FORMING GEL STRUCTURES USING MICROFLUIDIC CHANNELS

FIELD OF INVENTION The present invention relates generally to articles and methods for forming structures in microfluidic channels, and more specifically, to articles and methods for forming structures comprising gels in microfluidic channels.

RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application Serial

No. 61/001,923, filed November 5, 2007, entitled "Forming and Using Structures in Microfluidic Channels," by Wong, et ah, and U.S. Provisional Patent Application Serial No. 61/008,810, filed December 21, 2007, entitled "Forming and Using Structures in Microfluidic Channels," by Wong, et ah, each incorporated herein by reference.

BACKGROUND

Microfluidic devices are flow systems miniaturized to dimensions as small as a few micrometers (μm), or even smaller in some instances. These devices can be used to manipulate and handle small fluid samples, to pattern biological materials such as proteins and cells, and to produce gel structures in the microchannels of the device. Reduction of the dimensions of gel structures has been a challenge. Several techniques have demonstrated that microfabrication makes it possible to reduce the dimensions of the structures of gel. For example, poly(dimethylsiloxane) (PDMS) stamps fabricated by soft lithography can mold layers of most types of gel, including biologically-derived ones that gel thermally, but typically they can generate only planar patterns — or layered stacks of them. Certain photolithographic techniques (those that use photochemical initiators, followed by exposure to UV light, to define the regions of crosslinked gel) can produce more complex structures than those based on molding, but may be limited to synthetic hydrogels that cure by UV light. Accordingly, although methods for fabricating gel structures exist, systems and methods that would allow for more flexible designs of gel structures, are compatible with biologically-derived gels, and/or allow easier integration of components within the gels would be beneficial.

SUMMARY OF THE INVENTION

Articles and methods involving microfluidic channels and gels are provided. In one embodiment, a method is provided. The method comprises flowing a first fluid and a second fluid simultaneously in a microfluidic channel, and forming a gel structure comprising substantially all of the first fluid in the microfluidic channel. The gel may have a length of at least 0.5 times, at least 0.75 times, or the same length of the microfluidic channel. The may have a width of, for example, 50 microns, 100 microns, or 200 microns. The method may include, in some embodiments, flowing a third fluid in the microfluidic channel simultaneously with the first and second fluids. The second fluid may be flowed between the first and third fluids. In some cases, the third fluid is a gel precursor, and, optionally, the method may further comprise forming a gel comprising substantially all of the third fluid in the microfluidic channel. The method may further comprise forming a gel comprising substantially all of the second fluid in the microfluidic channel. The forming step may comprise applying heat to the microfluidic channel, for example, the gel may be formed at 37 degrees Celsius. In other embodiments, the forming step comprises crosslinking by the reaction of at least two reactants. The method may further comprising forming a gradient of at least one component, e.g., a soluble factor, in the gel. An article may be formed by the method described above. In another embodiment, an article is provided. The article comprises a microfluidic channel and a first structure comprising a thermocurable gel that occupies a portion, but not all of a microfluidic channel. The structure extends the length of the channel, wherein the thermocurable gel includes at least a first side and a second side that are not in contact with a surface of the channel. In another embodiment, a method is provided. The method comprises providing a microfluidic channel including a first cell disposed in a first gel portion and a second cell disposed in a second gel portion. The first and second gel portions are separated by a portion that does not contain cells. At least one gel portion comprises a thermocurable gel. The method also involves allowing interaction between the first and second cells. Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying figures. In cases

where the present specification and a document incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and/or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. In the figures: Fig. IA shows the partitioning of a microfluidic channel, which can be used to form one or more gel structures, in one embodiment of the invention.

Fig. IB shows a photograph of an experimental set-up for the partitioning system shown in Fig. IA according to one embodiment of the invention.

Fig. 1C is a schematic diagram of the experimental set-up shown in Fig. IB according to one embodiment of the invention.

Fig. ID is a schematic diagram of an incubator that may be employed in some embodiments of the invention.

Figs. IE- II show various examples of gel cross-sections according to one embodiment of the invention. Figs. 2A-2C show various designs of microchannels according to one embodiment of the invention.

Fig. 2D shows an example of a cross-sectional view of a microchannel according to one embodiment of the invention.

Fig. 3 is a photograph showing laminar flow in a microchannel when the liquids have the same viscosity according to one embodiment of the invention.

Figs. 4A-4C are photographs showing laminar flow in a microchannel when the liquids have different viscosities according to one embodiment of the invention.

Figs. 5A-5F show various examples of gel structures formed in microfluidic channels according to one embodiment of the invention.

Figs. 6A-6B show cells disposed within gel structures according to one embodiment of the invention. Figs. 7A-7D show various examples of arrangements of subchannels within microchannels according to one embodiment of the invention.

Fig. 8A is a schematic diagram of a five-inlet channel according to one embodiment of the invention.

Fig. 8B shows one example of a gel structure that may be formed when employing a five-inlet channel according to one embodiment of the invention. The figure also shows cells embedded in the gel structure.

Fig. 8C shows migration of cells cultured within a gel structure according to one embodiment of the invention.

Figs. 9A-9D show gradients that may be produced in microchannels according to one embodiment of the invention.

Fig. 9E is a graph of the fluorescence intensity of the gradients shown in Figs. 9A-9D according to one embodiment of the invention.

Fig. 10 is a table of results collected from a study directed at shearing cells, according to one embodiment of the invention.

DETAILED DESCRIPTION

Articles and methods for forming structures in microfluidic channels are provided. Methods described herein may include the use of spatially-defined flows of fluid within microchannels to form portions of gel (e.g., gel structures) inside the microchannels. The gel structures may be formed by flowing, e.g., laminarly, one or more streams of fluid in a microfluidic channel, at least one of the streams including a gel precursor. The stream(s) of gel precursor can be polymerized (e.g., gelled) to form one or more gel structures by various methods such as by application of heat. Advantageously, the dimensions of the gel structures may be varied, for example, by applying different flow rates to the fluid streams, choosing different viscosities of the fluids, and/or by varying the dimensions of the microchannel. Using such methods, different configurations of gel structures in microfluidic channels can be formed. For

example, in one embodiment, a stream of gel precursor is flanked by two streams of spacing solution (e.g., non-gel precursors), which may be flowed into a microchannel simultaneously. Upon polymerization of the gel precursor, a self-standing gel structure may be formed between the two streams of spacing solution inside the microchannel. Optionally, the spacing solutions may be rinsed out and replaced by other solutions. Gel structures having different components encapsulated therein may also be formed.

Articles and methods described herein may involve, in some embodiments, i) culturing cells within or on surfaces of the gel structures, ii) patterning different types of cells on or in adjacent gel structures, and/or iii) applying gradients of soluble factors across the cell-containing gel structures. Such structures may be used for studying intercellular communication between cells cultured within biologically-derived, 3-D matrices of microscopic size.

In one aspect of the invention, a method includes flowing a first fluid and a second fluid in a microfluidic channel, at least one of the first and second fluids being a gel precursor. Optionally, a third, fourth, or fifth, etc. fluid may also be flowed in the channel. The gel precursor may be polymerized to form a gel structure that occupies a portion, but not all of the volume of the channel. For example, as shown in the embodiment illustrated in Fig. 1, device 10 includes a microfluidic channel 12 containing fluid streams 16, 18, and 20 which form portions, or subchannels, 26, 28, and 30, respectively. Fluid streams 16, 18, and 20 may be flowed simultaneously in microfluidic channel 12 so as to form multiple, laminar streams of fluid. Under conditions of laminar flow, at least one fluid may flow parallel to the walls of the microchannel, such that there is little or no turbulence between the adjacent streams of fluid. In some such embodiments, mixing between the streams occurs only by diffusion. According to one embodiment, at least one of fluid streams 16, 18, and 20 contains a gel precursor. That is, all or a portion of the gel precursor-containing fluid stream can be polymerized to form a gel structure. In some embodiments, the gel structure has a substantially similar configuration and dimensions of the gel precursor used to form the structure. In addition, the gel structure may occupy a portion, but not all, of the volume of the microfluidic channel. For example, in one embodiment, fluid stream 18 of Fig. 1 comprises a gel precursor and fluid streams 16 and 20 are non-gel precursors, also referred to herein as spacing solutions. Upon polymerization of fluid

stream 18, subchannel 28 may contain a gel structure while the spacing solutions remain unpolymerized. These non-polymerized portions can be used to deliver fluids and/or components to one or more sides of the gel structure. In this embodiment, fluid stream 18 can be formed into a gel such that at least a first side and at least a second side of the gel is not in contact with a surface of the channel (e.g., the sides of fluid stream 18 that are in contact with fluid stream 16 and fluid stream 20 are not in contact with a surface of the channel, and therefore, the sides of the gel that forms from fluid stream 18 are also not in contact with a surface of the channel). Of course, in other embodiments, fluid stream 16 and/or fluid stream 20 may include a gel precursor so as to form gels in subchannels 26 and/or 30 of microfluidic channel 12.

When two or more gels (e.g., a first gel and a second gel) are present in a microchannel, the gels may be formed of the same or a different polymeric composition. The difference in the polymeric compositions may be attributed to the addition of cells or other components, the type of material used to form the gel, the degree of crosslinking of the gel, or the like.

As used herein, the term "gel" is given its ordinary meaning in the art and refers to a material comprising a polymer network that is able to trap and contain fluids. The gel may comprise polymer chains that are crosslinked, either directly or via a crosslinking agent. The degree of crosslinking may be varied, in some cases, to tailor the extent to which the gel absorbs or retains fluids. Those of ordinary skill in the art would be able to select appropriate materials suitable for use as gels. In some cases, the gel precursor comprises a material that forms a gel upon reaction of at least two components. In another embodiment, the gel precursor comprises a material that forms a gel upon application of light to the material. Different types of gels that can be used in accordance with the present invention are described in more detail below.

In one particular embodiment, a gel precursor comprises a material that forms a thermocurable gel upon the application of heat. In some such embodiments, all or a portion of the gel precursor is exposed to an environment having a first temperature prior to forming a gel and a second temperature during formation of the gel. For example, device 10 containing the gel precursor may be kept at a first temperature prior to and/or during flowing of the gel precursor in the channel. To polymerize the gel precursor, the

gel precursor may be exposed to an environment having a second temperature. The second temperature may be greater than the first temperature.

Figs. IB-ID describe one method for forming a device including a microfluidic channel and a gel structure according to one embodiment of the invention. As shown in the embodiment illustrated in Fig. IB, a device 10 including a microchannel may be placed on a thermally-conductive disc 32, which may facilitate conduction of heat away from or towards the microchannel. Such a configuration is especially useful for polymerizing thermocurable gels. One or more outlet(s) 33 of the microchannel may be connected to a syringe pump via tubing 34. In some embodiments, the microchannel may be formed from a polymer 35 positioned on a surface 36 (e.g., a glass surface). Optionally, surface 36 may be positioned on top of thermally-conductive disc 32 and a material 37, e.g., tissue paper, may be positioned between the surface and the thermally- conductive disc. The thermally-conductive disc may keep the device at a temperature below the gelling temperature of the gel. Material 37 may aid in the visualization and/or identification of certain components in the system, as discussed below. In certain embodiments, e.g., when thermally cured gels are used, the conductive disc may be an aluminum plate that is cooled, for example, by ice 38.

Fluid streams may be introduced into the microfluidic channel(s) by any suitable method. In some cases, the fluid streams are flowed into the microfluidic channel by applying a reduced pressure (e.g., vacuum) at outlet 33. In one particular embodiment, a droplet of gel precursor and/or of spacing solution is deposited at each of inlets 41-45. As illustrated, the solutions may be pulled into the channel by applying a vacuum to the outlet of the channel using a syringe 39 or other suitable device; however, in other embodiments, a positive pressure may be applied to one or more inlets of the device, as described in more detail below.

In certain cases, as soon as the channel is filled with one or more gel precursors and/or spacing solutions, the tubing at the outlet may be disconnected from the syringe to immediately stop the flow. This may be done, for example, by cutting the tubing with scissors. The gel precursor may be allowed to gel after the flow has stopped, or the act of polymerization itself may cause the flow to stop. When thermally-cured gels are used, polymerization of the gel may be performed by placing the conductive disc in an incubator or sterile bath 53 having a temperature suitable for crosslinking the gel

precursor. Of course, other methods of polymerization may be used, such as exposure of the device to light to initiate polymerization of a photopolymerizable gel precursor.

In some instances, after gelation, the inlets for delivering a gel precursor may become plugged and may no longer pass fluid. The inlets used for delivering spacing solutions may remain open and could be used afterwards to access the parts of the channel not filled with gel. In other instances, however, the inlets for delivering a gel precursor are not plugged; for example, the gel may be crosslinked only at a main portion of a channel and not at the inlets. Such local crosslinking may be performed for thermocurable gels, for example, by applying heat locally to certain portions of the channel. In yet other instances, inlets that are plugged with a gel precursor can become unplugged, e.g., by removing all or portions of the gel. Removal can be performed, for example, by melting a gel portion (e.g., by placing the gel into a cooler environment, e.g., 4 degrees Celsius to liquefy the gel), dissolving a gel portion (e.g., using an enzyme), and/or physically removing a gel portion (e.g., by cutting). For example, to dissolve or break down certain thermal-curable gels (e.g., biologically-derived gels), a solution of enzyme such as a collagenase (e.g., dispase) can be introduced into a fluid stream adjacent a gel and the enzyme can diffuse into the gel. Additionally or alternatively, the enzyme solution can be introduced into an inlet of a channel containing a gel structure and the enzyme can be allowed to diffuse into the gel. It should be understood that all or portions of a gel structure can be removed in a microfluidic channel (e.g., a main channel) by using such or other methods.

In certain embodiments, such and other methods can remove all or portions of a gel structure while maintaining integrity of any components embedded in the gel structure. For instance, where cells are embedded in a gel structure, removal of a gel can be performed without substantially changing the morphology of the cell, without causing the cell to die, and/or without causing denaturation of any components.

In some embodiments, substantially all of a fluid (e.g., a gel precursor) in a channel is used to form the gel. For example, a gel precursor in fluid stream 18 may form a gel structure in the form of subchannel 28 upon polymerization of the precursor, and substantially all of the fluid of fluid stream 18 present in the channel may be used to form this gel structure. In embodiments where two or more fluids are gel precursors,

substantially all of the two or more fluids present in the channel form a gel in the channel. In such and other embodiments, different portions of the gel across the width of the channel can be formed simultaneously and are not limited by diffusion. By contrast, in other embodiments, a gel structure is formed at the interface between first and second fluids. For instance, a first fluid stream may include a first gel precursor and a second fluid stream may include a second gel precursor, the first and second gel precursors forming a gel structure upon interaction of the precursor components. In such embodiments, only portions, but not all, of the first and second fluids present in the microchannel are used to form the interfacial gel structure. The gel precursor that does not interact with a necessary component to form the gel flows out of the channel. In this particular embodiment, different portions of the gel across the width of the channel are not formed simultaneously since the gel first forms at the interface of the precursor fluids, and then the gel can become wider as diffusion of the precursor takes place. The number of inlets and/or outlets of the channel and/or the distribution of solutions at the inlets may be additional factors that influence the final arrangement of gel structures inside the channel. For instance, the distribution of gel precursor solution(s) and spacing solution(s) can be arranged such that first and second gel structures are positioned immediately adjacent to one another or adjacent to one another via a non-gel portion. In one particular embodiment, fluid streams 16 and 18 include gel precursors, and upon crosslinking of the gels, the gels in subchannels 26 and 28 are formed immediately adjacent to one another. Subchannel 30 may include a fluid stream in one embodiment; in another embodiment, fluid stream 20 may be used to form a solid or other non-gel structure. In some cases, fluid stream 20 is replaced with a gas (e.g., air). Microfluidic channels including a first and a second gel may, therefore, be positioned directly adjacent to one another, or may be separated from one another by non-gel component(s). As described in more detail below, a series of gel structures can be formed within a single microfluidic channel by methods described herein.

As illustrated in several embodiments, a gel structure formed in a microfluidic channel may extend the length of the channel. In other embodiments, however, a gel structure may be formed at discrete points along the channel. For example, a channel may include discrete conductive elements (e.g., electrodes, films, meshes, and the like) that can cause local polymerization of a thermocurable gel precursor. The conductive

elements may be positioned at a surface of the channel or may extend into the channel, and may be separated from one another by non-conductive elements. By flowing a gel precursor in a channel and applying heat to discrete portions of the channel, gel precursors only at those portions may be polymerized. Gel structures in a microfluidic channel may be formed having any suitable cross section. Figs. 1E-1H show exemplary cross sections of a microfluidic channel containing a gel structure. As shown in the embodiment illustrated in Fig. IE, gel structure 40 may extend from a top surface 46 to a bottom surface 48 of microfluidic channel 12. Portions 50 and/or 54 of microfluidic channel 12 may include a fluid, a second gel structure, or a solid, in some embodiments. As shown in the embodiment illustrated in Fig. IF, microfluidic channel 12 may include a gel structure 40 that is not in physical contact with top portion 46, bottom portion 48, side portion 60, or side portion 62 of the microfluidic channel at least one point along the length of the channel. Such an embodiment may be formed, for example, by flowing top and bottom streams, as well as side streams, so as to encapsulate gel structure 40. Fig. IG shows another example of a gel structure 40 that occupies a portion, but not all of a microfluidic channel. As illustrated in Fig. IH, gel structure 40 may be formed such that it is in contact with a side portion 62 of a microfluidic channel. A gel structure 40 may be in contact with a single bottom (or top) portion, as shown in Fig. II. It should be understood that Figs. IE- II re exemplary and that other configuration of gels can be formed in microfluidic channels using methods described herein.

In some embodiments, after formation of at least one gel structure in a microfluidic channel, all or a portion of the microfluidic channel can be removed, e.g., leaving behind a gel structure positioned on a surface. The gel structure can then be manipulated as desired. For instance, in one embodiment, a second microfluidic channel is positioned on top of or adjacent to the at least one gel structure. In another embodiment, the gel structure is encapsulated in a second gel.

Using various methods described herein, a gel structure in a microfluidic channel may have any suitable width. The width of the gel structure may be, for example, at least 25 microns, at least 50 microns, at least 100 microns, at least 200 microns, at least 300 microns, at least 500 microns, or at least lmm. In addition, the gel may have any suitable length within a microfluidic channel. In some cases, the gel may be the same

length than that of the microfluidic channel. In other embodiments, the gel may have a different length than that of the microfluidic channel. For example, the gel may have a length of at least 0.25 times, at least 0.50 times, or at least 0.75 times the length of the microfluidic channel. In other embodiments, the width of a microfluidic channel may be chosen, in part, by the number of inlets in fluid communication with the channel. For example, the width of a main channel (W), e.g., where the inlet channels may converge (e.g., microfluidic channel 12 of Fig. IA), may be W = n ^■ w,-, where n is the number of inlets of each design (for example, n = 3, 4, or 5). Figs. 2A-2C show examples of various designs of microchannels where n = 3, 4 and 5, respectively. Fig. 2D shows an example of a cross-sectional view of a microchannel 10.

In one embodiment, the dimensions of the microfluidic channels and/or the operating conditions (e.g., flow rate) are chosen such that the Reynolds number is less than 2000, less than 1000, less than 100, or less than 1. Laminar flow may be defined when the Reynolds numbers is less than about 2000. In fluid mechanics, the Reynolds number is the ratio of internal forces to viscous forces and consequently it quantifies the relative importance of these two types of forces for given flow conditions. In some cases, the Reynolds numbers for an aqueous solution flowing at 15 μL/min in these microchannels may be less than 1 and the resulting flows may be laminar. Matching the viscosity of the precursor gel and the spacing solutions may minimize the dependence of the width of the streams flowing in microchannels upon the differences in viscosity between the fluids forming each stream, as discussed herein.

As shown in the embodiment illustrated in Fig. 1 , one or more fluids may be introduced and/or flowed into a channel by applying a reduced pressure (e.g., vacuum) at one or more outlets of the device. In some cases, the inlet channels may comprise one or more wells, which can act as reservoirs for one or more fluids. Fluid(s), such as a gel precursor or a spacer solution, may be introduced into each well and upon application of a reduced pressure at the outlet, the fluids can flow into the channel. The reduced pressure may be applied using a syringe pump, a syringe, a microfluidic pump, or any other suitable device. In other instances, one or more fluids may be introduced and/or flowed in a channel by applying positive pressure at one or more inlets. The positive pressure may be applied using a syringe pump, a syringe, or a microfluidic pump, for example. In some cases, the fluids may continue to flow while the gel is being cured.

For instance, the gel forming step may be initiated and/or completed while at least one of the fluids (e.g., the gel precursor or a non-gel precursor) is flowing. In other cases, the gel forming step is initiated and/or completed while at least one of the fluids is not flowing. Advantageously, the width and/or configuration of the gel structure(s) formed in a microfluidic channel may be varied by a variety of methods including viscosity and flow rate. In one embodiment, the width of a gel structure is varied by applying different flow rates to fluid streams 16, 18, and 20 of Fig. IA. For example, by applying equal flow rates to fluid streams 16, 18, and 20, the fluid streams may have substantially the same width across microfluidic channel 12. If a high flow rate is applied to fluid stream 18 compared to fluid streams 16 and 20, fluid stream 18 may have a larger width than fluid streams 16 and 20. As such, if fluid stream 18 includes a gel precursor, a gel structure may be formed in subchannel 28 so as to have a larger width than subchannels 26 and 30. Likewise, if a lower flow rate is applied to fluid stream 18 than fluid streams 16 and 20, a narrower gel structure in the form of subchannel 28 may be formed compared to subchannels 26 and 30. Each of fluid streams 16, 18, and 20 may also be flowed at different flow rates to form gel structures having various dimensions. Thus, by choosing appropriate dimensions of microfluidic channel 12 and flow rates of fluid streams, a gel structure having any suitable width (and/or height) may be formed in a microfluidic channel.

The width of a microchannel may be chosen, at least in part, based on the differences in viscosity between the fluids forming each stream. In certain embodiments, where the viscosity of the gel precursor is greater than the viscosity of the spacer solution, the width of the gel precursor subchannels may increase with increasing flow rate. In certain embodiments where the viscosity of the precursor gel is less than the viscosity of the spacer solution, the width of the gel precursor subchannels may decrease with increasing flow rate. As a specific example, as shown in the illustrative embodiment of Fig. 3, a four inlet system was designed wherein two inlets were filled with spacer solutions (251 and 253), and the other two inlets were filled with spacer solutions plus dye (250 and 252). The viscosities of the streams were substantially the same, and therefore, the widths of the streams may be approximately substantially the same. Another example is shown in Fig. 4. Fig. 4A shows a three inlet system wherein

the center inlet 302 includes a more viscous material than the spacer solutions 301 and 303. Particularly in this case, the viscosity of the center fluid is approximately three times that of the spacer fluid. The flow rate Fl of the fluid in Fig. 4A is greater than the flow rate F2 of the fluid in Fig. 4B, which is greater than the flow rate F3 of the fluid in Fig. 4C.

Fig. 5 shows various examples of gel structures formed in microfluidic channels. Schematic diagrams of the distribution of flowing solutions (e.g., precursor gel and spacing solution) to form one (Fig. 5A) or two (Fig. 5B) gel structures in microchannels are also shown. In the exemplary embodiment shown in Fig. 5A, spacing solutions 65 and 67 may be flowed on either side of gel precursor 66. The example shown in Fig. 5B shows spacing solutions 68, 70 and 72 being flowed in conjunction with precursor gel solutions 69 and 71. Fig. 5E and Fig. 5F show the portions of gel 66, 69, and 71 dividing microchannel 12 after curing the gel portions into subchannels that were accessible through the corresponding inlets. The gels may be cured after or during flow of the gel precursor solutions . In one particular example, a suspension of fluorescent 50-nm Nile Red polystyrene beads 73 and 50-nm Yellow Green polystyrene beads 74 were added to the spacing solutions. As shown in Figs. 5C and 5D, the beads remained in their respective subchannel compartments. In some cases, the beads can remain in their respective subchannel compartments for various amounts of time (e.g., more than 30 minutes, 1 hour, 1 day, 1 week, etc.) as the suspensions are flowing or after stopping the flow of the fluids.

As described herein, the present invention provides methods for i) culturing cells either within the gel structures or on one or more surfaces of the gel structures, ii) patterning different types of cells in adjacent gel structures, and iii) applying gradients of soluble factors across the gel structures containing cells. It should be understood, however, that while the discussion herein primarily focuses on cells associated with gel structures, other components such as nanoparticles, beads, soluble components, or other entities may be used in place of or in conjunction with cells in certain embodiments. Much of cell biology has developed based on studies of cells grown in glass or polystyrene Petri dishes. The limitations of these systems, and their differences from 3D, in-vivo biology, are well accepted but the need to produce useful systems that provide a

practical, and more biologically-relevant environment for cell and tissue culture is still required.

There are three key issues to growing cells in 3D culture: i) Cells cannot be more than approximate 150-300 microns from the source of nutrients or they may suffer from hypoxia, glucose deprivation, and other forms of starvation. Cells in the center of microtumors (diameter < 1 -2 mm) develop survival mechanisms (arguably due to starvation) that seem to cause those cells to be resistant to chemo- and radiotherapy, ii) Cells require exposure to gradients of signaling molecules to survive, proliferate, migrate, or differentiate normally, iii) The mechanical compliance of the medium must be appropriate for the cells.

In-vivo cellular behavior is complex: cells integrate and respond to numerous signals that originate both from their local microenvironment, and from distant sources. These signals come from direct contact with other local cells (such as gap junctions in myocytes), from soluble factors secreted by neighboring or distant cells (such as growth factors and hormones), and from the surrounding extracellular matrix (ECM), which contributes both chemical and mechanical signals. Intercellular communication is necessary for coordinating collective responses among multiple cells. One example is the type of inflammatory response in the immune system in which macrophages, recruited to a site of injury, secrete TNF (tumor necrosis factor) to activate leukocytes. A second example occurs during wound healing, where fibroblasts and epidermal cells secrete IGF (insulin-like growth factor) to induce the formation of granulation tissue and the reepithelialization of the wound.

Several experimental methods are useful in studying cellular communication within three-dimensional (3-D) matrices. In transmigration assays, cells cultured in an upper chamber coated with gel respond to a gradient of cytokine that diffuses from a lower chamber through pores in the base of the upper chamber. The bases of the two chambers are typically several millimeters apart. Cells suspended in uncured gel can also be plated on Petri dishes, and — after gelation — exposed to a solution of cytokine that covers the layer of gel. Suspending cells in liquid gel followed by curing makes it possible to co-culture different types of cells.

The effectiveness of these methods to deconstruct the dependence of cellular behavior on the concentration of specific soluble biomolecules may be limited, because

they may not be able to i) control the position of and distance between cells on a length scale similar to that observed in tissue (e.g., a few to hundreds of microns), or ii) expose the cells in culture to steady gradients of soluble factors that are not disturbed by convective flow. The gel structures described herein and methods associated therewith can overcome the above-mentioned challenges and may allow some or all of the following features: i) the cultivation of mammalian cells in 3D environments; ii) exposing these cells to chemical signals, especially gradients; iii) observing them (using optical microscopy) in real time; iv) separating them from other cells; v) the use of a matrix that is relevant to in-vivo cell biology.

As shown in the embodiment illustrated in Figs. 6A and 6B, in some cases a microfluidic channel including a gel disposed therein includes at least one cell. The cell may be disposed in or on a gel portion of the channel, or in or on a non-gel portion of the channel. As illustrated in Fig. 6A, microfluidic channel 100 includes portions, or subchannels 110, 112, and 114. Subchannel 112 may include a gel 113 which may optionally include one or more cells disposed therein. Subchannel 114 may include, for example, a fluid, optionally having one or more cells disposed therein. In some cases, the cells in subchannel 114 are attached to at least one surface of microfluidic channel 100. The surface portion of the channel having cell(s) attached thereto, in one embodiment, is not in physical contact with gel 113. As shown in Figs. 6A and 6B, the cells attached to a surface of the channel, such as those shown in Fig. 6A, may have a different morphology than the cells that are disposed in the gel, as shown in Fig. 6B. Using such structures, interactions between cells that have different morphologies can be determined. In one specific embodiment, NIH/3T3 fibroblasts were cultured on the surface of a subchannel 114 adjacent to gel 113. Fig. 6B shows a phase-contrast image of NIH/3T3 fibroblasts after two days in culture within gel in a microchannel.

In some cases, cells can be allowed to interact with one another, with a chemical substance, and/or with a physical force, e.g., under flowing conditions or non-flowing conditions. A change in characteristic of at least one cell as a result of the interaction(s) may be determined. For instance, gel 113 including at least one cell disposed therein may be adjacent a fluid flowing in subchannel 110. The flowing stream of fluid may have a suitable flow rate so as to apply a shearing stress to the at least one cell disposed

in gel 112. In some cases, a characteristic of a cell as a result of the applied shearing stress may be determined. For example, a change in morphology of a cell may be determined using articles and methods described herein. In other embodiments, the effect of exposing a cell to a chemical substance (e.g., a toxin) can be determined. Advantageously, such effects can be determined controllably while the cells are positioned within biologically-derived, 3-D matrices of microscopic size that can mimic in vivo systems.

In another embodiment of the invention, an article comprises a microfluidic channel including at least one first cell and at least one second cell, the cells associated with a surface of the channel and/or a gel structure within the channel. For example, the microfluidic channel may comprise a first gel including at least a first cell disposed therein and a second gel including at least a second cell disposed therein. The first and second cells may be the same cells in some embodiments, or may be different cells in other embodiments. In some embodiments, the first and second cells have at least one different characteristic (e.g., different cell surface receptors, different exposure to a substance, etc.).

In embodiments in which a microfluidic channel includes at least two gel structures, e.g., a first and a second gel structure, the first and second gel structures may be positioned directly adjacent to each other. In other cases, the first and second gel structures are not positioned directly adjacent to each other; e.g., at least one liquid and/or an intervening gel structure may be positioned between the first and second gel structures. Additional channel portions or subchannels within the microchannel may be or comprise a liquid (e.g., in the form of a fluid stream) or a gel, which may or may not comprise cells. The additional subchannels with the microchannel may be formed of the same substance as the first and second gel structures, or may be formed of different substances.

In instances where three subchannels are formed within a microchannel, non- limiting examples of arrangement of the substances within the subchannels are shown in Fig. 7A. Similarly, when four or five subchannels are formed within a microchannel, non-limiting examples of arrangement of the substances within the subchannels are given in Fig. 7B or 7C, respectively. Fig. 7D depicts non-limiting arrangements for five subchannels formed within a microchannel and when three cell types are present. A

method involving such and other structures may include allowing interaction between the first and second cells, and determining a change in characteristic of at least one cell as a result of the interaction.

As a specific example, as shown in the embodiments illustrated in Figs. 8A-8C, microfluidic channel 160 may include subchannels 162, 164, 166, 168, and 170, which may be formed by the laminar flow of fluids in each of the channels. In one particular embodiment, subchannel 162 includes a fluid and subchannel 164 includes a first gel (or first gel precursor) including a first cell or set of first cells 163 disposed therein. Subchannel 166 includes a second gel (or second gel precursor) that does not include a cell disposed therein. Subchannel 168 includes a third gel (or third gel precursor) including a second cell or set of second cells 165 disposed therein, and subchannel 170 includes a fourth gel (or fourth gel precursor) that does not comprise a cell disposed therein. Upon flowing of the fluid streams in the subchannels, the gel precursors can be crosslinked to form gels in the respective subchannels (Fig. 8B). As shown in the embodiment illustrated in Fig. 8C, the first and second cells can be allowed to interact with one another by allowing the cells to migrate from one subchannel to another. The cells may migrate between different gel portions, from gel portions to non-gel portions, or from non-gel portions to gel portions. Such experiments can be done under different conditions, such as under flowing conditions, non-flowing conditions, without nutrients (e.g., conditions that starve the cells), or with various buffers, solvents, or other components (e.g., drugs or toxins).

In one particular example, two murine cell lines were studied, in particular, adherent, macrophage-like BAC1.2F5 cells (BAC cells) and weakly-adherent, monocyte- derived LADMAC cells. BAC cells require the presence of CSF-I, a growth factor secreted by LADMAC cells that stimulates the survival and proliferation of macrophages. A five-inlet microchannel was fabricated, such as the one shown in Fig. 8. In this example, subchannels 162, 164, 166, 168 and 170 were filled with five differing solutions, for example, a spacer fluid in subchannel 162 and gel-precursor fluids in the four adjacent subchannels. The gel portions contained: i) BAC cells embedded in a gel within subchannel 164, ii) a gel portion without cells in subchannel 166 iii) a gel portion containing LADMAC cells in subchannel 168, and another gel portion without cells in subchannel 170. The spacing solution in the subchannel may be replaced with culturing

medium (for example, alpha MEM supplemented with 10% newborn calf serum and 36 ng/ml of recombinant human CSF-I). The microchannel may then be incubated. In this example, after 12 hours of incubation, the cultering medium lacking recombinant CSF-I (starving medium) may be injected into subchannel 162. Therefore, under these conditions, the only source of CSF-I available to the BAC cells is the nearby LADMAC cells. The starving medium in the channels may be renewed (for example, daily), and in the presence of LADMAC cells, BAC cells were viable for a week in the channel.

In a control example, a similar microchannel was prepared as mentioned above: i) BAC cells embedded in subchannel 164 ii) a gel portion without cells in subchannel 166 iii) a gel portion without cells in subchannel 168 and iv) another gel portion without cells in subchannel 170. In this example, the BAC cells died within two days after the addition of starving media. These example may confirm that the presence of LADMAC cells was necessary for the proliferation of BAC cells in starving medium.

In yet another embodiment of the invention, a gradient of components can be formed in at least one gel structure positioned in a microfluidic channel. For example, as shown in the embodiment illustrated in Figs. 9A-9E, microfluidic channel 180 may include subchannels 182, 184, and 186 that may be formed, for example, by laminar flow. In one embodiment, subchannel 184 includes a gel structure and subchannels 182 and 186 includes one or more fluids. The fluid in subchannel 182 may include a first component and the fluid in subchannel 186 may include a second component different from the first component. As the fluids are allowed to diffuse, a gradient may be formed across the width of the gel structure. For instance, subchannel 182 including the first component may have a higher concentration of the first component near portion 190 of the gel structure compared to portion 192 of the gel structure. Similarly, subchannel 186 including the second component may include a higher amount of the second component at portion 192 of the gel structure compared to portion 190 of the gel structure. The gel structure can, therefore, comprise two different concentration gradients at different portions of the structure. The gradient formed in the gel structure may depend on factors such as, for example, the cross linking density of the gel, and the physical characteristics of the first and second components such as the size and molecular weight of the first and second components. For instance, as shown in Fig. 9B, a high molecular weight

component in subchannel 182 may diffuse slower through the gel structure compared to a low molecular weight component, as shown in Fig. 9D, all other factors being equal. In one particular example, a solution of TRITC-labeled dextran (Fig. 9B = 40 kDa, Fig. 9C = 70 kDa, or Fig. 9D = 155 kDa) at 0.5 mg/ml in PBS was injected into subchannel 182, and PBS was injected into subchannel 186. In certain cases, the flow rates of the fluids may be the same, for example, 5 μL/min. In other cases, the flow rate of the fluids in adjacent channels may be different. Figs. 9B, 9C, and 9D show fluorescent images of the microchannel after injecting the solutions into the subchannels for 300 minutes. Fig. 9E shows the fluorescence intensity profiles reached by diffusion of TRITC-dextran with different molecular weights (after 300 minutes) plotted against the width of the channel. The calculated values of the diffusion constant for 40-kDa, 70- kDa, and 155-kDa TRITC-dextran through the gel structures (Matrigel) were determined to be 0.45, 0.34, and 0.18 μm ² /s, respectively. Equations for calculating the diffusion constants are given below. These data suggest that it may be possible to use the gel structures as semi-permeable barriers to exclude embedded cells from biomolecules of particular sizes (e.g., 155-kD antibodies), while permitting their exposure to other small cytokines (e.g., insulin, TGF-β). Accordingly, in some embodiments, gel structures can be act as barriers to molecules having a size of greater than or equal to 30 kD, 50 kD, 75 kD, 100 kD, 150 kD, 200 kD, or 300 kD, which sizes can be modified by the particular type of gel, the crosslinking density, etc.

As mentioned above, other components such as nanoparticles, beads, soluble components, or other entities may be used in place of or in conjunction with cells in certain embodiments. The gel structures, surfaces of channels, and/or fluid streams employed in embodiments described herein may also involve the use of such entities. As described herein, gel structures may include polymer gels which are typically characterized by long chain polymer molecules that are crosslinked to form a network. This network can trap and hold fluid, which can give gels properties somewhere between those of solids and liquids. Depending on the level of crosslinking, various properties of a particular gel can be tailored. For example, a highly crosslinked gel generally is structurally strong and a lightly crosslinked gel may be weaker structurally. In the design of gels for a particular application, the degree of crosslinking may be adjusted to achieve the desired compromise between, for example, speed of curing and level of

structural integrity. In some cases, crosslinking may be adjusted to allow for a certain controlled rate of release of components (e.g., drugs, cells, etc.) from the gel. Those of ordinary skill in the art would be able to identify methods for modulating the degree of crosslinking in such gels. In some embodiments, a gel described herein may be a gel, including a crosslinkable gel. The term "gel" refers to water-soluble polymer chains that are crossl inked in the presence of water to form a network. A short description of the properties and behavior of certain hydrogels is provided below, which hydrogels may be suitable for use in certain embodiments of the invention. It should be noted that the list is not exhaustive, and those of ordinary skill in the art may readily select or form other suitable gel materials using available information regarding, for example, curing temperature of various materials, and/or using routine experimentation and simple screening tests.

Examples of polymers capable of forming hydrogels include, but are not limited to, silicon-containing polymers, polyacrylamides, crosslinked polymers (e.g., polyethylene oxide, poly AMPS and polyvinylpyrrolidone), polyvinyl alcohol, acrylate polymers (e.g., sodium polyacrylate), and copolymers with an abundance of hydrophilic groups. In some embodiments, the gel may be a sol-gel. A "sol-gel" refers to a colloidal suspension capable of being gelled to form a solid. In some cases, the sol-gel may be formed from a mixture of solid particles (e.g., inorganic salts) suspended in a liquid, wherein a series of reactions including hydrolysis and polymerization reactions may be performed to form a colloidal suspension.

In some cases, the gel may be an organogel, wherein the polymer may be swollen by addition of an organic solvent. In certain embodiments, the gel is a thermocurable gel (i.e., a gel that can be cured by application of heat). In some cases, the gel is cured by raising the temperature from a lower temperature to a higher temperature, for example, 4 ⁰ C to 37 ⁰ C. In other instances, the temperature may be raised from a lower temperature to at least 15 ⁰ C, 20 ⁰ C, 22 ⁰ C, 25 ⁰ C, 30 ⁰ C, 35 ⁰ C, 40 ⁰ C, or to any other suitable temperature that may cause the gel to cure partially or completely. In some cases, the temperature is chosen in part such that the cells contained within the gel remain viable. When the gel used is a thermocurable gel, in some embodiments, the gel may be returned to a gel precursor by

altering the temperature. For instance, in some cases the temperature may be decreased, e.g., to cause at least a portion of the gel to return to a more fluid state. In one particular example, the temperature may be decreased from 37 ⁰ C to 4 ⁰ C. By redissolving the gel, the cells or other entities that are in the gel may be recovered. For example, one or more cells have been encapsulated in a gel (and, optionally, have been allowed to interact with each other or subject to a physical and/or chemical stimuli), the gel may be returned to a gel precursor liquid and the cell(s) may be collected and/or analyzed (e.g., counted, subjected to certain techniques such as Western Blotting, further expanded in cell culture). In another embodiment, the gel is a natural gel; that is, a biologically-derived gel.

A natural gel may include, for example, collagen I, collagen IV, fibrin, laminin, and combinations thereof. In one particular embodiment, Matrigel (MG), a combination of collagen IV and laminin, is used. Benefits that may be attributed to MG and other gels useful in embodiments described herein include, but are not limited to, i) it is biologically-derived as it is a soluble extract of the basement membrane of murine tumoral epithelia, mainly composed of laminin and collagen IV; ii) it is commercially available, and has been used extensively both in vivo and in vitro in the culture of many types of cells; iii) it is liquid at 4 ⁰ C, but gels within 15 minutes at 22 ⁰ C - 37 ⁰ C, and iv) it is sufficiently stiff after gelling to form self-standing structures of gel. In some embodiments, Matrigel may be employed in its purified form, i.e. Growth Factor Reduced Matrigel.

Certain types of polymers are known to form crosslinking bonds under appropriate conditions. Non-limiting examples of crosslinkable polymers include: polyvinyl alcohol, polyvinylbutryl, polyvinylpyridyl, polyvinyl pyrrolidone, polyvinyl acetate, acrylonitrile butadiene styrene (ABS), ethylene-propylene rubbers (EPDM), EPR, chlorinated polyethylene (CPE), ethelynebisacrylamide (EBA), acrylates (e.g., alkyl acrylates, glycol acrylates, polyglycol acrylates, ethylene ethyl acrylate (EEA)), hydrogenated nitrile butadiene rubber (HNBR), natural rubber, nitrile butadiene rubber (NBR), certain fluoropolymers, silicone rubber, polyisoprene, ethylene vinyl acetate (EVA), chlorosulfonyl rubber, flourinated poly(arylene ether) (FPAE), polyether ketones, polysulfones, polyether imides, diepoxides, diisocyanates, diisothiocyanates, formaldehyde resins, amino resins, polyurethanes, unsaturated polyethers, polyglycol

vinyl ethers, polyglycol divinyl ethers, polysaccharides, copolymers thereof, and those described in U.S. Patent No. 6,183,901. In one set of embodiments, at least a portion of the polymeric structure may comprise polyvinyl pyridine or quaternized polyvinyl N- methyl-pyridine. Those of ordinary skill in the art can choose appropriate polymers that can be crosslinked, as well as suitable methods of crosslinking, based upon general knowledge of the art in combination with the description herein.

As mentioned above, in some cases, gels described herein include one or more types of particles such as cells (e.g., mammalian or bacterial), beads, nanoparticles, nanotubes, colloids, etc. The particles may have any suitable particle size. As used herein, "particle size" refers to the largest characteristic dimension (i.e. of a line passing through the geometric center of the particle e.g., diameter) that can be measured along any orientation of a particle (e.g., a polymer particle). Particle size as used herein may be measured or estimated, for example, using a sieve analysis, wherein particles are passed through openings of a standard size in a screen. The particle-size distribution may be reported as the weight percentage of particles retained on each of a series of standard sieves of decreasing size, and the percentage of particles passed of the finest size. That is, the average particle size may correspond to the 50% point in the weight distribution of particles.

In some embodiments, methods described herein involve binding between at least two entities. The term "binding" refers to the interaction between a corresponding pair of components that exhibit mutual affinity or binding capacity, typically specific or non-specific binding or interaction, including biochemical, physiological, and/or pharmaceutical interactions. Biological binding defines a type of interaction that occurs between pairs of components including proteins, (e.g., adhesion proteins), nucleic acids, glycoproteins, carbohydrates, hormones and the like. Specific examples include cell (e.g., cell surface receptor)/protein, adhesion protein/integrin, antibody/antigen, antibody/hapten, enzyme/substrate, enzyme/inhibitor, enzyme/cofactor, binding protein/substrate, carrier protein/substrate, lectin/carbohydrate, receptor/hormone, receptor/effector, complementary strands of nucleic acid, protein/nucleic acid repressor/inducer, ligand/cell surface receptor, virus/ligand, etc.

In certain embodiments, binding of a sample component includes interaction between two or more different corresponding pairs of components that exhibit mutual

affinity or binding capacity, such as two or more of the corresponding pairs listed above. As discussed above, the interaction of at least two corresponding pairs of binding partners, e.g., binding of both an adhesion molecule and an antibody with the sample component, can cause effective recognition and capture of the sample component from a solution, resulting in immobilization of the component at a surface. For example, in one particular embodiment, binding of a sample component (e.g., a cell) may include interaction between an integrin (a receptor in the plasma membrane of the cell) and an adhesion molecule (e.g., ICAM-I and/or LF A-3), as well as interaction between an antibody of the cell (e.g., anti-CD3, anti-CD4, anti-CD8, and/or anti-CD5) and its corresponding antigenic binding partner (e.g., CD3, CD4, CD8, and/or CD5, respectively).

"Signaling entity" means an entity that is capable of indicating its existence in a particular sample or at a particular location. Signaling entities of the invention can be those that are identifiable by the unaided human eye, those that may be invisible in isolation but may be detectable by the unaided human eye if in sufficient quantity (e.g., colloid particles), entities that absorb or emit electromagnetic radiation at a level or within a wavelength range such that they can be readily detected visibly (unaided or with a microscope including an electron microscope or the like), optically, or spectroscopically, entities that can be detected electronically or electrochemically, such as redox-active molecules exhibiting a characteristic oxidation/reduction pattern upon exposure to appropriate activation energy ("electronic signaling entities"), or the like. Examples include dyes, pigments, electroactive molecules such as redox-active molecules, fluorescent moieties (including, by definition, phosphorescent moieties), up- regulating phosphors, chemiluminescent entities, electrochemiluminescent entities, or enzyme-linked signaling moieties including horseradish peroxidase and alkaline phosphatase. "Precursors of signaling entities" are entities that, by themselves, may not have signaling capability but, upon chemical, electrochemical, electrical, magnetic, or physical interaction with another species, become signaling entities. An example includes a chromophore having the ability to emit radiation within a particular, detectable wavelength only upon chemical interaction with another molecule. Precursors of signaling entities are distinguishable from, but are included within the definition of, "signaling entities" as used herein. In some embodiments, such as the one depicted in

Fig. IA, a material 37 (for example, tissue paper) may be positioned between conductive disc 32 and surface 36 to aid in the visualization and/or identification of the signaling entities.

In some cases, the spacing solution in these experiments employed may be a solution of PEG. Benefits that may be observed when using PEG include, PEG is nontoxic and biocompatible and for certain examples, the viability of certain cells cultured in the media may not be affected.

In some, but not all embodiments, all components of the systems and methods described herein are microfluidic. "Microfluidic," as used herein, refers to a device, apparatus or system including at least one fluid channel having a cross-sectional dimension of less than 1 mm, and a ratio of length to largest cross-sectional dimension of at least 3:1. A "microfluidic channel," as used herein, is a channel meeting these criteria.

The "cross-sectional dimension" of the channel is measured perpendicular to the direction of fluid flow. Most fluid channels in components of the invention have maximum cross-sectional dimensions less than 2 mm, and in some cases, less than 1 mm. In one set of embodiments, all fluid channels containing embodiments of the invention are microfluidic or have a largest cross sectional dimension of no more than 2 mm or 1 mm. In another embodiment, the fluid channels may be formed in part by a single component (e.g., an etched substrate or molded unit). Of course, larger channels, tubes, chambers, reservoirs, etc. can be used to store fluids in bulk and to deliver fluids to components of the invention. In one set of embodiments, the maximum cross-sectional dimension of the channel(s) containing embodiments of the invention are less than 500 microns, less than 200 microns, less than 100 microns, less than 50 microns, or less than 25 microns. A "channel," as used herein, means a feature on or in an article (substrate) that at least partially directs the flow of a fluid. The channel can have any cross-sectional shape (circular, oval, triangular, irregular, square or rectangular, or the like) and can be covered or uncovered. In embodiments where it is completely covered, at least one portion of the channel can have a cross-section that is completely enclosed, or the entire channel may be completely enclosed along its entire length with the exception of its inlet(s) and outlet(s). A channel may also have an aspect ratio (length to average cross sectional dimension) of at least 2:1, more typically at least 3:1, 5:1, or 10:1 or more. An open

channel generally will include characteristics that facilitate control over fluid transport, e.g., structural characteristics (an elongated indentation) and/or physical or chemical characteristics (hydrophobicity vs. hydrophilicity) or other characteristics that can exert a force (e.g., a containing force) on a fluid. The fluid within the channel may partially or completely fill the channel. In some cases where an open channel is used, the fluid may be held within the channel, for example, using surface tension (i.e., a concave or convex meniscus). A channel may have form any suitable shape and may be, for example, linear, curved, serpentine, or may form a circle, square, or an irregular shape.

The channel may be of any size, for example, having a largest dimension perpendicular to fluid flow of less than about 5 mm or 2 mm, or less than about 1 mm, or less than about 500 microns, less than about 200 microns, less than about 100 microns, less than about 60 microns, less than about 50 microns, less than about 40 microns, less than about 30 microns, less than about 25 microns, less than about 10 microns, less than about 3 microns, less than about 1 micron, less than about 300 nm, less than about 100 nm, less than about 30 nm, or less than about 10 nm. In some cases the dimensions of the channel may be chosen such that fluid is able to freely flow through the article or substrate. The dimensions of the channel may also be chosen, for example, to allow a certain volumetric or linear flowrate of fluid in the channel. Of course, the number of channels and the shape of the channels can be varied by any method known to those of ordinary skill in the art. In some cases, more than one channel or capillary may be used. For example, two or more channels may be used, where they are positioned inside each other, positioned adjacent to each other, positioned to intersect with each other, etc.

The geometry of the microfluidic channel may provide for the laminar flow of fluids through the channel, even at relatively high flow rates. Alternatively, turbulent flow may be employed for example, by using even faster flow rates, wider channels, or devices such as microfluidic mixers. Such mixing may provide for a greater amount of contact between potential binding partners.

A channel may also include a number of different inlets and/or outlets. For example, a channel may have 1, 2, 3, 4, 5, 6, etc. inlets and/or outlets. Microfluidic systems described herein may optionally include one or more platforms for performing chemical reactions, combining and separating fluids, diluting

samples, and generating gradients, such as those described in US Patent No. 6,645,432, hereby incorporated by reference herein.

A microfluidic device described herein can be fabricated of a polymer, for example an elastomeric material such as poly(dimethylsiloxane) (PDMS) using rapid prototyping and soft lithography. For example, a high resolution laser printer may be used to generate a mask from a CAD file that represents the channels that make up the fluidic network. The mask may be a transparency that may be contacted with a photoresist, for example, SU-8 photoresist (MicroChem), to produce a negative master of the photoresist on a silicon wafer. A positive replica of PDMS may be made by molding the PDMS against the master, a technique known to those skilled in the art. To complete the fluidic network, a flat substrate, for example, a glass slide, silicon wafer, or polystyrene surface may be placed against the PDMS surface and may be held in place by van der Waals forces, or may be fixed to the PDMS using an adhesive. To allow for the introduction and receiving of fluids to and from the network, holes (e.g., 1 millimeter in diameter) may be formed in the PDMS by using an appropriately sized needle. To allow the fluidic network to communicate with a fluid source, tubing, for example of polyethylene, may be sealed in communication with the holes to form a fluidic connection. To prevent leakage, the connection may be sealed with a sealant or adhesive such as epoxy glue. Examples of methods of manufacturing a microfluidic device are provided in U.S. Patent No. 6,645,432, incorporated by reference in its entirety herein.

In a specific embodiment, the microchannels may be fabricated by soft lithography using standard protocols to generate replicas of a mold. The mold may be chosen from any common lithography material which includes, but is not limited to, poly(dimethylsiloxane) (PDMS). In some cases, the replicas may be sealed to glass slides irreversibly. The replicas-on-glass microchannels may be sterilized by rinsing with a selected solution which may include, but is not limited to, 70% ethanol and/or sterile phosphate-buffered saline (PBS).

The following references are herein incorporated by reference: U.S. Provisional Patent Application Serial No. 61/001,923, filed November 5, 2007, entitled "Forming and Using Structures in Microfluidic Channels," by Wong, et ah, and U.S. Provisional Patent Application Serial No. 61/008,810, filed December 21, 2007, entitled "Forming and Using Structures in Microfluidic Channels," by Wong, et al.

The following examples are included to demonstrate various embodiments of the invention. Those of ordinary skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Accordingly, the following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention.

EXAMPLES Cell culture. NIH/3T3 fibroblasts were obtained from American Type Culture

Collection (ATCC; CRL- 1658). Upon arrival, the frozen aliquot of cells was immediately thawed in a 37 ⁰ C water bath, transferred to a 25-mm ² tissue-culture flask (Falcon) containing 13 ml of culture medium (Dulbecco's Modified Eagle's Medium (DMEM, Gibco) supplemented with 4 mM L-glutamine, 4.5 g/1 glucose, and 10% fetal bovine serum), and cultured at 10% CO ₂ . When fibroblasts reached about 70% confluency, they were passaged by removing the culture medium and adding about 0.5 ml of 0.05% Trypsin-EDTA (Gibco) for about 3 minutes at 37 ⁰ C. After the cells detached from the surface, we centrifuged the cells (for 5 minutes at 1100 rpm), removed the supernatant, and replated about 30% of the cells in a new 25-mm ² flask with fresh culture medium.

Monocyte-derived LADMAC cells, which constitutive Iy secrete colony stimulating factor- 1 (CSF-I), were purchased from ATCC (CRL-2420). The frozen aliquot was thawed in a 37 ⁰ C water bath, transferred to a 25-mm ² tissue-culture flask containing 13 ml of culture medium ¹⁰ (Eagle's Minimum Essential Medium (MEM) with 1.5 g/1 sodium bicarbonate and 10% fetal bovine serum), and cultured at 5% CO ₂ . These weakly-adherent cells were passaged every 3 days by resuspending about 20% of the volume within the flask in fresh culturing medium in a new 25-mm ² flask.

BAC1.2F5 macrophages (donated by Dr. E. R. Stanley, Albert Einstein College of Medicine, New York) were cultured at 5% CO ₂ in alpha MEM (Eagle's Minimum Essential Medium, Alpha modification; Invitrogen) supplemented with 10% newborn calf serum (Invitrogen) and 36 ng/ml recombinant human CSF-I (also a gift of R. Stanley). The culture medium was replaced every 2 days to renew. At 70% confluence,

BAC cells were gently scraped from the tissue-culture treated flask (Falcon) with a cell scraper (Falcon) and replated at -30% confluence. BAC cells were not cultured for more than 20 passages.

BAC and LADMAC cells were co-cultured in alpha MEM (Eagle's Minimum Essential Medium, Alpha modification; Invitrogen) supplemented with 10% newborn calf serum (Invitrogen) and 36 ng/ml recombinant human CSF-I or alpha MEM (Eagle's Minimum Essential Medium, Alpha modification; Invitrogen) supplemented with 10% newborn calf serum (Invitrogen), a starving medium. As controls, prior to co-culture, BAC cells were grown in L-conditioned medium (alpha MEM with 10% newborn calf serum and 10% medium in which LADMAC cells had been cultured for two days) and LADMAC cells in starving medium for a week. The proliferation and survival of BAC and LADMAC cells in the controls were not affected by the changes of medium.

Fabrication of PDMS-on-glass microchannels. Designs for the channels were generated in a Computer-Aided Design (CAD) program (CIe Win, WieWeb Software). The designs for the microchannels were printed on a 10,000 dpi-resolution transparency by a commercial printer (CAD/Art Services, Inc.) from the CAD files. Negative photoresist (SU-8 50; MicroChem Corp.) was spin-coated (2100 rpm, 30s) on a silicon wafer (3-inch test-grade wafers; Silicon Sense) and baked to drive off solvent (10 min at 65 ⁰ C, and 30 min at 95 ⁰ C). The photolithography was performed on the photoresist with the transparency film as the photomask (30-s exposure time) to generate features about 75 μm high. After the post-exposure baking step (1 min at 65 ⁰ C and 10 min at 95 ⁰ C), the microfeatures were developed on the silicon wafer in propylene glycol methyl ether acetate (PGMEA) to remove the unexposed areas of photoresist. The surface of the SU-8 master was exposed to a vapor of (tridecafluoro-l,2,2-tetrahydrooctyl)-l- trichlorosilane (United Chemical Technologies, www.unitedchem.com) in a vacuum desiccator for about 8 h to prevent the adhesion of the PDMS to the silicon wafer.

The poly(dimethylsiloxane) prepolymer (PDMS; Sylgard 184 from Dow Corning) was poured onto the silanized master, degassed it for about 30 min in a vacuum desiccator, and allowed it to cure overnight in an oven at 60 ⁰ C to minimize the quantity of un-crosslinked oligomers. After peeling the PDMS replicas off the master, holes were punched through the slabs of PDMS for the inlets and outlets of the microchannels.

New glass slides (VWR) were submerged in an aqueous 0.1% (w/v) Triton X-100 solution for 5 min, then rinsed twice with distilled water, rinsed once with 100% ethanol, and dried overnight in an oven at 120 ⁰ C. The surfaces of both a PDMS replica and a clean glass slide were exposed to an oxidizing plasma for 1 min and 5 min, respectively, in an air plasma cleaner (about 1 Torr, 1000 W; Harrick Scientific) and brought into contact. The surfaces adhered to one another irreversibly and generated the system of closed channels that was only accessible through the holes punched previously for the inlets and outlets. For good adhesion, it is important that both surfaces are extremely clean before oxidation. To improve the adhesion of the parts, the channels were left on a hot plate at 120 ⁰ C for about 30 min. At this point, the channels were ready for immediate use.

Formation of portions of gel in microchannels. Structures of gel were formed inside the microchannels by injecting the gel in its liquid phase and gelling it in situ, thermally. Since Matrigel (Growth Factor-Reduced Matrigel (MG); BDBiosciences) is liquid at about 4 ⁰ C and gels at about 25 - 37 ⁰ C, during the formation of the gel portions, all required tools (channels, pipette tips, aluminum tube holders (VWR), etc.) and solutions (Matrigel, spacing solution, and cells in suspension) were stored at about 4 ⁰ C to prevent premature curing. Fig. IB shows an example of an experiment set-up. A flat, aluminum disc 32 placed on top of ice 38 in a thermally-insulated container (VWR) served as the chilled working surface on which the microchannels rested during fabrication; for ease in visualization of the channel, paper tissue 37 can be placed on the aluminum disc 32. All these elements were sterilized (with 70% ethanol) of the experimental setup before bringing them into a sterile, laminar-flow hood; a syringe pump (PHD 2000 from Harvard Apparatus) was the only element of the setup that remained outside the hood.

The solutions of Matrigel (MG) were prepared to form the different structures of gel inside the microchannels. For structures of gel containing cells, we first prepared a concentrated solution of cells in the appropriate, chilled medium. The suspension of cells was mixed with Matrigel at 80:20 in volume (MGxell suspension). The final concentration of cells in Matrigel was ~10 ⁵ cells/ml.

A microchannel — filled with PBS — was placed on the chilled, aluminum disc 32 and rinsed thoroughly with PBS. The outlet 33 of the channel was connected to a gas-

tight syringe (Hamilton) in the syringe pump (Harvard Apparatus) with polyethylene tubing 34 (PE60; Becton Dickinson) that was prefilled with PBS to maintain a uniform pressure drop throughout the tubing. After connecting the tubing, the channel was rinsed once again by depositing droplets of PBS on the inlets and delivering them into the channel by the action of the syringe pump; this additional rinsing step ensured that no bubbles were trapped in the channel. Excess PBS was removed from the inlets and a droplet (6 μl) of liquid Matrigel solution or of spacing solution (220 mg/ml PEG-8000 in PBS; PEG-8000 bought from Sigma Aldrich) was deposited to the inlets 41-45. Immediately after adding the droplets the syringe pump 39 was started in its refill mode at a flow rate of 15 μl/min to drive the droplets into the channel by suction (Fig. 1C). As soon as the channel was filled with Matrigel and PEG solutions, scissors were used to cut the tubing at the outlet — which instantaneously stopped the flow — and partially submerged the channel in a sterile, warm PBS bath 53 (Fig. ID) at 37 ⁰ C; the warm PBS bath consisted of a Petri dish (VWR) filled with enough PBS to cover the side walls of the microchannel after it was placed in the bath. After 10 minutes in the warm bath, the spacing solutions were rinsed out with PBS, and additional drops of PBS were deposited on every inlet and outlet to prevent the hydrogels from drying out, and kept the microchannels in the incubator for further experiments.

Formation of portions of gel containing viable cells. A concentrated suspension of cells in chilled culturing medium were prepared, and mixed with liquid Matrigel at a ratio of 80:20 in volume (MGxell solution). 6-μl droplets of either the suspension of cells in liquid Matrigel, or of the spacing solution were deposited on each inlet, and delivered into the microchannel by applying vacuum at the outlet with a syringe pump. Characterization of the portions of gel. To determine if the portions of gel extended top to bottom of the channel, suspensions of 50-nm Nile Red polystyrene beads (λabs about 485 nm, λ _em s about 525 nm; Polysciences) and 50-nm Yellow Green polystyrene beads (λ _abs about 441 nm, λ _em s about485 nm; Polysciences) were added to the subchannels and the samples were imaged under an epi-fluorescent microscope (Leika DM IRB). The widths of 10 different gel structures formed in microchannels having 300-μm-wide inlet channels were measured and the width of the structures was found to be 315 μm ± 21 μm.

Fluorescent staining of cells. To assess the viability of cells, they were stained with propidium iodide (PI; 3,8-Diamino-5-[3-(diethylmethylammonio)propyl]-6- phenylphenanthridinium diiodide, Sigma). PI increases its fluorescence about 20 fold (λ _a bs ⁼ 493 nm, λ _em s ⁼ 630 nm) when it binds to nucleic acids; this binding only occurs with cells having their cytoplasmic membrane compromised. Immediately before imaging, about 20 μl of PI solution (100 μg/ml in the appropriate culturing medium) were added to the inlets of the microchannels, and PI (0.668 kDa) was allowed to diffuse into the channel and portion of gel for 15 minutes.

CellTracker Green (5-chloromethylfluorescein diacetate; Invitrogen) was used to fluorescently label LADMAC cells. CellTracker Green is a cell-permeable dye that becomes fluorescent (λ _abs about 480 nm, λ _ems about 525 nm) upon cleavage by cytosolic esterases. The stock solution was diluted to 1OmM in dimethylsulfoxide (DMSO), and stored it at -20 ⁰ C. The cells were labeled by suspending them in a solution of CellTracker in culture medium (5 μM) for 15 minutes. The cells were rinsed twice with culture medium and the samples were imaged.

The membrane of the cells were stained with Vybrant DiI (Invitrogen) for imaging them by confocal microscopy (Olympus 1X81 with FluoView 1000 and spectral scanner). Vybrant DiI is a lipophilic dye that becomes highly fluorescent upon incorporation into cellular membranes (λ _abs about 549 nm, λ _ems about 565 nm). The cells were pelleted by centrifugation (5 min at 1100 rpm) and resuspended in 1 ml of culturing medium. To this solution was added 5 μl of the stock solution of Vybrant DiI. After an incubation period of 15 min at 37 ⁰ C, the cells were pelleted again and mixed with liquid Matrigel.

Study of the effect of shear stress on cells. Murine fibroblasts within cultured within microgel structures of MG to study the effect of shear stress on the cells in liquid MG. In pressure-driven flows, velocity may not be constant over the cross section of the channel, resulting in shear stress on the cells as they flow. Shear stress on cells (5-15 dyn/cm ² ; 10 μN = 1 dyn/cm ² ) occurs in vivo, and is necessary for endothelial cells to develop properly. Excessive shear stress, however, can damage the cellular membrane, and compromise the viability of the cells. Shear stress can be decreased by lowering the flow rate. In some cases, however, decreasing the flow rate at which solutions were delivered may result in gel structures that did not extend from top to bottom of the

channel. Therefore, the flow rate may be adjusted to ensure that the cells remain viable as well as the gel structures to extend from top to bottom of the channel. The table in Figure 10 summarizes the results of a particular experiment which is discussed in detail below. Six-μl droplets of either a suspension of cells in liquid MG (20% v/v; ~10 ⁵ cells/ml), or of spacing solution, were deposited individual inlets of a microchannel and the solutions were delivered into the microchannel by applying vacuum to the outlet with a syringe pump at flow rates between 5 and 110 μl/min. To assess the viability of the cells embedded in the gel structures, a solution of propidium iodide (PI) in culturing medium (100 μg/ml) was added to the subchannels and incubated for 30 min. PI can be employed to characterize the cells because it reports the effect of shear stress on the integrity of the plasma membrane of cells. Suspensions of nanometer-sized beads were also injected into the subchannels (as described herein) to determine if the structures of hydrogel extended the entire height of the channel. In these experiments, flow rates between 10 and 20 μl/min yielded hydrogel structures that extended from top and maximized the viability of the embedded cells (> 90%).

Microscopy. The channels were imaged using a charge-coupled device camera (CCD camera) on an inverted, epifluorescence microscope and the images were processed with MetaMorph and Adobe Photoshop. The fluorescence of Figs. 4 and 8 was false-colored that the contrast was increased using Photoshop.

Image Analysis. To obtain the profiles of concentration of TRITC-dextran as it diffused through the portions of gel, images (16-bit TIFF images) of the samples at several timepoints were taken and converted to 8-bit JPEG (511 x 639 pixels) images with Adobe Photoshop. With MATLAB, 10 randomly-distributed, horizontal lines were chosen per JPEG image and the light intensity of each pixel was extracted. The intensity of each pixel was average-summed for the 10 lines so that one intensity profile characterized each image. The background was then subtracted and the intensity profile of each image was normalized by dividing the intensity of each pixel by the maximum intensity of the image. Diffusion constants were obtained from correlating the normalized intensity profiles and equations describing diffusion through gel structures. For the experiments presented in Fig. 9, a continuous flow was kept in the subchannels and the concentration

of TRITC-dextran may be considered constant in the subchannels. The concentration was maximum (cø) in the subchannel delivering the solution of TRITC-dextran (182 in Fig. 9A) and zero in the subchannel delivering buffer (186 in Fig. 9A). Diffusion through a gel structure under the conditions in Fig.9 can then be described using Equations 1-4. c(x = 0,t > O) = C ₀ (Eq. 2) c(x = w,t > O)= O (Eq. 3) c(θ ≤ x ≤ w,t = O) = O (Eq. 4) where D is the diffusion constant, c stands for the concentration of TRITC-dextran in the microchannel, x for the position in the gel structure along the direction perpendicular to the edge of the gel structure (w is the thickness of the gel structure), and t stands for time. Equation 1 is the general equation for diffusive transport and equations 2 - 4 are the boundary conditions for Eq. 1 according to the experiments in Fig. 9. The solution of Eqs. 1 -4 inside of the gel structure is

— c = (Eq. 5) Using MATLAB, equation 5 was calculated for different (hypothetical) values of the diffusion constant. The calculated curves were compared with the experimental data (normalized intensity profiles) and the values of the diffusion coefficient of 40-kDa, 70- kDa, and 155-kDa TRITC-dextran were determined.

While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

What is claimed is: