Systems and apparatuses for cell culture

Abstract

A cell culture vessel includes a cell culture container having a cell culture compartment. The cell culture vessel also includes a cover, where the cell culture container and the cover are formed from a gas impermeable material. The cell culture vessel also includes a gasket assembly disposed in a contact plane between the cell culture container and the cover and forming a gas impermeable seal.

Description

SYSTEMS AND APPARATUSES FOR CELL CULTURECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and priority to, U.S. Provisional Patent Application Serial No. 63 / 515,411 filed on July 25, 2023, and U.S. Provisional Patent Application Serial No. 63 / 515,408 filed on July 25, 2023. The entire contents of the foregoing applications are incorporated by reference herein.BACKGROUND

[0002] Cell culture plates are an essential tool for cell biology research. They are used to grow cells in a controlled environment, which allows for study of the effects of different conditions on cell growth and development. The plates are typically made of plastic or glass and may have one or more wells, each of which can hold a small amount of cell culture media. The media provides the cells with the nutrients the cells need to grow and divide. Cell culture plates may be used in incubators to grow cells in a controlled environment as well as in glove boxes. The incubator provides the cells with the necessary conditions for growth, including a constant temperature, humidity, and atmosphere.

[0003] Conventional cell culture plates are susceptible to evaporation, which causes increased osmolarity of cell culture media. This in turn causes unrealistic growth of cells as well as potential differences between wells due to uneven evaporation. In addition, evaporation causes increased concentration of the salts involved in electrical signaling of electrically active cell types, changing the ionic gradients across the cell membrane, thereby affecting all characteristics of the initiation, transmission (and computation) in electrically active cells such as cardiac or neuronal cells.

[0004] It is also difficult to maintain desired dissolved gas concentration with standard cell culture plates. This generally requires use of a compressed gas system, which uses gas regulators, sensors that are expensive and have limited lifetimes, and feedback control as well as a glove box for culture and / or handling.

[0005] An incubator is used to maintain the desired temperature of the cell culture plates. The incubator impedes access to the cultures for feeding, for microscopy, etc. Furthermore, observation equipment for use inside an incubator needs to be designed to resist incubator conditions (e.g., body temperature heat and humidity). Incubators also take up significant space and packing of incubators in a laboratory is space-inefficient relative to the form factor of the cell culture plates. As the number of cell culture plates in a single incubator increases, the ability of the incubator to perform its function decreases, since there is a minimum number of times an incubator may be accessed per week per cell culture vessel. However, every time the incubator is accessed, it is unable to perform its functions for a prolonged period of time, e.g., over 30 minutes.

[0006] A further technical problem is that cell culture devices that use an air gap for gas exchange have an increased risk of microbial contamination via that air gap. This makes it difficult to perform manual cell culture experiments over the course of months without contamination. In addition, cross-contamination is more likely if multiple different experiments are being performed in the same laboratory. Thus, there is a need for cell culture vessels and systems that overcome these problems.SUMMARY

[0007] The present disclosure provides cell culture vessels that are configured to regulate gases (e.g., oxygen) and pH (via carbon dioxide) without any direct air contact with the cell culture media or air exchange with the outside environment. The vessels use nonporous, gas permeable membranes to exchange gas with a regulating environment without water evaporation. These vessels are also totally sealed and do not require standard incubator equipment, making them compact and versatile devices for culturing cells, bacteria, etc.

[0008] According to one embodiment of the present disclosure, a cell culture vessel is disclosed. The cell culture vessel includes a cell culture container having a base surrounded by sidewalls extending from the base and defining a cell culture compartment. The cell culture vessel also includes a gas permeable film having a first side and a second side and disposed over the cell culture compartment. The first side of the gas permeable film contacts a cell culture media disposed inside the cell culture compartment without an air gap in between.

[0009] Implementations of the above embodiment may include one or more of the following features. According to one aspect of the above embodiment, the cell culture container may be formed from a polymer. The polymer may be one of polycarbonate, polystyrene, polyethylene terephthalate, and combinations thereof. The gas permeable film may be formed from a polymer, which may be one of polyolefins, perfluorodioxolanes, polysiloxanes, perfluoroalkoxy alkanes, perfluorinated polymers, fluorosilicones, and combinations thereof. The gas permeable film may be formed from polymethylpentene. The gas control fluid container may be coupled to the cell culture container in a gastight manner such that the second side of the gas permeable film contacts the gas control fluid. The gas control fluid may be a buffer solution and may include at least one of carbonic acid, bicarbonate, or carbonate. The gas control fluid and the cell culturemedia may be in a carbon dioxide / carbonic acid equilibrium. The gas permeable film may be formed from a polymer permeable to carbon dioxide. The cell culture container may include a plurality of fluid access ports fluidly coupled to the cell culture compartment. The cell culture vessel may include a bumper disposed over gas permeable film and the sidewalls.

[0010] According to another embodiment of the present disclosure, a cell culture system is disclosed. The cell culture system includes a gas impermeable cell culture container and a gas exchanger, which includes a gas control fluid container having a gas control fluid. The gas exchanger also includes a media container coupled to the gas control fluid container in a gastight manner. The media container includes a base surrounded by sidewalls extending from the base and defining a media compartment. The media container is fluidly coupled to the cell culture container. The media container and the cell culture container are filled with a cell culture media. The gas exchanger further includes a gas permeable film having a first side and a second side. The film is disposed between the gas control fluid container and the media container, where the first side of the gas permeable film contacts the gas control fluid, and the second side of the gas permeable film contacts the media.

[0011] Implementations of the above embodiment may include one or more of the following features. According to one aspect of the above embodiment, at least one of the cell culture container, the gas control fluid container, or the media container may be formed from a polymer. The polymer may be one of polycarbonate, polystyrene, polyethylene terephthalate, and combinations thereof. The gas permeable film may be formed from a polymer, which may be one of polyolefins, perfluorodioxolanes, polysiloxanes, perfluoroalkoxy alkanes, perfluorinated polymers, fluorosilicones, and combinations thereof. The gas permeable film may be formed from polymethylpentene. The gas permeable film may be formed from a polymer permeable tocarbon dioxide. Each of the cell culture container, the gas control fluid container, and the media container may include a plurality of fluid access ports. The gas control fluid may be a buffer solution and may include at least one of carbonic acid, bicarbonate, or carbonate. The gas control fluid and the media may be in a carbon dioxide / carbonic acid equilibrium.

[0012] According to a further embodiment of the present disclosure, a cell culture vessel is disclosed. The cell culture vessel includes a cell culture container having a cell culture compartment. The cell culture vessel also includes a cover, where the cell culture container and the cover are formed from a gas impermeable material. The cell culture vessel also includes a gasket assembly disposed in a contact plane between the cell culture container and the cover and forming a gas impermeable seal.

[0013] Implementations of the above embodiment may include one or more of the following features. According to one aspect of the above embodiment, the gasket assembly may include an inner gasket adjacent and around the cell culture compartment and configured to provide a non- cytotoxic liquid seal for the cell culture compartment and an outer gasket disposed around the inner gasket and configured to provide the gas impermeable seal for the cell culture compartment. The inner gasket may be formed from a biocompatible polymer, which may be selected from polyolefins, perfluorodioxolanes, polysiloxanes, perfluoroalkoxy alkanes, perfluorinated polymers, fluorosilicones, FFKM perfluoroelastomers, and combinations thereof. The outer gasket may be formed from isobutylene isoprene rubber. One of the cell culture container or the cover may include an inner cutout configured to secure the inner gasket and an outer cutout configured to secure the outer gasket. The cover may include a plurality of access ports fluidly coupled to the cell culture compartment. The cell culture container and the covermay be formed from a polymer, which may be selected from polycarbonate, polystyrene, polyethylene terephthalate, and combinations thereof.

[0014] According to yet another embodiment of the present disclosure, a cell culture vessel is disclosed. The cell culture system includes a cell culture vessel having a cell culture container with a cell culture compartment. The cell culture vessel also includes a cover. The cell culture container and the cover are formed from a gas impermeable material. The cell culture vessel also includes a gasket assembly disposed in a contact plane between the cell culture container and the cover. The gasket forms a gas impermeable seal. The cell culture system also includes a gas exchanger having a gas control fluid container with a gas control fluid. The gas exchanger also includes a media container coupled to the gas control fluid container in a gastight manner. The media container includes a base surrounded by sidewalls extending from the base and defining a media compartment. The media container is fluidly coupled to the cell culture container, and the media container and the cell culture container are filled with a cell culture media. The gas exchanger further includes a gas permeable film having a first side and a second side. The gas permeable film is disposed between the gas control fluid container and the media container, where the first side of the gas permeable film contacts the gas control fluid, and the second side of the gas permeable film contacts the media.

[0015] Implementations of the above embodiment may include one or more of the following features. According to one aspect of the above embodiment, one of the cell culture container, the cover, the gas control fluid container, or the media container may be formed from a polymer, which may be selected from polycarbonate, polystyrene, polyethylene terephthalate, and combinations thereof. The gas permeable film may be formed from a polymer, which may be selected from polyolefins, perfluorodioxolanes, polysiloxanes, perfluoroalkoxy alkanes,perfluorinated polymers, fluorosilicones, and combinations thereof. The gas permeable film may be formed from polymethylpentene. The gas permeable film may be formed from a polymer permeable to carbon dioxide. Each of the cell culture container, the gas control fluid container, the media container may include a plurality of fluid access ports. The gas control fluid may be a buffer solution and may include at least one of carbonic acid, bicarbonate, or carbonate. The gas control fluid and the media may be in a carbon dioxide / carbonic acid equilibrium. The media compartment may have a serpentine configuration. The cell culture system may include a pump configured to move the media between the media container and the cell culture container. The media container may be fluidly coupled to the cell culture container via one or more fluid lines. The pump may be a peristaltic pump coupled to the fluid line(s). The media compartment may include a magnetic stir bar disposed therein.BRIEF DESCRIPTION OF DRAWINGS

[0016] Various embodiments of the present disclosure are described herein below with reference to the figures wherein:

[0017] FIG. 1 is a side, cross-sectional view of an air gap free cell culture vessel according to an embodiment of the present disclosure;

[0018] FIG. 2 is a side, cross-sectional view of an air gap free cell culture vessel according to another embodiment of the present disclosure;

[0019] FIG. 3 is a top, cross-sectional view of a cell culture container of the air gap free cell culture vessel of FIG. 2;

[0020] FIG. 4 is a side, cross-sectional view of the cell culture container of FIG. 3;

[0021] FIG. 5 is a front, cross-sectional view of the cell culture container of FIG. 3;

[0022] FIG. 6 is a side, cross-sectional view of an incubator free (i.e., sealed) cell culture vessel according to an embodiment of the present disclosure;

[0023] FIG. 7 is a side, cross-sectional view of a sealed cell culture vessel according to another embodiment of the present disclosure;

[0024] FIG. 8 is a schematic of chemical processes and equilibria involved in cell culture vessels of FIGS. 6 and 7;

[0025] FIG. 9 is a side, cross-sectional view of an incubator free cell culture system with a separated gas exchanger according to an embodiment of the present disclosure;

[0026] FIG. 10 is a side, cross-sectional view of a cell culture vessel according to an embodiment of the present disclosure;

[0027] FIG. 11 is a side, cross-sectional view, with parts separated, of the cell culture vessel of FIG. 10;

[0028] FIG. 12 is a top view of a gasket assembly of the cell culture vessel of FIG. 10;

[0029] FIG. 13 is a recirculating cell culture system for use with the cell culture vessel of FIG. 10;

[0030] FIG. 14 is a top, interior view of a media container of a recirculating gas exchanger of the recirculating system of FIG. 13;

[0031] FIG. 15 is top, interior view of a fluid container of the recirculating gas exchanger of the recirculating system of FIG. 13;

[0032] FIG. 16 is an open fluid cell culture system for use with the cell culture vessel of FIG. 10;

[0033] FIG. 17 shows plots of relative sodium concentration change over time for a Petri dish and a filter flask;

[0034] FIG. 18 shows plots of relative sodium concentration change over time for the air gap free cell culture vessel of FIGS. 1-5 and the incubator free cell culture vessel of FIGS. 6 and 7;

[0035] FIG. 19 shows plots of pH vs days of cell cultures;

[0036] FIG. 20 shows plots of dissolved carbon dioxide vs days of cell cultures;

[0037] FIG. 21 shows plots of dissolved oxygen vs days of cell cultures;

[0038] FIG. 22 is a photograph of the recirculating cell culture system of FIG. 13;

[0039] FIG. 23 is a plot of pH vs time in a non-recirculating loop;

[0040] FIG. 24 is a plot of pH vs time in a recirculating loop;

[0041] FIG. 25 shows a side, schematic cross-sectional view, with parts separated of a cell culture vessel according to another embodiment of the present disclosure;

[0042] FIG. 26 is a perspective, with parts separated of the cell culture vessel of FIG. 25;

[0043] FIG. 27 is perspective, cross-sectional view, with parts separated, of the cell culture vessel of FIG. 25;

[0044] FIG. 28 is a side, cross-sectional view of the cell culture vessel of FIG. 25;

[0045] FIG. 29 is a schematic of gas exchange in a conventional plate and a bubbled reservoir;

[0046] FIG. 30 is a schematic of chemical processes and equilibria involved in the cell culture vessel of FIG. 25;

[0047] FIG. 31 is a plot of oxygen saturation over time in the cell culture vessel of FIG. 25;

[0048] FIG. 32 shows dot plots of metabolic activity in the cell culture vessel of FIG. 25 and a conventional well plate in an incubator;

[0049] FIG. 33 shows dot plots of glucose consumption, lactate production, and aerobic quotient in the cell culture vessel of FIG. 25 and a conventional well plate in an incubator;

[0050] FIG. 34 is a side, cross-sectional view of a sealed cell culture vessel according to another embodiment of the present disclosure;

[0051] FIG. 35 is a perspective, cross-sectional view of the sealed cell culture vessel of FIG. 34;

[0052] FIG. 36 is a perspective view of a cell culture vessel assembly including a plurality of the sealed cell culture vessel of FIG. 34;

[0053] FIGS. 37A-D show plots of evaporation over time (36 hours) in a 96 well plate;

[0054] FIGS. 38A-C show plots of evaporation over time (36 hours) in a 10 cm well plate;

[0055] FIGS. 39A-B show plots of evaporation over time (36 hours) in a T25 flask;

[0056] FIG. 40 shows a plot of evaporation over time (36 hours) in the sealed cell culture vessel of FIG. 34;

[0057] FIG. 41 shows a plot of pH and pCCE vs sodium carbonate concentration in a gas control fluid according to an embodiment of the present disclosure; and

[0058] FIG. 42 shows a dot plot of metabolic activity in the sealed cell culture vessel of FIG. 34 and a conventional 12 well plates inside and outside an incubator.DETAILED DESCRIPTION

[0059] With reference to FIG. 1, an air gap free cell culture vessel 10 includes a cell culture container 12 having a base 14 surrounded by sidewalls 16 extending from the base 14 to define a cell culture compartment 18. A cell culture, microorganisms, or organoids are placed in a media inside the culture compartment 18. The cell culture vessel 10 also includes a film 20 disposed over the cell culture container 12 enclosing the cell culture compartment 18.

[0060] The cell culture container 12 may be formed from any gas impermeable, biocompatible (i.e., not harmful to living tissue) material, which may be glass, metal (e.g., titanium, cytocompatible steels including but not limited to 316 and 254 SMO grades, nickel-chromium- based alloys such as Inconel, etc.) and / or a polymer, including but not limited to one or more of polyarylethersulfones, polyaryletherketones polycarbonates, polystyrenes, polyethyleneterephthalates, and the like. The use of gas impermeable thermoplastics maximizes efficiency by eliminating gas equilibration with the outside environment. The cell culture container 12 may be constructed using manufacturing techniques such as subtractive manufacturing (e.g., computer numerical control (CNC) milling, machining, laser cutting), additive manufacturing (e.g., 3D printing), injection molding, thermal or ultrasonic welding of walls, and the like.

[0061] The film 20 may be attached to the cell culture container 12 by laminating the film 20 onto the sidewalls 16, i.e., flange formed by the sidewalls. In embodiments, the film 20 may be used to form one or more of the surfaces of the cell culture container 12 or portions of the sidewalls 16. This film 20 is in contact with the cell culture media such that gases may diffuse across the film 20 between the cell culture compartment 18 and the atmosphere on the opposite side of the film 20 or with a container holding a gas control fluid as shown in FIGS. 6 and 7. The film 20 is nonporous and is gas permeable to oxygen, carbon dioxide, and any other gases (e.g., nitrogen) and may be formed from a biocompatible polymer, including but not limited to polyolefins (e.g., polymethylpentene), perfluorodioxolanes (e.g., poly-perfluoro(2-methylene-4- methyl- 1,3 -dioxolane), polysiloxanes (e.g., polydimethylsiloxane), perfluoroalkoxy alkanes, perfluorinated polymers (e.g., fluorinated ethylene propylene), and combinations thereof.

[0062] The film 20 separates an otherwise sealed cell culture compartment 18 from the outside environment. As such, no water vapor escapes through the film 20, however the cell culture vessel 10 may still depend on its surrounding environment having actively regulated oxygen and carbon dioxide levels. The gas atmosphere may also be temperature regulated since gas dissolution equilibria are generally temperature dependent.

[0063] As shown FIG. 2 the air gap free cell culture vessel 10 may include a bumper 22 disposed over film 20 laminated over the sidewalls 16, to protect the film 20 and secure it to the sidewalls16. With reference to FIGS. 2-5, the cell culture vessel 10 may have any suitable shape such as a rectangle as shown in FIG. 3, circle, etc. The cell culture compartment 18 may have a base surface area of about 21.5 cm2and a depth of about 2 mm to match dimensions and volume of standard 60 mm cell culture plates. The cell culture vessel 10 includes a plurality of (i.e., two or more) access ports 24, which may be airtight Luer lock ports, providing fluid access to the cell culture compartment 18 for loading the cell culture (i.e., cells and corresponding liquid media). The access ports 24 are in fluid communication with the cell culture compartment 18 via fluidic channels 26, which may enter the cell culture compartment 18 from its base due to the shallow depth of the compartment 18 and to accommodate the threaded access ports 24 through the sidewalls 16. During loading, the cell culture may be deposited in the cell culture 18 through one of the ports 24, while the other of the ports 24 is open to allow for air to escape as the cell culture compartment 18 is filled. The ports 24 may be sealed with Luer locks or any other stoppers, e.g., an airtight gasketed lid.

[0064] As noted above, the cell culture vessel 10 uses a gas permeable film 20 placed directly over the media (i.e., containing media) without any intervening air filling that space. Thus, the cell culture compartment 18 is devoid of air since the cell culture compartment 18 is completely filled with the cell culture media and as a result there is no ullage or an air gap present. As a result, apart from the film 20, there is no other site of ingress or egress of gas from the device since the cell culture container 12 is formed from a gas impermeable material since the access ports 24 are sealed after the cell culture is loaded. The film 20 has a low surface energy such that even though the immersion of the film surface in the media presents an additional potential surface on which for cells to adhere, adhesion is energetically unfavorable.

[0065] With reference to FIG. 6, a sealed (i.e., incubator free) cell culture vessel 40 includes a gas control fluid container 42 disposed over the cell culture container 12 of FIGS. 1-5. The fluid container 42 may be formed from the same material(s) as the cell culture container 12 and includes a cover 44 surrounded by sidewalls 46 extending from the cover 44 to define fluid compartment 48, which stores a gas control fluid. The gas control fluid may include a carbonic acid - bicarbonate - carbonate buffer solution. The sidewalls 46 contact the sidewalls 16 of the cell culture container 12 in a gastight manner. Thus, the cell culture compartment 18 and the control fluid compartment 48 are enclosed within a gastight cell culture vessel 40 and are separated from each other by the gas permeable film 20. Although the embodiment of FIG. 6 shows the gas control fluid container 42 being disposed over the cell culture container 12, it is envisioned that the arrangement may be reversed or the containers may be disposed side-by-side, while the film 20 is used to form a sidewall that separates the compartments.

[0066] With reference to FIG. 7, the cell culture vessel 40 may be placed over a heater 60 to maintain desired temperature of the cell culture. The film 20 may be laminated between the cell culture container 12 and the fluid container 42.

[0067] The fluid container 42 includes a plurality of (i.e., two or more) access ports 50, which are substantially similar to the access ports 24, and are used to fill the fluid container 42 with the buffer solution. The fluid container 42 may be filled after cell culture is loaded into the cell culture container 12 and the fluid container 42 is attached to the cell culture container 12. Just like the cell culture compartment 18, the gas control fluid compartment 48 is devoid of air since the gas control fluid compartment 48 is completely filled with the gas control fluid and there is no ullage or an air gap present.

[0068] The gas control fluid compartment 48 provides a passive buffer system across the film 20, which provides dissolved gas regulation, removing the need for a gas-controlled atmosphere surrounding the cell culture vessel 40. FIG. 8 shows schematic chemical processes and equilibria in the cell culture vessel 40. Inside the gas control fluid compartment 48, a carbonic acid - bicarbonate - carbonate based buffer system is established to provide a buffered concentration of carbonic acid, which is in equilibrium with carbon dioxide. At and around physiological pH this equilibrium favors carbon dioxide. Diffusion and convection dispose the dissolved carbon dioxide throughout the buffer in the gas control fluid compartment. At the interface between the gas control fluid and the film 20, the carbon dioxide is in equilibrium between dissolution in the fluid and in the film 20. Carbon dioxide diffuses freely through the film 20 and is in equilibrium with the other side of the film 20, i.e., the cell culture compartment 18. Carbon dioxide is also disposed throughout the cell culture media by diffusion and convection. Here, the dissolved carbon dioxide completes the carbonic acid - bicarbonate - carbonate buffer system via its interconversion with carbonic acid. As a result, this buffer system maintains physiological pH despite the generation of ammonium, lactic acid and other acid species by cellular metabolism.

[0069] FIG. 9 shows a cell culture system 70, which includes a cell culture container 72 fluidly coupled to a gas exchanger 90 via a fluid line 82. The fluid line 82 may be formed from any gas impermeable material such as unplasticized polyvinylidene fluoride, polytetrafluoroethylene, polyvinylchloride, polypropylene, or polycarbonate.

[0070] The cell culture container 72 is substantially similar to the cell culture container 12 with the exception of the film 20 being replaced by a cover 80. The cell culture container 72 includes a base 74 surrounded by sidewalls 76 extending from the base 74 to define a cell culture compartment 78. The cell culture is placed in a media inside the culture compartment 78 viaaccess ports 84 as described above with respect to access ports 24. The cell culture container 72 may be formed from the same gas impermeable material(s) as the cell culture container 12.

[0071] The gas exchanger 90 is structurally similar to the cell culture vessel 40 of FIG. 6. The gas exchanger 90 includes a gas control fluid container 102 disposed over a media container 92, which is substantially similar to cell culture container 12 of FIGS. 1-5 but is used to store the cell culture media.

[0072] The media container 92 includes a base 94 surrounded by sidewalls 96 extending from the base 94 to define a media compartment 98 storing the cell media. The media container 92 also includes a film 100 disposed over the media container 92 enclosing the media compartment 98. The media container 92 includes a plurality of (i.e., two or more) access ports 99 that provide access to the media compartment 98 for filling with the cell culture media. The access ports 99 are also used to couple to the fluid line 82 to fluidly couple the media compartment 98 with the culture compartment 78. The media compartment 98 may have any suitable shape that follows the outline of the media container 92. In embodiments, the media compartment 98 may have a serpentine shape as shown schematically in FIG. 13 and in a plan view shown in FIG. 14.

[0073] The media container 92 and the fluid container 102 may be formed from the same material(s) as the cell culture container 12 of FIGS. 1-5. The fluid container 102 includes a cover 104 surrounded by sidewalls 106 extending from the cover 104 to define fluid compartment 108, which stores a gas control fluid as described above with respect to the fluid container 42 of FIG. 4. The media compartment 98 and the control fluid compartment 108 are enclosed within the gastight gas exchange vessel 90 and are separated from each other by the gas permeable film 100. The media container 92 also includes a plurality of (i.e., two or more)access ports (not shown), which are substantially similar to the access ports 50 or 24, and are used to fill the fluid container 102 with the buffer solution.

[0074] Gas exchange happens separately from the cell culture container 72, and the media is conditioned using the gas permeable film 100 and the passive gas control fluid in the control fluid compartment 108 in the manner described above with respect to FIG. 8. The preconditioned media stored in the media compartment 98 is then circulated through the cell culture container 72, e.g., via automated fluidic actuators. The cell culture system 70 of FIG. 9 provides a more biomimetic method by regulating the gas concentration of the immediate cell culture environment via gas exchange with another aqueous compartment, akin to the gas exchange between interstitial fluid and the bloodstream. The cell culture compartment 78, the media compartment 98, and the fluid compartment 108 are devoid of air since they are completely filled with cell culture media or the gas control fluid, respectively, and as a result, there is no ullage or an air gap present in these compartments. In embodiments, the gas exchanger 90 may only include the media container 92 with the film 100 being similarly exposed as the cell culture container 12.

[0075] With reference to FIGS. 10 and I l a cell culture vessel 200 includes a cell culture container 212 having a base 214 surrounded by sidewalls 216 extending from the base 214 to define a cell culture compartment 218. The cell culture compartment 218 may be subdivided into a plurality of partitions 218a and 218b. Cells or organoids are placed in a media inside the culture compartment 218 and their partitions 218a and 218b. The cell culture vessel 200 also includes a cover 220 disposed over the cell culture container 212 enclosing the cell culture compartment 218. The cover 220 includes a plurality of (i.e., two or more) access ports 224a and 224b, which may be airtight Luer lock ports, providing fluid access to the cell culturecompartment 218. The access ports 224a and 224b are in fluid communication with the cell culture compartment 218 via fluidic channels 226a and 226b, respectively. The cell culture container 212 and the cover 220 may be formed from the same material(s) as the cell culture container 12.

[0076] With reference to FIGS. 11 and 12, a gasket assembly 240 may be disposed between the cover 220 and the cell culture compartment 218 to provide for a gastight seal for the cell culture compartment 218. The gasket assembly 240 may include a plurality of (i.e., two or more) gaskets, an inner gasket 242 and an outer gasket 244 disposed in a boundary (i.e., contact) plane between the cover 220 and the cell culture compartment 218.

[0077] The cell culture compartment 218 may include a plurality of (i.e., two or more) partitions, an inner cutout 252 and an outer cutout 254 defined in a contact (i.e., top) surface of the cell culture compartment 218. The cutouts 252 and 254 are configured to seat the inner and outer gaskets 242 and 244, respectively. In embodiments, the partitions may be defined in the contact (i.e., bottom) surface of the cover 220. The inner gasket 242 provides a non-cytotoxic liquid seal for the cell culture compartment 218. The inner gasket 242 may be formed from biocompatible, gas permeable elastomer, including but not limited to polyolefins (e.g., polymethylpentene), perfluorodioxolanes (e.g., poly-perfluoro(2-methylene-4-methyl- 1,3 -di oxolane), polysiloxanes (e.g., polydimethylsiloxane), perfluoroalkoxy alkanes, perfluorinated polymers (e.g., fluorinated ethylene propylene), fluorosilicones, FFKM perfluoroelastomers (such as KALREZ™ fluoropolymer elastomers available from DuPont of Wilmington, DE) and combinations thereof. Gas permeable elastomers generally all relatively gas permeable with some exceptions, such as isobutylene isoprene rubber. However, isobutylene isoprene rubber may release leachates by direct contact with water which are also known to be toxic to multiple cell lines and organoids.Thus, the outer gasket 244 is formed from gas impermeable, flexible material, such as isobutylene isoprene rubber, and surrounds the inner gasket 242 thereby providing a gastight seal between the cover 220 and the cell culture compartment 218. The inner and outer gaskets 242 and 244 may be constructed using manufacturing techniques such as subtractive manufacturing (e.g., CNC drag knife cutting, CNC rotary knife cutting, manual die punching, CNC routing, machining, laser cutting), additive manufacturing (e.g., 3D printing), injection molding, and the like.

[0078] The cell culture vessel 200 may be disposed in contact with a heating block 230, which may be formed from any heat conducting material, e.g., metal. The heating block 230 includes sidewalls 232 defining a cavity 234 configured to receive the cell culture container 212. The cover 220 may be secured to the cell culture container 212 via a plurality of bolts 228, which are configured to be threaded into corresponding threaded channels 229 defined in the sidewalls 232 of the heating block 230. The heating block 230 may be placed on the heater 60 to regulate the temperature of the cell culture vessel 200.

[0079] As shown in FIG. 12, the cell culture vessel 200 (i.e., the cell culture container 212 and the cover 220) may have a substantially rectangular shape and the cell culture compartment 218 may have a circular shape. The inner gasket 242 may have a ring shape and outer gasket 244 may have an inner perimeter that is circular, i.e., matches the shape of the inner gasket 242 and the cell culture compartment 218 and an outer perimeter that is rectangular, i.e., matches the shape of the cell culture container 212. The channels 229 pass through the comers of the cell culture container 212, the cover 220, and the outer gasket 244.

[0080] The cell culture vessel 200 may be used with a gas exchanger using a similar setup as shown in FIG. 9. The organoid and cell culture vessels according to the present disclosure maybe used with separate gas exchangers using the following configurations of FIGS. 13 and 16, although other configurations are also possible. In FIG. 13, recirculated media is used, which flows through an external gas exchanger. This is media-efficient since it reduces waste but increases and therefore dilutes the effective cell / organoid culture media volume. Alternatively, it is possible to precondition media prior to injection into the cell culture compartment, then regularly cycle the media to waste as shown in FIG. 16. This is media-inefficient but reduces the active media volume “in use” at any given time.

[0081] FIG. 13 shows a cell culture system 270, which includes the cell culture vessel 200 fluidly coupled to a recirculating gas exchanger 290 via fluid lines 282a and 282b, which may be substantially similar to the fluid line 82.

[0082] The gas exchanger 290 is structurally similar to the gas exchanger 90 of FIG. 9. The gas exchanger 290 includes a gas control fluid container 302 disposed over a media container 292, which is substantially similar to cell culture container 92 of FIG. 9 but is used to store the cell culture media.

[0083] With reference to FIGS. 13 and 14, the media container 292 includes a base 294 surrounded by sidewalls 296 extending from the base 294 to define a media compartment 298 storing the cell media. The media compartment 298 has a serpentine shape shown schematically in FIG. 13 and in a plan view of the prototype media compartment 298 shown in FIG. 14. The media container 292 also includes a film 300 disposed over the media container 292 enclosing the media compartment 298. The serpentine shape of the media compartment 298 has a larger surface area (i.e., than a straight channel) that is in contact with the film 300 and in turn with the gas control fluid stored in the fluid container 302, which provides for a larger gas exchange surface.

[0084] The media container 292 includes a plurality of (i.e., two or more) access ports 299a and 299b that provide access to the media compartment 298 for filling with the cell culture media. The access ports 299a and 299b are coupled via the fluid lines 282a and 282b to the access ports 224a and 224b of the cell culture vessel 200, respectively. The fluid line 282b is inserted into a pump 285, which may be a stepper motor based, peristaltic pump configured to circulate the media through the fluid circuit defined by the fluid lines 282a and 282b, the media compartment 298, and the cell culture compartment 218. The access ports 299a and 299b are fluidly coupled to media compartment 298.

[0085] The media container 292 and the fluid container 302 may be formed from the same material(s) as the cell culture container 12 of FIGS. 1-5. As shown in FIGS. 13 and 15, the fluid container 302 includes a cover 304 surrounded by sidewalls 306 extending from the cover 304 to define fluid compartment 308, which stores a gas control fluid as described above with respect to the fluid container 92 of FIG. 9. The surface area of the fluid compartment 308 may fully encompass the serpentine media compartment 298 to maximize the overlap between the media compartment 298 and the fluid container 302.

[0086] The media compartment 298 and the control fluid compartment 308 are enclosed within the recirculating gas exchanger 290 and are separated from each other by the gas permeable film 300. The media container 292 also includes a plurality of (i.e., two or more) access ports (not shown), which are substantially similar to the access ports 50 or 24 and are used to fill the fluid container 302 with the buffer solution. Gas exchange happens separately from the cell culture vessel 200, and media is conditioned using the gas permeable film 300 and the passive gas control fluid in the control fluid compartment 308 in the manner described above with respect to FIG. 8. The preconditioned media stored in the media compartment 298 is then circulatedthrough the cell culture container 72 via the pump 285. The sealed culture system 70 of FIG. 9 provides a more biomimetic method by regulating the gas concentration of the immediate cell culture environment via gas exchange with another aqueous compartment, akin to the gas exchange between interstitial fluid and the bloodstream. The cell culture compartment 218, the media compartment 298, and the fluid compartment 308 are devoid of air since they are completely fdled with cell culture media or the gas control fluid, respectively, and as a result, there is no ullage or an air gap present in these compartments.

[0087] FIG. 16 shows an intermittent feeding (i.e., open) cell culture system 370, which includes the cell culture vessel 200 fluidly coupled to a gas exchanger 390 via fluid lines 282a and 282b. The cell culture system 370 is substantially similar to the cell culture system 270 of FIG. 13 and only the differences are described below.

[0088] The media compartment 298 rather than having a serpentine shape may have a rectangular or any other suitable shape to accommodate a stir bar 372. The gas exchanger 390 may be placed on a magnetic stirring device (not shown), which rotates the stir bar 372 to enable faster equilibration of the media in the gas exchanger 390. In addition, the cell culture system 370 may be configured as an open fluidic system to allow for intermittent feeding of the cell culture disposed in the cell culture vessel 200. The pump 285 pumps the feeding media stored in a media source (not shown) through a fluid line 282c entering the gas exchanger 390 where the media is stirred by the stir bar 372 and equilibrated with gas control fluid. The media then enters the cell culture vessel 200 through the fluid line 282a and exits through the fluid line 282b coupled to the access port 224b of the cell culture vessel 200. The fluid line 282b may dispose of the media exiting the cell culture vessel 200 in a separate container (not shown).

[0089] One advantage of air gap-free cell cultures is in preventing the evaporation. As a result, cell culture devices no longer need to be kept in a humidified environment. While mixed gas blends with elevated carbon dioxide levels are used to achieve carbonate bicarbonate buffer pH regulation, this removes one of the major constraints in cell culturing, which forces the use of incubators or incubator equivalents. Another benefit is that stricter osmolarity may be maintained than possible with even a perfectly humidified incubator.

[0090] FIGS. 25-28 show a cell culture vessel 400 that is similar to the cell culture vessel 200 of FIGS. 10 and 11 with some modifications that are described below. The cell culture vessel 400 includes a cell culture container 412 having a base 414 surrounded by sidewalls 416 extending from the base 414 to define a cell culture compartment 418. The cell culture compartment 418 may be subdivided into a plurality of partitions 418a-d, which may be arranged in any suitable configuration, e.g., a 2 x 2 matrix (FIG. 26). Cells or organoids are placed in a media inside the culture compartment 418 and their partitions 418a-d. The cell culture vessel 400 also includes a cover 420 disposed over the cell culture container 412 enclosing the cell culture compartment 418.

[0091] As shown in FIGS. 25 and 28, the cover 420 includes a plurality of (i.e., two or more) access ports 424a and 424b, which may be airtight Luer lock ports, providing fluid access to the cell culture compartment 418. The access ports 424a and 424b are in fluid communication with the cell culture compartment 418 via fluidic channels 426a and 426b, respectively.

[0092] The cell culture container 412 and the cover 420 may be formed from the same material(s) as the cell culture container 12, which may be any gas impermeable, biocompatible (i.e., not harmful to living tissue) material, such as glass, metal (e.g., titanium, cytocompatible steels including but not limited to 316 and 254 SMO grades, nickel-chromium-based alloys suchas Inconel, etc.) and / or a polymer, including but not limited to one or more of polyarylethersulfones, polyaryletherketones polycarbonates, polystyrenes, polyethylene terephthalates, and the like. The use of gas impermeable thermoplastics maximizes efficiency by eliminating gas equilibration with the outside environment. In further embodiments, the cell culture container 412 and the cover 420 may be formed from the same materials as each other or from different materials. For example, the cell culture container 412 may be formed from metal, e.g., titanium, and the cover 420 may be formed from glass.

[0093] The cell culture container 412 and the cover 420 may be constructed using manufacturing techniques such as subtractive manufacturing (e.g., computer numerical control (CNC) milling, machining, laser cutting), additive manufacturing (e.g., 3D printing), injection molding, thermal or ultrasonic welding of walls, and the like.

[0094] With reference to FIGS. 25-28, a gasket assembly 440 may be disposed between the cover 420 and the cell culture compartment 418 to provide for a gastight seal for the cell culture compartment 418. The gasket assembly 440 may include a plurality of (i.e., two or more) gaskets, an inner gasket 442 and an outer gasket 444 disposed in a boundary (i.e., contact) plane between the cover 420 and the cell culture compartment 418.

[0095] As shown in FIGS. 25 and 27, the cell culture compartment 418 may include a plurality of (i.e., two or more) cutouts, an inner cutout 452 and an outer cutout 454 defined in a contact (i.e., top) surface of the cell culture compartment 418 in a concentric manner, such that the inner cutout 452 is inside the outer cutout 454. The cutouts 452 and 454 are configured to seat the inner and outer gaskets 442 and 444 (FIGS. 25, 27, and 28), respectively. In embodiments, the cutouts may be defined in the contact (i.e., bottom) surface of the cover 420.

[0096] The inner gasket 442 provides a non-cytotoxic liquid seal for the cell culture compartment 418. The inner gasket 442 may be formed from biocompatible, gas permeable elastomer, including but not limited to polyolefins (e.g., polymethylpentene), perfluorodioxolanes (e.g., poly-perfluoro(2-methylene-4-methyl- 1,3 -di oxolane), polysiloxanes (e.g., polydimethylsiloxane), perfluoroalkoxy alkanes, perfluorinated polymers (e.g., fluorinated ethylene propylene), fluorosilicones, FFKM perfluoroelastomers (such as KALREZ™ fluoropolymer elastomers available from DuPont of Wilmington, DE) and combinations thereof. Gas permeable elastomers generally all relatively gas permeable with some exceptions, such as isobutylene isoprene rubber. However, isobutylene isoprene rubber may release leachates by direct contact with water which are also known to be toxic to multiple cell lines and organoids. Thus, the outer gasket 444 is formed from gas impermeable, flexible material, such as isobutylene isoprene rubber, and surrounds the inner gasket 442 thereby providing a gastight seal between the cover 420 and the cell culture compartment 418. The inner and outer gaskets 442 and 444 may be constructed using manufacturing techniques such as subtractive manufacturing (e.g., CNC drag knife cutting, CNC rotary knife cutting, manual die punching, CNC routing, machining, laser cutting), additive manufacturing (e.g., 3D printing), injection molding, and the like.

[0097] The cell culture vessel 400 may be disposed in contact with a heating block 430, which may be formed from any heat conducting material, e.g., metal. The heating block 430 includes sidewalls 432 defining a cavity 434 configured to receive the cell culture container 412. The heating block 430 may be placed on the heater 60 to regulate the temperature of the cell culture vessel 400.

[0098] With reference to FIGS. 26-28, a heating insert 431 , which acts as a heat conducting interface, may be used. The heating insert 431 is disposed between the cell culture container 412 and the heating block 430. The cover 420 may be secured to the cell culture container 412 via a plurality of bolts 428, which are configured to be threaded into corresponding threaded channels 429 defined in the heating insert 431 (FIGS. 26 and 27). The cavity 434 of the heating block 430 has the same shape as the heating insert 431 to allow for close contact between the cavity 434 and the heating insert 431.

[0099] With reference to FIGS. 25 and 27, the cutouts 452 and 454 may have a serpentine shape, such as a cross-like shape, defined by four extending arms symmetrically arranged about a central axis (i.e., having four inner corners and eight outer corners). The shape of the cutouts 452 and 454 allows for passage of the bolts 428 through the cell culture container 412. The gaskets 442 and 444 may be circular gaskets configured to conform to the cutouts 452 and 454. In embodiments, the gaskets 442 and 444 may be formed to match the shape of the cutouts 452 and 454. Once placed inside the cutouts 452 and 454, the gaskets 442 and 444 are compressed by the cover 420 using the bolts 428.

[0100] The cell culture vessel 400 may be used with a gas exchanger using a similar setup as shown in FIG. 9. The organoid and cell culture vessels according to the present disclosure may be used with separate gas exchangers using the following configurations of FIGS. 13 and 16, although other configurations are also possible. In FIG. 13, recirculated media is used, which flows through an external gas exchanger. This is media-efficient since it reduces waste but increases and therefore dilutes the effective cell / organoid culture media volume.Alternatively, it is possible to precondition media prior to injection into the cell culturecompartment, then regularly cycle the media to waste as shown in FIG. 16. This is mediainefficient but reduces the active media volume “in use” at any given time.

[0101] FIG. 29 shows schematic chemical processes and equilibria in a conventional cell culture plate and a bubbled reservoir, in which oxygen and carbon dioxide are diffused through a buffer solution and water from the solution is evaporated.

[0102] FIG. 30 shows schematic chemical processes and equilibria in the cell culture vessel 200 or 400. Inside the gas control fluid compartment (e.g., compartment 308), a carbonic acid - bicarbonate - carbonate based buffer system is established to provide a buffered concentration of carbonic acid, which is in equilibrium with carbon dioxide. At and around physiological pH this equilibrium favors carbon dioxide. Diffusion and convection dispose the dissolved carbon dioxide throughout the buffer in the gas control fluid compartment. At the interface between the gas control fluid and the film (e.g., film 300), the carbon dioxide is in equilibrium between dissolution in the fluid and in the film. Carbon dioxide diffuses freely through the film and is in equilibrium with the other side of the film, i.e., the cell culture compartment 200 or 400. Carbon dioxide is also disposed throughout the cell culture media by diffusion and convection. Here, the dissolved carbon dioxide completes the carbonic acid - bicarbonate - carbonate buffer system via its interconversion with carbonic acid. As a result, this buffer system maintains physiological pH despite the generation of ammonium, lactic acid and other acid species by cellular metabolism. Additionally, oxygen may also be present in in the buffer solution and be provided to the cells or organoid. (See Example 11 below.)

[0103] FIGS. 34 and 35 show a cell culture vessel 500 that is similar to the cell culture vessel 40 of FIGS. 6 and 7 with some modifications that are described below. The cell culture vessel 500 is a sealed (i.e., incubator free) cell culture vessel and includes a gas control fluidcontainer 542 disposed over a cell culture container 512, which is similar to the cell culture container 12 of FIGS. 1-5. The cell culture container 512 includes a base 514 surrounded by sidewalls 516 extending from the base 514 to define a cell culture compartment 518. A cell culture, microorganisms, or organoids are placed in a media inside the culture compartment 518. The cell culture vessel 500 also includes the film 20 disposed over the cell culture container 512 enclosing the cell culture compartment 518.

[0104] The fluid container 542 and the cell culture container 512 may be formed from the same material(s) as the cell culture vessel 400. The fluid container 542 includes a cover 544 surrounded by sidewalls 546 extending from the cover 544 to define fluid compartment 548, which stores a gas control fluid. The gas control fluid may include a carbonic acid - bicarbonate - carbonate buffer solution. The sidewalls 546 contact the sidewalls 516 of the cell culture container 512 in a gastight manner. Thus, the cell culture compartment 518 and the control fluid compartment 548 are enclosed within a gastight cell culture vessel 500 and are separated from each other by the gas permeable film 20. Although the embodiment of FIGS. 34 and 35 show the gas control fluid container 542 being disposed over the cell culture container 512, it is envisioned that the arrangement may be reversed or the containers may be disposed side-by-side, while the film 20 is used to form a sidewall that separates the compartments. The cell culture vessel 500 may be placed over a heater to maintain desired temperature of the cell culture. The film 20 may be laminated between the cell culture container 12 and the fluid container 42.

[0105] The cell culture vessel 500 also includes a plurality of (i.e., two or more) access ports 524, which may be airtight Luer lock ports, providing fluid access to the cell culture compartment 18 for loading the cell culture (i.e., cells and corresponding liquid media). The access ports 524 are in fluid communication with the cell culture compartment 518 via fluidicchannels 526, which may enter the cell culture compartment 518 from its base due to the shallow depth of the compartment 518 and to accommodate the threaded access ports 524 through the sidewalls 516. During loading, the cell culture may be deposited in the cell culture 518 through one of the ports 524, while the other of the ports 524 is open to allow for air to escape as the cell culture compartment 518 is fdled. The ports 524 may be sealed with Luer locks or any other stoppers, e.g., an airtight gasketed lid.

[0106] The fluid container 542 also includes a plurality of (i.e., two or more) access ports 550, which are substantially similar to the access ports 524, and are used to fill the fluid container 542 with the buffer solution. The fluid container 542 may be filled after cell culture is loaded into the cell culture container 512 and the fluid container 542 is attached to the cell culture container 512. Just like the cell culture compartment 518, the gas control fluid compartment 548 is devoid of air since the gas control fluid compartment 548 is completely filled with the gas control fluid and there is no ullage or an air gap present. The gas control fluid compartment 548 provides a passive buffer system across the film 20, which provides dissolved gas regulation, removing the need for a gas-controlled atmosphere surrounding the cell culture vessel 500 as described above with respect to FIG. 30.

[0107] Presently disclosed cell culture devices and fluid containers may be assembled using medical device primers such as LOCTITE® SF 7701 and medical cyanoacrylates such as LOCTITE® 4311, e.g., coupling the gas control fluid container 42 to the cell culture container 12.

[0108]

[0109] The disclosed designs also differ from conventional polydimethylsiloxane(PDMS) microfluidic devices. While in general, conventional PDMS devices have the samebenefits of being able to exchange carbon dioxide and oxygen through a microbially impermeable layer, the material is exceptionally permeable to water vapor, causing widespread issues with osmolarity shifts, potentially causing cell death or clogging of lines if the devices are operated in a heated but non-humidified environment.

[0110] Removing the air gap also provides significant benefits for antisepsis. Contamination of the cultures by inadvertent introduction of microorganisms or other material is much less possible than with conventional vessels. Specifically, the only ingress is through normally closed access ports. Furthermore, it is easier to handle access ports aseptically than other devices. Having a sealed, nonporous design also allows for routine decontamination of the outside of cell culture vessels. In an air gap design, the conventional laboratory decontamination methods of spraying the exterior of objects with 70% alcohol or with diluted bleach cannot be done without exposing the contents to mists of these chemicals, which are also cytotoxic to the cells under study.

[0111] This also means that secondary containment is not needed for handling, transport, or storage of live cell cultures. Provided biosafety requirements are met, it would be possible to perform cell culturing using the disclosed cell culture vessels and systems outside of a laboratory environment without any need for containment. This has immense practical applications for transport and for laboratory design.

[0112] It would also be possible to build laboratories where only the areas used for active manipulation of cultures would need to be kept clean since only actively opened cell culture devices would be susceptible to contamination. This would potentially make laboratory design both cheaper and more flexible. This could also potentially allow for more cell culture in resource-limited settings.

[0113] Furthermore, to take the concept of microbial barrier a step further, it would allow a particularly efficient method of heat maintenance, i.e., by direct immersion of cell culture vessels in a water bath instead of a closed sterile incubator. Water has a specific heat capacity which rivals metals but at a much lower price. In addition, it has the advantage of convection as a heat transfer mechanism, which allows for very uniform heat distributions, even in complex geometries. In comparison, solids that rely only on the conduction of heat have their evenness of heating highly dependent on geometry.

[0114] An air gap-free cell culture ecosystem would provide an additional engineering control against direct exposure of laboratory workers to microorganisms from inside the cell culture device, potential release of these microorganisms into the immediate laboratory environment or beyond, or against cross-contamination between microorganisms contained in separate vessels.

[0115] Another advantage of simple, local gas exchange devices of the present disclosure is their simplicity, compactness and lack of need for microfluidic pumps. The advantage of the external gas conditioning designs is that they maximize the available real estate for cell culture observation and manipulation hardware, which is one of the primary goals of developing a new cell culture system for highly instrumented cell culture. Thus, one principal advantage of incubator-free cell culture is removal of impediments to using external devices such as microscopes, electrophysiology equipment, optical or electrical sensors or other microfluidic liquid sampling and assay devices in concert with cell culture. Some of these impediments arise from the physical bulk and inflexibility of the cell culture incubator, which is not suited to observation of the cell cultures contained within using arbitrary instrumentation. Some of these impediments are also related to the challenges of using devices to observe the cells while stillmaintaining desired gas concentrations, osmolarity, temperature and sterility. Incubator-free cell culture removes the physical bulk of the incubator and allows factors such as temperature or gas concentration to be controlled with devices (e.g., using heat blocks, magnetic plates, or gas exchange modules) whose positioning can easily be adjusted to accommodate instrumentation.

[0116] The second set of advantages, which are in a sense an accidental consequence of the idea of getting rid of the issues caused by the incubator, is that incubator-free cell culture can be more precise or versatile than conventional air gap cell culture when it comes to controlling gas concentrations or other environmental factors of the cells. Better precision and environmental fidelity can be achieved by eliminating the gas, humidity and temperature leakage inherent to the cell culture vessels having an air gap. Using a sealed, gas control fluid-based system allows simplicity and flexibility in maintaining chemical composition, temperature and pressure.

[0117] Using a liquid solution rather than an air chamber to provide dissolved gas blends to the cell culture compartment can simplify the maintenance of target gas concentrations. Dissolved gas concentration in the liquid solution can be in equilibrium with a chemical system that can include non-gas components. For example, carbon dioxide dissolved gas concentrations can be maintained by using an aqueous carbonic acid - bicarbonate - carbonate buffer system as a carbon dioxide generator. In this system, the non-gas species carbonic acid is in turn in equilibrium with its reversible decomposition to carbon dioxide and the equilibrium vastly favors carbon dioxide at and around standard temperature and pressure. This allows the concentration of carbon dioxide gas to be maintained by participation in a chemical buffer system. This is much simpler and more cost effective than the two standard methods of maintaining carbon dioxide concentrations different from room air. The first, more common method uses a carbon dioxidesensor and active metering of compressed carbon dioxide gas to maintain a gas concentration setpoint. The sensors and actuators involved are expensive, complex, and susceptible to failure. The second method uses actively replaced premixed compressed gas blends. While this does not depend on expensive and failure-prone sensors and controllers, it is not cost effective to routinely purchase and continually vent custom blends of compressed gas.

[0118] The same principles also apply to using a liquid gas control solution to provide lower than room air (i.e., physoxic, hypoxic, anoxic) oxygen concentrations. Rather than using an active feedback control of oxygen, oxygen can be controlled by one of two methods. For anoxia, a scavenger such as sodium sulfite can be used.

[0119] An air gap-free cell culture ecosystem would provide an additional engineering control against direct exposure of laboratory workers to microorganisms from inside the cell culture device, potential release of these microorganisms into the immediate laboratory environment or beyond or against cross-contamination between microorganisms contained in separate vessels.

[0120] Another advantage of simple, local gas exchange devices of the present disclosure is their simplicity, compactness and lack of need for microfluidic pumps. The advantage of the external gas conditioning designs is that they maximize the available real estate for cell culture observation and manipulation hardware, which is one of the primary goals of developing a new cell culture system for highly instrumented cell culture. Thus, one principal advantage of incubator-free cell culture is removal of impediments to using external devices such as microscopes, electrophysiology equipment, optical or electrical sensors or other microfluidic liquid sampling and assay devices in concert with cell culture. Some of these impediments arise from the physical bulk and inflexibility of the cell culture incubator, which is not suited toobservation of the cell cultures contained within using arbitrary instrumentation. Some of these impediments are also related to the challenges of using devices to observe the cells while still maintaining desired gas concentrations, osmolarity, temperature and sterility. Incubator-free cell culture removes the physical bulk of the incubator and allows factors such as temperature or gas concentration to be controlled with devices (e.g., using heat blocks, magnetic plates, or gas exchange modules) whose positioning can easily be adjusted to accommodate instrumentation.

[0121] The presently disclosed sealed systems can also function with gas blends other than those commonly used in standard cell culture. The system may be used to achieve gas blends which are impossible outside of costly and impractical fully sealed glovebox culture systems. For example, it would be possible to study cells in carbon dioxide buffered yet completely anoxic conditions. In glovebox systems, low concentrations of oxygen are possible using exuberant nitrogen flushes and access through an airlock, but this sort of cell culturing is cost-ineffective and difficult for technicians to manipulate. Furthermore, any brief exposure to an oxygen-containing environment (e g., during feeding or other manipulations) may cause oxygen to dissolve. This cannot be easily removed since negative pressure pulls other gases out of solution, sparging is impractical and likely to disturb the culture irreparably, and oxygen scavenging involves chemical agents that are toxic to cell cultures. Rather than active feedback control of oxygen, oxygen can be controlled by one of two methods using the disclosed devices and systems. For anoxia cell culturing, a scavenger such as sodium sulfite can be used. In the gas exchangers of the present disclosure, gas control fluid may be replaced with an oxygen scavenger on the other side of the film in order to draw all oxygen out of the system. In embodiments, suitable oxygen scavenger may be cobalt-catalyzed sodium sulfite to completely eliminate oxygenation.

[0122] The cell culture principles underlying incubator-free cell culture are also possible with water-soluble gases other than oxygen, nitrogen and carbon dioxide. Toxicological studies of dissolved gases such as carbon monoxide, ammonia, methane, or nitrous oxide may be used in the gas exchangers without the risk of releasing these species into the laboratory environment. These sorts of cell culturing are currently possible, but only in a glove box environment. Similarly, cell cultures in the presence of volatile toxins such as alcohols, organophosphates or organic solvents may also be performed using the disclosed incubator-free systems and devices.

[0123] The closed system also allows the study of tissue culture models under hydrostatic pressures other than atmospheric pressure. This may be used in the study of important disease states involving elevated pressures in tissue compartments, e.g., neoplasms, pericardial effusion, certain modes of heart failure, brain inflammation in the context of stroke, infection or seizure, among others. It is also well known that many tissues have hydrostatic pressures other than atmospheric pressure under normal conditions. Therefore, incubator-free cell culture vessels may also allow for more realistic models of organ systems from a pressure point of view, even for organs in their healthy state.

[0124] The following Examples illustrate embodiments of the present disclosure. TheseExamples are intended to be illustrative only and are not intended to limit the scope of the present disclosure.EXAMPLE 1

[0125] This Example describes experiments for determining performance of air gap free vessels according to the present disclosure.

[0126] The disclosed air gap device of FIGS. 1-5 and incubator free cell culture device of FIGS. 6-7 were compared against the prototypical air gap cell culture devices: a cell culture dish and the vented cell culture flask. Specifically, in order to match the cell growth surface area of our devices, a 60 mm Petri cell culture dish and a T25 format cell culture flask with a 0.2 pm microporous filter vent were used. Non-gas exchange controls of the T25 flask were sealed with a “plug” cap and were used to compare with a sealed variant of the cell culture vessels, which was completely sealed to gas exchange on all sides rather than incorporating any membrane. Although the T25 flask lacked gas exchange with the incubator environment, the flask included a trapped gas volume exceeding the media volume with which to exchange gases.

[0127] A multi-day cell culture experiment with comparisons between various experimental conditions was conducted to determine whether the non-air gap devices may be used to achieve cell cultures comparable in performance to conventional air gap devices. This was determined based on comparisons of viability and growth rate. The second determination was whether the devices achieved parity in physiological variables. And the third determination was whether the non-air gap devices outperformed air gap devices and osmolarity was addressed by taking time point series measurements of physiological analytes over a multi-day cell culture experiment.

[0128] The experiments were performed using a standard and fast-growing cell line, the NIH / 3T3 murine embryonic fibroblast. Standard cell culture conditions were implemented, using Gibco brand Dulbecco’s Modified Eagle Medium, a media which is representative of common media blends used in mammalian cell culture.EXAMPLE 2

[0129] This Example describes cell cultures used in the experiments.

[0130] NIH-3T3 murine embryonic fibroblasts which were obtained at passage number 151 from Sigma Aldrich. Media used was Dulbecco’s Modified Eagle Medium (DMEM) with GlutaMAX from Gibco supplemented with 10% donor bovine serum (Gibco NQ 16030074, New Zealand Origin.) Gibco DMEM uses the standard 3.7 g / L sodium bicarbonate concentration. Prior to beginning the experiments, the cells were expanded to generate homogeneous stocks to use for experiments. This process used at most 10 additional passages beyond the passage number 151 at which the cell line was obtained. In each experiment, all replicates of all conditions were seeded at equal seeding densities from cells at the same passage number grown from the same plates.

[0131] All cell handling operations were performed using aseptic technique in a Class II biosafety cabinet. Except where otherwise noted, cell culture was performed in a shared humidified carbon dioxide incubator with temperature setpoint of 37 °C and carbon dioxide setpoint of 5%. This carbon dioxide setpoint was used due to the laboratory being a shared facility, even though it is not appropriate for standard DMEM.EXAMPLE 3

[0132] This Example describes analyte measurement setup.

[0133] Point measurements of pH and of concentrations of oxygen, carbon dioxide, sodium bicarbonate, sodium ion and chloride ion were taken using a Beckman Coulter Vi-CELLMetaFlex Bioanalyte Analyzer. Measurements were taken from 0.2 mL aliquots gathered in 1 mL syringes. Measurements were taken as soon as possible after aliquot collection; if more than 2-3 minutes elapsed between sample collection and measurement, syringes were capped tightly with polypropylene Luer-lock caps to prevent gas exchange with room air. Any bubbles aspirated were expelled prior to sample loading, and care was taken to avoid bubbles of air contaminating any sample.

[0134] Analyte analyzer measurements were not taken from closed-lid T25 flask samples except for initial and terminal measurements, since daily opening and closing of their lids for sampling would effectively cause gas replenishment of their internal chamber, confounding potential differences between conditions. For all other conditions, 0.2 mL aliquots were collected with a 1 mL polypropylene Luer syringe for analyte measurements on a daily basis.EXAMPLE 4

[0135] This Example describes initial cell seeding conditions.

[0136] The disclosed cell culture devices were loaded as follows. First, both Luer caps of the cell culture compartment of the device were removed to allow for pressure equalization, then the vessel was laid on its side. Cells were deposited into the well via a pipette filler. The well was then filled with media by the same port and the air bubbles were allowed to escape via the other port. The cell compartment was then closed by re-insertion and locking of the Luer caps.

[0137] For air gap free devices, the seeding protocol was adapted from standard protocols for culture of NIH 3T3 cells using standard laboratory plasticware. The cell culture chamber was uncapped. Cell culture chambers were rinsed in Dulbecco’s phosphate-buffered saline via the access ports. An aliquot of 350,000 cells, suspended in 4-4.5 mL of media was loaded into the cell culture component and the media was subsequently topped off to fdl the cellculture component, if necessary. The access ports to the cell culture chamber were closed, and the plates were placed in a cell culture incubator. The device was placed with the cell culture surface downwards such that the cells could settle and adhere, and the plate was kept in this position for the remainder of the experiment. The plate was kept undisturbed in the incubator for 24 hours after which time the media was exchanged and the experiment started.

[0138] On day four after plating, at which point the cell cultures in the devices had reached 80 to 90% confluency, experiments were terminated. Cells were counted using a BIORAD TC 20 automated cell counter after dissociation under standard protocols using 1 mL TrypLE Express dissociation reagent (Gibco). After a 7-8 minute incubation with TrypLE Express, the cells were diluted to a total volume of 5 mL with media, collected, centrifuged for 3 minutes at 210 g and resuspended in 1 mL of media, after which automated cell counting was performed.EXAMPLE 5

[0139] This Example describes cell culture using standard cell culture labware.

[0140] Control groups were cultured in either Thermo Scientific NQ 12-556-001 60 mm tissue culture (i.e., Petri) dishes, VWR brand NQ 10861-570 T25 closed lid tissue culture flasks or VWR brand NQ 10861-572 T25 microporous membrane vented lid tissue culture flasks. Plating cell counts, media volumes, media change schedule and experimental endpoints were identical to air gap free vessels. Tissue culture dish and flask cultures were grown in the same cell culture incubator shared with other experimental conditions.EXAMPLE 6

[0141] This Example describes incubator-free plate experimental procedure.

[0142] Aqueous gas control solution was prepared from solid sodium bicarbonate and sodium carbonate (Sigma) in water. Stock mixtures were not used due to the possibility of decomposition and subsequent carbon dioxide loss from stock solutions which would cause the experiment parameters to deviate from those intended. The default mixture was 0.8 M sodium bicarbonate, 0.7 M sodium carbonate resulted in a carbon dioxide concentration of approximately 7% and provided a cell culture media pH on the order of 7.4.

[0143] The first step in the gas control solution preparation was to ensure an oxygen concentration in equilibrium with room air at 37 °C. First, Type I deionized water was heated to 37 °C in an open vessel under magnetic stirring for 15 minutes, at a rate sufficient to generate a vortex. This allowed the water to attain a partial pressure of oxygen approximately in equilibrium with room air at physiological temperature.

[0144] After 15 minutes, dry reagent was added and the vessel was sealed to prevent carbon dioxide loss. The dry reagent was stirred for an additional 5 minutes to ensure dissolution. A 300 pF aliquot was collected fresh to confirm pCh and pCCh measurements using the MetaFlex analyte analyzer.

[0145] A 10 mL syringe was filled with gas control fluid. The two Luer caps of the gas control fluid compartment were opened and a syringe was attached to one of the Luer fitting. The fluid was injected into the compartment, expelling the air through the other port.

[0146] Prior to loading of the cell culture devices, the heating system was turned on and set to 40 °C which was found to give an average internal temperature of about 37 °C in the well. This was left to heat up and equilibrate during the cell culture device loading process. Once loaded, the cell culture devices were placed, cell culture chamber down, on the heat block previously described.

[0147] Media changes were performed analogously to the initial loading. Both Luer caps to the cell culture compartment were unscrewed to allow for free flow of media. The device was placed on its side and media was removed from the bottom Luer port. The media was then refilled via the same port with the air bubble being allowed to escape via the other port.EXAMPLE 7

[0148] This Example describes analyte and gas exchange results.

[0149] Conventional air gap cell culture in Petri dishes and T25 flasks caused an increase in media osmolarity via evaporation. Media sodium concentrations were measured using the MetaFlex analyte analyzer. Hypernatremia was used as a quantitative proxy for extent of water loss via evaporation, since sodium ion concentration in an evaporation-susceptible environment is an accurate proxy for measuring evaporation in cell cultures.

[0150] With reference to FIG. 17, which shows plots of relative sodium concentration change over a four-day period, air gap cultures grown in 6 cm Petri dishes showed significant day-over-day increases in sodium, indicating evaporation. On average, over the four days of cell culture, sodium concentration in these dishes increased by 13 mM over a baseline of 151 m, corresponding to an average volume loss of 9.4%. This was expected, based on the humidity gradient and opportunity for free diffusion of water vapor that is inherent to air gap designs.

[0151] This effect was significantly blunted when using filter-cap T25 flasks and Petri dishes, showing volume depletion limited to 1.8% over four days. This was likely due to a relatively lower effective cross-sectional area of the air gap venting conduit in the filter cap versus the completely barrier-less air gaps of the conventional plate.

[0152] With reference to FIG. 18, which shows plots of relative sodium concentration change over time for the air gap free cell culture vessel of FIGS. 1-5 and the incubator free cellculture vessel of FIGS. 6 and 7, fluid-based respiration cell culture devices maintained stable carbon dioxide concentrations and pH values within target ranges. Physiological human arterial pH is tightly regulated in the range of 7.36-7.44. Thus, cell culture laboratories often set their incubator setpoints to 5% carbon dioxide: however, if 5% carbon dioxide can truly be maintained, then absent acid products of metabolism, expected pH is about 7.67 based on the Henderson-Hasselbalch equation, assuming a pKa of approximately 6.1 for carbonic acid. In long-term cell cultures, accumulation of acid products of metabolism, primarily lactic acid and ammonium, decreases this pH marginally. However, the 5% carbon dioxide setpoint was expected to produce an alkalotic media pH.

[0153] With reference to FIG. 19, which shows plots of pH vs days of cell cultures, it was observed that, apart from on day 0, where the pH measured is of the fresh media with a 7% dissolved carbon dioxide content, neither the air gap cell culture vessels (Petri dish or filter flask) nor the air gap free vessel (which exchanges carbon dioxide with the incubator mixed gas via its polymethylpentene gas permeable film) achieved any pH in the physiological range. The media buffer was Gibco DMEM with 3.7 g / L bicarbonate buffer. The incubator setpoint was 5% carbon dioxide. The incubator-free vessel used an 800 mM sodium bicarbonate, 70 mM sodium carbonate buffer for gas generation and regulation. The media buffer was Gibco DMEM with 3.7 g / L bicarbonate buffer.

[0154] With reference to FIG. 20, which shows plots of dissolved carbon dioxide vs days of cell cultures, it was also observed that the dissolved carbon dioxide level in these vessels reached the incubator setpoint of 5%. The incubator-free vessel again used an 800 mM sodium bicarbonate, 70 mM sodium carbonate buffer for gas generation and regulation. Gibco DMEM nominally requires 5-10% carbon dioxide setpoint to achieve physiological pH. It was noted thatno device exchanging gas with the incubator environment was able to maintain a partial pressure of CO2 at the setpoint. The average dissolved carbon dioxide partial pressure was about 4.40% and the maximum observed was about 4.83%. This suggested that the underprovision of carbon dioxide at the level of the incubator mixed gas setpoint was worsened by incubator openings and / or inhomogeneity, as has been observed with humidity levels. While the elevated pH seen in all three of these conditions was primarily indicative of the fact that a 5% CO2 setpoint is not appropriate for a standard DMEM buffer system, it also suggested two other advantages of an incubator-free cell culture system.

[0155] First, the difference between the measured dissolved carbon dioxide concentrations and the intended incubator setpoint indicates that incubator openings are a major barrier to achieving precise carbon dioxide (and therefore pH) control. Second, using shared cell culture incubators requires that all cell culture vessels use the same gas blend. Often a cultural default such as 5% carbon dioxide is chosen rather than choosing a concentration which is actually ideal for their experiment. The pH and carbon dioxide data also shows that the air gap free cell culture vessel, which uses a fluidically sealed cell culture chamber equipped with a nonporous polymethylpentene gas permeable film, freely exchanges dissolved gas with the incubator mixed gas environment. This is shown by the concordance between the dissolved gas levels of these air gap free vessels and the petri dishes and filter flasks. In contrast, incubator free vessels employing an 800 mM sodium bicarbonate, 70 mM sodium carbonate buffer for gas generation and regulation showed a pH within the physiological range of 7.36 to 7.44 for the duration of the experiment.

[0156] Non air gap, fluid-based respiration cell culture devices maintained stable oxygen concentrations within target ranges. With reference to FIG. 21, which shows dissolved oxygen vsdays of cell cultures, measurement of dissolved oxygen content of the cell culture devices did not show clear trends or differences between experimental conditions. Dissolved oxygen trends were approximately equal between all four vessel types: standard air gap petri dish and filter flask vessels, as well as air gap free and incubator-free vessels. At atmospheric pressure and 37 °C, water in equilibrium with room air had a partial pressure of 20.9% oxygen. Neither air gap free (membrane respiration of incubator mixed gas) nor incubator free (membrane respiration of gas control fluid) devices showed differences in oxygen trends as compared to conventional air gap devices. Without being limited to any particular theory, it is believed that near-atmospheric oxygen concentrations have excess of oxygen relative to metabolic needs for there to be any measurable consumption in any cell culture system tested.EXAMPLE 8

[0157] This Example describes construction of a recirculating cell culture system according to the present disclosure.

[0158] The recirculating gas exchange device was milled in two components from polycarbonate. The top subcomponent included two ’A” -28 UNF fluidic ports feeding into a high aspect ratio serpentine channel. The channel had a 400 pm depth and a 6.4 mm width. The bottom subcomponent included a large reservoir for gas control fluid, also equipped with two ’A” -28 UNF fluidic ports. The open base of the gas control fluid reservoir and the serpentine channel occupy rectangles of the same dimensions with the same wide flange around them. The flange is used to laminate a polymethylpentene gas exchange film covering these rectangular regions between the two subcomponents.

[0159] A peristaltic pump was assembled from a stepper motor peristaltic pump and 1 / 16” inner diameter Pharmed BPT peristaltic pump tubing. A Pololu Tic T825 stepper motor controller, controlled via computer via USB was used to control the peristaltic pump. The stepperdriver was set to 1 / 32 micro stepping to reduce fluctuations in fluid velocity and noise and to 500 mA current limiting to limit heat.

[0160] With reference to FIG. 22, a loop circuit was designed and constructed, consisting of the cell culture well, the peristaltic pump, the membrane oxygenator and inline sensors. Either 1 / 16” inner diameter Tygon E-3606 non-DEHP plasticized polyvinylchloride fluidic tubing, or 1 / 16” inner diameter poly vinylidene fluoride non plasticized tubing was used for the circuit components. Luer lock to 1 / 16” barb fluidic connectors, !4”-28 UNF compression fittings and adapters between the two were used for tubing assembly.

[0161] In FIG. 22, the following components are shown: A) Membrane respiration device. B) Polyvinylidene fluoride (gas impermeable) tubing. C) ’A” -28 UNF compression fitting for polyvinylidene fluoride tubing and adapter to hose barb fitting for PharMed BPT peristaltic pump tubing. D) Stepper motor based, computer controlled peristaltic pump, designed and assembled as described previously. PharMed BPT peristaltic pump tubing. Controlled via computer using USB stepper driver module. E) Resistive heating apparatus and heat block for organoid wells under computer monitoring and control via USB interface. F) Organoid well, attached to circuit via1 / 4,’-28 UNF chromatography compression fittings. G) Inline pH monitoring module. H) Ika RCT safety hotplate used as a heating device for maintaining temperature of membrane respiration device.

[0162] EXAMPLE 9

[0163] This Example describes pH regulation of cell culture media via gas preconditioning chamber in a non-recirculating loop.

[0164] A gas preconditioning device incorporating a pH monitoring apparatus was assembled as described in Example 8. A microcontroller controlling a peristaltic pump actuatorof the type previously described was set to replace the fluid in the media chamber of the device on four-hour intervals. Media provided was Gibco DMEM supplemented with Primocin whose dissolved carbon dioxide concentration had been preequilibrated with room air by vigorous magnetic stir ring in a conical tube at 500 rpm for one hour. The media was kept at room temperature prior to loading into the gas preconditioning device to avoid excess dissolution of carbon dioxide. The gas control fluid chamber of the device was filled with freshly prepared gas control fluid consisting of 0.8 M sodium bicarbonate and 60 mM sodium carbonate. The device was maintained at approximately 37 °C with 100 rpm magnetic stirring using an IKA RCT magnetic hotplate. The pH was measured at 1 second intervals using an Atlas Scientific Micro pH probe and reference sensors.

[0165] With reference to FIG. 23, which shows a pH vs time plot of the gas preconditioning chamber, initial media pH following media change exceeded 8, as expected on account of low carbon dioxide concentration of the media following equilibration with room air. Immediately following media change, a monotonic decrease in pH was observed as gas is exchanged with the gas control fluid chamber across the polymethylpentene film. The pH asymptotically approached a value of 7.2. The approximately steady-state pH was established at approximately 180 minutes and remains steady to 240 minutes at which time the media in the gas preconditioning chamber is replaced.EXAMPLE 10

[0166] This Example describes pH regulation of cell culture media via membrane respiration in recirculating fluidic loop.

[0167] An inline pH meter apparatus was constructed and inserted in the recirculating fluidic loop of Example 8. The peristaltic pump, controlled by a desktop computer using a PololuTic T825 USB motor driver, was driven at a constant rate of approximately 96 rotations per minute. The recirculating gas exchange device was inserted into the circuit, with the media path flowing through the high aspect ratio serpentine chamber such that it was spread out in a thin layer in contact with the polymethylpentene film. The gas control fluid chamber of the recirculating gas exchange device was filled with a freshly prepared mixture of 0.8 M sodium bicarbonate and 60 mM sodium carbonate. The circuit was primed with Gibco DMEM supplemented with Primocin whose dissolved carbon dioxide concentration had been preequilibrated with room air by vigorous magnetic stirring in a conical tube at 500 rpm for one hour. The device was maintained at approximately 37 °C with 100 rpm magnetic stirring using an IKA RCT magnetic hotplate. The pH was measured at 1 second intervals.

[0168] With reference to FIG. 24, which shows a pH vs time plot of the recirculating fluidic loop, similarly to the pH response in the static gas preconditioning chamber, the recirculating gas exchange device showed a monotonic decrease in pH which started immediately after exposure of the media to the gas control fluid across the polymethylpentene film and asymptotically approached the target pH of 7.2. The pH equilibration kinetics and steady state pH were also similar, with a steady state pH of approximately 7.2 reached by approximately 180 minutes and thereafter maintained for the remaining 2 hours of the experiment.EXAMPLE 11

[0169] This Example describes oxygen concentration in a closed system according to the present disclosure (e.g., FIG. 13).

[0170] FIG. 31 shows a plot of oxygen saturation over time in the cell culture vessel ofFIG. 25 in combination with the system of FIG. 13. Maintenance of oxygen concentration in theclosed perfusion system using a separate gas exchanger (e.g., the fluid container 302), was measured using a fluorescence lifetime intensity based continuous oxygen monitoring probe. The plot shows that oxygen concentration was maintained for approximately over 72 hours.EXAMPLE 12

[0171] This Example describes metabolic activity of an organoid inside a closed system according to the present disclosure (e.g., FIG. 13) and a conventional plate disposed in an incubator.

[0172] In order to provide a direct comparison of metabolic activity, multiple cell culture vessels of FIG. 25 with identical well bottom surface areas and media volumes were used. Experiments were performed in parallel and cell suspensions were maintained under magnetic stirring at approximately 500 rpm in a 25 mL conical tube using an SP Spinvane stirrer in order to prevent systematic error due to cell suspension settling.

[0173] Metabolic activity was compared using XTT assays for NAD(P)H oxidoreductase activity on cells grown in the cell culture vessel of FIG. 25 in combination with the system of FIG. 13 outside of an incubator and cells grown in static conditions in a conventional well plate inside an incubator. Metabolic activity of cells grown using the presently disclosed system was much higher (e.g., more than double) than the metabolic activity of cells grown using conventional means as shown in the dot plots of FIG. 32. In addition, glucose consumption was increased along with aerobic quotient, while lactate production decreased, as shown in dot plots of FIG. 33.EXAMPLE 13

[0174] This Example describes tracking evaporation over a course of a single media change interval of three days inside a closed system according to the present disclosure (e.g., cellculture vessel of FIGS. 34 and 35) and a conventional cell culture vessels disposed in an incubator.

[0175] Evaporation was measured for multiple wells of a 96 well plate (e.g., corner and middle wells). The plots of FIGS. 37A and C show the evaporation for corner wells and illustrate a relatively high linear rate of evaporation, reaching about 25% after three days. The plots of FIGS. 37B and D show the evaporation for middle wells and illustrate a lower rate of evaporation than the corner wells, reaching about 10% after three days. Similarly, evaporation plots of a 10 cm cell culture plates placed at different racks inside the incubator had similar linear rates of evaporation reaching about 10% after three days as shown in plots of FIGS. 38A- C. A T25 flask had lower rates of evaporation reaching 5% when unsealed and about 2.5% when sealed as shown in the plots of FIGS. 39A and B. In contrast, the evaporation in the closed cell culture vessel of FIGS. 34 and 35 is barely registered as shown in the plot of FIG. 40.EXAMPLE 14

[0176] This Example describes measurement of pH, pCO2, and sodium carbonate concentration in the gas control fluid used in a closed system according to the present disclosure (e.g., FIGS. 34 and 35).

[0177] The gas control fluid was used in the closed system with the cell culture vessel of FIGS. 34 and 35. During use, pH, pCO2, and sodium carbonate concentration in the gas control fluid were measured. With reference to FIG. 41, the plot of pH and pCCE vs sodium carbonate concentration shows linear relationship between pH and sodium carbonate concentration. EXAMPLE 15

[0178] This Example describes metabolic activity of cells in a closed system according to the present disclosure (e.g., FIGS. 34 and 35) and a conventional plate disposed in an incubator.

[0179] In order to provide a direct comparison of metabolic activity, multiple cell culture vessels of FIGS. 34 and 35 with identical well bottom surface areas and media volumes were used. Experiments were performed in parallel and cell suspensions were maintained under magnetic stirring at approximately 500 rpm in a 25 mL conical tube using an SP Spinvane stirrer in order to prevent systematic error due to cell suspension settling.

[0180] Comparative metabolic activity, as a proxy for viability of cells cultured in conventional 12 well plates inside an incubator and cells cultured in cell culture vessel of FIGS. 34 and 35 outside of an incubator was assessed using the XTT assay for NAD(P)H oxidoreductase activity. Metabolic activity of cells grown using the presently disclosed system was much higher (e.g., more than double) than the metabolic activity of cells grown using conventional means as shown in the dot plots of FIG. 42.

[0181] It will be appreciated that of the above-disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also, various presently unforeseen or unanticipated alternatives, modifications, variations, or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims. Unless specifically recited in a claim, steps, or components according to claims should not be implied or imported from the specification or any other claims as to any particular order, number, position, size, shape, angle, or material.

Claims

WHAT IS CLAIMED IS:

1. A cell culture vessel comprising: a cell culture container including a cell culture compartment; a cover, wherein the cell culture container and the cover are formed from a gas impermeable material; and a gasket assembly disposed in a contact plane between the cell culture container and the cover and forming a gas impermeable seal.

2. The cell culture vessel according to claim 1, wherein the gasket assembly including: an inner gasket adjacent and around the cell culture compartment configured to provide a non-cytotoxic liquid seal for the cell culture compartment; and an outer gasket disposed around the inner gasket and configured to provide the gas impermeable seal for the cell culture compartment.

3. The cell culture vessel according to claim 2, wherein the inner gasket is formed from a biocompatible polymer selected from the group consisting of polyolefins, perfluorodioxolanes, polysiloxanes, perfluoroalkoxy alkanes, perfluorinated polymers, fluorosilicones, FFKM perfluoroelastomers, FFKM perfluoroelastomers, and combinations thereof.

4. The cell culture vessel according to claim 2, wherein the outer gasket is formed from isobutylene isoprene rubber.

5. The cell culture vessel according to claim 2, wherein at least one of the cell culture container or the cover includes an inner cutout configured to secure the inner gasket and an outer cutout configured to secure the outer gasket.

6. The cell culture vessel according to claim 5, wherein the inner cutout is inside the outer cutout and each of the inner cutout and the outer cutout have a serpentine shape.

7. The cell culture vessel according to claim 1, wherein the cover includes a plurality of access ports fluidly coupled to the cell culture compartment.

8. The cell culture vessel according to claim 1, wherein the cell culture container and the cover are formed from a polymer selected from the group consisting of polycarbonate, polystyrene, polyethylene terephthalate, and combinations thereof.

9. The cell culture vessel according to claim 1, wherein the cell culture container is formed from a metal and the cover is formed from glass or a polymer.

10. A cell culture system comprising: a cell culture vessel including: a cell culture container including a cell culture compartment; a cover, wherein the cell culture container and the cover are formed from a gas impermeable material; anda gasket assembly disposed in a contact plane between the cell culture container and the cover and forming a gas impermeable seal; and a gas exchanger including: a gas control fluid container having a gas control fluid; a media container coupled to the gas control fluid container in a gas tight manner, the media container having a base surrounded by sidewalls extending from the base and defining a media compartment, wherein the media container is fluidly coupled to the cell culture container, and the media container and the cell culture container are filled with a cell culture media; and a gas permeable film having a first side and a second side, the gas permeable film is disposed between the gas control fluid container and the media container, wherein the first side of the gas permeable film contacts the gas control fluid, and the second side contacts the media.

11. The cell culture system according to claim 10, wherein at least one of the cell culture container, the cover, the gas control fluid container, or the media container is formed from a polymer selected from the group consisting of polycarbonate, polystyrene, polyethylene terephthalate, and combinations thereof.

12. The cell culture vessel according to claim 10, wherein the cell culture container is formed from a metal and the cover is formed from glass or a polymer.

13. The cell culture system according to claim 10, wherein the gas permeable film is formed from a polymer selected from the group consisting of polyolefins, perfluorodioxolanes, polysiloxanes, perfluoroalkoxy alkanes, perfluorinated polymers, fluorosilicones, and combinations thereof.

14. The cell culture system according to claim 10, wherein the gas permeable film is formed from polymethylpentene.

15. The cell culture system according to claim 10, wherein the gas permeable film is formed from a polymer permeable to carbon dioxide.

16. The cell culture system according to claim 10, wherein each of the cell culture container, the gas control fluid container, and the media container includes a plurality of fluid access ports.

17. The cell culture system according to claim 10, wherein the gas control fluid is a buffer solution including at least one of carbonic acid, bicarbonate, or carbonate.

18. The cell culture system according to claim 10, wherein the gas control fluid and the media are in a carbon dioxide / carbonic acid equilibrium.

19. The cell culture system according to claim 10, wherein the media compartment has a serpentine configuration.

20. The cell culture system according to claim 10, further comprising a pump configured to move the media between the media container and the cell culture container.

21. The cell culture system according to claim 20, wherein the media container is fluidly coupled to the cell culture container via at least one fluid line.

22. The cell culture system according to claim 21, wherein the pump is a peristaltic pump coupled to the at least one fluid line.

23. The cell culture system according to claim 10, wherein the media compartment includes a magnetic stir bar disposed therein.

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