Microcavity bioreactors and systems for 3D cell culture

Abstract

A microcavity bioreactor is provided that allows cell growth and cell differentiation in the same vessel, such that higher order three-dimensional cell cultures such as organoids can be generated in a singular vessel. The microcavity bioreactor may be part of a microcavity bioreactor system that allows for perfusion based cell culture and perfusion based cell harvesting. The microcavity bioreactor includes at least one, and preferably at least two, fluid distributor structures in the housing vessel of the microcavity bioreactor.

Description

Atty. Docket No. SP24-300MICROCAVITY BIOREACTORS AND SYSTEMS FOR 3D CELL CULTURECROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority under 35 U.S.C. §119 of U.S. Provisional Application Serial No. 63 / 722,354 filed on November 19, 2024, the content of which is relied upon and incorporated herein by reference in its entirety.FIELD

[0002] The present disclosure relates to three-dimensional cell cultures and their production on a large scale.BACKGROUND

[0003] Three-dimensional (3D) cell culture is becoming increasingly used in today’s cell culture platforms as 3D cell culture better mimics in vivo physiology compared to traditionally used two-dimensional (2D) cell culture systems. Three-dimensional cell cultures are emerging as a new tool in early drug discovery and as potential therapeutics to treat disease. Current scaffold- free 3D cell culture systems are limited to small footprints such as petri dishes, microtiter plates, small flasks, and open plates. These systems work for smaller scale applications in basic research and drug discovery. However, there exists a need for large scale production of 3D spheroids and organoids to move beyond the limited applications small scale 3D cultures can provide. The present disclosure provides a solution to this need.SUMMARY

[0004] According to some aspects of the present disclosure, a cell culture apparatus for three-dimensional cell cultures is provided. In some aspects of the present disclosure, a cell culture system for producing three-dimensional cell cultures is provided. In yet some other aspects of the present disclosure, a method for producing three-dimensional cell cultures is provided.

[0005] Aspect 1. A cell culture apparatus for three-dimensional cell cultures is provided that comprises (1) a housing vessel, (2) an inlet port and an outlet port on the housing vessel, (3) an open frame structure comprising a plurality of pores, (4) a fluid distributor structure, and (5) aAtty. Docket No. SP24-300 plurality of microcavity substrates. The fluid distributor structure is between either the inlet port or outlet port and the open frame structure. Each microcavity substrate is adjacent to the open frame structure, and each microcavity substrate sits on, is attached to, or both sits on and is attached to at least one protrusion on the open frame structure.

[0006] Aspect 2. The cell culture apparatus of aspect 1, wherein the open frame structure is a first open frame structure and the fluid distributer structure is a first fluid distributor structure, and wherein the cell culture apparatus further comprises a second open frame structure and a second fluid distributor structure.

[0007] Aspect 3. The cell culture apparatus of aspect 2, wherein each fluid distributor structure is between either the inlet port or outlet port and either the first open frame structure or the second open frame structure.

[0008] Aspect 4. The cell culture apparatus of any one of aspects 1-3, the housing vessel further comprising sidewalls, wherein at least one of the sidewalls is approximately perpendicular to a long side of each of the plurality of microcavity substrates. The sidewall of the housing vessel is attached to the long side of each of the plurality of microcavity substrates.

[0009] Aspect 5. The cell culture apparatus of aspect 4, wherein the sidewall comprises a plurality of protrusions that each of the microcavity substrates sits on, that each of the microcavity substrates is attached to, or that each of the microcavity substrates sits on and is attached to.

[0010] Aspect 6. The cell culture apparatus of any one of aspects 1-5, wherein the microcavity substrates are ultra-low attachment microcavity substrates.

[0011] Aspect 7. A cell culturing system for three-dimensional cell culture is provided that comprises (1) a microcavity bioreactor, and (2) a media conditioning vessel. The microcavity bioreactor comprises (a) a housing vessel, (b) an inlet port and an outlet port on the housing vessel, (c) an open frame structure, (d) a fluid distributor structure, and (e) a plurality of microcavity substrates. The fluid distributor structure is between either the inlet port or outlet port and the open frame structure. Each microcavity substrate is adjacent to the open frame structure, and each microcavity substrate sits on or is attached to at least one protrusion on the open frame structure.Atty. Docket No. SP24-300

[0012] Aspect 8. The cell culturing system of aspect 7, wherein the open frame structure is a first open frame structure and the fluid distributer structure is a first fluid distributor structure. The cell culture apparatus further comprises a second open frame structure and a second fluid distributor structure.

[0013] Aspect 9. The cell culturing system of aspect 8, wherein each fluid distributor structure is between either the inlet port or outlet port and either the first open frame structure or the second open frame structure.

[0014] Aspect 10. The cell culturing system of any one of aspects 7-9, the housing vessel further comprising sidewalls, wherein at least one of the sidewalls is approximately perpendicular to a long side of each of the plurality of microcavity substrates. The sidewall of the housing vessel is attached to the long side of each of the plurality of microcavity substrates.

[0015] Aspect 11. The cell culturing system of aspect 10, wherein the sidewall comprises a plurality of protrusions that each of the microcavity substrates sits on, that each of the microcavity substrates is attached to, or that each of the microcavity substrates sits on and is attached to.

[0016] Aspect 12. The cell culturing system of any one of aspects 7-11, wherein the microcavity substrates are ultra-low attachment microcavity substrates.

[0017] Aspect 13. A method of culturing three-dimensional cell cultures is provided, comprising the steps of (1) providing a cell culture apparatus for culturing three-dimensional cell cultures, (2) providing cells of a cell type that form three-dimensional cell cultures, and (3) culturing the cells in the microcavities of the microcavity bioreactor. The cell culture apparatus comprises (a) a housing vessel, (b) an inlet port and an outlet port on the housing vessel, (c) an open frame structure, (d) a fluid distributor structure, and (e) a plurality of microcavity substrates comprising microcavities. The fluid distributor structure is between either the inlet port or outlet port and the open frame structure. Each microcavity substrate is adjacent to the open frame structure, and each microcavity substrate sits on or is attached to at least one protrusion on the open frame structure.Atty. Docket No. SP24-300

[0018] Aspect 14. The method of aspect 13, wherein the open frame structure is a first open frame structure and the fluid distributer structure is a first fluid distributor structure. The cell culture apparatus further comprises a second open frame structure and a second fluid distributor structure.

[0019] Aspect 15. The method of aspect 14, wherein each fluid distributor structure is between either the inlet port or outlet port and either the first open frame structure or the second open frame structure.

[0020] Aspect 16. The method of any one of aspects 13-15, the housing vessel further comprising sidewalls, wherein at least one of the sidewalls is approximately perpendicular to a long side of each of the plurality of microcavity substrates. The sidewall of the housing vessel is attached to the long side of each of the plurality of microcavity substrates.

[0021] Aspect 17. The method of aspect 16, wherein the sidewall comprises a plurality of protrusions that each of the microcavity substrates sits on, that each of the microcavity substrates is attached to, or that each of the microcavity substrates sits on and is attached to.

[0022] Aspect 18. The method of any one of aspects 13-17, wherein the microcavity substrates are ultra-low attachment microcavity substrates.

[0023] Aspect 19. The method of any one of aspects 13-18, wherein the step of culturing cells in the microcavities of the microcavity bioreactor further comprises the steps of proliferating the cells of a cell type and differentiating the cells of a cell type.

[0024] Aspect 20. The method of any one of aspects 13-19, further comprising the step of harvesting the cultured cells from the microcavity bioreactor by perfusion, by rotation of the microcavity bioreactor, or a combination thereof.

[0025] Additional features and advantages 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 as described herein, including the detailed description which follows, the claims, as well as the appended drawings.Atty. Docket No. SP24-300

[0026] It is to be understood that both the foregoing general description and the following detailed description are merely exemplary and are intended to provide an overview or framework to understanding the nature and character of the claims. The accompanying drawings are included to provide a further understanding and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiment(s), and together with the description serve to explain principles and operation of the various embodiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The following is a description of the figures in the accompanying drawings, given purely by way of non-limiting example. The figures are not necessarily to scale, and certain features and certain views of the figures may be shown exaggerated in scale or in schematic in the interest of clarity and conciseness.

[0028] FIG. 1 is a long-axis side view of a microcavity bioreactor for 3D cell culture, according to some aspects of the present disclosure.

[0029] FIG. 2 is a short-axis side view of the microcavity bioreactor for 3D cell culture from FIG. 1, according to some aspects of the present disclosure.

[0030] FIG. 3 is a perspective view of an open frame structure, according to some aspects of the present disclosure.

[0031] FIG. 4 is a schematic drawing of a microcavity bioreactor system, according to some aspects of the present disclosure.DETAILED DESCRIPTION

[0032] The various aspects and embodiments will now be fully described herein. These aspects and embodiments may, however, be embodied in many different forms and should not be construed as limiting; rather, these embodiments are provided so the disclosure will be thorough and complete, and will fully convey the scope of the present subject matter to those skilled in the art. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.Atty. Docket No. SP24-300

[0033] Modifications of the disclosure will occur to those skilled in the art and to those who make or use the disclosure. Therefore, it is understood that the embodiments shown in the drawings and described above are merely for illustrative purposes and not intended to limit the scope of the disclosure, which is defined by the following claims, as interpreted according to the principles of patent law, including the doctrine of equivalents.A. Definitions

[0034] Unless defined otherwise, all terms and phrases used herein include the meanings that the terms and phrases have attained in the art, unless the contrary is clearly indicated or clearly apparent from the context in which the term or phrase is used. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, particular methods and materials are now described.

[0035] As used herein the terms “the,” “a,” or “an,” mean “at least one,” and should not be limited to “only one” unless explicitly indicated to the contrary. Thus, for example, reference to “a component” includes embodiments having two or more such components unless the context clearly indicates otherwise.

[0036] Unless otherwise stated, the use of individual numerical values are stated as approximations as though the values were preceded by the word “about” or “approximately.” Similarly, the numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word “about” or “approximately.” In this manner, variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. As used herein, the terms “about” and “approximately” when referring to a numerical value shall have their plain and ordinary meanings to a person of ordinary skill in the art to which the disclosed subject matter is most closely related or the art relevant to the range or element at issue. The amount of broadening from the strict numerical boundary depends upon many factors. For example, some of the factors which may be considered include the criticality of the element and / or the effect a given amount of variation will have on the performance of the claimed subject matter, as well as other considerations known to those of skill in the art. As used herein, the use of differing amounts of significant digits for different numericalAtty. Docket No. SP24-300 values is not meant to limit how the use of the words “about” or “approximately” will serve to broaden a particular numerical value or range. Thus, as a general matter, “about” or “approximately” broaden the numerical value. Also, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values plus the broadening of the range afforded by the use of the term “about” or “approximately.” Consequently, recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually recited herein.

[0037] As used herein, the term “and / or,” when used in a list of two or more items, means that any one of the listed items can be employed by itself, or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing components A, B, and / or C, the composition can contain A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[0038] As used herein, “have,” “having,” “include,” “including,” “comprise,” “comprising” or the like are used in their open ended sense, and generally mean “including, but not limited to.”

[0039] “Optional” or “optionally” means that the subsequently described element, component or circumstance may or may not occur, so that the description includes instances where the element, component, or circumstance occurs and instances where it does not.

[0040] In this document, relational terms, such as first and second, top and bottom, and the like, are used solely to distinguish one entity or action from another entity or action, without necessarily requiring or implying any actual such relationship or order between such entities or actions.

[0041] All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.B. IntroductionAtty. Docket No. SP24-300

[0042] Three-dimensional (3D) cell culture platforms for small scale productions have been applied to basic research, drug discovery, clinical research and therapeutics. These platforms can be classified into scaffold-based, scaffold-free, and 3D printing, enabling a range of 3D culture systems including spheroids, mamospheres, organoids, and organs-on-chips.

[0043] Currently existing scaffold-free based 3D cell culture is limited to petri dishes, microtiter plates (2-well, 6-well, 24-well, 96-well, 384-well or 1536-well), flasks and open plate formats, which generally allows for only static culturing methods. However, due to potential therapeutic implications with 3D cell cultures, the 3D cell culture industry needs large scale bioproduction of 3D cell cultures, including for spheroid and organoid cultures. Large scale 3D cell culture systems offer not only a solution for scale-up cell production, but also new forms of tissue engineering, regenerative medicine, and therapeutics for treating many different diseases. For example, stem cell spheroids have been proposed as a new form of regenerative medicine as spheroid cultures have been reported to improve the efficacy of mesenchymal stem cell (MSC)- based therapeutics. Further, spheroid cultures have been used to enrich patient-specific stem cells for disease treatment, and to scale up stem cell products for the use in clinical trials.

[0044] The present disclosure provides microcavity bioreactors and systems for creating large scale 3D cell cultures (such as spheroids, organoids, mammospheres, embryonic bodies, etc.) and their byproducts (e.g., extracellular vesicles), using perfusion culturing conditions. The microcavity bioreactors include centered, vertically stacked microcavity substrates, which are attached to an open frame structure and may be sandwiched between two fluid distributor plate structures. The present disclosure also provides a microcavity bioreactor system, such as one comprising a microcavity bioreactor, a media conditioning vessel (MCV), and a mechanical pump to drive medium flow recirculation from the media conditioning vessel to the bioreactor, and then back to the media conditioning vessel.

[0045] The bioreactor system of the present disclosure permits perfusion culture and medium exchange with little or minimal disturbance to the 3D cell cultures within the microcavities, which allows culturing for longer periods of times than with static-based culturing systems. It also permits automated harvesting of 3D cell cultures and / or their byproducts for downstream applications.Atty. Docket No. SP24-300

[0046] Additional features and advantages will be set forth in the detailed description which follows and will be apparent to those skilled in the art from the description or recognized by practicing the embodiments as described in the following description, together with the claims and appended drawings.C. Microcavity Bioreactors and Systems

[0047] The microcavity bioreactor of the present disclosure has a housing vessel, at least one inlet port, at least one outlet port, and a plurality of substrates for culturing 3D cell cultures that can be stacked in a vertical formation. The microcavity substrates are held in position by at least one open frame structure at the ends of the substrate that allows for the vertical stacking formation of the microcavity substrates. The one or more open frame structures have openings such that fluid may flow across the frame through the openings. When at least two open frame structures are present, fluid may flow into the inlet port of the microcavity bioreactor, through a first open frame structure, across or through the microcavity substrates, then through a second open frame structure, and then through the outlet port of the microcavity bioreactor. The open frame structures have a plurality of protrusions that help position the microcavity substrates.

[0048] The inlet and outlet ports may be anywhere on the housing vessel. Referring to the inlet port, the inlet port may be anywhere on the housing vessel that allows the inlet port to fluidly connect to a first open frame structure where fluid can flow through the frame and to at least one of the microcavity substrates. In embodiments where at least two open frame structures are present, the outlet port may be anywhere on the housing vessel that allows the outlet port to fluidly connect to a second open frame structure where fluid can flow from at least one of the microcavity substrates and through the second frame and to the outlet port. In some embodiments, flow may be reversible such that an inlet port can become an outlet port and an outlet port can become an inlet port.

[0049] In some embodiments, the microcavity bioreactor may further have at least one fluid distribution structure between the frame(s) and the inlet port and / or outlet port. A fluid distribution structure may alter the flow of the fluid entering the housing vessel through an inlet port to distribute the fluid through a first open frame structure of the microcavity vessel. Likewise, a fluid distribution structure may alter the flow of fluid exiting through a second open frameAtty. Docket No. SP24-300 structure of the microcavity vessel to connect to an outlet port on housing vessel so fluid may exit the microcavity bioreactor. The microcavity bioreactor may have a fluid distribution structure between the inlet port and the first open frame structure, a fluid distribution structure between the second open frame structure and the outlet port, or a combination thereof.

[0050] FIGS. 1-2 illustrate schematics of exemplary microcavity bioreactors. Microcavity bioreactor 100 comprises a housing vessel 110, an inlet port 120, an outlet port 130, and a plurality of microcavity substrates 140 that are vertically stacked. Each microcavity substrate 140 has a plurality of microcavities across the microcavity substrate 140. The microcavity substrates 140 are held in position by two open frame structures 150a, 1506 having a plurality of protrusion structures 152. Fluid distributor structures 160a and 1606 are between the ports of the housing vessel 110 and two open frame structures 150a and 1506. The first fluid distributor structure 160a is positioned between the inlet port 120 of the housing vessel 110 and the first open frame structure 150a, while the second fluid distributor structure 1606 is positioned between the second open frame structure 1506 and the outlet port 130 of the housing vessel 110. In some embodiments, the first and second open frame structures 150a, 1506 are independent structures from each other, meaning they are not connected to each other. In some embodiments, the first and second open frame structures 150a, 1506 are connected to each other, meaning they are either part of a singular structure or are otherwise held together by a frame or other type of connector (e.g., horizontal support structure such as a rod, square bar, etc.).

[0051] The microcavity substrates 140 are held in position by protrusions 152 on the open frame structures 150a, 1506 and / or through protrusions or attachment means on the side wall of the housing vessel 110. The attachment means may be adhesive, ultrasonic welding, or using any other means for attachment known to those of ordinary skill in the art. It is not necessary to form a tight seal between the plurality of microcavity substrates 140 and the protrusion structures 152 of the open frame structures, or with the side wall of the housing vessel 110.

[0052] The microcavity substrates may be a plastic film or a plastic plate, or may be glass or other materials known to those of ordinary skill in the art. If plastic, the microcavity substrates may be formed from polystyrene, polypropylene, polyethylene, polylactic acid, polyhydroxyalkanoates, polyvinylchloride, polycarbonate, combinations thereof, or any otherAtty. Docket No. SP24-300 appropriate polymer known to those of ordinary skill in the art. The microcavities of the microcavity substrate can be formed by any technique known to those of ordinary skill in art, including but not limited to hot embossing, hot plate pressing, laser microfabrication process, and self-assembled microsphere monolayer-assisted polymer casting.

[0053] In some embodiments, the diameter of each microcavity may be between 100 pm and 1500 pm. As used herein, the “diameter” of the microcavity refers to the average distance across the top of the microcavity. The microcavity may have any shape when viewed from the top (from above), such as circular, oval, square, rectangular, pentagonal, hexagonal, or even an irregular shape. In some embodiments, the diameter of the microcavity may be between 100 pm and 1000 pm, between 200 pm and 1200 pm, between 300 pm and 1400 pm, between 400 pm and 1500 pm, between 800 pm and 1500 pm, between 700 pm and 1400 pm, between 600 pm and 1300 pm, between 500 pm and 1200 pm, between 400 pm and 1100 pm, between 300 pm and 1000 pm, between 200 pm and 900 pm, between 100 pm and 800 pm, between 100 pm and600 pm, or between 400 pm and 1200 pm, or at any value or in any range between 100 pm and 1500 pm. The diameter and depth may be chosen based on the desired application. For example, if the application is for smaller spheroid cultures, the microcavities may have a smaller diameter and smaller depth (e.g., 100-500 pm diameter and 100-500 pm depth). If the application is, for example, larger mamospheres or larger organoid cultures, the microcavities may instead have a larger diameter and larger depth (e.g., 500-1300 pm diameter and 500-1300 pm depth).

[0054] The bottoms of the microcavities may be flat, may be rounded, or may any other geometry such as cone-shaped, pointed, or angled. In a preferred embodiment, the bottoms of the microcavities are rounded. In one specific embodiment, the bottoms of the microcavities are hemispherical. In another specific embodiment, the bottoms of the microcavities are flat. In yet another specific embodiment, the bottoms of the microcavities are not flat.

[0055] The microcavities of the microcavity substrate may be treated (in part or in their entirety) to help encourage 3D cell culture formation. For example, a coating may be applied to the microcavities that comprises an ultralow attachment chemistry. As used herein, an “ultralow attachment” chemistry refers to molecules that repels cells from adhering onto the surface of aAtty. Docket No. SP24-300 substrate, such as a plastic film. Any ultralow attachment chemistries known to those of ordinary skill in the art may be employed. In one nonlimiting example, the plurality of microcavities include a coating on their surface, and the coating comprises 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer.

[0056] Referring to FIG. 3, the open frame structure 150 has a plurality of protrusion structures 152. In some embodiments, the number of microcavity substrates 140 is the same as the number of protrusion structures 152. In other embodiments, the number of microcavity substrates 140 is less than the number of protrusion structures 152. The open frame structure 150 has a plurality of open pores 155 that allow fluid and air to flow through from one side to the other of the open frame structure 150. Each microcavity substrate 140 is held in position by the protrusion structure 152 that the microcavity substrate 140 sits on. As shown in FIG. 1, when two open frame structures are used, one end of a microcavity substrate 140 sits on a first protrusion structure 152 at a particular height from the bottom of the first open frame structure 150a, and the other end of microcavity substrate 140 sites on a second protrusion structure 152 at the same height from the bottom of the second open frame structure 1506.

[0057] Referring back to FIG. 1, during perfusion culture, media or other aqueous solutions will enter the housing vessel 110 through an inlet port 120. The media or other aqueous solutions will then pass sequentially through the first fluid distributor structure 160a, the first open frame structure 150a, the microcavity substrates 140, the second open frame structure 1506, and the second fluid distributor structure 1606, before reaching outlet port 130. The open frame structures 150a, 1506 can be made by any process known to those of ordinary skill in the art, such as injection molding, or 3D printing.

[0058] The protrusion structure 152 of the open frame structure 150 can be a flange, a trough, or any other shape that allows the microcavity substrate 140 to sit on the protrusion structure 152 or to secure the microcavity substrate 140 on the protrusion structure 152. In some embodiments, the protrusion structure 152 is a shelf, a flange, or a trough. The outer portions of the microcavity substrate 140 that contact the protrusion structure may be shaped to match the shape of the protrusion structure 152. The protrusion structure 152 of the open frame structure 150 is preferably a trough. In one embodiment, the microcavity substrate 140 is secured to a protrusionAtty. Docket No. SP24-300 structure 152 that is a trough by snapping a shaped outer portion of the microcavity substrate 140 into a matching receptacle in the trough.

[0059] The side walls of the housing vessel 110 may also have protrusions to aid in in holding microcavity substrate 140 in position. The protrusions on the side walls of the housing vessel 110 may be any shape that allows the microcavity substrate 140 to sit on the protrusion of the side wall or to secure the microcavity substrate 140 on the protrusion of the side wall. In one particular embodiment, the side wall of the housing vessel 110 has a trough along the side wall at the same height as the protrusion structure 152, such that open frame structure 150a and / or open frame structure 1506 can be snapped into the trough and help hold the microcavity substrate 140 in position. In one embodiment, the trough runs continuously along the entirety of the side wall. In another embodiment, the trough may be discontinuous along the side wall, such that there is a single trough that is less than the entirety of the side wall, or there are multiple shorter troughs along the side wall. It should be understood that for each microcavity substrate in the plurality of microcavity substrates, there may be corresponding protrusions along the side walls of the housing vessel.

[0060] The open pores 155 of the open frame structure 150 may have any shape. An individual pore may have the shape of circle, square, triangle, rectangle, hexagon, or any other geometry, including shapes such as trapezoids and parallelograms. The open pores 155 of the open frame structure 150 may form a web, a net, a slot, or any other configurations in the open frame structure 150.

[0061] The microcavity bioreactor may be part of a microcavity bioreactor system. The microcavity bioreactor system may include a media conditioning vessel, and one or more pumps for transferring fresh media to the microcavity bioreactor and for transferring spent media from the microcavity bioreactor. Instead of a media conditioning vessel, the microcavity bioreactor system may include a first vessel with fresh media and a second vessel for disposing spent media. Tubing or other connectors for fluid flow can be used to connect any of the vessels to the microcavity bioreactor through the inlet and outlet ports on the microcavity bioreactor. It should be understood that fluid can flow into or out of either port. For example, fluid may flow into a microcavity bioreactor through the outlet port and out of the microcavity bioreactor through theAtty. Docket No. SP24-300 inlet port. Alternatively, fluid may flow into the microcavity bioreactor through the inlet port and out of the microcavity bioreactor through the outlet port. In some embodiments, the flow direction into the microcavity bioreactor may alternate between the inlet and outlet ports. The microcavity bioreactor system may have additional components, including but not limited to single use pH sensors, oxygen sensors, pressure sensors, flow rate sensors, which can be incorporated at the appropriate location(s) in the bioreactor system depending on the applications and operations, as understood by those of ordinary skill in art. Other additional components, such as gas (oxygen, nitrogen, CO2) lines can also be incorporated into the bioreactor system in locations such as the media conditioning vessel, as understood by those of ordinary skill in the art.

[0062] An exemplary microcavity bioreactor system 200 is shown in FIG. 4. Microcavity bioreactor system 200 comprises microcavity bioreactor 100, a media conditioning vessel 210, and a mechanical pump 240. The media conditioning vessel 210 contains media 250, or other aqueous solutions used for cell culture. Mechanical pump 240 is used to drive fluid recirculation between the media conditioning vessel 210 and the microcavity bioreactor 100, thus enabling perfusion culture. A first tubing 220 connects the media conditioning vessel 210 to the inlet port of the microcavity bioreactor 100, while a second tubing 230 connects the media conditioning vessel 210 to the outlet port of the microcavity bioreactor 100. The fluid movement can begin in the media conditioning vessel and flow to the inlet of the microcavity bioreactor 100, or it can begin from the media conditioning vessel and flow to the outlet of the microcavity bioreactor 100, or the flow can alternate from the media conditioning vessel to the inlet and the outlet of the microcavity bioreactor 100.

[0063] The present disclosure also contemplates methods of using the microcavity bioreactors and systems described herein. The methods include the steps of (1) seeding cells into the microcavities of the microcavity substrates, (2) culturing the seeded cells in the microcavities by perfusion in the microcavity bioreactor into three-dimensional cell cultures, and (3) collecting the three-dimensional cell cultures from the microcavity bioreactor.

[0064] Regarding the step of seeding cells, any cell type that grows 3D cell cultures such as organoids, spheroids, embryoid bodies, or mamospheres can be used. Cells may be added into the media conditioning vessel to create a cell suspension solution. The cell suspension solutionAtty. Docket No. SP24-300 may then be loaded into the microcavity bioreactor through the inlet port. A pump connected to tubing that connects the media conditioning vessel to the inlet of the microcavity bioreactor may control the flow of the cell suspension solution into the microcavity bioreactor. Once past the inlet of the microcavity bioreactor, the cell suspension solution passes the first fluid distributor structure, which helps to distribute cells more evenly into the microcavities of the microcavity substrates. During this seeding step, the cell suspension solution can be loaded into the microcavities of the microcavity bioreactor with any flow rate that does not shear or otherwise damage the cells to be cultured.

[0065] Regarding the step of culturing, the cells may be cultured in the microcavities of the microcavity bioreactor under continuous perfusion or discontinuous perfusion, according to the protocol for the specific cell type, number of cells, and cell growth pattern. When needed, fresh media can be used to replace spent media in the media conditioning vessel at any time during the culture.

[0066] Certain types of cells, like stem cells, can both grow more of themselves (proliferation) and can differentiate into different types of cells (differentiation). The microcavity bioreactor described herein can be used for both proliferation and differentiation stages. In some embodiments, the step of culturing comprises culturing the seeded cells in proliferation media. Proliferation media is any cell culturing medium suitable for proliferating cells. In some embodiments, the step of culturing comprises culturing the seeded cells in proliferation media and in differentiation media. Differentiation media is any cell culturing medium suitable for differentiating cells. In this embodiment, a cell differentiation medium can replace the cell proliferation medium after cells form desired aggregates in the cell proliferation medium. The cell differentiation medium can then drive the aggregated cells into growing into an organoid.

[0067] After the step of culturing is completed, the formed three-dimensional cell cultures may be harvested or collected. The three-dimensional cell cultures, such as spheroids, mamospheres or organoids, can be collected from the microcavity bioreactor in several ways. In one embodiment, the three-dimensional cell cultures may be harvested from the microcavities of the microcavity bioreactor by rotating the microcavity bioreactor up to 90 degrees from a horizontal position. The horizontal position (i.e. 0 degrees) is when the bottom of the microcavityAtty. Docket No. SP24-300 bioreactor is parallel to the ground. In a further embodiment, the rotation of microcavity bioreactor further includes agitating the microcavity bioreactor during the rotation. In a different embodiment, the three-dimensional cell cultures may be harvested from the microcavities of the microcavity bioreactor by rotating the microcavity bioreactor to an angle greater than 90 degres from a horizontal position. In a further embodiment, the rotation of microcavity bioreactor further includes agitating the microcavity bioreactor during the rotation. Once the three-dimensional cell cultures have been dislodged from the microcavities, the three-dimensional cell cultures are perfused out of the microcavity bioreactor into a collection vessel, or even into the media conditioning vessel. This harvesting procedure works for both collection of three-dimensional cell cultures and any extracellular vesicles produced by the cell cultures.

[0068] While the present disclosure includes a limited number of embodiments, those skilled in the art, having benefit of this disclosure, will appreciate that other embodiments can be devised which do not depart from the scope of the present disclosure.

Claims

Atty. Docket No. SP24-300CLAIMSWhat Is Claimed Is:

1. A cell culture apparatus for three-dimensional cell cultures, comprising: a housing vessel; an inlet port and an outlet port on the housing vessel; an open frame structure comprising a plurality of pores; a fluid distributor structure; and a plurality of microcavity substrates; wherein the fluid distributor structure is between either the inlet port or outlet port and the open frame structure; and wherein each microcavity substrate is adjacent to the open frame structure, and each microcavity substrate sits on, is attached to, or both sits on and is attached to at least one protrusion on the open frame structure.

2. The cell culture apparatus of claim 1, wherein the open frame structure is a first open frame structure and the fluid distributer structure is a first fluid distributor structure, and wherein the cell culture apparatus further comprises a second open frame structure and a second fluid distributor structure.

3. The cell culture apparatus of claim 2, wherein each fluid distributor structure is between either the inlet port or outlet port and either the first open frame structure or the second open frame structure.

4. The cell culture apparatus of any one of claims 1-3, the housing vessel further comprising sidewalls, wherein at least one of the sidewalls is approximately perpendicular to a long side of each of the plurality of microcavity substrates; andAtty. Docket No. SP24-300 wherein the sidewall of the housing vessel is attached to the long side of each of the plurality of microcavity substrates.

5. The cell culture apparatus of claim 4, wherein the sidewall comprises a plurality of protrusions that each of the microcavity substrates sits on, that each of the microcavity substrates is attached to, or that each of the microcavity substrates sits on and is attached to.

6. The cell culture apparatus of any one of claims 1 -5, wherein the microcavity substrates are ultra-low attachment microcavity substrates.

7. A cell culturing system for three-dimensional cell culture, comprising: a microcavity bioreactor, wherein the microcavity bioreactor comprises: a housing vessel; an inlet port and an outlet port on the housing vessel; an open frame structure; a fluid distributor structure; and a plurality of microcavity substrates; and a media conditioning vessel; wherein the fluid distributor structure is between either the inlet port or outlet port and the open frame structure; and wherein each microcavity substrate is adjacent to the open frame structure, and each microcavity substrate sits on or is attached to at least one protrusion on the open frame structure.

8. The cell culturing system of claim 7, wherein the open frame structure is a first open frame structure and the fluid distributer structure is a first fluid distributor structure, and wherein the cell culture apparatus further comprises a second open frame structure and a second fluid distributor structure.Atty. Docket No. SP24-3009. The cell culturing system of claim 8, wherein each fluid distributor structure is between either the inlet port or outlet port and either the first open frame structure or the second open frame structure.

10. The cell culturing system of any one of claims 7-9, the housing vessel further comprising sidewalls, wherein at least one of the sidewalls is approximately perpendicular to a long side of each of the plurality of microcavity substrates; and wherein the sidewall of the housing vessel is attached to the long side of each of the plurality of microcavity substrates.

11. The cell culturing system of claim 10, wherein the sidewall comprises a plurality of protrusions that each of the microcavity substrates sits on, that each of the microcavity substrates is attached to, or that each of the microcavity substrates sits on and is attached to.

12. The cell culturing system of any one of claims 7-11, wherein the microcavity substrates are ultra-low attachment microcavity substrates.

13. A method of culturing three-dimensional cell cultures, comprising the steps of: providing a cell culture apparatus for culturing three-dimensional cell cultures, the cell culture apparatus comprising: a housing vessel; an inlet port and an outlet port on the housing vessel; an open frame structure; a fluid distributor structure; and a plurality of microcavity substrates comprising microcavities; providing cells of a cell type that form three-dimensional cell cultures; and culturing the cells in the microcavities of the microcavity bioreactor;Atty. Docket No. SP24-300 wherein the fluid distributor structure is between either the inlet port or outlet port and the open frame structure; and wherein each microcavity substrate is adjacent to the open frame structure, and each microcavity substrate sits on or is attached to at least one protrusion on the open frame structure.

14. The method of claim 13, wherein the open frame structure is a first open frame structure and the fluid distributer structure is a first fluid distributor structure, and wherein the cell culture apparatus further comprises a second open frame structure and a second fluid distributor structure.

15. The method of claim 14, wherein each fluid distributor structure is between either the inlet port or outlet port and either the first open frame structure or the second open frame structure.

16. The method of any one of claims 13-15, the housing vessel further comprising sidewalls, wherein at least one of the sidewalls is approximately perpendicular to a long side of each of the plurality of microcavity substrates; and wherein the sidewall of the housing vessel is attached to the long side of each of the plurality of microcavity substrates.

17. The method of claim 16, wherein the sidewall comprises a plurality of protrusions that each of the microcavity substrates sits on, that each of the microcavity substrates is attached to, or that each of the microcavity substrates sits on and is attached to.

18. The method of any one of claims 13-17, wherein the microcavity substrates are ultra-low attachment microcavity substrates.

19. The method of any one of claims 13-18, wherein the step of culturing cells in the microcavities of the microcavity bioreactor further comprises the steps of proliferating the cells of a cell type and differentiating the cells of a cell type.

20. The method of any one of claims 13-19, further comprising the step of harvesting the cultured cells from the microcavity bioreactor by perfusion, by rotation of the microcavity bioreactor, or a combination thereof.

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