System and method for 3D tissue culture

Abstract

The present disclosure relates to cell culture vessels and methods of using the same that are capable of providing a laminar flow of cell culture media to cells in the cell culture vessels. The cell culture vessels include a plurality of wells in which cells reside as media moves across the top thereof. The cell culture system is further enclosed to maintain sterility. The media can be constantly renewed within the cell culture vessel, can be recirculated within the cell culture vessel, or a combination of both. Circulation of media around the cells residing therein can be particularly useful for 3D tissue culture, such as for producing spheroids or organoids.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 600,364, filed November 17, 2023, pursuant to 35 USC §119, the contents of which are used as the basis and are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure relates to systems and methods for establishing three-dimensional cell cultures in vitro. Background Technology

[0004] Compared to traditional monolayer culture models, 3D cell culture models better simulate the in vivo environment. The physiology of cells organizing themselves into 3D structures (such as spheroids or organoids) allows for excellent reproduction of the cell's natural environment. Many 3D cell culture methods for forming spheroids have been established, although these methods have been hampered by a lack of reproducibility, a wide distribution of the final spheroid size, and limitations on the number of spheroids that can be produced per container. Spheroid generation involves a high level of complexity and cost, and uniformity and reproducibility across spheroids are prerequisites for those skilled in the art to value the resulting data in order to provide a truly valuable in vivo environmental model.

[0005] Therefore, alternative systems and methods are needed to address the potential complexity and cost issues, which may present challenges in spheroid formation. Summary of the Invention

[0006] A first aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container for culturing cells, the container comprising: a top plate, a bottom plate, and at least one sidewall connecting the top plate to the bottom plate, wherein the top plate, the bottom plate, and the at least one sidewall define an internal volume; at least one well plate disposed within the internal volume of the container, the well plate comprising a plurality of microcavities for receiving cells, spheroids, or organoids; an inlet port extending into the internal volume at a first end of the internal volume; and an outlet port extending into the internal volume at a second end of the internal volume opposite to the first end, wherein: the well plate is positioned between the inlet port and the outlet port; and liquid culture medium introduced into the internal volume at the inlet port flows through the well plate and is extracted from the internal volume at the outlet port.

[0007] A second aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to the first aspect, wherein the plurality of microcavities comprise concave recesses in the upper surface of the base plate.

[0008] A third aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to the first aspect, wherein the inlet port is positioned toward a first end of the top plate and is configured to provide laminar flow of liquid culture medium through the plurality of orifices to the outlet port.

[0009] A fourth aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to the first aspect, wherein the inlet port is operatively connected to at least one nozzle, and wherein one end of the at least one nozzle is disposed above the base plate within the internal volume.

[0010] A fifth aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container as described in the fourth aspect, wherein at least two nozzles are aligned linearly across the width of the base plate.

[0011] The sixth aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to the fifth aspect, wherein at least eight nozzles are aligned across the width of the base plate.

[0012] A seventh aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to a fourth aspect, wherein the at least one nozzle is configured to provide a flow of liquid culture medium at or near the top plate of the orifice plate.

[0013] The eighth aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to the fourth aspect, which further comprises: an inlet conduit operatively connected to the inlet port; and a pump operatively connected to the inlet conduit.

[0014] A ninth aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to the first aspect, wherein the outlet port is operatively connected to at least one nozzle, and wherein one end of the at least one nozzle is disposed above the base plate within the internal volume.

[0015] The tenth aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container as described in the ninth aspect, wherein at least two nozzles are aligned linearly across the width of the base plate.

[0016] The 11th aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container as described in the 10th aspect, wherein at least eight nozzles are aligned across the width of the base plate.

[0017] The 12th aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to the 9th aspect, wherein the at least one nozzle is configured to remove liquid culture medium from the upper surface of the orifice plate.

[0018] The 13th aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to the 9th aspect, further comprising: an outlet conduit operatively connected to the outlet port; and a pump operatively connected to the outlet conduit.

[0019] The 14th aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to the 1st aspect, wherein at least a portion of the inner surface of each of the plurality of holes comprises a polymer coating.

[0020] A 15th aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container as described in aspect 14, which further comprises a screen disposed above the perforated plate.

[0021] The 16th aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to the 15th aspect, wherein the mesh screen is attached to the base plate, the at least one sidewall, or both.

[0022] The 17th aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to the 15th aspect, wherein the mesh screen comprises a magnetic or ferromagnetic portion such that a magnetic field placed beneath the perforated plate fixes the mesh screen above at least one of the plurality of microcavities.

[0023] The 18th aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to the 15th aspect, wherein the mesh screen comprises an opening with a diagonal length greater than or equal to 25 µm and less than or equal to 500 µm.

[0024] A 19th aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to a 15th aspect, wherein each opening in the mesh is larger than the diameter of a cell in at least one pore of the plurality of microcavities and smaller than the diameter of the spheroid or organoid of interest.

[0025] A 20th aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container as described in aspect 15, wherein the mesh screen further comprises a solid frame surrounding its periphery.

[0026] A 21st aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to a 15th aspect, wherein the mesh screen further comprises a polymer coating, wherein the polymer coating is the same as the polymer coating of the plurality of microcavities, wherein the polymer coating of the mesh screen inhibits cell adhesion to the mesh plate.

[0027] A 22nd aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to a 21st aspect, wherein the polymer coating of the mesh screen and the plurality of microcavities is selected from UV-crosslinkable PEG, poly(2-hydroxyethyl methacrylate), 2-methacryloyloxyethyl phosphorylcholine polymer, or agarose.

[0028] A 23rd aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to a 15th aspect, wherein the screen plate comprises a polymer coating, wherein the polymer coating is different from the polymer coating of the plurality of microcavities, wherein the polymer coating of the screen inhibits cell attachment to the screen.

[0029] The 24th aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to the 15th aspect, wherein the mesh screen is capable of being dissolved by enzymatic digestion.

[0030] A 25th aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to the first aspect, which further comprises a pump operatively connected to the inlet port and configured to pump a liquid culture medium through the inlet port onto the well plate.

[0031] A 26th aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to a 25th aspect, wherein the pump is operatively connected to the outlet port and configured to draw the liquid culture medium from the well plate through the outlet port.

[0032] A 27th aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to a 26th aspect, wherein the pump is configured to recirculate the liquid culture medium from the outlet port to the inlet port.

[0033] A 28th aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to the first aspect, which further comprises a pump operatively connected to the outlet port and configured to draw the cell culture medium from the well plate through the outlet port.

[0034] A 29th aspect of this disclosure, alone or in combination with any other aspect herein, relates to a method for replacing a liquid culture medium in a container according to the first aspect, the method comprising adding the liquid culture medium through the inlet port.

[0035] A 30th aspect of this disclosure, alone or in combination with any other aspect herein, relates to the method according to aspect 29, wherein the liquid culture medium is drawn through the outlet port.

[0036] A 31st aspect of this disclosure, alone or in combination with any other aspect herein, relates to a method for three-dimensional tissue culture, the method comprising: placing cells, spheroids, or organoids in a microcavity of a container according to the first aspect; allowing a liquid culture medium to flow into the container through the inlet port, wherein the liquid culture medium contacts the cells or spheroids; and withdrawing the liquid culture medium through the outlet port.

[0037] A 32nd aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to a 31st aspect, wherein the liquid culture medium flows into the inlet port at a first flow rate.

[0038] A 33rd aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to a 32nd aspect, wherein the liquid culture medium is drawn at the outlet port at a second flow rate.

[0039] A 34th aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container as described in aspect 33, wherein the first flow and the second flow are the same.

[0040] A 35th aspect of this disclosure, alone or in combination with any other aspect herein, relates to a container according to a 33rd aspect, wherein the first flow rate and the second flow rate establish laminar flow of the liquid culture medium through the orifice plate.

[0041] Additional features and advantages of the 3D tissue culture containers and methods of using described herein will be set forth in the detailed description below, and these features and advantages will be apparent in part from the description or will be recognized by those skilled in the art through practice of the embodiments described herein, including the detailed description below, the claims, and the drawings.

[0042] It should 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 characteristics of the claimed subject matter. The accompanying drawings are included to provide a further understanding of the various embodiments, and are incorporated in and form a part of this specification. The drawings illustrate the various embodiments described herein and, together with the description, serve to explain the principles and operation of the claimed subject matter. Attached Figure Description

[0043] Figure 1 An elevated side-view of an embodiment of the present disclosure is shown.

[0044] Figure 2A It shows Figure 1 This is part of an embodiment.

[0045] Figure 2B It shows Figure 1 A separate side view of the base plate of the embodiment.

[0046] Figure 3 It shows an additional component for circulating culture medium. Figure 1 A top view of an embodiment.

[0047] Figure 4 The following is shown: A container configured for replacing and / or recirculating the culture medium within the vessel. Figure 1 Examples of implementations.

[0048] Figure 5 The image shows a comparison between a reticulated plate (left) and multiple cellular spheres in multiple microcavities, each microcavity having a sphere at its bottom.

[0049] Figure 6 The cumulative EV yields from MSCs cultured for 2 days in 2D (the endpoint) and 14 days in 3D are shown.

[0050] Figure 7 The image shows bright-field images of MSCs in 2D on day 2 (top) and bright-field images of spheroids harvested on day 14 (bottom), taken under a microscope with a magnification degree set to 4X.

[0051] Figure 8 The results showed no statistically significant differences in cell viability (left) and EV diameter (right) between mesenchymal stem cells cultured for 2 days in 2D and 14 days in 3D (data showed mean ± standard deviation, n = 3). Detailed Implementation

[0052] Reference will now be made in detail to embodiments of the containers described herein, examples of which are shown in the accompanying drawings. Wherever possible, the same reference numerals will be used throughout the drawings to refer to the same or similar parts. In some aspects, this disclosure relates to a cell culture container including a top plate, a bottom plate, and at least one sidewall connecting the top plate to the bottom plate. The top plate, the bottom plate, and the at least one sidewall define an internal volume. The container also includes a perforated plate disposed within the internal volume of the container, the perforated plate having a plurality of polymer pores thereon for receiving cells or spheroids. The container further includes an inlet port extending into the internal volume at a first end of the internal volume; and an outlet port extending into the internal volume at a second end of the internal volume opposite to the first end. The perforated plate is positioned between the inlet port and the outlet port. Liquid culture medium (such as tissue culture medium) may be introduced into the internal volume at the inlet port, allowed to flow through the perforated plate, and then extracted from the internal volume at the outlet port. This article will describe in further detail various embodiments of containers for culturing cells and their methods of use with reference to the accompanying drawings.

[0053] In this document, a range may be expressed as from “about” a specific value and / or to “about” another specific value. Another embodiment of expressing such a range includes from one specific value and / or to another specific value. Similarly, when a value is expressed as an approximation using the antecedent “about,” it should be understood that a specific value forms another embodiment. It should be further understood that the endpoints of each range are significant relative to and independent of the other endpoint.

[0054] As used herein, directional terms (e.g., up, down, right, left, front, back, top, bottom) refer only to the descriptions in the accompanying drawings and are not intended to imply absolute orientation.

[0055] Unless otherwise expressly stated, it is never intended to interpret any method set forth herein as requiring its steps to be performed in a particular order, nor is it intended to require any particular orientation of any device. Therefore, it is never intended to infer any order or orientation in any respect where a method claim does not actually describe the order in which its steps are followed, or where any device claim does not actually describe the order or orientation of individual components, or where the claims or description do not otherwise specifically specify that the steps are limited to a particular order, or where a particular order or orientation of the components of the device is not described. This applies to any possible non-expressive basis for interpretation, including: logical questions relating to the arrangement of steps, the flow of operations, the order of components, or the orientation of components; simple meanings derived from grammatical organization or punctuation; and the number or type of embodiments described in the description.

[0056] As used herein, unless the context explicitly indicates otherwise, the singular forms “a / an” and “the” include plural indicators. Thus, for example, unless the context explicitly indicates otherwise, a reference to a “a / an” component includes aspects having two or more such components.

[0057] This disclosure relates to systems and methods for culturing cells. In some aspects, the systems and methods relate to 3D cell culture. It will be understood that while the examples of work described herein may involve the consistent generation of spheroids, the systems and methods can be readily applied to other aspects of culturing cells and / or tissues. The systems and methods described herein are agnostic to many cell culture vessels; provide multiple modes for forming and maintaining 3D cultures; are scalable to provide closed-system perfusion with in-line sampling ports that do not contact the cell culture area; are suitable for mechanical agitation / dynamic culture medium movement; and allow collection of cell conditioned media without disturbing 3D culture.

[0058] In some aspects, this disclosure relates to systems for culturing cells. As understood in the art, cultured cells are susceptible to microbial infection, and therefore, it is desirable to provide an isolated or enclosed environment for the cultured cells. For access to or even placement of cells, the enclosure requires an access point or operable port or removable wall. In some aspects, the access point should be sealable or form a seal with the enclosure to prevent unwanted air from entering the interior. In some aspects, the enclosure comprises a container. In some aspects, the container is a sealable container.

[0059] In some aspects, the container needs to be arranged such that cells can reside in the cell-receiving surface. In some aspects, the container has a flat surface serving as a base, which in turn allows cells within the cell-receiving surface to remain horizontally or orally oriented. In some aspects, the cell-receiving surface is part of the inner surface of the base. While the shape of the container is not necessarily limited, for example, the base wall can be considered a rectangular prism with horizontally oriented rectangular faces, its top surface forming the inner surface of the container, and its base being flat or partially flat to allow the container to rest on a horizontal surface. The base can be further adjusted according to desired conditions. For example, rounding the surface on which the container rests would allow for the application of rocking motion. It will also be understood that the cell-receiving surface need not be a direct part of the base wall, but can be an insulated connection or stationary substrate in contact with the inner portion of the base wall. It will be apparent that the cell-receiving surface can have a rounded bottom, a square bottom, or any other shape of bottom. In some aspects, the container can include stacked cell-receiving surfaces, wherein at least one second cell-receiving surface is stacked or placed above a first cell-receiving surface. It is understood that doing so can improve scalability. In some respects, the container is configured to be stackable with another container.

[0060] In some aspects, the container has two ends: a first end and a second end opposite the first end. The two ends are located at opposite walls or points on the container and are configured such that fluid entering from one port passes over the cell-receiving surface before being able to flow out through the other port. While the inlet can be located on the end wall of the container, this is not mandatory. For example, the top plate of the container may include one or more of the ports configured to be located on one side of the cell-receiving surface. Similarly, ports may extend through the bottom plate and be configured in a similar manner. In some aspects, the distal port may be located between the wall of the first end and the cell-receiving portion of the surface. Similarly, the port may be located between the wall of the second end and the cell-receiving portion of the surface. In some aspects, one port may serve as an inlet toward the first end, and the other port may serve as an outlet toward the second end, and vice versa. Such an arrangement can thereby allow or provide cell culture medium to flow through the container between the inlet and the outlet.

[0061] In various aspects, a cell-receiving surface is a microcavity, pore, or recess within the base of a container in which cells can settle. In some aspects, the container includes a first horizontal end, a second horizontal end opposite the first horizontal end, and a cell-receiving surface having an inlet port and an outlet port located therebetween, the inlet port being positioned between the pore / recess and the first horizontal end, and the outlet port being positioned between the pore / recess and the second horizontal end.

[0062] In some respects, the cell-receiving surface includes multiple microcavities, pores, or depressions. This allows for the isolation of cells within the container. For example, as illustrated in the examples herein, the container can be used to generate spheroids or organoids. The presence of isolating microcavities, pores, or depressions within the cell-receiving surface allows for the generation of multiple independent 3D cultures within the same container. It is understood that the microcavities, pores, or depressions can be concave depressions on the cell-receiving surface.

[0063] In some aspects, the inlet port is positioned toward a first horizontal end and configured to provide a flow of liquid culture medium through one or more holes in the cell receiving surface of the base plate to an outlet. In some aspects, the outlet port is positioned toward a second end and configured to receive a flow of liquid culture medium from the cell receiving surface. It is understood that the inlet and outlet ports may be reversed. It is also understood that a certain volume of liquid culture medium fills or at least partially fills the container. In these aspects, it is understood that not all of the supplied liquid culture medium will enter the cell receiving surface, and the culture medium will continue to flow through the container.

[0064] Reference will now be made in detail to embodiments of the container described herein, examples of which are shown in the accompanying drawings. Wherever possible, the same reference numerals will be used throughout the drawings to refer to the same or similar parts. One embodiment of the container is shown in… Figure 1 In the middle, and throughout the entire document, it is generally indicated by the figure reference 10. Figure 1 An elevation side view of an embodiment of the container 10 of this disclosure, including a container body 12, is shown. The container body 12 includes a first end wall 14 and a second end wall 15 opposite to the first end wall 14. Side walls 17 and 18 extend between the first end wall 14 and the second end wall 15. In some embodiments, the first end wall 14 and the second end wall 15, or portions thereof, are substantially parallel, and a pair of side walls 17 and 18, or portions thereof, are substantially parallel, thereby forming a slightly rectangular perimeter 19 of the cell culture chamber 11. Although a rectangular perimeter 19 is shown, any suitable perimeter shape can be used. The container 10 also includes an inlet port 20, an outlet port 30, a bottom plate 40, a top plate 50, and a cap 60 optionally threadedly connected to a discharge port 61 formed in the second end wall 15. The inlet port 20 and the outlet port 30 are further operatively connected to one or more culture medium dispensers 25, 35, respectively. Culture medium dispensers 25, 35 may include one or more nozzles 26, 36, which may be positioned close to the base plate 40 to allow cell culture medium to flow through the inlet port 20 and the inlet culture medium dispenser 25 and the inlet nozzle 26, across the base plate 40, and then be drawn from the container via the outlet nozzle 36 and the outlet culture medium dispenser 35 and the outlet port 30. One or more nozzles 26, 36 may be linearly aligned across the width of the container 10. There may be two, three, four, five, six, seven, eight, or more nozzles linearly aligned across the width of the container 10. Cells (not depicted) for culture may be retained or placed on the base plate 40 and between the inlet nozzle 26 and the outlet nozzle 36. The inlet port 20 may be further operatively connected to the inlet conduit 21, and the outlet port 30 may similarly be connected to the outlet conduit 31 to allow liquid inflow and outflow without damaging the cell culture chamber 11 or exposing it to air and potential contaminants therein. As depicted, inlet port 20 and outlet port 30 are located on top plate 50, although in other embodiments not depicted, they may be located on sidewalls 17, 18 or first endwall 14 and second endwall 15.

[0065] Turning Figure 2A and 2B An enlarged cross-sectional view of the cell culture chamber of the container is depicted. Figure 2A ) and side view of base plate 40 ( Figure 2B Nozzles 26 and 36 extend from the inlet culture medium dispenser 25 and the outlet culture medium dispenser 35 toward the base plate 40, respectively. Figure 2AAs seen, multiple inlet nozzles 26 allow the inlet culture medium dispenser 25 to allow liquid to flow across the entire width of the cell culture chamber 11 into the cell culture chamber.

[0066] The base plate 40 includes a cell culture medium plate 41 positioned between the inlet nozzle 26 and the outlet nozzle 36. The cell culture medium plate 41 may include a well plate 42 comprising a plurality of microcavities (or pores or recesses) 43, the size and shape of which are configured to receive at least one cell or form 3D cultures, such as spheroids or organoids. The microcavities 43 may be shaped as concave recesses within the well plate 42. Thus, the well plate 42 is a cell culture region configured to promote cell growth and development within the cell culture chamber 11. Additionally, gas permeability is a property that can contribute to a 3D cell culture environment. Cell growth can be promoted by allowing gas permeability within the microcavities 43 of the cell culture container. The microcavity containers are unique in their geometry and formation because they are formed from a base plate 40 in which the microcavities 43 are disposed. Due to the thickness of the base plate 40, the microcavities 43 are gas-permeable, wherein gas permeability occurs because the base plate can be formed of a very thin polystyrene material with a thickness of about 28 micrometers to about 72 micrometers. Each microcavity 43 may include an inner cavity having a circular bottom 44 that does not adhere to cells. Thus, the microcavity container described herein is a cell culture device that facilitates 3D cell culture by allowing cells seeded into the microcavities 43 to self-assemble or attach to each other to form spheroids in each microcavity. The microcavities 43 may be shallow, allowing cell culture medium to simultaneously cover spheroids, organoids, or 3D cell aggregates in all cavities, which improves ease of handling. Each microcavity 43 may have any suitable size. For example, the diameter or width of each microcavity 43 may range from about 500 micrometers to about 5 millimeters. The depth of each microcavity 43 may range from about 500 micrometers to about 6 millimeters. In some embodiments, the depth of each microcavity 43 may be from about 500 micrometers to about 650 micrometers. In some aspects, the depth of each microcavity 43 may be about 1.6 millimeters. In some respects, the microcavities 43 can be formed within a thicker base plate 40 because the container 10 is compatible with perfusion culture (i.e., the culture medium flows continuously during the culture duration), and dissolved oxygen can be supplied through the culture medium circulation; therefore, gas permeability of the well plate 42 is not a prerequisite. The use of the thick base plate 40 allows for the fabrication of the microcavities 43 by injection molding or any other high-throughput process, thereby reducing costs.

[0067] Figure 2BOptional screen 120 is also shown. In some aspects, screen 120 includes a mesh that defines an array of microcavities 43. Screen 120 itself may include a mesh membrane having multiple openings. The mesh membrane may be selected from plastic woven mesh, plastic nonwoven mesh, glass fiber woven mesh, glass fiber nonwoven mesh, or any plastic membrane having an array of openings or pores.

[0068] In some respects, the mesh screen 120 may be positioned above the well plate 42 or the microcavities 43 therein. It is understood that this will allow liquid culture medium to flow through the microcavities 43 and for single cells to pass through and enter the microcavities 43, while providing a physical barrier that helps retain 3D cell cultures therein. While in some respects the mesh screen 120 may simply be positioned above the well plate 42, anchoring the mesh screen 120 to prevent displacement may be beneficial, especially considering that the culture medium flows through the entire container. In some respects, the mesh screen 120 may be anchored or attached to the sidewalls 17, 18 and / or the first end wall 14 or the second end wall 15. Figure 1 Examples of such attachment or anchoring include using adhesives to bond the mesh screen 120 or using heat to weld or melt the mesh screen 120 to a portion of the base plate 40 of the container or to the walls 14, 15, 17, 18. In some aspects, the mesh screen 120 may include magnetic or ferromagnetic portions therein or on it, such that placing a magnetic field below the cell culture chamber 11 will hold the mesh screen 120 above the microcavity 43 or the well plate 42. In some aspects, the mesh screen 120 may be retractable or removable, thereby allowing a user to access the 3D cell culture within the well plate 42 or the microcavity 43. In some aspects, the mesh screen 120 may include a solid frame (not depicted) surrounding its periphery or a portion thereof. It is understood that the presence of the solid frame can act as an anchor or weight to hold the mesh screen 120 in place above the well plate 42 or the microcavity 43. In some aspects, the screen 120 may include at least one retaining structure that extends to the outside of the top of the container, such that the screen 120 can be pushed against the top of the base plate 40 or moved away from the base plate 40 when needed (e.g., by rotating around the extension of the solid frame using screws). In this way, the user can then collect spheroids or organoids when needed.

[0069] Figure 5A size comparison between a mesh screen 120 (left) and a microcavity 43 (right) is depicted, wherein the opening of the mesh screen is smaller than the opening of the microcavity 43. In some aspects, the mesh screen 120 includes openings of a predetermined size that allow individual cells to pass through and help retain cell spheres within the cell receiving surface. In some aspects, the size of the mesh openings is smaller than the diameter or intended diameter of the spheres therein. In other aspects, the mesh openings may be larger than the diameter of the retained spheres; however, the presence of the mesh itself can mitigate sphere loss. In some aspects, the diagonal length of the mesh screen 120 is greater than or equal to 25 µm and less than or equal to 500 µm. In some aspects, the diagonal length of the mesh openings is greater than or equal to 40 µm and less than or equal to 250 µm. In some aspects, the diagonal length of the mesh openings is greater than 100 µm and less than or equal to 250 µm. In some aspects, the size of each opening in the mesh screen 120 is selected to be larger than the diameter of the cell but smaller than the diameter of the sphere of interest within the pore.

[0070] Refer again Figure 2A and 2B In some aspects, the system of this disclosure includes a flow of liquid culture medium through container 10. To facilitate the flow of the liquid culture medium, inlet port 20 may be further operably connected (e.g., fluidly connected) to inlet conduit 21, which in turn is operably connected (e.g., fluidly connected) to a sealed culture medium source (not shown and see, for example...). Figure 4 This allows for the delivery of sterile and / or recirculated culture media. Similarly, outlet port 30 is operatively connected (e.g., fluidly connected) to outlet conduit 31 to allow for the extraction of culture media from cell culture chamber 11 without exposure to ambient air that allows the system to remain closed. In some aspects, oxygen and CO2 gas lines are integrated into the sealed culture medium source to control dissolved oxygen concentration and pH. In some aspects, outlet conduit 31 leads to a waste container ( Figure 4In other respects, the inlet conduit 21 of inlet port 20 and the outlet conduit 31 of outlet port 30 are operatively connected (e.g., fluidly connected) to provide recirculation of culture medium in a closed system. Liquid culture medium can be circulated or flowed onto cells residing or resting on or within the substrate 40 by adding liquid culture medium via inlet port 20 and withdrawing liquid culture medium from outlet port 30. In some respects, inlet port 20 and / or outlet port 30 may include nozzles or a series of nozzles, as described herein. In some respects, inlet nozzle 26 is positioned close to a surface, such as the sidewall 17 or 18 of the substrate 40 of container 10, such that when culture medium flows out from inlet nozzle 26, the culture medium does not splash onto the substrate 40 or otherwise cause undesirable interference to the circulating culture medium and / or cells in the well plate 42. Similarly, placing the outlet nozzle 36 associated with outlet port 30 at or near the substrate 40 of container 10 will allow a user to withdraw all or almost all of the culture medium from container 10. In some respects, the inlet nozzle 26 and / or the outlet nozzle 36 may be movable to allow the user to configure their placement relative to the base plate 40.

[0071] In some respects, the inlet nozzle 26 is positioned or configured to provide a flow of liquid culture medium at or near the upper surface of the orifice plate 42.

[0072] Go to Figure 3 and 4 The figure illustrates an optional embodiment of container 10 with respect to pipes 21, 31. As shown, pipes 21, 31 may optionally include one or more fittings 22, 32 to allow multiple fluids to flow into inlet port 20 or optionally change the source or material flowing into inlet port 20. It is also understood that, although not depicted, including valves in pipes 21, 31 can allow control and / or shut-off of flow. Similarly, clamps 23, 33 may be used to restrict flow in a portion of pipes 21, 31. Figure 4 As seen, container 10 can be configured to circulate and / or replace culture medium within the cell culture chamber. Inlet pipe 21 is operatively connected to connector 22, which allows connection to culture medium container 24 and outlet pipe 31. Outlet pipe 31 is similarly connected to connector 22 to allow additional flow to waste container 34. Inlet pipe 21 and outlet pipe 31 are routed via pump 70 to allow culture medium to be recirculated through cell culture chamber 11.

[0073] It is understood that liquid culture medium can be introduced via pump 70 (e.g., a peristaltic pump). Similarly, liquid culture medium can be extracted via pumping or a vacuum device. It is understood that while the flow into container 10 through inlet port 20 does not need to match the outflow rate, it is beneficial to keep both rates the same or nearly the same over an extended period to prevent the removal of all culture medium from the cells or to prevent all culture medium from overflowing from container 10. In some aspects, inlet port 20 and outlet port 30 can be connected to the same pumping device. In some aspects, inlet port 20 can provide fresh culture medium, and outlet port 30 can deliver removed culture medium to waste container 34. In other aspects, inlet port 20 and outlet port 30 can be operatively connected to enable the recirculation of culture medium through container 10. In some aspects, outlet pipe 31 can be separated to allow for the recirculation of a portion of the culture medium and the treatment of the remaining culture medium. Similarly, inlet pipe 21 can be connected to both the supply and recirculation of fresh culture medium.

[0074] One aspect of this disclosure also provides laminar flow of culture medium over and to cultured cells, and collection of valuable cellular byproducts from the culture medium. In some aspects, providing laminar flow to cells in the microcavities 43 of the well plate 42 can promote the growth and / or generation of extracellular vesicles (EVs). EVs have garnered significant preclinical attention for regenerative medicine therapies over the past decade. "Extracellular vesicles" is a broad term used to refer to a population of non-replicable particles defined by a lipid bilayer that are naturally released from cells. EVs are attractive to researchers and clinicians because they provide information for cells to communicate with each other and facilitate physiological responses via biomolecular information sent from one cell to another. One of the biggest challenges facing EV therapy is large-scale manufacturing. Unused or unmodified EVs are collected from stem cells via used cell culture media. Due to the limited standardization of various technologies and tools currently available, batch-to-batch variability can be a significant concern. Furthermore, there are currently no methods to support in vivo-like conditions to facilitate appropriate EV production, as scale-out and scale-up methods are more suited to 2D cell cultures, or 2.5D if microcarriers are used. One aspect of this disclosure is overcoming these obstacles and enabling the reproducible production of EVs by providing a laminar flow of culture medium to cells in containers and systems as described herein. It is well known that cells, especially stem cells, behave significantly differently in vivo than they do in 2D monolayers cultured in vitro, which subsequently negatively impacts the therapeutic value of the generated EVs. As shown in the working examples, systems and methods of this disclosure are provided for the bulk collection of EVs from used culture medium flowing laminarly through mesenchymal stem cells (MSCs). Figure 6 The results show that, compared with standard 2D culture, the laminar flow perfusion 3D cultured cells in the system and method described in this paper can continuously generate EVs for about two weeks, which is longer than that of the 2D counterpart. The EVs accumulated at 15 days are more than four times the EVs obtained in two days by 2D culture. Figure 7 An overview of 2D-grown cells after two weeks and cells perfused using the systems and methods of this disclosure is shown. Perfused cells are individual spherical bodies, compared to the flattened cells in 2D culture. Figure 8 The systems and methods of this disclosure show that they have no negative impact on the cell viability or mean mode diameter of the resulting EVs.

[0075] In some aspects, the system may include a material that prevents cell adhesion. The material may at least constitute the area in contact with the cells, such as a cell-receiving surface and / or a mesh screen, or may be a coating applied thereon. In some aspects, the coating is a polymer coating. In some aspects, the cell-receiving surface (e.g., at least a portion of the surface of the pores) and / or the mesh screen are coated with an ultra-low adhesion chemical (ULA) (see U.S. Patent 5,002,582) or other non-binding chemical (e.g., a 2-methacryloyloxyethylphosphorylcholine (MPC) polymer). In some aspects, the mesh screen and the cell-receiving surface (e.g., at least a portion of the surface of the pores) are coated with the same polymer coating. In some aspects, the coating may be selected from UV-crosslinkable polyethylene glycol (PEG), poly(2-hydroxyethyl methacrylate), and / or agarose. It is also understood that, in embodiments, the polymer coating of the mesh screen may differ from the coating of the pores of the cell-receiving surface and inhibit cell adhesion to the mesh screen. As shown in the working example, using a larger mesh opening allows cells to pass through (…) once introduced into the container. Figure 5 (As illustrated in the example) the opening settles into the bottom of the cell-receiving surface and then begins to grow into a 3D sphere or organoid (see example...). Figure 7 (Bottom image). Once the spheroids grow to a size larger than the opening of the mesh screen, the mesh screen acts as a physical constraint on the spheroids or organoids, and thus the spheroids or organoids remain within each microcavity or pore of the cell receiving surface during culture exchange, culture perfusion, or mechanical agitation. Even if the size of the spheroids is smaller than the opening of the mesh screen, the mesh screen still provides some constraint on the spheroids. Therefore, users can actively perfuse culture, or easily perform culture exchange, and / or apply mechanical agitation such as lateral shaking without disturbing the culture.

[0076] In some respects, dissolving the mesh may be advantageous, such as if the mesh is permanently or semi-permanently attached or adhered to or above a cell-receiving surface. Once the container's purpose is achieved, as in the working example, the solubility of the mesh allows the user to retrieve the cells within it as desired spheres are formed. For example, a permanent or semi-permanent mesh may have openings large enough for a single cell to pass through. A culture medium flow can then be established, allowing spheres to form within the container. Once sufficient and / or the desired size is reached, the mesh can be dissolved to retrieve the cells. In these respects, the mesh can be a material that can be dissolved by enzymatic digestion. In some respects, the mesh is made of polygalacturonic acid (PGA) or cellulose.

[0077] In some aspects, this disclosure also relates to methods using containers and systems as described herein. In some aspects, the methods include using a container for three-dimensional cell culture. In some aspects, the methods may include adding one or more cells to a cell-receiving surface or well as described herein. In some aspects, a mesh screen may then be placed over the cell-receiving surface or well, or pre-positioned to allow cells to fall between the mesh openings. The methods may then include a process of establishing a flow of liquid culture medium through the cells by adding liquid culture medium through an inlet port and withdrawing liquid culture medium from an outlet. In some aspects, the liquid culture medium may be a tissue culture medium and / or a cell culture medium, such as a medium supplemented with salts, buffers, growth factors, antibiotics, cytokines, sugars, and combinations thereof. In some aspects, the liquid culture medium is recycled or recirculated over the cells in the cell-receiving surface or well. It is understood that in instances of multiple wells, each well may be seeded with one or more cells to initiate 3D culture therein. Separation between wells allows each well to self-grow. In some aspects, the methods include providing a flow of liquid culture medium through an inlet port. As illustrated herein, nozzles allow liquid culture media to flow into a container from one side of the orifice, with an outlet drawing the media from the opposite side of the orifice. In some aspects, the drawn media can be recycled wholly or partially back to the inlet. In other aspects, the media from the outlet can be directed wholly or partially to a waste container or similar container.

[0078] In some aspects, this disclosure includes a method for three-dimensional tissue culture, the method comprising the steps of: placing cells or spheroids in wells of a system as described herein, and allowing tissue culture medium to flow into the system through an inlet, wherein the tissue culture medium contacts the cells or spheroids; and then aspirating the tissue culture medium through an outlet port. In some aspects, the system container can be inoculated with a single-cell suspension. With the aid of gravity and a non-adhesive coating, cells will be driven to settle at the lowest point of the well plate. Depending on the cell concentration and the size of the settling area (e.g., microcavities), the result is single-celled, small multicellular formations (spheroids), or large multicellular / tissue fragments (organoids).

[0079] In some aspects, the method of this disclosure includes providing tissue culture medium through an inlet port at a first rate. In some aspects, the method may include aspirating tissue culture medium at an outlet port at a second rate. In some aspects, the first rate and the second rate are the same. In some aspects, the first flow rate and the second flow rate establish a laminar flow of tissue culture medium across a cell-receiving surface.

[0080] Example

[0081] The container described herein is arranged to establish a laminar flow of recirculating culture medium through mesenchymal cells in the wells of a plate. A mesh screen is placed above the wells to retain the cells within. A control arrangement for preparing spheroids using conventional 2D cell culture is also provided for comparison. The benefits of bulk collection of EVs from used culture medium flowing through a laminar flow through mesenchymal stem cells (MSCs) are described in [link to relevant documentation]. Figure 6 . Figure 6 The results show that, compared with standard 2D culture, the laminar flow perfusion 3D cultured cells in the system and method described in this paper can continuously generate EVs for about two weeks, which is longer than that of the 2D counterpart. The EVs accumulated at 15 days are more than four times the EVs obtained in two days by 2D culture. Figure 7 An overview of 2D-grown cells after two weeks and cells perfused using the systems and methods of this disclosure is shown. Perfused cells exhibit a 3D clustered appearance compared to flattened cells in 2D culture. Figure 8 The systems and methods of this disclosure show that they have no negative impact on the cell viability or mean mode diameter of the resulting EVs.

[0082] While specific aspects have been shown and described herein, it should be understood that various other changes and modifications may be made without departing from the spirit and scope of the claimed subject matter. Furthermore, although various aspects of the claimed subject matter have been described herein, these aspects need not be used in combination. Therefore, it is intended that all such changes and modifications within the scope of the claimed subject matter be covered in the appended claims.

[0083] It should be understood that, unless otherwise stated, all reagents are available from sources known in the art.

[0084] It should also be understood that this disclosure is not limited to the specific aspects and methods described herein, as specific components and / or conditions can certainly vary. Furthermore, the terminology used herein is for the purpose of describing specific aspects of this disclosure only and is not intended to be limiting in any way. It will also be understood that although the terms “first,” “second,” “third,” etc., may be used herein to describe various elements, components, regions, layers, and / or portions, these elements, components, regions, layers, and / or portions should not be limited by these terms. These terms are used only to distinguish one element, component, region, layer, or portion from another element, component, region, layer, or portion. Therefore, without departing from the teachings herein, “first element,” “component,” “region,” “layer,” or “part” discussed below may be referred to as a second (or other) element, component, region, layer, or portion. Similarly, as used herein, unless the content clearly indicates otherwise, the singular forms “a / an” and “the” are intended to include the plural forms, including “at least one.” “Or” means “and / or.” As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. It should be further understood that the terms "comprises" and / or "comprising" or "includes" and / or "including," when used in this specification, specify the presence of the stated features, areas, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, areas, integers, steps, operations, elements, components, and / or groups thereof. The term "or a combination thereof" means a combination including at least one of the foregoing elements.

[0085] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. It should be further understood that terms such as those defined in commonly used dictionaries should be interpreted as having meanings consistent with their meanings in the relevant field and in the context of this disclosure, and will not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0086] Reference is made in detail to the exemplary compositions, aspects, and methods of this disclosure, which constitute the best mode of practice currently known to the inventors. The drawings are not necessarily drawn to scale. However, it should be understood that the disclosed aspects are merely examples of this disclosure that may be embodied in various and alternative forms. Therefore, the specific details disclosed herein should not be construed as limiting, but merely as a representative basis for any aspect of this disclosure and / or as a representative basis for teaching those skilled in the art to employ this disclosure in different ways.

[0087] The patents, publications, and applications mentioned in this specification demonstrate the skill of a person skilled in the art to which this disclosure pertains. These patents, publications, and applications are incorporated herein by reference to the extent that each individual patent, publication, or application is specifically and individually incorporated herein by reference.

[0088] The foregoing description illustrates specific embodiments of this disclosure, but does not imply limitation on its practice. The following claims, including all their equivalents, are intended to define the scope of this disclosure.

Claims

1. A container for culturing cells, the container comprising: A top plate, a bottom plate, and at least one side wall connecting the top plate to the bottom plate, wherein the top plate, the bottom plate, and the at least one side wall define an internal volume; A perforated plate, the perforated plate being disposed within the internal volume of the container, the perforated plate comprising a plurality of microcavities for receiving cells, spheroids or organoids; An inlet port that extends into the internal volume at a first end of the internal volume; as well as An outlet port extends into the internal volume at a second end opposite to the first end, wherein: The orifice plate is positioned between the inlet port and the outlet port; and The liquid culture medium introduced into the internal volume at the inlet port flows through the well plate and is extracted from the internal volume at the outlet port.

2. The container of claim 1, wherein the plurality of microcavities comprise concave recesses in the upper surface of the base plate.

3. The container of claim 1, wherein the inlet port is positioned toward a first end of the top plate and configured to provide laminar flow of liquid culture medium through the plurality of microcavities to the outlet port.

4. The container of claim 1, wherein the inlet port is operatively connected to at least one nozzle, and wherein one end of the at least one nozzle is disposed above the bottom plate within the internal volume.

5. The container of claim 4, wherein at least two nozzles are aligned linearly across the width of the base plate.

6. The container of claim 5, wherein at least eight nozzles are aligned across the width of the base plate.

7. The container of claim 4, wherein the at least one nozzle is configured to provide a flow of liquid culture medium at or near the top plate of the orifice plate.

8. The container of claim 4, further comprising: An inlet conduit operatively connected to the inlet port; and A pump, which is operatively connected to the inlet pipe.

9. The container of claim 1, wherein the outlet port is operatively connected to at least one nozzle, and wherein one end of the at least one nozzle is disposed above the bottom plate within the internal volume.

10. The container of claim 9, wherein at least two nozzles are aligned linearly across the width of the base plate.

11. The container of claim 10, wherein at least eight nozzles are aligned across the width of the base plate.

12. The container of claim 9, wherein the at least one nozzle is configured to remove liquid culture medium from the upper surface of the orifice plate.

13. The container of claim 9, further comprising: An outlet pipe, operatively connected to the outlet port; and A pump, which is operatively connected to the outlet pipe.

14. The container of claim 1, wherein at least a portion of the inner surface of each of the plurality of microcavities comprises a polymer coating.

15. The container of claim 14, further comprising a mesh screen disposed above the perforated plate.

16. The container of claim 15, wherein the mesh screen is attached to the bottom plate, the at least one side wall, or both.

17. The container of claim 15, wherein the mesh screen comprises a magnetic or ferromagnetic portion such that a magnetic field is placed below the perforated plate to fix the mesh screen above at least one of the plurality of microcavities.

18. The container of claim 15, wherein the screen comprises an opening with a diagonal length greater than or equal to 25 µm and less than or equal to 500 µm.

19. The container of claim 15, wherein each opening in the mesh screen is larger than the diameter of a cell in at least one pore of the plurality of microcavities and smaller than the diameter of the spheroid or organoid of interest.

20. The container of claim 15, wherein the mesh screen further comprises a solid frame surrounding its periphery.

21. The container of claim 15, wherein the mesh screen further comprises a polymer coating, wherein the polymer coating is the same as the polymer coating of the plurality of microcavities, wherein the polymer coating of the mesh screen inhibits cell attachment to the mesh screen.

22. The container of claim 21, wherein the polymer coating of the mesh screen and the plurality of microcavities is selected from UV-crosslinkable PEG, poly(2-hydroxyethyl methacrylate), 2-methacryloyloxyethyl phosphorylcholine polymer, or agarose.

23. The container of claim 15, wherein the mesh screen comprises a polymer coating, wherein the polymer coating is different from the polymer coating of the plurality of microcavities, wherein the polymer coating of the mesh screen inhibits cell attachment to the mesh screen.

24. The container of claim 15, wherein the mesh screen is capable of being dissolved by enzymatic digestion.

25. The container of claim 1, further comprising a pump operably connected to the inlet port and configured to pump a liquid culture medium through the inlet port onto the well plate.

26. The container of claim 25, wherein the pump is operatively connected to the outlet port and configured to draw the liquid culture medium from the well plate through the outlet port.

27. The container of claim 26, wherein the pump is configured to recirculate the liquid culture medium from the outlet port to the inlet port.

28. The container of claim 1, further comprising a pump operably connected to the outlet port and configured to draw the liquid culture medium from the well plate through the outlet port.

29. A method for replacing a liquid culture medium in a container according to claim 1, the method comprising adding the liquid culture medium through the inlet port.

30. The method of claim 29, wherein the liquid culture medium is drawn through the outlet port.

31. A method for three-dimensional tissue culture, the method comprising: Cells, spheroids or organoids are placed in the microcavities of the container according to claim 1; Liquid culture medium is allowed to flow into the container through the inlet port, wherein the liquid culture medium contacts the cells or spheroids; and, The liquid culture medium is drawn through the outlet port.

32. The method of claim 31, wherein the liquid culture medium flows into the inlet port at a first flow rate.

33. The method of claim 32, wherein the liquid culture medium is drawn at the outlet port at a second flow rate.

34. The method of claim 33, wherein the first flow rate and the second flow rate are the same.

35. The method of claim 33, wherein the first flow rate and the second flow rate establish laminar flow of the liquid culture medium through the well plate.

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