PRIORITY CLAIM

The present application claims priority to U.S. Provisional Application serial number 62/072094, filed October 29, 2014, which is herein incorporated by reference.

FIELD

[0001] The present disclosure relates to apparatuses, systems and methods for culturing cells.

TECHNICAL BACKGROUND

[0002] Cells cultured in three dimensions, such as spheroids, can exhibit more in-vivo like functionality than their counterparts cultured in two dimensions as monolayers. In two dimensional cell culture systems, cells can attach to a substrate on which they are cultured. However, when cells are grown in three dimensions, such as spheroids, the cells interact with each other rather than attaching to the substrate. Accordingly, cell culture media exchanges for culture systems in which cells are cultured as spheroids that are mobile in suspension can present challenges.

BRIEF SUMMARY

[0003] In accordance with various embodiments of the present disclosure, cell culture inserts configured to allow exchange of culture media in wells containing spheroids without aspirating or damaging the spheroids are described. The cell culture inserts can include a frame having a first open end, a second open end, and at least one support extending between the first open end and the second open end. The inserts also include a fluid permeable mesh coupled to the frame and disposed across the opening of the second end. The mesh defines pores having an average pore size in a range from 10 micrometers to 200micrometers, e.g., 10, 20, 50, 100 or 200 micrometers, including ranges between any of the foregoing values. In embodiments, the pores are sized to allow individual cells to pass across or through the mesh, but to prevent spheroid cell clusters from passing.

[0004] In some embodiments, provided herein are cell culture well inserts comprising: a frame having a first open end, a second end defining an opening, and at least one support extending between the first open end and the second end, wherein the opening of the second end has a diameter between 9 mm and 0.25 mm (e.g., 9 ... 5 ... 3 ... 2 ... 0.75 ... 0.25 mm); and a fluid permeable mesh coupled to the frame and disposed across the opening of the second end, the mesh defining pores having an average pore size in a range from 10 micrometers to 200 micrometers (e.g., 10 ... 35 ... 75 ... 200 micrometers). In some embodiments, the frame is configured to be at least partially inserted into a well of a cell culture article such that: i) the mesh is positioned in the well a distance from a nadir of the well, and ii) such that a spheroid, when present in said well, is trapped in the well.

[0005] In some embodiments, provided herein are devices comprising: a well insert having a first open end, a second end having an opening, wherein said first open end and said second end define a cavity therebetween, wherein the opening of the second end has a diameter between 9 mm and 0.25 mm; and a fluid permeable mesh disposed across the opening of the second end, the mesh defining pores having an average pore size in a range from 10 micrometers to 200 micrometers, wherein said well insert is configured to be at least partially inserted into a well of a cell culture article such that: i) the mesh is positioned in the well a distance from a nadir of the well, and ii) such that a spheroid, when present in said well, is trapped in said well. In certain embodiments, the well insert is an integral molded device.

[0006] In particular embodiments, the frame comprises a plurality of supports extending from the first end to the second end. In other embodiments, the mesh extends from the first open end to the second end of the frame and defines a cavity within the frame. In further embodiments, the at least one support of the frame comprises an enclosing sidewall extending from the first open end to the second end and defining an interior of the frame. In some embodiments, the at least one support of the frame comprises sidewalls extending from the first open end to the second end and defining an interior of the frame. In further embodiments, the frame further comprises one or more flanges radially extending from the first open end. In additional embodiments, the frame is configured to be at least partially inserted into a well of a cell culture article such that the mesh is positioned in the well a distance from a nadir of the well.

[0007] In particular embodiments, provided herein are methods comprising: culturing a cell in a well, the well comprising a cell culture insert as described herein. In particular embodiments, the cell is in a spheroid and/or cells form a spheroid. In further embodiments, there is media above the insert and the methods further comprise removing the media from the well above the insert.

[0008] In certain embodiments, provided herein are cell culture assemblies comprising: a well defining an interior surface, the interior surface defining a nadir; and an insert according as described herein, wherein the insert is configured to be at least partially inserted into the well such that the fluid permeable mesh is positioned in the well a distance from the nadir. In some embodiments, the well is non-adherent to cells. In further embodiments, the well is configured such that cells cultured in the well form a spheroid. In additional embodiments, the pores of the mesh are of a size that allows passage of individual cells through the mesh and prevents passage of a spheroid formed therefrom through the mesh.

[0009] In certain embodiments, provided herein are cell culture assemblies comprising: a) a well defining an interior surface and configured such that cells cultured therein form a spheroid, wherein the interior surface defines a nadir; and b) an insert comprising: i) a fluid permeable mesh defining pores, wherein the pores define an average pore size in a range from 10 micrometers to 200 micrometers, and wherein the mesh is configured to be positioned within at least a portion of the well and a distance from the nadir, and ii) a frame coupled to the mesh and configured to extend away from the nadir.

[0010] In certain embodiments, the pores of the mesh are configured to prevent the spheroid from passing through the mesh. In other embodiments, the frame is configured to position the mesh a distance from the nadir. In additional embodiments, the well further defines an upper edge, wherein the frame contacts the upper edge to position the mesh a distance from the nadir. In other embodiments, assembly further comprises a support, wherein the frame contacts the support to position the mesh a distance from the nadir. In additional embodiments, the mesh defines pores, wherein the pores define an average pore size of less than or equal to 40 micrometers (e.g., 40 ... 35 ... 25 ... 15 ... or 10 micrometers). In certain embodiments, the well further defines an aperture opposite the nadir, wherein the interior surface of the well defines a conical shape from the aperture to the nadir. In some embodiments, the mesh defines a circular shape or square shape.

[0011] In further embodiments, the assembly further comprises a plurality of wells and wherein the insert further comprises a plurality of fluid permeable meshes, wherein the fluid permeable mesh and each of the plurality of fluid permeable meshes are configured to be positioned in at least a portion of a different one of the wells and the plurality of wells. In other embodiments, the interior surface of the well is coated with an ultra-low-adhesion material. In further embodiments, the mesh defines a mesh edge and the frame surrounds the mesh along the mesh edge and extends away from the nadir. In other embodiments, the frame comprises at least one support wire coupled to the fluid permeable mesh. In some embodiments, the well comprises at least one sub-well along the interior surface.

[0012] In particular embodiments, provided herein are methods for removing culture media from a well of a cell culture apparatus, the well defining an interior surface having a nadir, the method comprising: a) disposing an end of an insert into the well, the end of the insert defining an opening in fluid communication with a cavity of the insert, the insert having a fluid permeable mesh disposed across the opening, wherein the end of the insert is disposed within the well such that the mesh is positioned a distance from the nadir; b) inserting a tip of a fluid removal device into the cavity of the insert such that the tip is a distance from the nadir at least as far as the distance from the mesh to the nadir; and c) removing fluid from the well by withdrawing fluid via the fluid removal device.

[0013] In certain embodiments, provided herein are cell culture assemblies comprising: a first well defining an interior surface, wherein the interior surface defines a nadir; a fluid permeable mesh defining pores, wherein the pores define an average pore size in a range from 10 micrometers to 200 micrometers; and a frame coupled to the mesh and configured to maintain the mesh in a position over at least a portion of the first well.

[0014] In some embodiments, the mesh defines pores and wherein the pores define an average pore size in a range from 20 micrometers to 200 micrometers. In further embodiments, the fluid permeable mesh has a diameter between 9 mm and 0.25 mm (e.g., 9 ... 7 ... 4 ... 2 ... 1 ... 0.25 mm). In other embodiments, the mesh defines pores, wherein the pores define an average pore size less than or equal to 40 micrometers. In additional embodiments, the mesh is configured to be positioned a distance from the nadir within the at least a portion of the well. In other embodiments, the frame is configured to extend away from the nadir. In additional embodiments, the first well defines an upper edge, and wherein the mesh is configured to be disposed over the upper edge of the well. In further embodiments, the assembly comprises a second well, wherein the mesh is configured to be positioned over the second well. In particular embodiments, the mesh defines a mesh perimeter and the frame surrounds the mesh along the mesh perimeter.

[0015] In some embodiments, wells (e.g., microwells) have a cross-sectional shape approximating a sine wave. In such embodiments, the bottom of the well is rounded (e.g., hemispherically round), the side walls increase in diameter from the bottom of the well to the top and the boundary between wells is rounded. As such the top of the wells does not terminate at a right angle. In some embodiments, a well has a diameter D at the half-way point (also termed D _ha i _{f way} ) between the bottom and top, a diameter D _top at the top of the well and a height H from bottom to top of the well. In these embodiments, D _top is greater than D. An additional embodiment is shown in Fig. 7, where the width of the well is greater than the width of the barrier between contiguous wells. Such an embodiment permits a greater number of wells within a given area of a culture surface.

[0016] Additional features and advantages of the subject matter of the present disclosure will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the subject matter of the present disclosure as described herein, including the detailed description which follows, the claims, as well as the appended drawings.

[0017] It is to be understood that both the foregoing general description and the following detailed description present embodiments of the subject matter of the present disclosure, and are intended to provide an overview or framework for understanding the nature and character of the subject matter of the present disclosure as it is claimed. The accompanying drawings are included to provide a further understanding of the subject matter of the present disclosure, and are incorporated into and constitute a part of this specification. The drawings illustrate various embodiments of the subject matter of the present disclosure and together with the description serve to explain the principles and operations of the subject matter of the present disclosure. Additionally, the drawings and descriptions are meant to be merely illustrative, and are not intended to limit the scope of the claims in any manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The following detailed description of specific embodiments of the present disclosure can be best understood when read in conjunction with the following drawings, where like structure is indicated with like reference numerals and in which:

[0019] FIG. 1A is a perspective view of an embodiment of a cell culture apparatus having wells.

[0020] FIG. IB is a cross-sectional view of an embodiment of the apparatus depicted in FIG.

1A.

[0021] FIG. 1C is a schematic top view of cells grown as spheroids in wells of an embodiment of a structured surface;

[0022] FIG. 2A is a perspective view of an embodiment of a sphere trap or well insert;

[0023] FIG. 2B and 2C are top views of embodiments of the well insert of FIG. 2A depicting two different shaped meshes;

[0024] FIG. 3 is a schematic cross-sectional view of an embodiment of a plurality of wells and a plurality of well inserts;

[0025] FIG. 4 is a schematic cross-sectional view of an embodiment of a well insert and a well including a plurality of sub-wells;

[0026] FIG. 5 is perspective view of an embodiment of a cell culture apparatus having a plurality of wells and a well insert; and

[0027] FIG. 6 is a drawing of an array of wells having a sinusoidal or parabolic shape.

[0028] FIG. 7 is a side view of an array of wells containing spheroids, in an embodiment.

[0029] FIG. 8 is a schematic drawing illustrating an exemplary method for using an embodiment of an apparatus as described herein. DETAILED DESCRIPTION

[0030] Reference will now be made in greater detail to various embodiments of the subject matter of the present disclosure, some embodiments of which are illustrated in the accompanying drawings. Like numbers used in the figures refer to like components, steps and the like. However, it will be understood that the use of a number to refer to a component in a given figure is not intended to limit the component in another figure labeled with the same number. In addition, the use of different numbers to refer to components is not intended to indicate that the different numbered components cannot be the same or similar to other numbered components.

[0031] The present disclosure describes, among other things, cell culture apparatuses having a substrate defining one or more wells in addition to a cell culture well insert that may be positioned within at least one of the one or more of wells.

[0032] In some embodiments, the wells may be configured such that cells cultured in the wells form spheroids. For example, the wells can be non-adherent to cells to cause the cells in the wells to associate with each other and form spheroid clusters. The spheroids may expand to size limits imposed by the geometry of the wells. In some embodiments, the wells may be coated with an ultra-low binding material to make the wells non-adherent to cells. Because the cells are non-adherent to the surface of the wells, exchange of cell culture media without aspirating or disturbing the spheroid can be difficult.

[0033] Cells cultured in three dimensions, such as spheroids, can exhibit more in vivo like functionality than their counterparts cultured in two dimensions as monolayers. In two dimensional cell culture systems, cells can attach to a substrate on which they are cultured. However, when cells are grown in three dimensions, such as spheroids, the cells interact with each other rather than attaching to the substrate. Cells cultured in three dimensions more closely resemble in vivo tissue in terms of cellular communication and the development of extracellular matrices. Spheroids thus provide a superior model for cell migration, differentiation, survival, and growth and therefore provide better systems for research, diagnostics, and drug efficacy, pharmacology, and toxicity testing.

[0034] In some embodiments, the devices are configured such that cells cultured in the devices form spheroids. For example, the wells in which cells are grown can be non- adherent to cells to cause the cells in the wells to associate with each other and form spheres. The spheroids expand to size limits imposed by the geometry of the wells. In some embodiments, the wells are coated with an ultra-low binding material to make the wells non-adherent to cells.

[0035] Examples of non-adherent material include perfluorinated polymers, olefins, or like polymers or mixtures thereof. Other examples include agarose, non-ionic hydrogels such as polyacrylamides, polyethers such as polyethylene oxide and polyols such as polyvinyl alcohol, or like materials or mixtures thereof. The combination of, for example, non-adherent wells, well geometry (e.g., size and shape), and/or gravity induce cells cultured in the wells to self-assemble into spheroids. Some spheroids maintain differentiated cell function indicative of a more in vz o-like response relative to cells grown in a monolayer. Other cell types, such as mesenchymal stromal cells, when cultured as spheroids retain their pluripotency.

[0036] In some embodiments, the systems, devices, and methods herein comprise one or more cells. In some embodiments, the cells are cryopreserved. In some embodiments, the cells are in three dimensional culture. In some such embodiments, the systems, devices, and methods comprise one or more spheroids. In some embodiments, one or more of the cells are actively dividing. In some embodiments, the systems, devices, and methods comprise culture media (e.g., comprising nutrients (e.g., proteins, peptides, amino acids), energy (e.g., carbohydrates), essential metals and minerals (e.g., calcium, magnesium, iron, phosphates, sulphates), buffering agents (e.g., phosphates, acetates), indicators for pH change (e.g., phenol red, bromo-cresol purple), selective agents (e.g., chemicals, antimicrobial agents), etc.). In some embodiments, one or more test compounds (e.g., drug) are included in the systems, devices, and methods.

[0037] A wide variety of cell types may be cultured. In some embodiments, a spheroid contains a single cell type. In some embodiments, a spheroid contains more than one cell type. In some embodiments, where more than one spheroid is grown, each spheroid is of the same type, while in other embodiments, two or more different types of spheroids are grown. Cells grown as spheroids may be natural cells or altered cells (e.g., cell comprising one or more non-natural genetic alterations). In some embodiments, the cell is a somatic cell. In some embodiments, the cell is a stem cell or progenitor cell (e.g., embryonic stem cell, induced pluripotent stem cell) in any desired state of differentiation (e.g., pluripotent, multi-potent, fate determined, immortalized, etc.). In some embodiments, the cell is a disease cell or disease model cell. For example, in some embodiments, the spheroid comprises one or more types of cancer cells or cells that can be induced into a hyper-proliferative state (e.g., transformed cells). Cells may be from or derived from any desired tissue or organ type, including but not limited to, adrenal, bladder, blood vessel, bone, bone marrow, brain, cartilage, cervical, corneal, endometrial, esophageal, gastrointestinal, immune system (e.g., T lymphocytes, B lymphocytes, leukocytes, macrophages, and dendritic cells),liver, lung, lymphatic, muscle (e.g., cardiac muscle), neural, ovarian, pancreatic (e.g., islet cells), pituitary, prostate, renal, salivary, skin, tendon, testicular, and thyroid. In some embodiments, the cells are mammalian cells (e.g., human, mice, rat, rabbit, dog, cat, cow, pig, chicken, goat, horse, etc.).

[0038] The cultured cells find use in a wide variety of research, diagnostic, drug screening and testing, therapeutic, and industrial applications.

[0039] In some embodiments, the cells are used for production of proteins or viruses.

Systems, devices, and methods that culture large numbers of spheroids in parallel are particularly effective for protein production. Three-dimensional culture allows for increased cell density, and higher protein yield per square centimeter of cell growth surface area. Any desired protein or viruses for vaccine production may be grown in the cells and isolated or purified for use as desired. In some embodiments, the protein is a native protein to the cells. In some embodiments, the protein is non-native. In some embodiments, the protein is expressed recombinantly. Preferably, the protein is overexpressed using a non-native promoter. The protein may be expressed as a fusion protein. In some embodiments, a purification or detection tag is expressed as a fusion partner to a protein of interest to facilitate its purification and/or detection. In some embodiments, fusions are expressed with a cleavable linker to allow separation of the fusion partners after purification.

[0040] In some embodiments, the protein is a therapeutic protein. Such proteins include, but are not limited to, proteins and peptides that replace a protein that is deficient or abnormal (e.g., insulin), augment an existing pathway (e.g., inhibitors or agonists), provide a novel function or activity, interfere with a molecule or organism, or deliver other compounds or proteins (e.g., radionuclides, cytotoxic drugs, effector proteins, etc.). In some embodiments, the protein is an immunoglobulin such as an antibody (e.g., monoclonal antibody) of any type (e.g., humanized, bi-specific, multi-specific, etc.). Therapeutic protein categories include, but are not limited to, antibody-based drugs, Fc fusion proteins, anticoagulants, antigens, blood factor, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics. Therapeutic proteins may be used to prevent or treat cancers, immune disorders, metabolic disorders, inherited genetic disorders, infections, and other diseases and conditions.

[0041] In some embodiments, the protein is a diagnostic protein. Diagnostic proteins include, but are not limited to, antibodies, affinity binding partners (e.g., receptor- binding ligands), inhibitors, antagonists, and the like. In some embodiments, the diagnostic protein is expressed with or is a detectable moiety (e.g., fluorescent moiety, luminescent moiety (e.g., luciferase), colorimetric moiety, etc.).

[0042] In some embodiments, the protein is an industrial protein. Industrial proteins include, but are not limited to, food components, industrial enzymes, agricultural proteins, analytical enzymes, etc.

[0043] In some embodiments, the cells are used for drug discovery, characterization, efficacy testing, and toxicity testing. Such testing includes, but is not limited to, pharmacological effect assessment, carcinogenicity assessment, medical imaging agent characteristic assessment, half- life assessment, radiation safety assessment, genotoxicity testing, immunotoxicity testing, reproductive and developmental testing, drug interaction assessment, dose assessment, adsorption assessment, disposition assessment, metabolism assessment, elimination studies, etc. Specific cell types may be employed for specific tests (e.g., hepatocytes for liver toxicity, renal proximal tubule epithelial cells for nephrotoxicity, vascular endothelial cells for vascular toxicity, neuronal and glial cells for neurotoxicity, cardiomyocytes for cardiotoxicity, skeletal myocytes for rhabdomyo lysis, etc.). Treated cells may be assessed for any number of desired parameters including, but not limited to, membrane integrity, cellular metabolite content, mitochondrial functions, lysosomal functions, apoptosis, genetic alterations, gene expression differences, and the like.

[0044] In some embodiments, the cell culture devices are a component of a larger system. In some embodiments, the system comprises a plurality (e.g., 2, 3, 4, 5, 10, 20,

50, 100, 1000, etc.) of such cell culture devices. In some embodiments, the system comprises an incubator for maintaining the culture devices at optimal culture conditions (e.g., temperature, atmosphere, humidity, etc.). In some embodiments, the system comprises detectors for imaging or otherwise analyzing cells. Such detectors include, but are not limited to, fluorimeters, luminometers, cameras, microscopes, plate readers (e.g., PERKIN ELMER ENVISION plate reader; PERKIN ELMER VIEWLUX plate reader), cell analyzers (e.g., GE IN Cell Analyzer 2000 and 2200; THERMO/ CELLOMICS CELLNSIGHT High Content Screening Platform), and confocal imaging systems (e.g., PERKIN ELMER OPERAPHENIX high throughput content screening system; GE INCELL 6000 Cell Imaging System). In some embodiments, the system comprises perfusion systems or other components for supplying, re-supplying, and circulating culture media or other components to cultured cells. In some embodiments, the system comprises robotic components (e.g., pipettes, arms, plate movers, etc.) for automating the handing, use, and/or analysis of culture devices.

[0045] In some embodiments, the cell culture apparatuses with which an insert as described herein may be employed is a multi-well round bottom plate, such as a 96-well round bottom plate.

[0046] An embodiment of a cell culture container 100 is shown in FIGS. 1A and IB. FIG.

1A shows an embodiment that is a 96 well plate, having a plurality of wells 115. In general, a "well" means a macro well, such as those shown in FIG. 1A. However, any type of cell culture container may be provided. For example, a dish, a 6 well plate, a 12 well plate, a 24 well plate, a 96 well plate, a 384 well plate or a 1536 well plate may be provided. "Microwell" generally means small wells, structured to contain a single spheroid. The plurality of wells 115 may be provided a variety of different arrangements. For example the plurality of wells 115 may define a pattern that is stacked, hexagonal close-packed, etc. In embodiments, wells 115 are formed in major surface 112. Wells 115 may include an array of ordered well structures as shown in FIG. 1A. A side cross-sectional view of an embodiment of a multiwell cell culture container is shown in FIG. IB showing round bottom wells formed in a substrate 110. In this embodiment, each of the plurality of wells 115 drops below a major surface 112 of the cell culture apparatus 100 and has a round bottom which provides a position to form spheroids, as described above.

[0047] In embodiments, the cell culture container 100 may have macro-wells as shown in FIG. 1A and FIG. IB, suitable for containing an insert, as described herein. These macro-wells may be relatively small, as for example, a well in a 1536 well plate (which are generally 5.01 mm deep and the top diameter is 1.69 mm), but will still be much bigger than a microwell (which can be 500 μηι deep and about 500 μηι wide). In addition, in embodiments, a macro-well may have a cell culture surface (126 in FIG. 4) which contains microwells 125 (see also 125 in FIG. 4). These microwells are structured to accommodate a single spheroid in each microwell. The size and shape of microwells may induce the formation of spheroids, as described above. FIG. 1C is a schematic drawing showing cells 200 grown in microwells 125 (shown in FIG. 4 as 125) of an embodiment of a structured surface 113 including an array of hexagonal close-packed microwells. In some embodiments, the cells 200 within each well 125 form a single spheroid, as shown in FIG. 1C. Microwells may take any form or shape or geometry, including those shown in FIG. 1C.

[0048] FIG.s 2A-C illustrate embodiments of cell culture inserts which, in embodiments, may be inserted into wells 115 of cell culture containers 100. These are macro-well inserts.

[0049] Each of the plurality of wells 115 may include a spheroid trap insert, or well insert, or insert 150, as shown in FIG. 2A. The well insert 150 may be configured to be positioned in at least a portion of a well 115. The well insert 150, or portions thereof, may be made of any suitable material. Preferably, materials intended to contact cells or culture media are compatible with the cells and the media. Typically, cell culture components are formed from polymeric material. Examples of suitable polymeric materials include polystyrene, polymethylmethacrylate, polyvinyl chloride, polycarbonate, polysulfone, polystyrene copolymers, fluoropolymers, polyesters, polyamides, polystyrene butadiene copolymers, fully hydrogenated styrenic polymers, polycarbonate PDMS copolymers, and polyolefins such as polyethylene,

polypropylene, polymethyl pentene, polypropylene copolymers and cyclic olefin copolymers, and the like.

[0050] As shown in FIG. 2A, the well insert 150 may include a frame 160. The frame 160 may have a first end 161, a second end 162, and at least one support 163 extending between the first end 161 and the second end 162. As shown, the first end 161 may define an aperture or opening 164. The aperture 164 of the first end 161 may align with a top aperture of the well and allow access to at least a portion of the interior of the well. In some embodiments, the first end 161 may sit outside of the well and away from the top aperture of the well (see FIG. 4). The first end 161 of the frame 160 may be defined by a variety of shapes, e.g., circular, square, rectangular, diamond, hexagonal, etc. In some embodiments, the shape of the first end 161 may match the shape of the top aperture of the well.

[0051] As shown in FIG. 2 A ,the first end 161 of the frame 160 maybe configured such that the frame 160 is positioned in at least a portion of the well. For example, the frame 160 may extend from the top aperture of the well down into the well. In some embodiments, the shape of the first end 161 may assist in holding the frame 160 into position proximate the top aperture of the well. As shown in FIG. 2A, the frame 160 may include one or more flanges 168 extending from the first end 161 of the frame 160. The one or more flanges 168 may assist in positioning the frame 160 within at least a portion of the well. For example, the one or more flanges 168 may contact a portion proximate the well to position the frame 160 within at least a portion of the well. In some embodiments, the one or more flanges 168 may contact some other part of the cell culture apparatus to position the frame 160 within at least a portion of the well.

[0052] Support 163 may be a side wall. Support 163 of the frame 160 of the well insert 150 may extend between and be coupled to the first and second ends 161, 162 of the well insert 150, as shown in FIGS. 2A-2C. As shown in FIGS. 2B and 2C, the frame 160 includes four supports 166. The frame 160 may include less than four supports (e.g., one support, two supports, three supports, etc.), four supports, or more than four supports (e.g., five supports, six supports, eight supports, ten supports, etc.). The one or more supports 163 may include, e.g., wire, bars, rods, etc. The one or more supports 163 extending between the first and second ends 161, 162 may define a substantially open frame. In other words, the space between the first and second ends 161, 162 is generally open. In some embodiments, the one or more supports 163 may include a sidewall that extends from and is coupled to the first and second ends 161, 162. In some embodiments, the sidewall may completely surround an edge or perimeter of the second end 162. In other words, the sidewall may enclose an area between the first and second ends 161, 162 and, therefore, define a closed tubular structure of the frame 160. The sidewall support may be defined by a variety of different characteristics, e.g., solid, porous, fluid permeable. [0053] The second end 162 of the frame 160 may define an opening 165. The opening 165 of the second end 162 may be closer to a bottom of the well than the first end 161 is to the bottom of the well when the frame 160 is positioned in at least a portion of the well.

[0054] The well insert 150 may also include a fluid permeable mesh 170 coupled to the

frame 160 and disposed across the opening 165 of the second end 162. The mesh 170 may define pores 171. The pores 171 may define an average pore size of about, e.g., greater than or equal to 5 micrometers, greater than or equal to 10 micrometers, greater than or equal to 20 micrometers, greater than or equal to 35 micrometers, greater than or equal to 50 micrometers, etc. or, less than or equal to 200 micrometers, less than or equal to 90 micrometers, less than or equal to 75 micrometers, less than or equal to 60 micrometers, less than or equal to 45 micrometers, etc. In some embodiments, the pores may define an average pore size of about 40 micrometers. The pores 171 may be defined as being a size to prevent passage of a spheroid through the mesh 170. Also, the pores 171 may be defined as being a size that allows passage of individual cells through the mesh 170. The mesh may be made of a variety of different materials including but not limited to track-etched membrane ora woven or non- woven porous material. The material of the porous membrane may be treated or coated to make it more adherent, wettable, or more non- adherent to cells.

Treatment may be accomplished by any number of methods known in the art which include plasma discharge, corona discharge, gas plasma discharge, ion bombardment, ionizing radiation, and high intensity UV light. Coatings can be introduced by any suitable method known in the art including printing, spraying, condensation, radiant energy, ionization techniques or dipping. In certain embodiments, The coatings may then provide either covalent or non-covalent attachment sites. Such sites can be used to attach moieties, such as cell culture components (e.g., proteins that facilitate growth or adhesion). Further, the coatings may also be used to enhance the attachment of cells (e.g., polylysine). Alternatively, cell non-adherent coatings as described above can be used to prevent or inhibit cell binding. In some embodiments, the mesh comprises a nylon or polyester mesh.

[0055] In some embodiments, the mesh may also be disposed between the first and second ends. In such embodiments, in which the mesh or sidewall extends between the first and second ends, the frame may define an interior space or cavity of the frame. In other words, the interior space or cavity of the frame would be defined by the mesh disposed across the second opening and the sidewalk (e.g., mesh, solid, etc.) extending between the first and second ends.

[0056] The opening 165 of the second end 162, and thus the mesh 170, may be defined by a variety of shapes, e.g., square, rectangle, circle, hexagon, etc. As shown in FIG. 2B. the opening 165 of the second end 162 may be defined by a circle. As shown in FIG. 2C, the opening 165 of the second end 162 may be defined by a square. The shape of the second end 162 may be described as preventing the spheroid from exiting the well when the well insert 150 is positioned therein. In other words, the shape of the second end 162 of the frame 160 may correspond to the shape of the well such that any gap between the second end 162 of the frame 160 and a side of the well is not large enough to allow a spheroid to pass through. For example, the second end 162 may define a shape of the same size as defined by a side of the well and, thereby, eliminating any gap between the two.

[0057] In some embodiments, a cell culture container 100 may include one or more wells 115 and one or more well inserts 150, as shown in FIG. 3. As shown, each well 115 includes a corresponding well insert 150. The well insert 150 is configured to be positioned within at least a portion of the well 115. In other words, the well insert 150 may be inserted into at least a portion of the well 115 and the well insert 150 may be removed from at least a portion of the well 115. In some embodiments, the one or more well inserts 150 may be coupled to one another such that the one or more well inserts 150 may be positioned into and out of the one or more wells 115 at the same time. The one or more wells 115 may be coupled through the use of a frame 160. In other embodiments, each of the one or more well inserts 150 may be configured to be individually positioned into and out of a corresponding well 115. In other words, in embodiments, none of the one or more well inserts 150 are attached to one another. In yet other embodiments, the one or more well inserts may be coupled to one another in a variety of combinations based on application. For example, the one or more well inserts may be coupled in groups of about 384, 96, 48, 24, 12, 6, etc.

[0058] In additional embodiments, as shown in FIG. 4, the well 115 may have a cell culture surface 126 at the bottom of the well. In embodiments the cell culture surface has, at least in part, a structured surface 113 which has an array of microwells 125.

[0059] A structured surface 113 of a cell culture apparatus 100 as described herein may define any suitable number of microwells 125 that may have any suitable size or shape. The microwells 125 define a volume based on their size and shape. In many embodiments, one or more or all of the wells 125 are symmetrically rotatable around a longitudinal axis. In some embodiments, the longitudinal axes of one or more or all of the microwells 125 are parallel with one another. The microwells 125 may be uniformly or non-uniformly spaced. Preferably, the microwells 125 are uniformly spaced. One or more or all the microwells 125 may have the same size and shape or can have different sizes and shapes.

[0060] In certain embodiments, the wells 115 may be defined by substrate 110 that defines the bottom of the wells, extending below a major surface 112. Each well of the one or more wells 115 defines an interior surface 120, an exterior surface 114 and an upper aperture 118. In some embodiments, the wells 115 may be gas permeable through the substrate 110. The gas permeability of the wells 115 through the substrate 110 to exterior surface 114 will depend in part on the material of the substrate and the thickness of the substrate along the well 115. For example, the gas permeability of the wells may be as described in commonly-assigned U.S. provisional patent application no. 62/072088, which provisional patent application is hereby

incorporated herein by reference in its entirety to the extent that it does not conflict with the present disclosure.

[0061] The interior surface of the well defines a nadir 116, or a low point, that is opposite the upper aperture 118. Still with reference to FIG. 3, the wells 115 have a depth d defined by the height from nadir 116 to upper aperture 118. The wells 115 also have a diametric dimension w, such as a diameter, width, etc., across the well defined by the upper aperture 118. The wells may have any suitable depth d and diametric dimension w. In some embodiments, the depth d, diametric dimension w and shape of the well, along with the material forming the well, serve to define a volume in which cells can grow.

[0062] In some embodiments, the wells 115 described herein have a diametric dimension w in a range from about 200 micrometers to about 500 micrometers. Such diametric dimensions can control the size of a spheroid 130 grown therein such that cells at the interior of the spheroid 130 are maintained in a healthy state. In some embodiments, the wells 115 have a depth d in a range from about 100 micrometers to about 500 micrometers. Of course, other suitable dimensions may also be employed, such as up to 3000 micrometers or greater. [0063] In some embodiments, the inner surface of the wells 115 are non-adherent to cells.

The wells 115 may be formed from non-adherent material or may be coated with nonadherent material to form a non-adherent well. In some embodiments, the nonadherent material may be described as an ultra-low-adhesion material. Examples of non-adherent material include perfluorinated polymers, olefins, or like polymers or mixtures thereof. Other examples include agarose, non-ionic hydrogels such as polyacrylamides, or polyethers such as polyethyleneoxide or polyols such as polyvinylalcohol, or like materials or mixtures thereof. The combination of, for example, non-adherent wells, well geometry, and gravity can induce cells cultured in the wells to self-assembly into spheroids 130. Some spheroids 130 can maintain differentiated cell function indicative of a more in vivo like response relative to cells grown in a monolayer.

[0064] As seen in FIG. 4, the interior surface 120 may define a variety of different shapes from the upper aperture 118 to the nadir 116. For example, in some embodiments, one or more wells 115 may be defined by an arcuate surface, such as a hemi- spherical or concave surface, a conical surface having a rounded bottom, and the like surface geometries or a combination thereof. The nadir 116 of the well 115 may ultimately terminate, end, or bottom-out in a spheroid-conducive rounded or curved surface, such as a dimple, a pit, and like concave frusto-conical relief surfaces, or combinations thereof. Other shapes and construction of gas-permeable spheroid- conducive wells are described in commonly-assigned U.S. Patent Application No. 14/087,906, which application is hereby incorporated herein by reference in its entirety to the extent that it does not conflict with the present disclosure.

[0065] In some embodiments, the interior surface 120 may be flat or come to a point. The interior surface 120 may have any other suitable shape or dimension.

[0066] In some embodiments, the mesh 170 maybe configured such that individual cells may pass through the mesh 170 when the cells are being seeded into the wells 115. For example, the pores 171 may define an average pore size that is larger than an individual cell. However, after the cells form into spheroids 130, the spheroids 130 may be too large to pass back through the mesh 170. Once the spheroid 130 is positioned between the mesh 170 and the nadir 116 of the interior surface 120, the mesh 170 may also help increase user efficiency by reducing the potential for error. For example, the presence of the mesh 170 prevents a pipette from coming in contact with the spheroid 130. Additionally, the mesh 170 may help diffuse the flow of medium that is being introduced or withdrawn from the well 115. This diffusion may help to prevent eddies from disrupting the spheroid 130.

[0067] In some embodiments, the thickness and shape of the substrate around the well is configured to correct for refraction of light passing into the interior surface and out of the exterior surface. For example, the shape and thickness may be as described in commonly-assigned U.S. provisional patent application no. 62/072019, which provisional patent application is hereby incorporated herein by reference in its entirety to the extent that it does not conflict with the present disclosure.

[0068] In some embodiments, the well insert 150 may include a fluid permeable mesh 170 and a frame 160. As shown in FIG. 3, the well insert 150 is configured to be at least partially inserted into the well 115 such that the mesh 170 is positioned in the well 170 a distance 119 from the nadir 116. The frame 160 may be coupled to the mesh 170 and extend away from the nadir 116 when the well insert 150 is positioned within the well 115. The frame 160 may be used to help position the well insert 150 into and out of the well 115. In some embodiments, the frame 160 may be configured to position the mesh 170 the distance 119 from the nadir 116. For example, the frame 160 may contact an upper edge 121 of the well to position the mesh 170 the distance 119 from the nadir 116. In another example, the cell culture assembly 100 may include a support that contacts the frame 160 to ensure the mesh 170 is positioned a desired distance 119 from the nadir 116. In other embodiments, the mesh 170 may be in contact with the interior surface 120 of the well 115 and thereby controlling the distance 119 the mesh 170 is from the nadir 116.

[0069] As shown in FIG. 4, the well 115 may include at least one microwell 125 on a cell culture surface 126 along the interior surface of a well 115. In some embodiments, a plurality of microwells 125 are positioned along the interior surface 120 of the well 115. The microwell 125 may have similar characteristics as the wells 115 described above. Alternatively, the cell culture apparatus may include a reservoir and the reservoir may include a plurality of wells as described herein. Also shown in FIG. 4, the well 115 may contain a well insert 150 therein positioned between the interior surface 120 of the well 115 and the upper aperture of the well 115, similar to FIG. 3.

[0070] In some embodiments, a cell culture assembly 500 may include a reservoir 521 having an array of wells, 515, 525, a fluid permeable mesh 570 and a frame 560 coupled to the mesh 570, as shown in FIG. 5. The frame 560 may be configured to maintain the mesh 570 in a position over at least a portion of the first well 515. In some embodiments, the mesh 570 is configured to be disposed over an upper edge 520 of the first well 515. In some embodiments, the cell culture assembly 500 may include a second well 525 and the mesh 570 is configured to be positioned over the second well 525.

[0071] A well insert as described herein may be used to help contain spheroids in a cell culture container well 115, i.e., prevent spheroids from exiting the well 115 during processes such as exchanging cell culture media within the well 115. In some embodiments, the cell culture apparatus 100 maybe tilted to remove the cell culture medium when the well insert 150 (e.g., FIG. 2A) is positioned in at least a portion of the wells 115. In other embodiments, a pipette may be used to remove the cell culture medium when the well insert 150 is positioned in at least a portion of the wells 115. The use of the well insert 150 in combination with the pipette may reduce the risk of the pipette affecting the spheroid, e.g., the pipette cannot not aspirate the spheroid.

[0072] FIG. 6 illustrates an array of microwells 601 may have a sinusoidal or parabolic shape. This sinusoidal or parabolic well shape, has a rounded top and a rounded well bottom. As shown in FIG. 6, the well 615 has a top opening having a top diameter Dtop, a height from the bottom of the well 616 to the top of the well H, and a diameter of the well at a height half-way 613 between the top of the well and the bottom 616 of the well Dhalf-way. In embodiments, an array of wells of a cell culture container may additionally have an array of microwells, having structure such as that shown in FIG. 6 and FIG. 7, inside the array of wells, to induce the formation of spheroids once cells are trapped between the insert and the bottom surface or nadir of the cell culture well.

[0073] FIG. 7 is a schematic drawing of an array of microwells. FIG. 7 illustrates a plurality of microwells 615 arranged in an array in a structured surface 113. Also shown in FIG. 7 are a plurality of spheroids 500 residing in the plurality of microwells 615. In embodiments, the nadir or the cell culture surface of the cell culture container, for example the floor of the well of a 96 well plate, may be a substrate having an array of microwells, which provide induce the formation of spheroids. This is also called a structured bottom surface. [0074] A method 800 for removing culture media from a well of a cell culture apparatus is depicted in FIG. 8. The well defines an interior surface including a nadir. An end of an insert is disposed 810 into the well with the end of the insert defining an opening in fluid communication with a cavity of the insert. The insert includes a fluid permeable mesh disposed across the opening and the end of the insert is disposed within the well such that the mesh is positioned a distance from the nadir. A tip of a fluid removal device is inserted 820 into the cavity of the insert such that the tip is a distance from the nadir at least as far as the distance from the mesh to the nadir. Fluid is removed 630 from the well by withdrawing the fluid via the fluid removal device. FIG. 8 is a schematic drawing illustrating an exemplary method 800 for using an embodiment of an apparatus as described herein. First, in 810 the method disposes an end of an insert into the well such that the mesh is positioned a distance from the nadir. Second, 820, the method inserts a tip of a fluid removal device into the cavity of the insert such that the tip is at least the distance from the nadir. Third, 830, the method removes fluid from the well by withdrawing fluid via the fluid removal device.

[0075] A structured bottom surface as described herein can be formed in any suitable matter.

For example, a substrate can be molded to form the structured surface, a substrate film can be embossed to form the structured surface, or the like.

[0076] A structured bottom surface as described herein may be assembled into a cell culture apparatus in any suitable manner. For example, the structured bottom surface and one or more other components of the cell culture apparatus may be molded as a single part. In some embodiments, the structured bottom surface or a portion thereof is welded (e.g., thermal welding, ultrasonic welding, or the like), adhered or thermoformed to one or more other components of the cell culture apparatus.

[0077] The cell culture well inserts described herein may be used with a cell culture apparatus where, for example, media flows over a plurality of wells/chambers that contain the well inserts over the top of spheroids. In some embodiments, the cell culture inserts are integral (e.g., attached to, or formed part of) the cell culture apparatus. In certain embodiments, the well inserts prevent the spheroids in the wells from coming out of the well and entering the stream of media moving through the cell culture perfusion apparatus. In particular embodiments, as shown in FIG. 5, a single insert is employ to cover a plurality of wells. Such an insert could be inserted into, or be an integral part, of the cell culture apparatus. The cell culture apparatus may be employed with or without an oxygenator. An exemplary cell culture apparatus that does not require an oxygenator (e.g., the wells are gas permeable and air passages are provided in the apparatus) is described below with reference to Figures 8 and 9.

[0078] All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0079] As used herein, singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "structured bottom surface" includes examples having two or more such "structured bottom surfaces" unless the context clearly indicates otherwise.

[0080] As used in this specification and the appended claims, the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise. The term "and/or" means one or all of the listed elements or a combination of any two or more of the listed elements.

[0081] As used herein, "have", "has", "having", "include", "includes", "including", "comprise", "comprises", "comprising" or the like are used in their open ended inclusive sense, and generally mean "include, but not limited to", "includes, but not limited to", or "including, but not limited to".

[0082] "Optional" or "optionally" means that the subsequently described event, circumstance, or component, can or cannot occur, and that the description includes instances where the event, circumstance, or component, occurs and instances where it does not.

[0083] The words "preferred" and "preferably" refer to embodiments of the disclosure that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the inventive technology.

[0084] Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, examples include from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0085] Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Where a range of values is "greater than", "less than", etc. a particular value, that value is included within the range.

[0086] Any direction referred to herein, such as "top," "bottom," "left," "right," "upper," "lower," "above," below," and other directions and orientations are described herein for clarity in reference to the figures and are not to be limiting of an actual device or system or use of the device or system. Many of the devices, articles or systems described herein may be used in a number of directions and orientations. Directional descriptors used herein with regard to cell culture apparatuses often refer to directions when the apparatus is oriented for purposes of culturing cells in the apparatus.

[0087] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that any particular order be inferred. Any recited single or multiple feature or aspect in any one claim can be combined or permuted with any other recited feature or aspect in any other claim or claims.

[0088] It is also noted that recitations herein refer to a component being "configured" or "adapted to" function in a particular way. In this respect, such a component is "configured" or "adapted to" embody a particular property, or function in a particular manner, where such recitations are structural recitations as opposed to recitations of intended use. More specifically, the references herein to the manner in which a component is "configured" or "adapted to" denotes an existing physical condition of the component and, as such, is to be taken as a definite recitation of the structural characteristics of the component. [0089] While various features, elements or steps of particular embodiments may be disclosed using the transitional phrase "comprising," it is to be understood that alternative embodiments, including those that may be described using the transitional phrases "consisting" or "consisting essentially of," are implied. Thus, for example, implied alternative embodiments to an insert comprising a frame and a mesh include embodiments where an insert consists of a frame and a mesh and embodiments where an insert consists essentially of a frame and an insert.

[0090] It will be apparent to those skilled in the art that various modifications and variations can be made to the present inventive technology without departing from the spirit and scope of the disclosure. Since modifications, combinations, sub -combinations and variations of the disclosed embodiments incorporating the spirit and substance of the inventive technology may occur to persons skilled in the art, the inventive technology should be construed to include everything within the scope of the appended claims and their equivalents.