrogel barrier of each of the one or more reversibly capped chambers further comprises a permeability limit, thereby preventing subsequently introduced reagents having a size greater than the permeability limit from entering the one or more reversibly capped chambers.

[00783] Embodiment 181. The method of embodiment 179 or 180, wherein at least some of the one or more reversibly capped chambers contain one or more biological micro-objects, and the first reversible hydrogel barrier prevents the one or more biological micro-objects from exiting the one or more reversibly capped chambers.

[00784] Embodiment 182. The method of any one of embodiments 179 to 181, wherein when the plurality of chambers is a plurality of sequestration pens, the first reversible hydrogel barrier is formed within an isolation region of the one or more sequestration pens, forming an assay region or a biological micro-object maintenance region distal to the opening of the one or more reversibly capped chambers.

[00785] Embodiment 183. The method of any one of embodiments 179 to 182, wherein the first reversible hydrogel barrier of each of the one or more reversibly capped chambers prevents a biological micro-object from entering a region of each of the one or more reversibly capped chambers wherein biological micro-objects were disposed prior to introducing the first reversible hydrogel barrier.

[00786] Embodiment 184. The method of any one of embodiments 179 to 183 wherein forming the second hydrogel comprises forming a second reversible hydrogel at the opening of a subset of the plurality of chambers, wherein the subset of the plurality of chambers: is different from the one or more reversibly capped chambers; is the same chambers as the one or more reversibly capped chambers; is selected to have some or all of the reversibly capped chambers; or is selected to comprise some or all of the reversibly capped chambers and some or all of the remainder of the plurality of chambers; thereby forming a first group of one or more capped chambers reversibly capped by a first reversible hydrogel barrier and a second group of chambers reversibly capped by the second reversible hydrogel barrier.

[00787] Embodiment 185. The method of embodiment 184, further comprising: introducing a first reversing reagent configured to the first reversible hydrogel into the flow region of the microfluidic device; and contacting the first hydrogel barriers of the first group of capped chambers with the first reversing reagent; and reversing the first hydrogel barriers, thereby removing the capping and providing a first group of uncapped chambers.

[00788] Embodiment 186. The method of embodiment 185, further comprising unpenning the one or more biological micro-objects from at least one of the first group of uncapped chambers.

[00789] Embodiment 187. The method of any one of embodiments 184 to 186, further comprising: introducing a second reversing reagent configured to reverse the second reversible hydrogel into the flow region of the microfluidic device; and contacting the second hydrogel barriers of the second group of capped chambers with the second reversing reagent; and reversing the second hydrogel barriers, thereby providing a second group of uncapped chambers. [00790] Embodiment 188. The method of embodiment 187, further comprising unpenning one or more biological micro-objects from at least one of the second group of uncapped chambers.

[00791] Embodiment 189. The method of embodiment 164, wherein the first hydrogel has a different permeability from a permeability of the second hydrogel.

[00792] Embodiment 190. The method of embodiment 189, wherein the first hydrogel has a first permeability limit that is higher than a permeability limit of the second hydrogel.

[00793] Embodiment 191. The method of embodiment 190, wherein forming the first hydrogel comprises forming a first permeable hydrogel barrier within one or more chambers of the plurality of chambers.

[00794] Embodiment 192. The method of embodiment 191, wherein forming the first permeable hydrogel barrier further comprises forming the first permeable hydrogel barrier distal to, at a mid-region of, or at the opening of the one or more chambers, thereby producing one or more capped chambers.

[00795] Embodiment 193. The method of embodiment 192, wherein the first permeable hydrogel barrier of each of the one or more capped chambers prevents subsequently introduced reagents having a size greater than the permeability limit from entering the one or more capped chambers but permits subsequently introduced reagents having a size lower than the permeability limit to diffuse through the first permeable hydrogel barrier into an interior of the one or more capped chambers.

[00796] Embodiment 194. The method of embodiment 192 or embodiment 193, wherein at least some of the one or more capped chambers contain one or more biological micro-objects, and the first permeable hydrogel barrier prevents the one or more biological micro-objects from exiting the one or more reversibly capped chambers.

[00797] Embodiment 195. The method of any one of embodiments 192 to 194, wherein when the plurality of chambers is a plurality of sequestration pens, the first permeable hydrogel barrier is formed within an isolation region of the one or more sequestration pens, forming an assay region or a biological micro-object maintenance region distal to the opening of the one or more capped chambers.

[00798] Embodiment 196. The method of any one of embodiments 192 to 195, wherein the first permeable hydrogel barrier of each of the one or more capped chambers prevents a biological micro-object from entering a region of each of the one or more capped chambers wherein biological micro-objects were disposed prior to introducing the first permeable hydrogel barrier.

[00799] Embodiment 197. The method of any one of embodiments 192 to 196, wherein forming the second hydrogel comprises forming a second permeable hydrogel barrier at the opening of a subset of the plurality of chambers, wherein the subset of the plurality of chambers: is different from the one or more capped chambers; is the same chambers as the one or more capped chambers; is selected to have some or all of the one or more capped chambers; or is selected to comprise some or all of the one or more capped chambers and some of the remainder of the plurality of chambers, and further wherein: the second permeable hydrogel barrier at each opening of the subset of the plurality of chambers prevents a biological micro-object from entering or exiting each chamber of the subset of chambers.

[00800] Embodiment 198. The method of embodiment 197, wherein the second permeable hydrogel barrier of the subset of the plurality of chambers, permits subsequently introduced reagents having a size lower than the permeability limit of the second permeable hydrogel barrier to diffuse through the second permeable hydrogel barrier and into the interior of the subset of chambers.

[00801] Embodiment 199. The method of embodiment 198, wherein a reagent configured to diffuse through the first permeable hydrogel barrier is excluded from diffusing through the second permeable hydrogel barrier. [00802] Embodiment 200. The method of any one of embodiments 189 to 199, wherein one of the first permeable hydrogel barrier and the second permeable hydrogel barrier is a reversible hydrogel barrier, and the method further comprises reversing the reversible hydrogel barrier, thereby uncapping selected chambers. [00803] Embodiment 201. The method of embodiment 200, further comprising unpenning one or more biological micro-objects from at least one of the selected chambers.

[00804] Embodiment 202. The method of embodiment 164, wherein the first hydrogel has a shrinkability different from a shrinkability of the second hydrogel.

[00805] Embodiment 203. The method of embodiment 202, wherein forming the first hydrogel comprises forming a first shrinkable hydrogel barrier within one or more chambers of the plurality of chambers.

[00806] Embodiment 204. The method of embodiment 203, wherein forming the first shrinkable hydrogel barrier further comprises forming the first shrinkable hydrogel barrier distal to, at a mid-region of, or at the opening of the one or more chambers, thereby producing one or more capped chambers.

[00807] Embodiment 205. The method of embodiment 204, wherein at least some of the one or more capped chambers contain one or more biological micro-objects, and the first shrinkable hydrogel barrier prevents the biological micro-object from exiting the one or more capped chambers.

[00808] Embodiment 206. The method of any one of embodiments 203 to 205, wherein when the plurality of chambers is a plurality of sequestration pens, the first shrinkable hydrogel barrier is formed within an isolation region of the one or more sequestration pens, forming an assay region or a biological micro-object maintenance region distal to the opening of the one or more capped sequestration pens.

[00809] Embodiment 207. The method of any one of embodiments 204 to 206, wherein the first shrinkable hydrogel barrier of each of the one or more capped chambers prevents a biological micro-object from entering a region of each of the one or more capped chambers wherein biological micro-objects were disposed prior to introducing the first shrinkable hydrogel barrier.

[00810] Embodiment 208. The method of any one of embodiments 202 to 207, wherein forming the second hydrogel comprises forming a second shrinkable hydrogel barrier at the opening of a subset of the plurality of chambers, wherein the subset of the plurality of chambers: is different from the one or more capped chambers; is the same chambers as the one or more capped chambers; is selected to have some or all of the one or more capped chambers; or is selected to comprise some or all of the one or more capped chambers and some of the remainder of the plurality of chambers, and thereby forming a first group of one or more capped chambers capped by the first shrinkable hydrogel barrier and a second group of chambers capped by a second shrinkable hydrogel barrier.

[00811] Embodiment 209. The method of embodiment 208, further comprising increasing a temperature of the microfluidic device whereby the first shrinkable hydrogel barrier experiences a higher degree of shrinking than a shrinking that the second shrinkable hydrogel barrier experiences.

[00812] Embodiment 210. The method of embodiment 209, wherein increasing the temperature comprises projecting a light in an area comprising or proximal to the first shrinkable hydrogel barrier.

[00813] Embodiment 211. The method of embodiment 209, wherein projecting the light comprises avoiding an area comprising or proximal to the second shrinkable hydrogel barrier.

[00814] Embodiment 212. The method of any one of embodiments 209 to 211, wherein the shrinking of the first shrinkable hydrogel barrier uncaps and provides a first group of uncapped chambers.

[00815] Embodiment 213. The method of embodiment 212, further comprising unpenning one or more biological micro-objects from at least one of the first group of uncapped chambers. [00816] Embodiment 214. The method of any one of embodiments 159 to 213, wherein introducing a first photoactivatable flowable polymer composition comprises diffusing the first photoactivatable flowable polymer composition from the flow region into the chambers; and/or wherein introducing a second photoactivatable flowable polymer composition comprises diffusing the second photoactivatable flowable polymer composition from the flow region into the chambers.

[00817] Embodiment 215. The method of any one of embodiments 159 to 214, further comprising introducing a photoinitiator into the flow region, and permitting the photoinitiator to diffuse into the chambers.

[00818] Embodiment 216. The method of embodiment 215, wherein the photoinitiator is LAP.

[00819] Embodiment 217. The method of any one of embodiments 159 to 216, wherein forming the first hydrogel further comprises illuminating the first selected area for a selected period of time with a wavelength of light configured to induce crosslinking; and/or wherein forming the second hydrogel further comprises illuminating the second selected area for a selected period of time with a wavelength of light configured to induce crosslinking.

[00820] Embodiment 218. The method of any one of embodiments 159 to 217, wherein first photoactivatable flowable polymer composition and/or the second photoactivatable flowable polymer composition further comprises an inhibitor of crosslinking.

[00821] Embodiment 219. The method of embodiment 218, wherein the inhibitor is MEHQ, 4 hydroxy TEMPO, or sodium ascorbate.

[00822] Embodiment 220. The method of any one of embodiments 159 to 219, further comprising introducing a second portion of the inhibitor into the flow region of the microfluidic device.

[00823] Embodiment 221. A method of reducing a risk of loss of clonality within a microfluidic device, the method comprising: introducing a biological micro-object into one of a plurality of chambers of the microfluidic device, wherein the microfluidic device comprises an enclosure comprising a base, a cover, and microfluidic circuit material defining a fluidic circuit therein, and further wherein the fluidic circuit comprises a flow region and the plurality of chambers opening to the flow region; assaying or culturing the biological micro-object or a daughter micro-object thereof, thereby identifying a biological micro-object or daughter micro-object thereof of interest; identifying the chamber containing the biological micro-object or daughter micro-object of interest being an identified chamber; introducing a photoactivatable flowable polymer composition into the flow region of the microfluidic device; activating crosslinking of the photoactivatable flowable polymer composition in a selected area of the microfluidic device, thereby forming a hydrogel barrier at the opening of chambers other than the identified chamber, thereby producing a subset of capped chambers of the plurality of chambers; and unpenning and exporting the biological micro-object or daughter micro-object thereof of interest from the identified chamber while keeping the subset of capped chambers capped, thereby preserving clonality of the biological micro-object or daughter micro-object thereof of interest.

[00824] Embodiment 222. The method of embodiment 221, wherein the composition comprises modified polyethylene glycol moieties.

[00825] Embodiment 223. The method of embodiment 221 or 222, wherein the composition is the composition of any one of embodiments 1 to 61.

[00826] Embodiment 224. A method of reducing a risk of loss of clonality within a microfluidic device, the method comprising: introducing a biological micro-object into one of a plurality of chambers of the microfluidic device, wherein the microfluidic device comprises an enclosure comprising a base, a cover, and microfluidic circuit material defining a fluidic circuit therein, and further wherein the fluidic circuit comprises a flow region and the plurality of chambers opening to the flow region; introducing a first photoactivatable flowable polymer composition into the flow region of the microfluidic device; activating crosslinking of the first photoactivatable flowable polymer composition in a first selected area of the microfluidic device, thereby forming a first hydrogel barrier within a first sub-set of the plurality of chambers of the microfluidic device; assaying or culturing the biological micro-object or a daughter micro-object thereof, thereby identifying one or more biological microobjects or daughter micro-objects thereof of interest; identifying one or more chambers containing one or more biological micro-objects or daughter micro-objects of interest; introducing a second photoactivatable flowable polymer composition into the flow region of the microfluidic device; activating crosslinking of the second photoactivatable flowable polymer composition in a second selected area of the microfluidic device, thereby forming a second hydrogel barrier at the opening of chambers other than the identified chambers, thereby producing a second capped subset of chambers of the plurality of chambers; and unpenning and exporting the one or more biological micro-objects or daughter micro-objects thereof of interest from the identified chambers, thereby preserving clonality of the biological micro-object or daughter micro-object thereof of interest.

[00827] Embodiment 225. The method of embodiment 224, wherein the first photoactivatable flowable polymer composition and the second photoactivatable flowable polymer composition each comprises modified polyethylene glycol moieties.

[00828] Embodiment 226. The method of embodiment 224 or 225, wherein the first photoactivatable flowable polymer composition is the composition of any one of embodiments 1 to 61.

[00829] Embodiment 227. The method of any one of embodiments 224 to 226, wherein the second photoactivatable flowable polymer composition is the composition of any one of embodiments 1 to 59.

[00830] Embodiment 228. The method of any one of embodiments 224 to 227, wherein a viscosity of the first photoactivatable flowable polymer composition and a viscosity of the second photoactivatable flowable polymer composition are substantially the same.

[00831] Embodiment 229. The method of any one of embodiments 224 to 228, wherein the first photoactivatable flowable polymer composition comprises a crosslinker providing a non-reversible hydrogel once formed.

[00832] Embodiment 230. The method of embodiment 199, wherein the crosslinker has a formula: HS-LB4-SH Formula (5), wherein linker backbone LB4 comprises 3 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms.

[00833] Embodiment 231. The composition of embodiment 230, wherein the linker backbone LB4 has a linear backbone having carbon atoms.

[00834] Embodiment 232. The composition of embodiment 231, wherein the linear backbone of LB4 has no silicon, nitrogen, oxygen, sulfur, or phosphorus atoms.

[00835] Embodiment 233. The method of any one of embodiments 224 to 232, wherein the first hydrogel barrier creates a first area proximal to the opening of the chamber to the flow region and a second area distal to the opening, wherein the second area contains the biological micro-object or the daughter micro-object thereof. [00836] Embodiment 234. The method of any one of embodiments 224 to 233, wherein the first hydrogel barrier comprises one or more discrete sections, each of which is moveably connected to one or more surfaces of the chamber, that substantially prevents biological micro-object or the daughter micro-object thereof from crossing through the first hydrogel barrier. [00837] Embodiment 235. The method of embodiment 234, wherein application of a threshold pressure to the one or more discrete sections of the first hydrogel barrier moves at least one of the one or more discrete sections with respect to the one or more surfaces of the chamber and thereby creates an opening in the second distal area. [00838] Embodiment 236. The method of embodiment 235, wherein unpenning further comprises applying the threshold pressure to the one or more discrete sections of the first hydrogel barrier of the identified chambers, thereby unpenning the one or more biological micro-objects or daughter micro-objects thereof of interest.

[00839] Embodiment 237. The method of embodiment 236, wherein the applying the threshold pressure comprises illuminating a distal region of each of the identified chambers with laser illumination, thereby generating a dislodging force to move the one or more biological micro-objects or daughter micro-objects past the one or more discreet sections of the first hydrogel barrier to the proximal region of the chambers.

[00840] Embodiment 238. The method of embodiment 237, wherein the first photoactivatable flowable polymer composition comprises a crosslinker providing a reversible first hydrogel barrier once formed.

[00841] Embodiment 239. The method of any one of embodiments 224 to 238, wherein unpenning the one or more biological micro-objects or daughter micro-objects thereof of interest further comprises introducing a reversing reagent configured to reverse the first hydrogel barrier comprising the composition of the first reversible hydrogel into the flow region of the microfluidic device; contacting the first hydrogel barriers of the first group of capped chambers with the reversing reagent; and reversing the first hydrogel barriers, thereby providing a first group of uncapped chambers suitable for unpenning.

[00842] Embodiment 240. A method of functionalizing an in situ-generated hydrogel, within a microfluidic device, wherein the microfluidic device comprises an enclosure comprising a base, a cover, and microfluidic circuit material defining a fluidic circuit therein, and further wherein the fluidic circuit comprises a flow region and a plurality of chambers opening to the flow region, the method comprising: introducing at least one hydrogel barrier in at least some chambers of a plurality of chambers, wherein the at least one hydrogel barrier is the hydrogel barrier of embodiment 104 or embodiment 105 and the method of introducing the at least hydrogel barrier is performed according to a method of any one of embodiments 132 to 158; introducing a functionalization reagent comprising a functionalizing reaction pair moiety configured to react with the functionalizable moiety of the hydrogel barrier; and contacting the functionalization reagent with the hydrogel barrier thereby coupling the functionalization reagent to the hydrogel barrier.

[00843] Embodiment 241. The method of embodiment 240, wherein the functionalizable moiety comprises a biotin, an aldehyde, a succinimidyl moiety, or an oligonucleotide.

[00844] Embodiment 242. The method of embodiment 240 or 241, wherein the functionalizable moiety comprises a biotin, and the functionalizing reaction pair moiety of the functionalization reagent comprises a streptavidin functionalizing reaction pair moiety configured to couple with the biotin moiety of the hydrogel. [00845] Embodiment 243. The method of any one of embodiments 240 to 242, wherein the functionalizable moiety comprises a first oligonucleotide, and the functionalizing reaction pair moiety of the functionalization reagent comprises a second oligonucleotide complementary to the first oligonucleotide.

[00846] Embodiment 244. The method of any one of embodiments 240 to 243, wherein the functionalization reagent further comprises a capture moiety.

[00847] Embodiment 245. The method of embodiment 244, wherein the capture moiety is a protein, a nucleic acid, an organic molecule, a saccharide, a combination thereof.

[00848] Embodiment 246. The method of any one of embodiments 240 to 245, wherein the functionalization reagent further comprises a detectable label. [00849] Embodiment 247. A method of controlling diffusion of a molecule of interest within a microfluidic device, the method comprising: introducing a prepolymer composition configured for forming a hydrogel into a flow region of the microfluidic device, wherein the microfluidic device comprises an enclosure comprising a base, a cover, and microfluidic circuit material defining a fluidic circuit therein, and further wherein the fluidic circuit of the microfluidic device comprises a flow region and a chamber opening to the flow region; introducing a molecule of interest into the flow region of the fluidic circuit; activating crosslinking of the prepolymer composition in a selected area within the fluidic circuit of the microfluidic device thereby forming a hydrogel barrier between the flow region and the chamber; and diffusing the molecule of interest through the hydrogel barrier.

[00850] Embodiment 248. The method of embodiment 247, wherein the hydrogel barrier has a porosity that restricts but does not block passage (e.g. slows diffusion) of the molecule of interest.

[00851] Embodiment 249. The method of embodiment 247 or 248, wherein the hydrogel barrier is formed after introducing the molecule of interest.

[00852] Embodiment 250. The method of embodiment 249, wherein introducing a molecule of interest into the flow region comprising diffusing the molecule of interest into the chamber.

[00853] Embodiment 251. The method of embodiment 250, wherein diffusing the molecule of interest through the hydrogel barrier comprises diffusing the molecule of interest from the chamber to the flow region.

[00854] Embodiment 252. The method of embodiment 247 or embodiment 248, wherein the hydrogel barrier is formed before introducing the molecule of interest.

[00855] Embodiment 253. The method of embodiment 252, wherein diffusing the molecule of interest through the hydrogel barrier comprises diffusing the molecule of interest from the flow region to the chamber.

[00856] Embodiment 254. The method of any one of embodiments 247 to 253, wherein the chamber comprises an opening to the flow region, and the hydrogel barrier is formed at the opening.

[00857] Embodiment 255. The method of embodiment 254, wherein the hydrogel barrier caps the opening of the chamber.

[00858] Embodiment 256. The method of any one of embodiments 247 to 255, wherein the hydrogel barrier is formed within the chamber.

[00859] Embodiment 257. The method of any one of embodiments 247 to 256, wherein the prepolymer composition for forming a hydrogel comprises a photoactivatable flowable polymers.

[00860] Embodiment 258. The method of any one of embodiments 247 to 257, wherein the composition is the composition of any one of embodiments 1 to 43.

[00861] Embodiment 259. The method of any one of embodiments 247 to 258, wherein introducing a molecule of interest into the fluidic circuit comprises introducing a fluidic medium comprising the molecule of interest, wherein the molecule of interest is soluble in the fluidic medium.

[00862] Embodiment 260. A method of controlling permeability of a hydrogel to a molecule of interest, comprising: preparing a prepolymer composition for forming the hydrogel, wherein the prepolymer composition is the composition of any one of embodiments 1 to 61; and adjusting a working concentration of the first polyethylene glycol polymer molecule to be different from a pre -determined standard concentration thereof, and/or adjusting a working concentration of the second polyethylene glycol polymer molecule to be different from a pre -determined standard concentration thereof thereby controlling permeability of the hydrogel to a molecule of interest. [00863] Embodiment 261. The method of embodiment 260, wherein the method does not comprises adjusting a working concentration of the crosslinker to be different from a pre-determined standard concentration thereof. [00864] Embodiment 262. The method of embodiment 260 or embodiment 261, wherein adjusting a working concentration of the first polyethylene glycol polymer molecule to be different from a pre-determined standard concentration thereof comprises adjusting the working concentration of the first polyethylene glycol polymer molecule to be -95%, -90%, -80%, -70%, -60%, -50%, -40%, -30%, -20%, -10%, -5%, +5%, +10%, +20%, +30%, +40%, +50%, +60%, +70%, +80%, +90%, +100%, +150%, +175%, +200%, +225%, +250%, +275%, +300%, +350%, +400%, or higher, or any value therebetween of the pre-determined standard concentration. [00865] Embodiment 263. The method of any one of embodiments 260 to 262, wherein adjusting a working concentration of the second polyethylene glycol polymer molecule to be different from a pre-determined standard concentration thereof comprises adjusting the working concentration of the second polyethylene glycol polymer molecule to be -95%, -90%, -80%, -70%, -60%, -50%, -40%, -30%, -20%, -10%, -5%, +5%, +10%, +20%, +30%, +40%, +50%, +60%, +70%, +80%, +90%, +100%, +150%, +175%, +200%, +225%, +250%, +275%, +300%, +350%, +400%, or higher, or any value therebetween of the pre-determined standard concentration.

[00866] Embodiment 264. The method of any one of embodiments 261 to 263, wherein the method does not comprises adjusting a working concentration of the inhibitor to be different from a pre -determined standard concentration thereof.

[00867] Embodiment 265. The method of any one of embodiments 260 to 264, wherein the prepolymer composition further comprises a photoinitiator, and the method does not comprises adjusting a working concentration of the photoinitiator to be different from a pre-determined standard concentration thereof. [00868] Embodiment 266. A method for assaying a micro-object in a microfluidic device, comprising: introducing a first in situ-generated hydrogel into the microfluidic device according to the method of any one of embodiments 132 to 158, wherein the first in situ-generated hydrogel is according to embodiment 104 or embodiment 105 and is functionalized with a first capture moiety configured to bind a first molecule of interest; wherein the microfluidic device comprises an enclosure comprising a base, a cover, and microfluidic circuit material defining a fluidic circuit therein, and further wherein the fluidic circuit of the microfluidic device comprises a flow region and a chamber opening to the flow region, and the micro-object is disposed within the chamber; allowing a biomolecule produced by the micro-object to interact with the first capture moiety within the microfluidic device; and detecting a first interaction between the first capture moiety and the biomolecule.

[00869] Embodiment 267. The method of embodiment 266, wherein the first in situ-generated hydrogel comprises a structure of Formula (1): PEG1-CG1-L-CG1-PEG2 Formula (1), wherein PEG1 is the first polyethylene glycol moiety and PEG2 is the second polyethylene glycol moiety; CGI is a coupled group covalently linking each moiety; and L is the crosslinker moiety, comprising a linear portion wherein a backbone of the linear portion comprises 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms.

[00870] Embodiment 268. The method of embodiment 267, wherein the crosslinker moiety comprises a molecular formula (7): -LB-CH2-C(H)(OH)-C(H)(OH)-CH2-LB- Formula (7), wherein each instance of linker backbone LB is independently selected to comprise 0 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms.

[00871] Embodiment 269. The method of embodiment 267 or embodiment 268, wherein the crosslinker moiety comprises a molecular formula (9): -PEPT- Formula (9), wherein PEPT is a peptidyl moiety comprising about 4 to about 16 amino acids, wherein the peptidyl moiety is susceptible to enzymatic cleavage. [00872] Embodiment 270. The method of embodiment 268 or embodiment 269, wherein allowing a biomolecule produced by the micro-object to interact with the first capture moiety comprises capturing the biomolecule with the first capture moiety.

[00873] Embodiment 271. The method of embodiment 270, further comprising introducing a reversing reagent configured to react on the vic-diol moiety or the peptide moiety thereby reversing the hydrogel barrier and releasing the captured biomolecule from the first capture moiety.

[00874] Embodiment 272. The method of embodiment 271, wherein the reversing reagent is a periodate reagent or an enzyme configured to cleave the peptidyl moiety.

[00875] Embodiment 273. The method of embodiment 271, wherein the enzyme configured to cleave the peptidyl moiety is a trypsin enzyme or analog thereof.

[00876] Embodiment 274. The method of embodiment 273, wherein the analog of the trypsin enzyme is a TrypLETM enzyme.

[00877] Embodiment 275. The method any one of embodiments 266 to 274, further comprising introducing a second in situ-generated hydrogel into the microfluidic device according to a method of any one of embodiments 132 to 158, wherein the second in situ-generated hydrogel is according to embodiment 104 or embodiment 105 and is functionalized with a second capture moiety configured to bind a second molecule of interest; wherein the first in situ-generated hydrogel is disposed at a first location and the second in situ-generated hydrogel is disposed at a second location, and the first location and the second location are physically distinguishable; and allowing the biomolecule produced by the biological micro-object to interact with the second capture moiety within the microfluidic device; and detecting second interaction between the second capture moiety and the biomolecule.

[00878] Embodiment 276. The method of embodiment 275, wherein the biomolecule comprises a first biomolecule and a second biomolecule, and detecting the first interaction comprises detecting an interaction between the first capture moiety and the first biomolecule, and detecting the second interaction comprises detecting an interaction between the second capture moiety and the second biomolecule.

[00879] Embodiment 277. The method of embodiment 275 or embodiment 276, wherein: detecting the first interaction comprises introducing a first detection reagent comprising a first detectable label to a region adjacent to the first in situ-generated hydrogel; and detecting the second interaction comprises introducing a second detection reagent comprising a second detectable label to a region adjacent to the second in situ-generated hydrogel.

[00880] Embodiment 278. The method of embodiment 277, wherein the first detectable label and the second detectable label are independently a fluorescent label, a colorimetric label, or a luminescent label.

[00881] Embodiment 279. The method of embodiment 278, wherein the first detectable label and the second detectable label are spectrally distinct.

[00882] Embodiment 280. The method of any one of embodiments 275 to 279, wherein the first capture moiety and the second capture moiety are independently a protein, a nucleic acid, an organic molecule, a saccharide, a combination thereof.

[00883] Embodiment 281. The method of any one of embodiments 275 to 280, wherein the first in situ- generated hydrogel is functionalized with the first capture moiety by: introducing a first functionalization reagent comprising a first functionalizing reaction pair moiety and the first capture moiety, wherein the first functionalizing reaction pair moiety is configured to couple with a first functionalizable moiety of the first in situ- generated hydrogel; and contacting the first functionalization reagent with the first in situ-generated hydrogel thereby coupling the first functionalization reagent to the first in situ-generated hydrogel.

[00884] Embodiment 282. The method of any one of embodiments 275 to 281, wherein the second in situ- generated hydrogel is functionalized with the second capture moiety by: introducing a second functionalization reagent comprising a second functionalizing reaction pair moiety and the second capture moiety, wherein the second functionalizing reaction pair moiety is configured to couple with a second functionalizable moiety of the second in situ-generated hydrogel; and contacting the second functionalization reagent with the second in situ- generated hydrogel thereby coupling the second functionalization reagent to the second in situ-generated hydrogel.

[00885] Embodiment 283. The method of embodiment 282, wherein the first functionalizable moiety does not couple with the second functionalizing reaction pair moiety, and the second functionalizable moiety does not couple with the first functionalizing reaction pair moiety.

[00886] Embodiment 284. The method of any one of embodiments 281 to 283, wherein the first functionalizable moiety comprises a first oligonucleotide comprising a first sequence, and the first functionalizing reaction pair moiety comprises a first complementary oligonucleotide comprises a sequence complementary to the first sequence.

[00887] Embodiment 285. The method of any one of embodiments 282 to 284, wherein the second functionalizable moiety comprises a second oligonucleotide comprising a second sequence, and the second functionalizing reaction pair moiety comprises a second complementary oligonucleotide comprises a sequence complementary to the second sequence.

[00888] Embodiment 286. The method of any one of embodiments 282 to 285, wherein the first functionalization reagent and the second functionalization reagent are introduced together in a fluidic medium.