WO2025106382A1 - System and methods for 3d tissue culture - Google Patents

Abstract

The present disclosure concerns cell culture vessels and methods of the use thereof that are capable of providing a laminar flow of cell media to cells therein. The cell culture vessel include a plurality of wells within which cells reside as media moves across the top thereof. The cell culture systems are further enclosed to maintain sterility. The media can be constantly refreshing within the cell culture vessel, be recycled within the cell culture vessel, or a combination of both. The circulation of media about the cells residing therein can be particularly useful for 3D tissue culturing, such as producing spheroids or organoids.

Description

SYSTEM AND METHODS FOR 3D TISSUE CULTURE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. §119 of U.S. Provisional Application Serial No. 63/600,364 filed on November 17, 2023, the content of which is relied upon and incorporated herein by reference in its entirety.

FIELD

[0002] The present disclosure relates to systems and methods for establishing three- dimensional cell cultures in vitro.

TECHNICAL BACKGROUND

[0003] 3D cell culture models better mimic an in vivo environment compared with traditional monolayer culture models. The physiology in which cells can organize themselves into 3D structures such as spheroids or organoids allows for excellent recapitulation of the cells’ native environment. A number of 3D cell culturing methods have been established for the formation of spheroids, though these all struggle from a lack of reproducibility, wide distribution of final spheroid sizes, and limitations in quantity of spheroids that can be produced per vessel. The generation of spheroids involves a high level of complexity and cost and, in order to provide true value as a model for in vivo environments, generating uniformity and reproducibility across spheroids is a prerequisite for one skilled in the art to value data generated therefrom.

[0004] Accordingly, a need exists for alternative systems and methods for the potential complexity and cost, there can be challenges associated with these methods for spheroid formation.

SUMMARY

[0005] A 1^st aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to a vessel for culturing cells, 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 interior volume; at least one well plate disposed in the interior volume of the vessel, the well plate comprising a plurality of microcavities for receiving cells, spheroids, or organoids; an inlet port extending into the interior volume at a first end of the interior volume; and an outlet port extending into the interior volume at a second end of the interior volume opposite the first end, wherein: the well plate is positioned between the inlet port and the outlet port; and liquid media introduced into the interior volume at the inlet port flows over the well plate and is extracted from the interior volume at the outlet port.

[0006] A 2^nd aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 1^st aspect, wherein the plurality of microcavities comprise concave indentations in an upper surface of the bottom plate.

[0007] A 3^rd aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 1^st aspect, wherein the inlet port is positioned toward a first end of the top plate and is configured to provide a laminar flow of liquid media across the plurality of wells to the outlet port.

[0008] A 4^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 1^st aspect, wherein the inlet port is operably connected to at least one nozzle and wherein an end of the at least one nozzle is disposed above the bottom plate within the interior volume.

[0009] A 5^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 4^th aspect, wherein at least two nozzles are aligned in a linear fashion across a width of the bottom plate.

[0010] A 6^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 5^th aspect, wherein at least eight nozzles are aligned across the width of the bottom plate.

[0011] A 7^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 4^th aspect, wherein the at least one nozzle is configured to provide a flow of liquid media at or near the top plate of the well plate.

[0012] An 8^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 4^th aspect, further comprising: inlet tubing operably connected to the inlet port; and a pump operably connected to the inlet tubing.

[0013] A 9^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 1^st aspect, wherein the outlet port is operably connected to at least one nozzle and wherein an end of the at least one nozzle is disposed above the bottom plate within the interior volume. [0014] A 10^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 9^th aspect, wherein at least two nozzles are aligned in a linear fashion across a width of the bottom plate.

[0015] An 11^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 10^th aspect, wherein at least eight nozzles are aligned across the width of the bottom plate.

[0016] A 12^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 9^th aspect, wherein the at least one nozzle is configured to remove liquid media from an upper surface of the well plate.

[0017] A 13^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 9^th aspect, further comprising: outlet tubing operably connected to the outlet port; and a pump operably connected to the outlet tubing.

[0018] A 14^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 1^st aspect, wherein at least a portion of an interior surface of each of the plurality of wells comprises a polymer coating.

[0019] A 15^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 14^th aspect, further comprising a mesh screen disposed over the well plate.

[0020] A 16^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 15 ^th aspect, wherein the mesh screen is attached to the bottom plate, the at least one sidewall, or both.

[0021] A 17^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 15 ^th aspect, wherein the mesh screen comprises a magnetic or ferro-magnetic portion such that placement of a magnetic field underneath the well plate secures the mesh screen over at least one of the plurality of microcavities.

[0022] An 18 ^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 15 ^th aspect, wherein the mesh screen comprises openings comprising a diagonal length of greater than or equal to 25 pm to less than or equal to 500 pm.

[0023] A 19^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 15 ^th aspect, wherein each opening in the mesh screen is greater than a diameter of a cell and smaller than a diameter of a spheroid or organoid of interest in at least one well of the plurality of microcavities.

[0024] A 20^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 15^th aspect, wherein the mesh screen further comprises a solid frame about a perimeter thereof.

[0025] A 21^st aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 15^th aspect, wherein the mesh screen further comprises a polymer coating, wherein the polymer coating is the same as a polymer coating of the plurality of microcavities, wherein the polymer coating of the mesh screen inhibits cell attachment to the meshed sheet.

[0026] A 22^nd aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 21^st 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.

[0027] A 23^rd aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 15^th aspect, wherein the mesh screen sheet comprises a polymer coating, wherein the polymer coating is the different from a polymer coating of the plurality of microcavities, wherein the polymer coating of the mesh screen inhibits cell attachment to the mesh screen.

[0028] A 24^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 15^th aspect, wherein the mesh screen is dissolvable through enzymatic digestion.

[0029] A 25^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 1^st aspect, further comprising a pump operably connected to the inlet port and configured to pump a liquid media through the inlet port on to the well plate.

[0030] A 26^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 25^th aspect, wherein the pump is operably connected to the outlet port and configured to withdraw the liquid media from the well plate through the outlet port. [0031] A 27^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 26^th aspect, wherein the pump is configured to recirculate the liquid media from the outlet port to the inlet port.

[0032] A 28^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 1^st aspect, further comprising a pump operably connected to the outlet port and configured to withdraw the cell culture media from the well plate through the outlet port.

[0033] A 29^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to a method for replacing liquid media in the vessel of the 1^st aspect, comprising adding a liquid media through the inlet port.

[0034] A 30^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the method of the 29^th aspect, wherein the liquid media is withdrawn through the outlet port.

[0035] A 31^st aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to a method for three-dimensional tissue culture, comprising: placing a cell, a spheroid, or an organoid in a microcavity of the vessel of the 1^st aspect; flowing a liquid media into the vessel through the inlet port, wherein the liquid media comes into contact with the cell or spheroid; and, withdrawing the liquid media through the outlet port.

[0036] A 32^nd aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 31^st aspect, wherein the liquid media flows in the inlet port at a first flow rate.

[0037] A 33^rd aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 32^nd aspect, wherein the liquid media is withdrawn at the outlet port at a second flow rate.

[0038] A 34^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 33^rd aspect, wherein the first flow rate and the second flow rate are the same.

[0039] A 35^th aspect of the present disclosure, either alone or in combination with any other aspect herein, relates to the vessel of the 33^rd aspect, wherein the first flow rate and the second flow rate establish a laminar flow of the liquid media across the well plate. [0040] Additional features and advantages of the 3D tissue culture vessels and methods of using the same described herein will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the embodiments described herein, including the detailed description which follows, the claims, as well as the appended drawings.

[0041] 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

[0042] FIG. 1 shows an elevated side-view of an embodiment of the disclosure.

[0043] FIG. 2A shows a portion of the embodiment of FIG. 1 .

[0044] FIG. 2B shows an isolated side-view of the bottom plate of the embodiment of FIG. 1.

[0045] FIG. 3 shows a top view of the embodiment of FIG. 1 with additional components to circulate media.

[0046] FIG. 4 shows the embodiment of FIG. 1 configured for replacement and/or recycling of media within the vessel.

[0047] FIG. 5 shows a comparison between a meshed sheet (left) and the plurality of cell spheroids in a plurality of microcavities, one spheroid in the bottom of each microcavity.

[0048] FIG. 6 shows cumulative EV production from MSCs cultured in 2D for 2 days (end point) and 14 days in 3D.

[0049] FIG. 7 shows bright-field imaging of MSCs in 2D at day 2 (top) and harvested spheroids at day 14 (bottom) taken with the microscope degree of magnification set at 4X.

[0050] FIG. 8 shows there was no statistical difference in cell viability (left) and resultant EV diameter (right) from mesenchymal stems cells cultured for 2 days in 2D and 14 days in 3D (data shown mean ± standard deviation, n = 3). DESCRIPTION

[0051] Reference will now be made in detail to embodiments of the vessel described herein, examples of which are illustrated in the accompanying drawings. Whenever possible, the same reference numerals will be used throughout the drawings to refer to the same or like parts. In some aspects, the present disclosure concerns a cell culture vessel that includes 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 interior volume. The vessel also includes a well plate disposed in the interior volume of the vessel, the well plate having thereon a plurality of polymer wells for receiving cells or spheroids. The vessel also includes an inlet port extending into the interior volume at a first end of the interior volume and an outlet port extending into the interior volume at a second end of the interior volume opposite the first end. The well plate is positioned between the inlet port and the outlet point. Liquid media, such as tissue culture media, can be introduced into the interior volume at the inlet port, allowed to flow over the well plate and then be extracted from the interior volume at the outlet port. Various embodiments of vessels for culturing cells, and methods for using the same, will be described in further detail herein with specific reference to the appended drawings.

[0052] Ranges can 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.

[0053] 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.

[0054] 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.

[0055] 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” component includes aspects having two or more such components, unless the context clearly indicates otherwise.

[0056] The present disclosure relates to systems and methods for culturing cells. In some aspects, the systems and methods relate to 3D culturing of cells. It will be appreciated that while the working examples herein may concern consistent production of spheroids, the systems and the methods can readily be applied to other aspects of culturing cells and/or tissues. The systems and the methods set forth herein are agnostic to many cell culture vessels; provide multiple modalities for formation and retention of 3D cultures; are scalable provide a closed system perfusion with in-line sampling port that does not contact the cell culture area; are amenable to mechanical agitation / dynamic media movement; and, allow for the collection of cell conditioned media without disturbing the 3D cultures.

[0057] In some aspects, the present disclosure relates to systems of culturing cells. As is understood in the art, cells in culture are susceptible to microbial infection, and as such, it is desirable to provide an isolated or enclosed environment for the cells being cultured. In order to access the cells or even place the cells therein, the enclosure needs an access point or an operable port or removable wall. In some aspects, the access points should be sealable or form a seal with the enclosure to prevent undesired air accessing the interior. In some aspects, the enclosure includes a vessel. In some aspects, the vessel is a sealable vessel

[0058] In some aspects, the vessel needs to be arranged such that the cells can reside in a cell-receiving surface. In some aspects, the vessel has a flat surface to serve as a bottom plate which in turn allows the cells within the cell receiving surface to rest on or in a horizontal plane or orientation. In some aspects the cell-receiving surface is part of the interior surface of the bottom plate. While the vessel need not be restricted in shape, by way of example, the bottom wall may be considered as a rectangular prism, with a rectangular face oriented horizontally, the top surface of which forms an interior surface of the vessel and a bottom plate of which is flat or partially flat to allow for the vessel to rest on a level horizontal surface. The bottom plate may further be adapted depending on the desired conditions. For example, rounding the surface upon which the vessel rests will allow for a rocking motion to be applied. It will also be appreciated that the cell-receiving surface need not be directly part of the bottom wall, but instead may be an isolated connected or resting substrate in contact with the interior portion of the bottom wall. It will be apparent that the cell-receiving surface(s) can have a round bottom, a square bottom, or any other shaped bottom. In some aspects, the vessel may include stacked cell-receiving surfaces, wherein at least one second cell-receiving surface is stacked or placed over a first cell-receiving surface. It will be appreciated that such can advance the scalability. In some aspects, the vessel is configured to be stackable with a further vessel.

[0059] In some aspects, the vessel contains two ends: a first end and a second end opposite the first end. The two ends are at opposing walls or opposing points of the vessel and configured such that a fluid entering from one port will pass over the cell-receiving surface before being able to exit through the other port. While the inlets may reside on end walls of the vessel, such is not a necessity. For example, a top plate of the vessel may include one or more of the ports, configured to be on one side of the cell receiving surface. Similarly, the ports may be through the bottom plate, configured in a similar manner. In some aspects, the distal port(s) may be between the wall of the first end and the cell receiving portion of that surface. Similarly, the ports may be positioned between the wall of the second end and the cell receiving portion of that surface. In some aspects, the one port may serve as an inlet towards the first end and another port may serve as an outlet towards the second end, or vice versa. Such arrangements can thereby allow or provide a flow of cell media through the vessel between the inlet and outlet.

[0060] In aspects, the cell -receiving surface is a microcavity or a well or an indentation within a bottom plate of the vessel in which cells can settle. In some aspects, the vessel includes a first horizontal end, a second horizontal end opposite the first horizontal end, and a cellreceiving surface therebetween with an inlet port and an outlet port, the inlet port being positioned between the well/indentation and the first horizontal end and the outlet port being positioned between the well/indentation and the second horizontal end.

[0061] In aspects, the cell receiving surface includes multiple microcavities or wells or indentations. It will be appreciated that such allows for the isolation of cells within the vessel. For example, as set forth in the Examples herein, the vessel can be utilized to produce spheroids or organoids. The presence of isolated microcavities or wells or indentations within the cellreceiving surface allows for the production of multiple independent 3D cultures within the same vessel. It will be appreciated that a microcavity or well or indentation may be a concave indentation on the cell-receiving surface.

[0062] In some aspects, the inlet port is positioned toward the first horizontal end and is configured to provide a flow of liquid media across the well or the plurality of wells in the cell receiving surface of the bottom plate to the outlet. In some aspects, the outlet port is positioned toward the second end and is configured to receive the flow of liquid media from the cell receiving surface. It will be appreciated that the inlet and outlet ports may be reversed. It will also be appreciated that the volume of liquid media fills the vessel or at least partially fills the vessel. In such aspects, it will be appreciated that not all provided liquid media will enter into the cell-receiving surface, yet the flow of media through the vessel will continue.

[0063] Reference will now be made in detail to embodiments of the vessel described herein, examples of which are illustrated in the accompanying drawings. Whenever possible, the same reference numerals will be used throughout the drawings to refer to the same or like parts. One embodiment of the vessel is shown in FIG. 1, and is designated generally throughout by the reference numeral 10. FIG. 1 shows an elevated side view of an embodiment of the vessel 10 of the present disclosure that includes a vessel body 12. The vessel body 12 includes a first end wall 14 and a second end wall 15 that is opposite the first end wall 14. Sidewalls 17 and 18 extend between the first end wall 14 and the second end wall 15. In some embodiments, the first and second end walls 14 and 15 or portions thereof are substantially parallel and the pair of side walls 17 and 18 or portions thereof are substantially parallel forming a somewhat rectangular-shaped perimeter 19 of a cell culture chamber 11. While a rectangular-shaped perimeter 19 is illustrated, any suitable perimeter shape may be used. The vessel 10 further includes an inlet port 20, an outlet port 30, a bottom plate 40, a top plate 50, and optionally a cap 60 threadably connected to a discharge port 61 formed in the second end wall 15. The inlet port 20 and the outlet port 30 are further operably connected to one or more media dispensers 25, 35, respectively. The media dispensers 25, 35 may include one or more nozzles 26, 36 that can be positioned close to the bottom plate 40 to allow for cell media to flow through the inlet port 20 and the inlet media dispenser(s) 25 and the inlet nozzles 26, across the bottom plate 40 and then be withdrawn from the vessel via the outlet nozzle(s) 36 and the outlet media dispenser(s) 35 and the outlet port 30. The one or more nozzles 26, 36 may be aligned in a linear fashion across the width of the vessel 10. The one or more nozzles 26, 36 may be of 2, 3, 4, 5, 6, 7, 8, or more aligned in a linear fashion across the width of the vessel 10. On the bottom plate 40 and between the inlet nozzle(s) 26 and the outlet nozzle(s) 36 can reside or rest thereon cells for culturing (not depicted). The inlet port 20 can further be operably connected to inlet tubing 21 and the outlet port 30 can similarly be connected to outlet tubing 31 to allow for ingress and egress of liquid without disruption or exposure of the cell culture chamber 11 to air and potential contaminants therein. As depicted, the inlet port 20 and the outlet port 30 are positioned on the top plate 50, though in other non-depicted embodiments could be located on a sidewall 17, 18 or the first end wall 14 and the second end wall 15.

[0064] Turning to FIGS. 2A and 2B, an enlarged cutaway view of the cell culture chamber of the vessel (FIG. 2A) and a side view of the bottom plate 40 (FIG. 2B) are depicted. The nozzle(s) 26, 36 extend from the inlet media dispenser 25 and the outlet media dispenser 35, respectively, towards the bottom plate 40. As seen in FIG. 2A, the inclusion of multiple inlet nozzles 26 allows for the inlet media dispenser 25 to flow liquid into the cell culture chamber 11 across the entire width thereof.

[0065] The bottom plate 40 includes a cell culture substrate 41 positioned between the inlet nozzle(s) 26 and the outlet nozzle(s) 36. Cell culture substrate 41 may comprise a well plate 42 that includes a plurality of microcavities (or wells or indentations) 43 sized and shaped to receive at least one cell or to form a 3D culture, such as a spheroid or organoid, therein. The microcavities 43 may be shaped as concave indentations in a well plate 42. Accordingly, the well plate 42 is a cell culturing area that is configured to facilitate growth and development of the cells within cell culture chamber 11. Additionally, gas permeability is a property that can contribute to the 3D cell culture environment. By allowing for gas permeability within microcavities 43 of the cell culture vessel, cell growth may be encouraged. Microcavity vessels are unique in their geometry and formation in that they are formed from the substrate of the bottom plate 40 with microcavities 43 disposed therein. The microcavities 43 are gas permeable due to the thickness of the bottom plate 40, wherein gas permeability occurs because the substrate may be formed from a very thin polystyrene material, which has a thickness of about 28 micrometers to about 72 micrometers. Each microcavity 43 may include an inner cavity with a rounded bottom 44 that is non-adherent to cells. Thus, microcavity vessels as described herein are cell culture devices that facilitate 3D cell culture by allowing cells seeded into the microcavities 43 to self-assemble or attach to one another to form a spheroid in each microcavity. Microcavities 43 may be shallow and permit cell culture medium to cover the spheroids, organoids, or 3D cell aggregates in all cavities at once which improved ease of handling. The individual microcavities 43 may have any suitable dimensions. For example, the diameter or width of individual microcavities 43 may be in a range of about 500 microns to about 5 millimeters. The depth of individual microcavities 43 may be in a range of about 500 microns to about 6 millimeters. In some embodiments, a depth of the individual microcavities 43 may be about 500 microns to about 650 microns. In some aspects, a depth of the individual microcavities 43 may be about 1.6 millimeters. In some aspects, microcavities 43 can be formed in a thicker bottom plate 40 as the vessel 10 is compatible to perfusion culture (i.e., continuous flow of media over culture duration), dissolved oxygen can be supplied through the media circulation and the gas permeability of the well plate 42 is therefore not a prerequisite. The use of a thick bottom plate 40 allows for injection molding or any other high throughput process to make the microcavities 43, thus reducing cost.

[0066] Also shown in FIG. 2B is an optional mesh screen 120. In some aspects, the mesh screen 120 includes a web that constrains the array of microcavities 43. The mesh screen 120 itself may include a web film with a plurality of openings. The web film can be selected from a plastic woven mesh, a plastic non-woven mesh, a fiberglass woven mesh, a fiberglass non-woven mesh, or any plastic film having an array of openings or holes.

[0067] In some aspects, a mesh screen 120 may be positioned over the well plate 42 or microcavities 43 therein. It will be appreciated that the presence of such will allow for liquid media to flow through the microcavities 43 as well as single cells to pass through and enter the microcavities 43, while providing a physical barrier that aids in retaining 3D cell cultures therein. While in some aspects the mesh screen 120 can be simply disposed over the well plate 42, it can be of benefit to anchor the mesh screen 120 to prevent the mesh screen 120 from being dislodged, particularly in consideration of the media flow throughout the vessel. In some aspects, the mesh screen 120 may be anchored or attached to a side wall 17,18 and/or the first end wall 14 or the second end wall 15 (FIG. 1). Examples of such attachment or anchoring include use of an adhesive to affix the mesh screen 120 or thermal energy to weld or melt the mesh screen 120 to a portion of the bottom plate 40 or a wall 14,15,17,18 of the vessel. In some aspects, the mesh screen 120 may include a magnetic or ferro-magnetic portion therein or thereon such that placement of a magnetic field underneath the cell culture chamber 11 secures the mesh screen 120 over the microcavities 43 or well plate 42. In some aspects, the mesh screen 120 may be retractable or removable, thereby allowing a user access to 3D cell cultures within the well plate 42 or microcavities 43. In some aspects, the mesh screen 120 may include a solid frame about the perimeter thereof or a portion thereof (not depicted). It will be appreciated that the presence of a solid frame may act as an anchor or weight to hold the mesh screen 120 in place over the well plate 42 or microcavities 43. In some aspects, the mesh screen 120 may include at least one holding structure extending all the way to the outside of the top of the vessel, so that the mesh screen 120 can be pushed against the top of the bottom plate 40 when needed, or moved away from the bottom plate 40 (for instance, using a screw to turn around the extended structure of the solid frame). In this way, a user can then collect spheroids or organoids when desired.

[0068] FIG. 5 depicts a size comparison between a mesh screen 120 (left panel) and the microcavities 43 (right panel), wherein the openings of the meshed screen are smaller than the openings of the microcavities 43. In some aspects, the mesh screen 120 includes openings therein of a pre-determined size that allows individual cells can pass through and assist in retaining the cell spheroids within the cell-receiving surface. In some aspects, the mesh opening is of a size smaller than the diameter or expected diameter of the spheroids therein. In other aspects, the mesh opening may be larger than the diameter of the spheroids being retained, however, the presence of the mesh itself may mitigate against spheroids being lost. In some aspects, the mesh screen 120 comprises a diagonal length of greater than or equal to 25 pm to less than or equal to 500 pm. In some aspects, the diagonal length of the mesh opening is greater than or equal to 40 pm to less than or equal to 250 pm. In some aspects, the diagonal length of the mesh opening is greater than 100 pm to less than or equal to 250 pm. In some aspects, each opening in the mesh screen 120 is of a size selected to be greater than the diameter of a cell but smaller than the diameter of a spheroid of interest in the well.

[0069] Referring again to FIGS. 2A and 2B, in some aspects, the system of the present disclosure includes a flow of liquid media through the vessel 10. To facilitate the flow of liquid media, the inlet port 20 can further be operably connected (such as fluidly connected) to inlet tubing 21 that is in turn operably connected (such as fluidly connected) to a sealed media source (not pictured and see e.g. FIG. 4) to allow for delivery of sterile and/or recycled media. Similarly, the outlet port 30 is operably connected (such as fluidly connected) to outlet tubing 31 to allow for media to be withdrawn from the cell culture chamber 11 without exposure to environmental air which allows the systems to remain closed. In some aspects, oxygen and CO2 gas lines are incorporated into a sealed media source to control dissolved oxygen concentration and pH. In some aspects, the outlet tubing 31 leads to a waste container (FIG. 4). In other aspects the inlet tubing 21 of the inlet port 20 and the outlet tubing 31 of the outlet port 30 are operably connected (such as fluidly connected) to provide for recycling of the media in a closed system. By adding liquid media through the inlet port 20 and withdrawing the liquid media from the outlet port 30, the liquid media can circulate or flow against the cells residing or resting on or in the bottom plate 40. In aspects, the inlet and/or outlet ports 20, 30 may include a nozzle or series of nozzles, as described herein. In some aspects, the inlet nozzle(s) 26 are positioned close to a surface, such as a sidewall 17 or 18 of the bottom plate 40 of the vessel 10, such that as media flows from the inlet nozzle(s) 26, the media does not splash on the bottom plate 40 or otherwise cause unwanted disturbances to the circulating media and/or cells in the well plate 42. Similarly, placing the outlet nozzle(s) 36 of associated with the outlet port 30 at or near the bottom plate 40 of the vessel 10 will allow for a user to withdraw all or near all media from the vessel 10. In some aspects, the inlet nozzle(s) 26 and/or outlet nozzle(s) 36 may be moveable to allow a user to configure their placement relative to the bottom plate 40.

[0070] In some aspects, the inlet nozzle(s) 26 are positioned or configured to provide a flow of liquid media at or near the upper surface of the well plate 42.

[0071] Turning to FIGS. 3 and 4, shown are optional embodiments for the vessel 10 with regards to tubing 21 , 31. As shown, the tubing 21, 31 may optionally include one or more junction(s) 22, 32 to allow for multiple fluids to flow into the inlet port 20 or optionally to change the source or material flowing into the inlet port 20. It will also be appreciated that while not depicted, the inclusion of valves in the tubing 21, 31 can allow for the flow to be controlled and/or shut off. Similarly, clamp(s) 23, 33 can be utilized to restrict flow in a section of the tubing 21, 31. As seen in FIG. 4, the vessel 10 can be set up to recycle media and/or replace media within the cell culture chamber. The inlet tubing 21 is operably connected to a junction 22 that allows for connection to a media container 24 and to the outlet tubing 31. The outlet tubing 31 similarly is connected to a junction to allow additional flow to a waste container 34. The inlet tubing 21 and the outlet tubing 31 are routed through a pump 70 to allow for recirculation of media through the cell culture chamber 11.

[0072] It will be appreciated that liquid media may be introduced through the pump 70, such as a peristaltic pump. Similarly, liquid media may be withdrawn through a pumping or vacuuming means. It will be appreciated that while the flow into the vessel 10 through the inlet port 20 need not match the outflow rate, over prolonged periods of time it is beneficial to have the two rates be the same or near the same to prevent removing all media from the cells or from overflowing the vessel 10. In some aspects, the inlet port 20 and the outlet port 30 may be connected to the same pumping means. In some aspects, the inlet port 20 may provide fresh media and the outlet port 30 may deliver removed media to a waste container 34. In other aspects, the inlet port 20 and the outlet port 30 may be operably connected to provide for recirculating media through the vessel 10. In some aspects, the outlet tubing 31 may be divided to recirculate a portion of the media and to dispose of the remaining media. Similarly, the inlet tubing 21 may be connected to a supply of fresh media and to the recirculating stream.

[0073] It is also an aspect of the present disclosure to provide for a laminar flow of media against and over cells in culture and collect the media thereof for valuable cell byproducts. In some aspects, providing a laminar flow to cells in the microcavities 43 of the well plate 42 can promote the growth and/or production of extracellular vesicles (EVs). EVs for regenerative medicine therapeutics has garnered significant pre-clinical interest in the past decade. “Extracellular vesicles” is an encompassing term for a population of particles naturally released from cells that are delimited by a lipid bilayer and cannot replicate. EVs are enticing to researchers and clinicians as they are the messages cells use to communicate with one another and facilitate physiological responses via the biomolecular messages sent from one cell to another. The greatest challenge in EV therapeutics is large-scale manufacturing. Naive or unmodified EVs are collected and processed from stem cells via spent cell culture media. Due to the current a variety of techniques and tools with limited standards, there can be significant concerns with batch-to-batch variation. Furthermore, no current approach supports in vzvo-like conditions to facilitate appropriate EV production because scale-out and scale-up methods are more applicable to 2D cell culture, or 2.5D if microcarriers are used. It is an aspect of the present disclosure that these obstacles are overcome and that EVs can be reproducible produced by providing a laminar flow of mediate cells in the vessels and systems as described herein. It is well understood that cells, especially stem cells, behave much differently in vivo than they do in 2D monolayer in vitro culture which has cause for subsequent negative impacts on the therapeutic value of the EVs being generated. As demonstrated in the working examples, the system and methods of the present disclosure provided for batch collection of EVs from spent media streamed by laminar flow across mesenchymal stem cells (MSCs). FIG. 6 shows that in comparison to standard 2D culture, the laminar-flow perfused 3D cultured cells in the system and methods set forth herein were able to continue to produce EVs for almost two weeks longer than the 2D counter-parts, amassing over four times the EVs at fifteen days than the 2D culture achieved in two days. FIG. 7 shows an overview of 2D grown cells at two weeks and cells perfused with the system and methods of the present disclosure. The perfused cells are individual spheroids in comparison to the flat cells in the 2D culture. FIG. 8 shows that the system and methods of the present disclosure have no negative effect on cell viability or the averaged mode diameter of EVs produced.

[0074] In some aspects, the system may include materials to prevent cell adherence. The materials may make up at least regions in contact with cells, such as the cell-receiving surface and/or meshed screen, or 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 a surface of the wells) and/or the meshed screen are coated with an ultralow attachment chemistry (ULA) (see, US Pat. 5,002,582) or other non-binding chemistry (e.g., 2- methacryloyloxy ethyl phosphorylcholine (MPC) polymer). In some aspects, the meshed screen and the cell-receiving surface (e.g., at least a portion of a surface of the wells) are coated with the same polymer coating. In some aspects, the coating(s) may be selected from UV crosslinkable polyethylene glycol (PEG), poly(2-hydroxyethyl methacrylate), and/or agarose. It will also be appreciated that, in embodiments, the polymer coating of the mesh screen may be different from the coating for the wells of the cell receiving surface and inhibits cell attachment to the mesh screen. As shown in the working examples, use of a larger mesh opening can allow cells once introduced into the vessel to settle through the openings (exampled in FIG. 5) into the bottom of the cell-receiving surface(s) and then start growing into 3D spheroids or organoids (see, e.g., FIG. 7 bottom image). Once the spheroids grow into a size greater than the opening of the mesh screen, the mesh screen acts as a physical constraint to the spheroids or organoids and thus the spheroids or organoids remain within each microcavity or well of the cell-receiving surface during medium exchange, medium perfusion, or mechanical agitation. Even if the size of spheroids is smaller than the opening of the mesh screen, the mesh screen also provides certain constraints to the spheroids. Therefore, a user can actively perfuse media, or perform medium exchange easily, and/or apply mechanical agitation such as lateral shaking without disturbing the cultures.

[0075] In some aspects, it can be advantageous to dissolve the mesh screen, such as if the mesh screen is permanently or semi-permanently attached or affixed on or over the cellreceiving surface. Dissolvability of the mesh screen allows a user to retrieve cells therein once the purpose of the vessel is fulfilled, such as in the working examples upon the formation of a desired spheroid. For example, a permanent or semi-permanent mesh screen may possess openings sufficient for single cells to pass therethrough. The flow of media can then be established and spheroid(s) allowed to form within the vessel. Once of a sufficient and/or desired size, to retrieve such, the mesh screen may be dissolved. In such aspects, the mesh screen may be of a material dissolvable by enzymatic digestion. In some aspects, the mesh screen is of polygalacturonic acid (PGA) or cellulose.

[0076] In aspects, the present disclosure also concerns methods of using the vessel and systems as set forth herein. In some aspects, the methods include using the vessel 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 be in place ahead of time with the cells able to fall in between the mesh openings. The method may then include the process of establishing a flow of liquid media across the cells by adding liquid media through the inlet port and withdrawing from the outlet. In some aspects, the liquid media may be a tissue culture media and/or a cell -culture media, such as a media supplemented with salts, buffers, growth factors, antibiotic(s), cytokines, sugars, and combinations thereof. In some aspects, the liquid media is recycled or recirculated over the cell(s) in the cell-receiving surface or well. It will be appreciated that in the instance of multiple wells, each well may be seeded with one or more cells to initiate the 3D culturing therein. The separation of the wells allows for each well to grow unto itself. In some aspects, the methods include providing a flow of liquid media through the inlet port. As set forth herein, the inclusion of nozzle(s) may allow for the liquid media to flow into the vessel at one side of the well(s), with the outlet withdrawing media from the opposing side of the well(s). In some aspects, the withdrawn media may be entirely or partially recirculated back to the inlet. In some aspects, the media form the outlet may be entirely or partially directed to a waste or similar container.

[0077] In some aspects, the present disclosure includes methods for three-dimensional tissue culture that includes the steps of placing a cell or spheroid in the well of the system as set forth herein and flowing a tissue culture media into the system through the inlet, wherein the tissue culture media comes into contact with the cell or spheroid; then withdrawing the tissue culture media through the outlet port. In some aspects, the vessel of the system may be seeded with a suspension of single cells. With the assistance of gravity and non-attachment coatings would drive the cell to settle in the lowest points of the well plate. Depending on the concentration of cells and the size of the settling areas (such as microcavities) the result is single cells, small multicellular formations (spheroids), or large multicellular/tissue fragments (organoids). [0078] In some aspects, the methods of the present disclosure include providing the tissue culture media through the inlet port at a first rate. In some aspects, the methods may include withdrawing the tissue culture media at the 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 the tissue culture media across the cell-receiving surface.

EXAMPLES

[0079] The vessel as described herein was arranged to establish a laminar flow of recycled media across mesenchymal cells in the wells on the well plate. A mesh screen was dispatched over the wells to retain the cells therein. A control arrangement using conventional 2D cell culture for preparing spheroids was simultaneously arranged for comparison. The benefits of the batch collection of EVs from spent media streamed by laminar flow across mesenchymal stem cells (MSCs) is seen in FIG. 6. FIG. 6 shows that in comparison to standard 2D culture, the laminar-flow perfused 3D cultured cells in the system and methods set forth herein were able to continue to produce EVs for almost two weeks longer than the 2D counterparts, amassing over four times the EVs at fifteen days than the 2D culture achieved in two days. FIG. 7 shows an overview of 2D grown cells at two weeks and cells perfused with the system and methods of the present disclosure. The perfused cells are in 3D clusters in comparison to the flat cells in the 2D culture. FIG. 8 shows that the system and methods of the present disclosure have no negative effect on cell viability or averaged mode diameter of EVs produced.

[0080] While particular aspects have been illustrated 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. Moreover, although various aspects of the claimed subject matter have been described herein, such aspects need not be utilized in combination. It is therefore intended that the appended claims cover all such changes and modifications that are within the scope of the claimed subject matter.

[0081] It is appreciated that all reagents are obtainable by sources known in the art unless otherwise specified.

[0082] It is also to be understood that this disclosure is not limited to the specific aspects and methods described herein, as specific components and/or conditions may, of course, vary. Furthermore, the terminology used herein is used only for the purpose of describing particular aspects of the present disclosure and is not intended to be limiting in any way. It will be also understood that, although the terms “first,” “second,” “third” etc. may be used herein to describe various elements, components, regions, layers, and/or sections, these elements, components, regions, layers, and/or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer, or section from another element, component, region, layer, or section. Thus, “a first element,” “component,” “region,” “layer,” or “section” discussed below could be termed a second (or other) element, component, region, layer, or section without departing from the teachings herein. Similarly, as used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms, including “at least one,” unless the content clearly indicates otherwise. “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 will be further understood that the terms “comprises” and/or “comprising,” or “includes” and/or “including” when used in this specification, specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, 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.

[0083] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0084] Reference is made in detail to exemplary compositions, aspects and methods of the present disclosure, which constitute the best modes of practicing the disclosure presently known to the inventors. The Figures are not necessarily to scale. However, it is to be understood that the disclosed aspects are merely exemplary of the disclosure that may be embodied in various and alternative forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for any aspect of the disclosure and/or as a representative basis for teaching one skilled in the art to variously employ the present disclosure.

[0085] Patents, publications, and applications mentioned in the specification are indicative of the levels of those skilled in the art to which the disclosure pertains . These patents, publications, and applications are incorporated herein by reference to the same extent as if each individual patent, publication, or application was specifically and individually incorporated herein by reference.

[0086] The foregoing description is illustrative of particular embodiments of the disclosure, but is not meant to be a limitation upon the practice thereof. The following claims, including all equivalents thereof, are intended to define the scope of the disclosure.

Claims

CLAIMS We claim:

1. A vessel for culturing cells, 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 interior volume; a well plate disposed in the interior volume of the vessel, the well plate comprising a plurality of microcavities for receiving cells, spheroids, or organoids; an inlet port extending into the interior volume at a first end of the interior volume; and an outlet port extending into the interior volume at a second end of the interior volume opposite the first end, wherein: the well plate is positioned between the inlet port and the outlet port; and liquid media introduced into the interior volume at the inlet port flows over the well plate and is extracted from the interior volume at the outlet port.

2. The vessel of claim 1, wherein the plurality of microcavities comprise concave indentations in an upper surface of the bottom plate.

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

4. The vessel of claim 1, wherein the inlet port is operably connected to at least one nozzle and wherein an end of the at least one nozzle is disposed above the bottom plate within the interior volume.

5. The vessel of claim 4, wherein at least two nozzles are aligned in a linear fashion across a width of the bottom plate.

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

7. The vessel of claim 4, wherein the at least one nozzle is configured to provide a flow of liquid media at or near the top plate of the well plate .

8. The vessel of claim 4, further comprising: inlet tubing operably connected to the inlet port; and a pump operably connected to the inlet tubing.

9. The vessel of claim 1, wherein the outlet port is operably connected to at least one nozzle and wherein an end of the at least one nozzle is disposed above the bottom plate within the interior volume.

10. The vessel of claim 9, wherein at least two nozzles are aligned in a linear fashion across a width of the bottom plate.

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

12. The vessel of claim 9, wherein the at least one nozzle is configured to remove liquid media from an upper surface of the well plate.

13. The vessel of claim 9, further comprising: outlet tubing operably connected to the outlet port; and a pump operably connected to the outlet tubing.

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

15. The vessel of claim 14, further comprising a mesh screen disposed over the well plate.

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

17. The vessel of claim 15, wherein the mesh screen comprises a magnetic or ferromagnetic portion such that placement of a magnetic field underneath the well plate secures the mesh screen over at least one of the plurality of microcavities.

18. The vessel of claim 15, wherein the mesh screen comprises openings comprising a diagonal length of greater than or equal to 25 pm to less than or equal to 500 pm.

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

20. The vessel of claim 15, wherein the mesh screen further comprises a solid frame about a perimeter thereof.

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

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

24. The vessel of claim 15, wherein the mesh screen is dissolvable through enzymatic digestion.

25. The vessel of claim 1, further comprising a pump operably connected to the inlet port and configured to pump a liquid media through the inlet port on to the well plate.

26. The vessel of claim 25, wherein the pump is operably connected to the outlet port and configured to withdraw the liquid media from the well plate through the outlet port.

27. The vessel of claim 26, wherein the pump is configured to recirculate the liquid media from the outlet port to the inlet port.

28. The vessel of claim 1, further comprising a pump operably connected to the outlet port and configured to withdraw the liquid media from the well plate through the outlet port.

29. A method for replacing liquid media in the vessel of claim 1, comprising adding a liquid media through the inlet port.

30. The method of claim 29, wherein the liquid media is withdrawn through the outlet port.

31. A method for three-dimensional tissue culture, comprising: placing a cell, a spheroid, or an organoid in a microcavity of the vessel of claim 1; flowing a liquid media into the vessel through the inlet port, wherein the liquid media comes into contact with the cell or spheroid; and, withdrawing the liquid media through the outlet port.

32. The method of claim 31, wherein the liquid media flows in the inlet port at a first flow rate.

33. The method of claim 32, wherein the liquid media is withdrawn 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 a laminar flow of the liquid media across the well plate.