Cell culture vessels comprising dialysis membranes

Abstract

Disclosed herein are cell culture vessels including a microcavity compartment, a media reservoir compartment, and a dialysis membrane separating the microcavity compartment from the media reservoir compartment. The dialysis membrane may be porous and liquid permeable. The dialysis membrane may have a pore size greater than or equal to 1 nanometer and less than or equal to 100 nanometers. The dialysis membrane may include a plurality of microwells, each microwell having an interior surface defining an upper aperture and a nadir, the upper aperture facing the microcavity compartment.

Description

SP24-149CELL CULTURE VESSELS COMPRISING DIALYSIS MEMBRANESCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority under 35 U.S.C. §119 of U.S. Provisional Application Serial No. 63 / 691,057 filed on September 5, 2024, the content of which is relied upon and incorporated herein by reference in its entirety.BACKGROUNDField

[0002] The present specification generally relates to cell culture vessels and, in particular, cell culture vessels comprising dialysis membranes that allow low molecular weight nutrients and waste byproducts to be removed without disturbing the cell cultures and allow helpful high molecular weight growth factors and byproducts to be retained.Technical Background

[0003] Cells cultured in a three-dimensional (3D) cell culture environment exhibit more in vivo-like functionality than cells cultured in two-dimensional (2D) environments as monolayers. In 2D cell culture systems, adherent cells attach to a substrate on which they are cultured. In contrast, when grown in 3D systems, cells interact with each other forming 3D cell cultures, aggregates or spheroids.

[0004] To encourage cell growth, a nutrient formula may be introduced to the cells. As the cells consume the nutrients, byproducts and metabolites are produced. Some of the byproducts such as lactate and ammonia are toxic to cell growth. However, other byproducts, such as autocrine cell growth factors, may be helpful to cell growth.

[0005] Challenges exist when growing 3D cell cultures in a conventional culture apparatus. For example, current cell growth substrates may be of either a solid construction or a porous mesh construction. The solid construction may trap all of the byproducts, both helpful and hurtful, as well as the nutrient formula in a single medium reservoir together with the 3D cell cultures. This may require frequent changeover of the medium in order to reduce the presence of the hurtful byproducts and replace necessary nutrients for cell survival and growth. Such frequent replacement may decrease the efficiency and increase the cost of growing cells.SP24-149The mesh construction may allow byproducts of all molecular weights to diffuse through the cell culture, which, while removing the hurtful byproducts, also allows the helpful growth factors and byproducts to diffuse away from the cell growths, thereby slowing cell growth.

[0006] Therefore, a continuing need exists for 3D cell culture vessels that selectively allow diffusion of nutrients and allow low molecular weight waste byproducts to be removed without disturbing the cell cultures and allow helpful growth factors and byproducts to be retained.SUMMARY

[0007] Aspect 1. A cell culture vessel comprising a microcavity compartment, a media reservoir compartment, and a dialysis membrane separating the microcavity compartment from the media reservoir compartment. The dialysis membrane is porous and liquid permeable. The dialysis membrane comprises a pore size greater than or equal to 1 nanometer and less than or equal to 100 nanometers. The dialysis membrane comprises a plurality of microwells, each microwell having an interior surface defining an upper aperture and a nadir, the upper aperture facing the microcavity compartment.

[0008] Aspect 2. The cell culture vessel of aspect 1, wherein the pore size of the dialysis membrane is greater than or equal to 1 nanometer and less than or equal to 5 nanometers.

[0009] Aspect 3. The cell culture vessel of aspect 1 or aspect 2, wherein the dialysis membrane comprises a molecular weight cut-off greater than or equal to 0.02 kD and less than or equal to 1000 kD.

[0010] Aspect 4. The cell culture vessel of aspect 3, wherein the molecular weight cutoff of the dialysis membrane is greater than or equal to 20 kD and less than or equal to 200 kD.

[0011] Aspect 5. The cell culture vessel of any one of aspects 1 to 4, wherein the dialysis membrane comprises cellulose, cellulose acetate, polyamide, polysulfone (PSF), poly(ether sulfone) (PES), poly (hydroethyl methacrylate) (PHEMA), polyethylene glycol methacrylate (PEGMA), polyphosphobetaine methacrylate (PBMA), polysulfobetaine methacrylate (SBMA), olyvinylidene fluoride (PVDF), polymethyl methacrylate (PMMA), polyurethane (PU), poly(lactic acid) (PLA), or combinations thereof.SP24-149

[0012] Aspect 6. The cell culture vessel of any one of aspects 1 to 5, wherein the cell culture vessel comprises an open vessel and an insert, wherein the dialysis membrane comprises a bottom of the insert, and wherein the dialysis membrane is above a bottom surface of the open vessel.

[0013] Aspect 7. The cell culture vessel of aspect 6, wherein the insert further comprises a side wall and an edge, the dialysis membrane being attached to the side wall.

[0014] Aspect 8. The cell culture vessel of aspect 7, wherein the open vessel comprises a well plate, the dialysis membrane being disposed in the well plate.

[0015] Aspect 9. The cell culture vessel of any one of aspects 6 to 8, wherein the insert comprises a plurality of dialysis membranes.

[0016] Aspect 10. The cell culture vessel of any one of aspects 1 to 5, wherein the cell culture vessel comprises a closed vessel, the closed vessel comprising a bottom wall, a top surface opposite the bottom wall, and a plurality of side walls extending between the bottom wall and the top surface; thereby forming an internal cavity, wherein the dialysis membrane is disposed within the internal cavity.

[0017] Aspect 11. The cell culture vessel of aspect 10, wherein the closed vessel further comprises a fence, the fence being disposed between the plurality of side walls and the dialysis membrane.

[0018] Aspect 12. The cell culture vessel of aspect 10 or aspect 11, wherein the closed vessel comprises a plurality of the microcavity compartments, a plurality of the media reservoir compartments, and a plurality of the dialysis membranes, each dialysis membrane separating a microcavity compartment from a media reservoir compartment.

[0019] Aspect 13. The cell culture vessel of aspect 12, wherein the closed vessel further comprises a first manifold disposed within the internal cavity, the first manifold having a first aperture and a second manifold disposed within the internal cavity, the second manifold having a second aperture, wherein the first manifold is fluidly coupled to the plurality of microcavity compartments and the second manifold is fluidly coupled to the plurality of media reservoir compartments.SP24-149

[0020] Aspect 14. The cell culture vessel of any one of aspects 1 to 13, wherein the upper aperture of each microwell is greater than or equal to 100 micrometers and less than or equal to 2000 micrometers.

[0021] Aspect 15. The cell culture vessel of any one of aspects 1 to 14, wherein a depth of each microwell is greater than or equal to 100 micrometers and less than or equal to 5000 micrometers.

[0022] Aspect 16. The cell culture vessel of any one of aspects 1 to 14, wherein the interior surface of at least one of the plurality of microwells comprises a coating disposed thereon.

[0023] Aspect 17. The cell culture vessel of claim 16, wherein the coating comprises a copolymer of acrylamide methacrylate and a photoreactive group, a hydrophilic polymer, a non-ionic surfactant, a protein, or combinations thereof.

[0024] Aspect 18. A cell culture vessel comprising a microcavity compartment, a media reservoir compartment, and a dialysis membrane separating the microcavity compartment from the media reservoir compartment, wherein the dialysis membrane is porous and liquid permeable, and the dialysis membrane comprises a plurality of microwells, each microwell having an interior surface defining an upper aperture and a nadir, the upper aperture facing the microcavity compartment, wherein the interior surface of at least one of the plurality of microwells comprises a coating disposed thereon.

[0025] Aspect 19. The cell culture vessel of aspect 18, wherein the coating comprises a copolymer of acrylamide methacrylate and a photoreactive group, a hydrophilic polymer, a non-ionic surfactant, a protein, or combinations thereof.

[0026] Aspect 20. The cell culture vessel of aspect 18 or aspect 19, wherein the dialysis membrane comprises cellulose, cellulose acetate, polyamide, polysulfone (PSF), poly(ether sulfone) (PES), poly (hydroethyl methacrylate) (PHEMA), polyethylene glycol methacrylate (PEGMA), polyphosphobetaine methacrylate (PBMA), polysulfobetaine methacrylate (SBMA), olyvinylidene fluoride (PVDF), polymethyl methacrylate (PMMA), polyurethane (PU), poly(lactic acid) (PLA), or combinations thereof.SP24-149

[0027] Aspect 21. The cell culture vessel of any one of aspects 18 to 20, wherein the cell culture vessel comprises an open vessel and an insert, wherein the dialysis membrane comprises a bottom of the insert, and wherein the dialysis membrane is above a bottom of the open vessel.

[0028] Aspect 22. The cell culture vessel of aspect 21, wherein the insert further comprises a frame, the frame comprising a side wall and an edge, the dialysis membrane being attached to the frame.

[0029] Aspect 23. The cell culture vessel of aspect 22, wherein the cell culture vessel further comprises a well plate, the dialysis membrane being disposed in the well plate.

[0030] Aspect 24. The cell culture vessel of any one of aspect 21 to 23, wherein the dialysis membrane comprises a plurality of dialysis membranes.

[0031] Aspect 25. The cell culture vessel of any one of aspects 18 to 20, wherein the cell culture vessel comprises a closed vessel, the closed vessel comprising a bottom wall, atop surface opposite the bottom wall, and a plurality of side walls extending between the bottom wall and the top surface; thereby forming an internal cavity, wherein the dialysis membrane is disposed within the internal cavity.

[0032] Aspect 26. The cell culture vessel of aspect 25, the closed vessel further comprises a fence, the fence being disposed between the plurality of side walls and the dialysis membrane.

[0033] Aspect 27. The cell culture vessel of aspect 25 or aspect 26, wherein the cell culture vessel comprises a plurality of the microcavity compartments, a plurality of the media reservoir compartments, and a plurality of the dialysis membranes, each dialysis membrane separating a microcavity compartment from a media reservoir compartment.

[0034] Aspect 28. The cell culture vessel of aspect 27, wherein the closed vessel further comprises a first manifold disposed within the internal cavity, the first manifold having a first aperture and a second manifold disposed within the internal cavity, the second manifold having a second aperture, wherein the first manifold is fluidly coupled to the plurality of microcavity compartments and the second manifold is fluidly coupled to the plurality of media reservoir compartments.SP24-149

[0035] Aspect 29. The cell culture vessel of any one of aspects 18 to 28, wherein the upper aperture of each microwell is greater than or equal to 100 micrometers and less than or equal to 2000 micrometers.

[0036] Aspect 30. The cell culture vessel of any one of aspects 18 to 29, wherein a depth of each microwell is greater than or equal to 100 micrometers and less than or equal to 5000 micrometers.

[0037] Aspect 31. The cell culture vessel of any one of aspects 18 to 30, wherein the dialysis membrane comprises a molecular weight cut-off greater than or equal to 0.02 kD and less than or equal to 1000 kD.

[0038] Aspect 32. A method of making a dialysis membrane comprising disposing a dialysis membrane polymer onto a micropost tool, the micropost tool comprising a plurality of protrusions, each protrusion having a top surface and curing the dialysis membrane polymer to form the dialysis membrane, wherein the dialysis membrane comprises a plurality of microwells corresponding to the plurality of protrusions of the micropost tool, each microwell having an interior surface defining an upper aperture and a nadir, the dialysis membrane is porous and liquid permeable and the dialysis membrane comprises a pore size greater than or equal to 1 nanometer and less than or equal to 100 nanometers.

[0039] Aspect 33. The method of aspect 32, wherein the dialysis membrane polymer comprises cellulose, cellulose acetate, polyamide, polysulfone (PSF), poly(ether sulfone) (PES), poly (hydroethyl methacrylate) (PHEMA), poly(ethylene glycol methacrylate (PEGMA), polyphosphobetaine methacrylate (PBMA), polysulfobetaine methacrylate (SBMA), olyvinylidene fluoride (PVDF), polymethyl methacrylate (PMMA), polyurethane (PU), poly(lactic acid) (PLA), or combinations thereof.

[0040] Aspect 34. The method of aspect 32 or aspect 33, wherein the disposing the dialysis membrane polymer comprises direct casting, electrospinning, or thermal forming.

[0041] Aspect 35. The method of any one of aspect 32 to 34, wherein the top surface of each protrusion comprises a semi-circular shape, a pyramidal shape, or a rectangular shape.SP24-149

[0042] Aspect 36. The method of any one of aspects 32 to 35, wherein the upper aperture of each microwell is greater than or equal to 100 micrometers and less than or equal to 2000 micrometers.

[0043] Aspect 37. The method of any one of aspects 32 to 36, wherein a depth of each microwell is greater than or equal to 100 micrometers and less than or equal to 5000 micrometers.

[0044] It is to be understood that both the foregoing general description and the following detailed description describe various embodiments and are intended to provide an overview or framework for understanding the nature and character of the claimed subject matter. The accompanying drawings are included to provide a further understanding of the various embodiments, and are incorporated into and constitute a part of this specification. The drawings illustrate the various embodiments described herein, and together with the description serve to explain the principles and operations of the claimed subject matter.BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG. 1 depicts a cross-sectional view of a cell culture vessel, according to one or more embodiments of the present disclosure;

[0046] FIG. 2A depicts a cross-sectional view of a microwell, according to one or more embodiments of the present disclosure;

[0047] FIG. 2B depicts a cross-sectional view of a coated microwell, according to one or more embodiments of the present disclosure;

[0048] FIG. 3 depicts a cross-sectional view of a cell culture vessel comprising an open vessel, according to one or more embodiments of the present disclosure;

[0049] FIG. 4 depicts a cross-sectional view of a cell culture vessel comprising a well plate, according to one or more embodiments of the present disclosure;

[0050] FIG. 5 depicts a cross-sectional view of a cell culture vessel comprising a well plate and a plurality of dialysis membranes, according to one or more embodiments of the present disclosure;SP24-149

[0051] FIG. 6 depicts a cross-sectional view of a cell culture vessel comprising a closed vessel, according to one or more embodiments of the present disclosure;

[0052] FIG. 7 depicts a cross-sectional view of a cell culture vessel comprising a closed vessel and a plurality of dialysis membranes, according to one or more embodiments of the present disclosure;

[0053] FIG. 8 schematically depicts a cell culture vessel comprising a first manifold and a second manifold, according to one or more embodiments of the present disclosure; and

[0054] FIG. 9 depicts a micropost tool according to one or more embodiments of the present disclosure.DETAILED DESCRIPTION

[0055] Reference will now be made in detail to various embodiments of cell culture vessels comprising dialysis membranes and methods of making such dialysis membranes.

[0056] According to embodiments, the cell culture vessel comprises a microcavity compartment, a media reservoir compartment, and a dialysis membrane separating the microcavity compartment from the media reservoir compartment. The dialysis membrane may be porous and liquid permeable. The dialysis membrane may comprise a pore size greater than or equal to 1 nanometer and less than or equal to 100 nanometers. The dialysis membrane may comprise a plurality of microwells, each microwell having an interior surface defining an upper aperture and a nadir. The upper aperture may face the microcavity compartment.

[0057] According to embodiments, the cell culture vessel comprises a microcavity compartment, a media reservoir compartment, and a dialysis membrane separating the microcavity compartment from the media reservoir compartment. The dialysis membrane may be porous and liquid permeable. The dialysis membrane may comprise a plurality of microwells, each microwell having an interior surface defining an upper aperture and a nadir. The upper aperture may face the microcavity compartment. The interior surface of at least one of the plurality of microwells may comprise a coating disposed thereon.

[0058] According to embodiments, a method of making a dialysis membrane comprises disposing a dialysis membrane polymer onto a micropost tool, the micropost tool comprising a plurality of protrusions, each protrusion having atop surface. The method further comprisesSP24-149 curing the dialysis membrane polymer to form the dialysis membrane. The dialysis membrane comprises a plurality of microwells corresponding to the plurality of protrusions of the micropost tool, each microwell having an interior surface defining an upper aperture and a nadir. The dialysis membrane may be porous and liquid permeable. The dialysis membrane comprises a pore size greater than or equal to 1 nanometer and less than or equal to 100 nanometers.

[0059] Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes 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 embodiment. 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.

[0060] Directional terms as used herein - for example up, down, right, left, front, back, top, bottom - are made only with reference to the figures as drawn and are not intended to imply absolute orientation.

[0061] 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, nor that with any apparatus specific orientations be required. Accordingly, where a method claim does not actually recite an order to be followed by its steps, or that any apparatus claim does not actually recite an order or orientation to individual components, or it is not otherwise specifically stated in the claims or description that the steps are to be limited to a specific order, or that a specific order or orientation to components of an apparatus is not recited, it is in no way intended that an order or orientation be inferred, in any respect. This holds for any possible non-express basis for interpretation, including: matters of logic with respect to arrangement of steps, operational flow, order of components, or orientation of components; plain meaning derived from grammatical organization or punctuation, and; the number or type of embodiments described in the specification.

[0062] As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a” componentSP24-149 includes aspects having two or more such components, unless the context clearly indicates otherwise.

[0063] As described herein, challenges exist when growing 3D cell cultures in a conventional culture apparatus. For example, conventional cell growth substrates may be of either a solid construction or a porous mesh construction. The solid construction may trap all of the byproducts, both helpful and hurtful, as well as the nutrient formula in a single medium. This may require frequent changeover of the medium in order to reduce the presence of the hurtful byproducts. Such frequent replacement may decrease the efficiency and increase the cost of growing cells. The mesh construction may allow the byproducts to diffuse through the cell growths, which, while removing the hurtful byproducts, also allows the helpful byproducts to diffuse away from the cell growths, thereby slowing cell growth.

[0064] Disclosed herein are cell culture vessels that mitigate the aforementioned problems. Specifically, the cell culture vessels disclosed herein comprise a porous (e.g., pore size greater than or equal to 1 nanometer and less than or equal to 100 nanometers) and a liquid permeable dialysis membrane separating a microcavity compartment from a media reservoir compartment, which results in the diffusion of dissolved solutes and toxic byproducts from the microcavity compartment to the media reservoir compartment and the ability to remove and replace media from the media reservoir compartment without disturbing the 3D cell cultures and retained larger helpful byproducts.

[0065] Referring now to FIG. 1, a cell culture vessel 100 may comprise a microcavity compartment 110, a media reservoir compartment 120, and a dialysis membrane 130 separating the microcavity compartment 110 from the media reservoir compartment 120. Embodiments of cell culture vessels 100 described in more detail hereinbelow may be used for long term cell culture, low seeding density spheroid culture, organoid culture, extracellular vesicle production, and cell expansion, among other uses. In general, cells in a cell culture medium may be added to the microcavity compartment 110 and a medium containing nutrients may be added to the media reservoir compartment 120. The media in the media reservoir compartment 120 may contact the dialysis membrane 130 so that molecules may diffuse between the media in the media reservoir compartment 120 and the cell culture medium in the microcavity compartment 110. For example, low molecular weight dissolved solutes, waste products, and toxic byproducts may diffuse through the dialysis membrane 130 from the microcavity compartment 110 to the media reservoir compartment 120, while helpful byproducts, such asSP24-149 growth factors, may be retained within the microcavity compartment 110. The separation of the media in the media reservoir compartment 120 and the cell culture medium in the microcavity compartment 110 by the dialysis membrane 130 may allow media containing toxic byproducts to be removed from the cell culture vessel 100 without disturbing the cell cultures in the microcavity compartment 110.

[0066] In one or more embodiments, the dialysis membrane 130 may be porous and liquid permeable. As described herein, a membrane is “liquid permeable” when the membrane comprises pores that permit liquids to pass through the membrane. The pores of a liquid permeable dialysis membrane may be sized and shaped such that liquid may pass through the dialysis membrane.

[0067] In one or more embodiments, the dialysis membrane 130 may comprise a pore size greater than or equal to 1 nanometer (nm) and less than or equal to 100 nm. In one or more embodiments, the dialysis membrane 130 may comprise a pore size greater than or equal to 1 nm and less than or equal to 5 nm. In one or more embodiments, the dialysis membrane 130 may comprise a pore size less than or equal to 5 nm, less than or equal to 4 nm, less than or equal to 3 nm, or even less than or equal to 2 nm. For example, the dialysis membrane 130 may comprise a pore size greater than or equal to 1 nm and less than or equal to 100 nm, greater than or equal to 10 nm and less than or equal to 100 nm, greater than or equal to 20 nm and less than or equal to 100 nm, greater than or equal to 30 nm and less than or equal to 100 nm, greater than or equal to 40 nm and less than or equal to 100 nm, greater than or equal to 50 nm and less than or equal to 100 nm, greater than or equal to 60 nm and less than or equal to 100 nm, greater than or equal to 70 nm and less than or equal to 100 nm, greater than or equal to 80 nm and less than or equal to 100 nm, greater than or equal to 90 nm and less than or equal to 100 nm, greater than or equal to 1 nm and less than or equal to 90 nm, greater than or equal to 1 nm and less than or equal to 80 nm, greater than or equal to 1 nm and less than or equal to 70 nm, greater than or equal to 1 nm and less than or equal to 60 nm, greater than or equal to 1 nm and less than or equal to 50 nm, greater than or equal to 1 nm and less than or equal to 40 nm, greater than or equal to 1 nm and less than or equal to 30 nm, greater than or equal to 1 nm and less than or equal to 20 nm, greater than or equal to 1 nm and less than or equal to 10 nm, greater than or equal to 1 nm and less than or equal to 5 nm, greater than or equal to 1 nm and less than or equal to 4 nm, greater than or equal to 1 nm and less than or equal to 3 nm, greaterSP24-149 than or equal to 1 nm and less than or equal to 2 nm, or any range or combination of ranges formed from these endpoints.

[0068] As described herein, the “molecular weight cut-off’ of a membrane refers to the lowest molecular weight of the solute for which 90% of the solute is retained by the membrane. In one or more embodiments, the dialysis membrane 130 comprises a molecular weight cut-off greater than or equal to 0.02 kiloDaltons (kD) and less than or equal to 1000 kD. In one or more embodiments, the dialysis membrane 130 comprises a molecular weight cut-off greater than or equal to 20 kD and less than or equal to 200 kD. In one or more embodiments, the dialysis membrane 130 comprises a molecular weight cut-off greater than or equal to 20 kD and less than or equal to 50 kD. For example, the dialysis membrane 130 may comprise a molecular weight cut-off greater than or equal to 0.02 kD and less than or equal to 1000 kD, greater than or equal to 0. 1 kD and less than or equal to 1000 kD, greater than or equal to 0.5 kD and less than or equal to 1000 kD, greater than or equal to 1 kD and less than or equal to 1000 kD, greater than or equal to 10 kD and less than or equal to 1000 kD, greater than or equal to 20 kD and less than or equal to 1000 kD, greater than or equal to 30 kD and less than or equal to 1000 kD, greater than or equal to 40 kD and less than or equal to 1000 kD, greater than or equal to 100 kD and less than or equal to 1000 kD, greater than or equal to 300 kD and less than or equal to 1000 kD, greater than or equal to 500 kD and less than or equal to 1000 kD, greater than or equal to 700 kD and less than or equal to 1000 kD, greater than or equal to 900 kD and less than or equal to 1000 kD, greater than or equal to 0.02 kD and less than or equal to 900 kD, greater than or equal to 0.02 kD and less than or equal to 700 kD, greater than or equal to 0.02 kD and less than or equal to 500 kD, greater than or equal to 0.02 kD and less than or equal to 300 kD, greater than or equal to 0.02 kD and less than or equal to 200 kD, greater than or equal to 0.02 kD and less than or equal to 100 kD, greater than or equal to 0.02 kD and less than or equal to 90 kD, greater than or equal to 0.02 kD and less than or equal to 80 kD, greater than or equal to 0.02 kD and less than or equal to 70 kD, greater than or equal to 0.02 kD and less than or equal to 60 kD, greater than or equal to 0.02 kD and less than or equal to 50 kD, greater than or equal to 0.02 kD and less than or equal to 10 kD, greater than or equal to 0.02 kD and less than or equal to 1 kD, greater than or equal to 0.02 kD and less than or equal to 0.5 kD, greater than or equal to 0.02 kD and less than or equal to 0.1 kD, or any range or combination of ranges formed from these endpoints.SP24-149

[0069] Without intending to be bound by theory, when the pore size of the dialysis membrane 130 is greater than or equal to 1 nm and less than or equal to 100 nm, then toxic byproducts of cell growth, such as lactate and ammonia may pass through the dialysis membrane 130 and away from the cell culture, while helpful byproducts, such as autocrine cell growth factors may be retained by the dialysis membrane 130. Likewise, when the molecular weight cut-off of the dialysis membrane 130 is greater than or equal to 0.02 kD and less than or equal to 1000 kD, toxic byproducts of cell growth, such as lactate and ammonia may pass through the dialysis membrane 130 and away from the cell culture, while helpful byproducts, such as autocrine cell growth factors may be retained by the dialysis membrane 130.

[0070] The dialysis membrane 130 may be formed from any suitable material. For example, the dialysis membrane 130 may comprise cellulose, cellulose acetate, polyamide, polysulfone (PSF), poly(ether sulfone) (PES), poly (hydroethyl methacrylate) (PHEMA), poly(ethylene glycol methacrylate (PEGMA), polyphosphobetaine methacrylate (PBMA), polysulfobetaine methacrylate (SBMA), olyvinylidene fluoride (PVDF), polymethyl methacrylate (PMMA), polyurethane (PU), poly(lactic acid) (PLA), or combinations thereof.

[0071] Referring now to FIG. 2A, the dialysis membrane 130 may comprise a plurality of microwells 132. Each microwell 132 may have an interior surface 134 defining an upper aperture 136 and a nadir 138. As described herein, the term “nadir” refers to the point on the interior surface 134 of a microwell 132 that is furthest from the upper aperture 136. The upper aperture 136 may face the microcavity compartment 110.

[0072] The plurality of microwells 132 may have any suitable shape. In one or more embodiments, one or more of the plurality of microwells 132 may have a round bottom or a flat bottom. In some embodiments, one or more of the plurality of microwells 132 may have a hemispherical shape, a shape formed using part of a sphere, a frustum shape, a conical shape, a cylindrical shape, or any combination of these shapes. For example, a microwell 132 may have an upper portion having a cylindrical shape and a lower portion having a hemispherical shape. Each of the plurality of microwells 132 may be shaped to promote self-assembly of cells to form 3D cell cultures, aggregates, or spheroids. The plurality of microwells 132 may be shaped such that scaffold-free 3D cell cultures may form, where cells preferentially attach to each other versus the dialysis membrane 130.SP24-149

[0073] In one or more embodiments, the length of the upper aperture 136 of each microwell 132 may be greater than or equal to 100 micrometers (pm) and less than or equal to 2000 pm. As described herein, the “length” of the upper aperture 136 of a microwell 132 refers to the longest dimension of the upper aperture 136 passing through a center of the upper aperture 136. In embodiments where the upper aperture 136 is circular, the “length” of the upper aperture 136 is a diameter of the upper aperture 136. For example, the length of the upper aperture 136 of each microwell 132 may be greater than or equal to 100 pm and less than or equal to 2000 pm, greater than or equal to 300 pm and less than or equal to 2000 pm, greater than or equal to 500 pm and less than or equal to 2000 pm, greater than or equal to 700 pm and less than or equal to 2000 pm, greater than or equal to 900 pm and less than or equal to 2000 pm, greater than or equal to 1100 pm and less than or equal to 2000 pm, greater than or equal to 1300 pm and less than or equal to 2000 pm, greater than or equal to 1500 pm and less than or equal to 2000 pm, greater than or equal to 1700 pm and less than or equal to 2000 pm, greater than or equal to 1900 pm and less than or equal to 2000 pm, greater than or equal to 100 pm and less than or equal to 1800 pm, greater than or equal to 100 pm and less than or equal to 1600 pm, greater than or equal to 100 pm and less than or equal to 1400 pm, greater than or equal to 100 pm and less than or equal to 1200 pm, greater than or equal to 100 pm and less than or equal to 1000 pm, greater than or equal to 100 pm and less than or equal to 800 pm, greater than or equal to 100 pm and less than or equal to 600 pm, greater than or equal to 100 pm and less than or equal to 400 pm, greater than or equal to 100 pm and less than or equal to 200 pm, or any range or combination of ranges formed from these endpoints.

[0074] In one or more embodiments, the depth of each microwell 132 may be greater than or equal to 100 pm and less than or equal to 5000 pm. As described herein, the “depth” of a microwell 132 refers to a vertical distance from the nadir 138 to the upper aperture 136. For example, the depth of each microwell 132 may be greater than or equal to 100 pm and less than or equal to 5000 pm, greater than or equal to 500 pm and less than or equal to 5000 pm, greater than or equal to 1000 pm and less than or equal to 5000 pm, greater than or equal to 1500 pm and less than or equal to 5000 pm, greater than or equal to 2000 pm and less than or equal to 5000 pm, greater than or equal to 2500 pm and less than or equal to 5000 pm, greater than or equal to 3000 pm and less than or equal to 5000 pm, greater than or equal to 3500 pm and less than or equal to 5000 pm, greater than or equal to 4000 pm and less than or equal to 5000 pm, greater than or equal to 4500 pm and less than or equal to 5000 pm, greater than or equal to 100 pm and less than or equal to 4500 pm, greater than or equal to 100 pm and lessSP24-149 than or equal to 4000 pm. greater than or equal to 100 qm and less than or equal to 3500 qm, greater than or equal to 100 qm and less than or equal to 3000 qm, greater than or equal to 100 qm and less than or equal to 2500 qm, greater than or equal to 100 qm and less than or equal to 2000 qm, greater than or equal to 100 qm and less than or equal to 1500 qm, greater than or equal to 100 qm and less than or equal to 1000 qm, greater than or equal to 100 qm and less than or equal to 500 qm, or any range or combination of ranges formed from these endpoints.

[0075] Referring now to FIG. 2B, the interior surface 134 of at least one of the plurality of microwells 132 may comprise a coating 210 disposed thereon. The coating 210 may comprise a copolymer of acrylamide methacrylate and a photoreactive group, a hydrophilic polymer, a non-ionic surfactant, a protein or combinations thereof. Examples of hydrophilic polymers include, but are not limited to polyethylene glycol and polyvinyl alcohol. A nonlimiting example of a non-ionic surfactant is Pluronic® F-127 from Sigma-Aldrich®. Examples of suitable proteins include, but are not limited to, bovine serum albumin. The coating may have any composition that minimizes or even prevents the attachment of adherent cells to the coating. The coating may have a non-binding or low-binding chemistry to reduce the likelihood that cells adhere to the microwell 132.

[0076] The coating 210 may be formed by any suitable method. In one or more embodiments, the coating 210 may be physically adsorbed onto the dialysis membrane 130. In such embodiments, the coating 210 may comprise PHEMA or agarose. In one or more embodiments, a dialysis membrane 130 may be covalently modified by a surface -initiated atom transfer radical polymerization process, an in-situ polymerization process, a radical graft polymerization process, a carbodiimide free radical polymerization process, or a surface initiated activator regenerated activator regenerated by electron transfer atom transfer radical polymerization process, as described in A. Mollahosseini, et al. “Latest Advances in Zwitterionic Structures Modified Dialysis Membranes.” Materials Today Chemistry, 2020,15, 100027). In some embodiments, attachment chemistry described in U.S. Patent No. 5,002,582, the entirety of which is incorporated by reference herein, may be used to form the coating 210 on the dialysis membrane 130.

[0077] Referring now to FIG. 3, the cell culture vessel 100 may comprise an open vessel 310 and an insert 320. The dialysis membrane 130 may comprise the bottom of the insert 320. The insert 320 may be positioned in the open vessel 310. The dialysis membrane 130 may be above a bottom surface 312 of the open vessel 310. The open vessel 310 may have anySP24-149 suitable shape. For example, the open vessel 310 may have a polygonal shape, such as a rectangular shape or a square shape, an oval shape, a circular shape, or any other suitable closed shape. In one or more embodiments, the insert 320 may comprise a side wall 322 and an edge 324. The dialysis membrane 130 may be attached to the side wall 322. The side wall 322 may be attached to the edge 324. In one or more embodiments, the dialysis membrane 130 and the edge 324 may be attached to opposite ends of the side wall 322. In some embodiments, the edge 324 and the dialysis membrane 130 may be substantially parallel. As described herein, “substantially parallel” refers to an orientation within 10°, 5°, 2°, or even 1° of parallel. The edge 324 may hold the insert 320 in place in the open vessel 310. The edge 324 may also be used to handle the insert 320. The insert 320 may be removable from the open vessel 310 such that the insert 320 may be moved from the open vessel 310 to another open vessel (not depicted).

[0078] Still referring to FIG. 3, in one or more embodiments, the media reservoir compartment 120 may be between the insert 320 and the open vessel 310. The microcavity compartment 110 may be above the dialysis membrane 130. The microcavity compartment 110 may be at least partially defined by the dialysis membrane 130 and the side wall 322 of the insert 320. In one or more embodiments, media may be added to the open vessel 310 to a level where the media would contact the dialysis membrane 130. The insert 320 comprising the dialysis membrane 130 may be positioned in the open vessel 310 such that the media contacts the dialysis membrane 130. Cell culture medium may be added to the microcavity compartment 110 above the dialysis membrane 130. As toxic byproducts are produced by cell growth in the microcavity compartment 110 and diffuse through the dialysis membrane 130, media from the media reservoir compartment 120 may be removed from the open vessel 310. The media and cell culture medium may be added to or removed from the cell culture vessel 100 by any suitable means. For example, the media and cell culture medium may be added or removed by pouring, pipetting, pumping, or any other suitable means. In some embodiments, the insert 320 may be removed from the open vessel 310 so that media may be added to or removed from the media reservoir compartment 120.

[0079] Referring now to FIG. 4, the open vessel 310 may comprise a well plate 410. The dialysis membrane 130 may be disposed in the well plate 410. The well plate 410 may comprise any suitable number of wells 412. For example, the well plate 410 may comprise 6, 24, 48, 384, or any other suitable number of wells 412. In one or more embodiments, the insertSP24-149320 may be shaped to fit within one well 412 of the well plate 410. In such embodiments, the insert 320 may be moved from one well to another well of the well plate 410. In some embodiments, the dialysis membrane 130 may have a cross-sectional shape that is substantially the same as the cross sectional shape of the well 412 in which it is disposed. For example, the dialysis membrane 130 and the well 412 may both have a circular cross sectional shape in a horizontal cross section.

[0080] Still referring to FIG. 4, in one or more embodiments, the media reservoir compartment 120 may be within a well 412 of the well plate 410 between the well plate 410 and the insert 320. The microcavity compartment 110 may be at least partially defined by the dialysis membrane 130 and the side wall 322 of the insert 320.

[0081] Referring now to FIG. 5, the insert 520 may comprise a plurality of dialysis membranes 130. In one or more embodiments, the insert 520 may comprise an edge 524, a plurality of side walls 522 and a plurality of dialysis membranes 130. Each of the plurality of side walls 522 may be attached to the edge 524. Each of the plurality of dialysis membranes 130 may be attached to a side wall 522. The plurality of side walls 522 and the plurality of dialysis membranes 130 may be arranged such that each of the dialysis membranes 130 may be positioned in a well 412 of the well plate 410. In some embodiments, a dialysis membrane 130 may be positioned in each well 412 of the well plate 410.

[0082] Still referring to FIG. 5, the cell culture vessel 100 may comprise a plurality of media reservoir compartments 120 and a plurality of microcavity compartments 110. Each media reservoir compartment 120 may be positioned in a well 412 of the well plate 410. Each of the microcavity compartments 110 may be at least partially defined by a dialysis membrane 130 and one of the plurality of side walls 522 of the insert 520.

[0083] Referring now to FIG. 6, the cell culture vessel 100 may comprise a closed vessel 610. The closed vessel 610 may comprise a bottom wall 612, a top surface 614 opposite the bottom wall 612, and a plurality of side walls 616 extending between the bottom wall 612 and the top surface 614, thereby forming an internal cavity 618. In embodiments, the closed vessel 610 may comprise a flask. The dialysis membrane 130 may be disposed within the internal cavity 618. The closed vessel 610 may further comprise a fence 620. The fence 620 may be disposed between the plurality of side walls 616 and the dialysis membrane 130. In oneSP24-149 or more embodiments, the dialysis membrane 130 may be attached to one or more of the plurality of side walls 616.

[0084] In one or more embodiments, the media reservoir compartment 120 may be between the bottom wall 612 of the closed vessel 610 and the dialysis membrane 130. The microcavity compartment 110 may be between the dialysis membrane 130 and the top surface 614 of the closed vessel 610. In some embodiments, the microcavity compartment 110 may be at least partially enclosed by the dialysis membrane 130 and the fence 620. In some embodiments, the microcavity compartment 110 may be at least partially enclosed by the dialysis membrane 130, the fence 620, and one or more of the plurality of side walls 616.

[0085] Still referring to FIG. 6, the closed vessel 610 may comprise an opening 630. The opening 630 may be shaped so that media may be introduced into and removed from the closed vessel 610. The opening 630 may also be shaped such that a cap 640 or any other suitable device (e.g., a stopper) may be used to close the closed vessel 610.

[0086] In one or more embodiments, media may be added to the media reservoir compartment 120 of the closed vessel 610 to a level where the media contacts the dialysis membrane 130. Cell culture medium may be added to the microcavity compartment 110 above the dialysis membrane 130. As toxic byproducts are produced by cell growth in the microcavity compartment 110 and diffuse through the dialysis membrane 130, media from the media reservoir compartment 120 may be removed from the closed vessel 610. The media and cell culture medium may be added or removed from the closed vessel 610 by any suitable means. For example, the media and cell culture medium may each be added or removed by pouring, pipetting, pumping, or any other suitable means.

[0087] Referring now to FIG. 7, the closed vessel 610 may comprise a plurality of the microcavity compartments 110, a plurality of the media reservoir compartments 120, and a plurality of the dialysis membranes 130. Each dialysis membrane 130 may separate a microcavity compartment 110 from a media reservoir compartment 120. In one or more embodiments, the plurality of the dialysis membranes 130 may be stacked within the closed vessel 610. The number of dialysis membranes 130 in the closed vessel 610 is not necessarily limited. For example, the closed vessel 610 may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more dialysis membranes 130.SP24-149

[0088] Still referring to FIG. 7, the cell culture vessel 100 may comprise a plurality of trays 710. Each tray 710 may be positioned between dialysis membranes 130. Each tray 710 may be formed of a solid material and shaped such that a media reservoir compartment 120 may be positioned beneath each dialysis membrane 130 in the closed vessel 610. Each tray 710 may be attached to one or more of the plurality of side walls 616 of the closed vessel 610. In one or more embodiments, each dialysis membrane 130, each tray 710 and the bottom wall 612 of the closed vessel 610 may be substantially parallel. As described herein, “substantially parallel” refers to an orientation within 10°, 5°, 2°, or even 1° of parallel.

[0089] In one or more embodiments, media may be added to each of the media reservoir compartments 120 of the closed vessel 610 to a level where the media in each media reservoir compartment 120 contacts one of the plurality of dialysis membranes 130. Cell culture medium may be added to each of the plurality of microcavity compartments 110 above each dialysis membrane 130. As toxic byproducts are produced by cell growth in the plurality of microcavity compartments 110 and diffuse through each of the plurality of dialysis membranes 130, media from each of the plurality of media reservoir compartments 120 may be removed from the closed vessel 610. The media and cell culture medium may be added or removed from the closed vessel 610 by any suitable means. For example, the media and cell culture medium may each be added or removed by pipetting, pumping, or any other suitable means. In one or more embodiments, tubes, pipes, or other suitable conduits (not depicted) may enter the closed vessel 610 through the opening 630 and may be in fluid communication with the plurality of microcavity compartments 110 and the plurality of media reservoir compartments 120 to facilitate addition and removal of media or cell culture medium to the plurality of compartments in the closed vessel 610. In some embodiments, as described in more detail below with respect to FIG. 8, media or cell growth medium may be routed to the plurality of microcavity compartments 110 or the plurality of media reservoir compartments 120 through a manifold within the closed vessel 610.

[0090] Referring now to FIG. 8, the closed vessel 610 may further comprise a first manifold 810 and a second manifold 820. The first manifold 810 may be disposed within the internal cavity 618. The first manifold 810 may be fluidly coupled to the plurality of microcavity compartments 110. In the embodiment depicted in FIG. 8, the first manifold 810 is positioned on the left side of the closed vessel 610, and the microcavity compartments 110 are shaded to indicate that the first manifold 810 is in fluid communication with the pluralitySP24-149 of microcavity compartments 110. The first manifold 810 may comprise a first aperture 812. The first manifold 810 and the first aperture 812 may be shaped such that media, cell cultures, and other such material may be passed through the first aperture 812 and the first manifold 810 to or from the plurality of microcavity compartments 110.

[0091] In one or more embodiments, the second manifold 820 may also be disposed within the internal cavity 618. The second manifold 820 may be fluidly coupled to the plurality of media reservoir compartments 120. In the embodiment depicted in FIG. 8, the second manifold 820 is positioned on the right side of the closed vessel 610, and the media reservoir compartments 120 are shaded to indicate that the second manifold 820 is in fluid communication with the plurality of media reservoir compartments 120. The second manifold 820 may comprise a second aperture 822. The second manifold 820 and the second aperture 822 may be shaped such that media may be passed through the second manifold 820 and the second aperture 822 to or from the plurality of media reservoir compartments 120. In one or more embodiments, the first manifold 810 and the second manifold 820 may be separated by a divider 850. The divider 850 may be a solid wall positioned between the first manifold 810 and the second manifold 820 to prevent the exchange of fluid from first manifold 810 to the second manifold 820 and vice versa.

[0092] Embodiments of the dialysis membranes 130 described hereinabove may be made by following methods. In one or more embodiments, a method of making a dialysis membrane 130 may comprise disposing a dialysis membrane polymer onto a micropost tool. In one or more embodiments, the dialysis membrane polymer may comprise cellulose, cellulose acetate, polyamide, polysulfone (PSF), poly(ether sulfone) (PES), poly (hydroethyl methacrylate) (PHEMA), polyethylene glycol methacrylate (PEGMA), polyphosphobetaine methacrylate (PBMA), polysulfobetaine methacrylate (SBMA), olyvinylidene fluoride (PVDF), polymethyl methacrylate (PMMA), polyurethane (PU), poly(lactic acid) (PLA), or combinations thereof.

[0093] An embodiment of a micropost tool is depicted in FIG. 9. Referring now to FIG. 9, the micropost tool 900 may comprise a plurality of protrusions 910, each protrusion 910 having a top surface 912. The micropost tool 900 may be formed from any suitable material. For example, the micropost tool 900 may be formed from silicone or a metal, such as nickel. The micropost tool 900 may be formed by any suitable process, such as micromachining. The protrusions 910, and the top surface 912 of each protrusion 910 may be shaped such thatSP24-149 microwells 132 of the desired shape and dimensions may be formed on the dialysis membrane 130. Embodiments of shapes and dimensions for the microwells 132 are described in detail hereinabove.

[0094] Disposing the dialysis membrane polymer onto the micropost tool 900 may comprise direct casting, electrospinning, or thermal forming. Direct casting may comprise dissolving the dialysis membrane polymer into a solvent. The solution may be degassed under vacuum and then spin coated onto the micropost tool 900. The dialysis membrane polymer may be cured to form the dialysis membrane 130. The dialysis membrane 130 may be removed from the micropost tool 900. In one or more embodiments, the dialysis membrane 130 may be washed, for example, soaked in alcohol, and subsequently dried.

[0095] In some embodiments, the dialysis membrane polymer may be disposed onto the micropost tool 900 by electrospinning. The dialysis membrane polymer may be cured to form the dialysis membrane 130. The dialysis membrane 130 may be removed from the micropost tool 900. In one or more embodiments, the dialysis membrane 130 may be washed, for example, soaked in alcohol, and subsequently dried.

[0096] In one or more embodiments, the dialysis membrane polymer may be in the shape of a membrane without microwells. The dialysis membrane polymer may be disposed onto the micropost tool 900 and heated in a thermal forming process to form the dialysis membrane 130 comprising a plurality of microwells 132. In such embodiments, the micropost tool 900 may be a metal micropost tool 900, such as a nickel micropost tool 900.

[0097] The present disclosure is directed to various embodiments of cell culture vessels comprising dialysis membranes and methods of making such dialysis membranes. The cell culture vessel may comprise a microcavity compartment, a media reservoir compartment, and a dialysis membrane separating the microcavity compartment from the media reservoir compartment. The dialysis membrane may be porous and liquid permeable, which may result in the diffusion of dissolved solutes and toxic byproducts from the microcavity compartment to the media reservoir compartment and the ability to remove and replace media from the media reservoir compartment without disturbing the 3D cell cultures and retained larger helpful byproducts.

[0098] It will be apparent to those skilled in the art that various modifications and variations can be made to the embodiments described herein without departing from the spiritSP24-149 and scope of the claimed subject matter. Thus, it is intended that the specification cover the modifications and variations of the various embodiments described herein provided such modification and variations come within the scope of the appended claims and their equivalents.

Claims

SP24-149CLAIMS1. A cell culture vessel comprising: a microcavity compartment; a media reservoir compartment; and a dialysis membrane separating the microcavity compartment from the media reservoir compartment, wherein: the dialysis membrane is porous and liquid permeable; the dialysis membrane comprises a pore size greater than or equal to 1 nanometer and less than or equal to 100 nanometers; and the dialysis membrane comprises a plurality of microwells, each microwell having an interior surface defining an upper aperture and a nadir, the upper aperture facing the microcavity compartment.

2. The cell culture vessel of claim 1, wherein the pore size of the dialysis membrane is greater than or equal to 1 nanometer and less than or equal to 5 nanometers.

3. The cell culture vessel of claim 1 or claim 2, wherein the dialysis membrane comprises a molecular weight cut-off greater than or equal to 0.02 kD and less than or equal to 1000 kD.

4. The cell culture vessel of claim 3, wherein the molecular weight cut-off of the dialysis membrane is greater than or equal to 20 kD and less than or equal to 200 kD.

5. The cell culture vessel of any one of claims 1 to 4, wherein the dialysis membrane comprises cellulose, cellulose acetate, polyamide, polysulfone (PSF), poly(ether sulfone) (PES), poly (hydroethyl methacrylate) (PHEMA), poly(ethylene glycol methacrylate (PEGMA), polyphosphobetaine methacrylate (PBMA), polysulfobetaine methacrylate (SBMA), olyvinylidene fluoride (PVDF), polymethyl methacrylate (PMMA), polyurethane (PU), poly(lactic acid) (PLA), or combinations thereof.

6. The cell culture vessel of any one of claims 1 to 5, wherein the cell culture vessel comprises an open vessel and an insert, wherein the dialysis membrane comprises a bottom of the insert, and wherein the dialysis membrane is above a bottom surface of the open vessel.SP24-1497. The cell culture vessel of claim 6. wherein the insert further comprises a side wall and an edge, the dialysis membrane being attached to the side wall.

8. The cell culture vessel of claim 7, wherein the open vessel comprises a well plate, the dialysis membrane being disposed in the well plate.

9. The cell culture vessel of any one of claims 6 to 8, wherein the insert comprises a plurality of dialysis membranes.

10. The cell culture vessel of any one of claims 1 to 5, wherein the cell culture vessel comprises a closed vessel, the closed vessel comprising: a bottom wall; a top surface opposite the bottom wall; and a plurality of side walls extending between the bottom wall and the top surface; thereby forming an internal cavity, wherein: the dialysis membrane is disposed within the internal cavity.

11. The cell culture vessel of claim 10, wherein the closed vessel further comprises a fence, the fence being disposed between the plurality of side walls and the dialysis membrane.

12. The cell culture vessel of claim 10 or claim 11, wherein the closed vessel comprises a plurality of the microcavity compartments, a plurality of the media reservoir compartments, and a plurality of the dialysis membranes, each dialysis membrane separating a microcavity compartment from a media reservoir compartment.

13. The cell culture vessel of claim 12, wherein the closed vessel further comprises: a first manifold disposed within the internal cavity, the first manifold having a first aperture; and a second manifold disposed within the internal cavity, the second manifold having a second aperture, wherein: the first manifold is fluidly coupled to the plurality of microcavity compartments and the second manifold is fluidly coupled to the plurality of media reservoir compartments.SP24-14914. The cell culture vessel of any one of claims 1 to 13, wherein the upper aperture of each microwell is greater than or equal to 100 micrometers and less than or equal to 2000 micrometers.

15. The cell culture vessel of any one of claims 1 to 14, wherein a depth of each microwell is greater than or equal to 100 micrometers and less than or equal to 5000 micrometers.

16. The cell culture vessel of any one of claims 1 to 15, wherein the interior surface of at least one of the plurality of microwells comprises a coating disposed thereon.

17. The cell culture vessel of claim 16, wherein the coating comprises a copolymer of acrylamide methacrylate and a photoreactive group, a hydrophilic polymer, a non-ionic surfactant, a protein, or combinations thereof.

18. A cell culture vessel comprising: a microcavity compartment; a media reservoir compartment; and a dialysis membrane separating the microcavity compartment from the media reservoir compartment, wherein: the dialysis membrane is porous and liquid permeable; and the dialysis membrane comprises a plurality of microwells, each microwell having an interior surface defining an upper aperture and a nadir, the upper aperture facing the microcavity compartment, wherein the interior surface of at least one of the plurality of microwells comprises a coating disposed thereon.

19. The cell culture vessel of claim 18, wherein the coating comprises a copolymer of acrylamide methacrylate and a photoreactive group, a hydrophilic polymer, a non-ionic surfactant, a protein, or combinations thereof.

20. The cell culture vessel of claim 18 or claim 19, wherein the dialysis membrane comprises cellulose, cellulose acetate, polyamide, polysulfone (PSF), poly(ether sulfone) (PES), poly (hydroethyl methacrylate) (PHEMA), poly(ethylene glycol methacrylate (PEGMA), polyphosphobetaine methacrylate (PBMA), polysulfobetaine methacrylateSP24-149(SBMA), olyvinylidene fluoride (PVDF), polymethyl methacrylate (PMMA), polyurethane (PU), poly(lactic acid) (PLA), or combinations thereof.

21. The cell culture vessel of any one of claims 18 to 20, wherein the cell culture vessel comprises an open vessel and an insert, wherein the dialysis membrane comprises a bottom of the insert, and wherein the dialysis membrane is above a bottom of the open vessel.

22. The cell culture vessel of claim 21, wherein the insert further comprises a frame, the frame comprising a side wall and an edge, the dialysis membrane being attached to the frame.

23. The cell culture vessel of claim 22, wherein the cell culture vessel further comprises a well plate, the dialysis membrane being disposed in the well plate.

24. The cell culture vessel of any one of claims 21 to 23, wherein the dialysis membrane comprises a plurality of dialysis membranes.

25. The cell culture vessel of any one of claims 18 to 20, wherein the cell culture vessel comprises a closed vessel, the closed vessel comprising: a bottom wall; a top surface opposite the bottom wall; and a plurality of side walls extending between the bottom wall and the top surface; thereby forming an internal cavity, wherein: the dialysis membrane is disposed within the internal cavity.

26. The cell culture vessel of claim 25, the closed vessel further comprises a fence, the fence being disposed between the plurality of side walls and the dialysis membrane.

27. The cell culture vessel of claim 25 or claim 26, wherein the cell culture vessel comprises a plurality of the microcavity compartments, a plurality of the media reservoir compartments, and a plurality of the dialysis membranes, each dialysis membrane separating a microcavity compartment from a media reservoir compartment.

28. The cell culture vessel of claim 27, wherein the closed vessel further comprises:SP24-149 a first manifold disposed within the internal cavity, the first manifold having a first aperture; and a second manifold disposed within the internal cavity, the second manifold having a second aperture, wherein: the first manifold is fluidly coupled to the plurality of microcavity compartments and the second manifold is fluidly coupled to the plurality of media reservoir compartments.

29. The cell culture vessel of any one of claims 18 to 28, wherein the upper aperture of each microwell is greater than or equal to 100 micrometers and less than or equal to 2000 micrometers.

30. The cell culture vessel of any one of claims 18 to 29, wherein a depth of each microwell is greater than or equal to 100 micrometers and less than or equal to 5000 micrometers.

31. The cell culture vessel of any one of claims 18 to 30, wherein the dialysis membrane comprises a molecular weight cut-off greater than or equal to 0.02 kD and less than or equal to 1000 kD.

32. A method of making a dialysis membrane comprising: disposing a dialysis membrane polymer onto a micropost tool, the micropost tool comprising a plurality of protrusions, each protrusion having a top surface; and curing the dialysis membrane polymer to form the dialysis membrane, wherein: the dialysis membrane comprises a plurality of microwells corresponding to the plurality of protrusions of the micropost tool, each microwell having an interior surface defining an upper aperture and a nadir; the dialysis membrane is porous and liquid permeable; and the dialysis membrane comprises a pore size greater than or equal to 1 nanometer and less than or equal to 100 nanometers.

33. The method of claim 32, wherein the dialysis membrane polymer comprises cellulose, cellulose acetate, polyamide, polysulfone (PSF), poly(ether sulfone) (PES), poly (hydroethyl methacrylate) (PHEMA), polyethylene glycol methacrylate (PEGMA), polyphosphobetaine methacrylate (PBMA), polysulfobetaine methacrylate (SBMA), olyvinylidene fluorideSP24-149(PVDF), polymethyl methacrylate (PMMA), polyurethane (PU), poly(lactic acid) (PLA), or combinations thereof.

34. The method of claim 32 or claim 33, wherein the disposing the dialysis membrane polymer comprises direct casting, electrospinning, or thermal forming.

35. The method of any one of claims 32 to 34, wherein the top surface of each protrusion comprises a semi-circular shape, a pyramidal shape, or a rectangular shape.

36. The method of any one of claims 32 to 35, wherein the upper aperture of each microwell is greater than or equal to 100 micrometers and less than or equal to 2000 micrometers.

37. The method of any one of claims 32 to 36, wherein a depth of each microwell is greater than or equal to 100 micrometers and less than or equal to 5000 micrometers.

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