WO2024030965A2 - Récipient renforcé perméable aux gaz - Google Patents

Récipient renforcé perméable aux gaz Download PDF

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Publication number
WO2024030965A2
WO2024030965A2 PCT/US2023/071528 US2023071528W WO2024030965A2 WO 2024030965 A2 WO2024030965 A2 WO 2024030965A2 US 2023071528 W US2023071528 W US 2023071528W WO 2024030965 A2 WO2024030965 A2 WO 2024030965A2
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WO
WIPO (PCT)
Prior art keywords
cell culture
gas
permeable
culture apparatus
permeable membrane
Prior art date
Application number
PCT/US2023/071528
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English (en)
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WO2024030965A3 (fr
Inventor
Dayu Teng
Original Assignee
Drugarray, Inc.
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Filing date
Publication date
Application filed by Drugarray, Inc. filed Critical Drugarray, Inc.
Publication of WO2024030965A2 publication Critical patent/WO2024030965A2/fr
Publication of WO2024030965A3 publication Critical patent/WO2024030965A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/24—Gas permeable parts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis

Definitions

  • the present disclosure relates generally to the field of cell culture and, in particular, gas-permeable devices, kits, systems, and methods for culturing cells.
  • a culture such as cell culture can be aerated by one, or a combination, of the following methods: surface aeration, sparging, membrane diffusion, medium diffusion, increasing the partial pressure of oxygen, and/or increasing the atmospheric pressure.
  • certain existing methods such as open gas exchange risk contamination of the cell culture and may be time consuming, low in aeration efficiency, and/or associated with high costs.
  • improved methods and devices for cell culture especially for large, industrial-scale cultures
  • gas-permeable membrane and/or container for cell culture may be used to allow gas exchange.
  • the gas permeability is inversely proportional to the thickness of the membrane or film: the thinner the membrane or film is, the better or greater the gas permeability the membrane or film has.
  • a gas-permeable cell culture apparatus comprising: at least a top surface and a bottom surface, an interior suitable for containing cell culture media, a gas-permeable membrane layer, a supportive layer in contact with the gas-permeable membrane layer to form a water-impermeable barrier comprising a plurality of openings, wherein each of the opening defines a gas-permeable window, and wherein the plurality of gas-permeable windows form at least a portion of a surface of the gas-permeable cell culture apparatus.
  • the thickness of the gas-permeable membrane is about 1 pm to about 1 mm, preferably about 5 pm to 500 pm. In some aspects, the thickness of the supportive layer is about 10 pm to about 5 mm, preferably about 100 pm to about 2 mm.
  • the gas-permeable membrane layer and the supportive layer are sealed together thermally, chemically, mechanically, or by lamination.
  • the water- impermeable barrier comprising a plurality of gas-permeable windows is formed by laminating the gas-permeable membrane layer to the supportive layer comprising a plurality of openings.
  • the gas-permeable membrane layer and the supportive layer comprising a plurality of openings are sealed together by thermal-bonding lamination.
  • the gas-permeable membrane layer and the supportive layer comprising a plurality of openings are sealed together by adhesive-bonding lamination, preferably wherein the adhesive is a medical grade glue.
  • the plurality of gas-permeable windows in the water-impermeable barrier is formed by molding. In some variations, the plurality of gas-permeable windows in the water- impermeable barrier is formed by pressing, preferably wherein the pressing is mechanical, thermal, or a combination thereof.
  • gas-permeable windows have a shape selected from the group consisting of circular, triangle, square, rectangle, diamond, pentagon, hexagon, or any combinations thereof. In some variations, the gas-permeable windows have a uniform shape and/or size. In some variations, the gas-permeable windows have different shapes and/or sizes. In some variations according to any of the gas-permeable cell culture apparatus provided herein, each of the gas- permeable windows have an area of about 1 pm 2 to about 30 cm 2 .
  • the gas-permeable membrane layer comprises one or more materials selected from the group consisting of: polyethylene, polyvinyl, polyvinyl chloride, polyurethane, silicone, polyethylene terephthalate glycol, polycarbonate, polystyrene, polypropylene, polytetrafluoroethylene, and acrylic.
  • the gas-permeable membrane layer comprises a single polymer.
  • the gas-permeable membrane layer comprises a copolymer.
  • the supportive layer comprises a material selected from the group consisting of: polyethylene, polyvinyl, polyvinyl chloride, polyurethane, silicone, polyethylene terephthalate glycol, polycarbonate, polystyrene, polypropylene, polytetrafluoroethylene, and acrylic.
  • the supportive layer comprises a single polymer.
  • the supportive layer comprises a copolymer.
  • the supportive layer and the gas-permeable membrane layer are of different materials. In some variations, the supportive layer and the gas-permeable membrane layer are of the same material.
  • the plurality of gas-permeable windows are transparent for observing the cell culture.
  • the gas-permeable membrane layer is on the interior of the cell culture apparatus relative to the supportive layer. In some variations, the gas-permeable membrane layer is on the exterior of the cell culture apparatus relative to the supportive layer.
  • the gas-permeable membrane layer and/or the supportive layer support adherent cell culture.
  • some of the surfaces of apparatus comprise one or more gas-permeable windows. In some variations, all of the surfaces of apparatus comprise one or more gas- permeable windows.
  • the apparatus further comprises a coating on the interior, wherein the coating support cell attachment for adherent cell culture, facilitate cell growth, or prevent cell attachment for suspension cell culture.
  • the interior of the cell culture apparatus is smooth. In some variations the interior of the cell culture apparatus is patterned. In some variations, the interior of the cell culture apparatus comprises microfluidic channels. In some variations, the interior of the cell culture apparatus comprises one or more wells.
  • the cell culture apparatus further comprises one or more ports suitable for inflow and outflow of the cell culture and/or media.
  • the one or more ports comprise valves.
  • a cell culture system comprising: a) a cell culture apparatus comprising at least a top surface and a bottom surface, an interior suitable for containing cell culture media, a gas-permeable membrane layer, a supportive layer in contact with the gas-permeable membrane layer to form a water-impermeable barrier comprising a plurality of openings, wherein each of the opening defines a gas-permeable window, and wherein the plurality of gas-permeable windows form at least a portion of a surface of the gas- permeable cell culture apparatus; and b) one or more cell cultures.
  • the cell culture system further comprises c) a gas chamber for communicating the gas with the surfaces of the cell culture apparatus at sufficient pressure to maintain a gas phase in the gas-permeable apparatus.
  • the cell culture system further comprises d) a liquid chamber in fluid communication with the interior of the cell culture apparatus for sending and receiving liquid.
  • the gas-permeable membrane layer permits exchange of CO2 and/or O 2 .
  • the cell culture system may comprise any of the cell culture apparatus provided herein.
  • a method for culturing cells comprising the steps of: a) providing a cell culture apparatus comprising at least a top surface and a bottom surface, an interior suitable for containing cell culture media, a gas-permeable membrane layer, a supportive layer in contact with the gas-permeable membrane layer to form a water- impermeable barrier comprising a plurality of openings, wherein each of the opening defines a gas-permeable window, and wherein the plurality of gas-permeable windows form at least a portion of a surface of the gas-permeable cell culture apparatus, b) adding a suitable combination of cells and medium to the interior of the cell culture apparatus, and c) incubating the cells under conditions suitable to propagate cell growth, wherein the thickness and the area of the gas- permeable window allows for gas exchange between the interior and the exterior of the cell culture apparatus.
  • the method comprises the steps: a) providing a cell culture apparatus comprising at least a top surface and a bottom surface, an interior suitable for containing cell culture media, a gas-permeable membrane layer, a supportive layer in contact with the gas- permeable membrane layer to form a water- impermeable barrier comprising a plurality of openings, wherein each of the opening defines a gas-permeable window, and wherein the plurality of gas-permeable windows form at least a portion of a surface of the gas-permeable cell culture apparatus, b) adding a suitable combination of cells and medium to the interior of the cell culture apparatus, and c) incubating the cells under conditions suitable to sustain cell viability, wherein the thickness and the area of the gas-permeable window allows for gas exchange between the interior and the exterior of the cell culture apparatus.
  • the cells are blood cells. In some variations, the cells are cultured using a cell culture apparatus disclosed herein for cell or gene therapy.
  • a cell culture medium storage system comprising: a) a cell culture medium apparatus comprising at least a top surface and a bottom surface, an interior suitable for containing cell culture media, a gas-permeable membrane layer, a supportive layer in contact with the gas-permeable membrane layer to form a water-impermeable barrier comprising a plurality of openings, wherein each of the opening defines a gas-permeable window, and wherein the plurality of gas-permeable windows form at least a portion of a surface of the gas-permeable cell culture medium apparatus; b) one or more cell culture media.
  • the cell culture medium storage system further comprises c) a gas chamber for communicating the gas with the surfaces of the cell culture medium apparatus at sufficient pressure to maintain a gas phase in the gas-permeable apparatus.
  • the cell culture medium storage system further comprises d) a liquid chamber in fluid communication with the interior of the cell culture medium apparatus for sending and receiving liquid.
  • the gas-permeable membrane layer permits exchange of CO2 and/or O 2 .
  • the cell culture medium storage system further comprises a cell culture apparatus connected or configured to be connected to the cell culture medium apparatus.
  • the cell culture apparatus is gas permeable.
  • the cell culture apparatus comprises at least a top surface and a bottom surface, an interior suitable for containing cell culture media, a gas-permeable membrane layer, a supportive layer in contact with the gas-permeable membrane layer to form a water-impermeable barrier comprising a plurality of openings, wherein each of the opening defines a gas-permeable window, and wherein the plurality of gas-permeable windows form at least a portion of a surface of the gas- permeable cell culture apparatus.
  • the cell culture apparatus is not gas permeable, and the interior of the cell culture apparatus is or is configured to be in fluid communication with the interior of the cell culture medium apparatus.
  • the interior of the cell culture apparatus comprises a cell in suspension and/or an adherent cell.
  • the interior of the cell culture medium apparatus does not comprise a cell in suspension and/or an adherent cell.
  • the connection between the cell culture medium apparatus and the cell culture apparatus comprises a filter configured to prevent cells from entering the cell culture medium apparatus.
  • the cell culture medium apparatus and the cell culture apparatus are fluidly configured in tandems, in parallels, or in any combination thereof.
  • the cell culture medium apparatus contains a mixture of a freshly prepared cell culture medium and a recycled cell culture medium from a cell culture.
  • the cell culture medium apparatus further comprises one or a plurality of ports.
  • the one or the plurality of ports further comprise valves to control the inflow/outflow of the cell culture, gas, and/or media.
  • a cell culture kit comprising: a) a cell culture apparatus comprising at least a top surface and a bottom surface, an interior suitable for containing cell culture media, a gas-permeable membrane layer, a supportive layer in contact with the gas-permeable membrane layer to form a water-impermeable barrier comprising a plurality of openings, wherein each of the opening defines a gas-permeable window, and wherein the plurality of gas-permeable windows form at least a portion of a surface of the gas- permeable cell culture medium apparatus; b) one or more cell cultures.
  • a cell culture medium kit comprising: a) a cell culture medium apparatus comprising at least a top surface and a bottom surface, an interior suitable for containing cell culture media, a gas-permeable membrane layer, a supportive layer in contact with the gas-permeable membrane layer to form a water-impermeable barrier comprising a plurality of openings, wherein each of the opening defines a gas-permeable window, and wherein the plurality of gas-permeable windows form at least a portion of a surface of the gas- permeable cell culture medium apparatus; b) one or more cell culture media.
  • a multi-layer, gas-permeable container comprising: at least a top surface and a bottom surface, an interior suitable for containing a fluid, a gas- permeable membrane layer, a supportive layer in contact with the gas-permeable membrane layer to form a water-impermeable barrier comprising a plurality of openings, wherein each of the openings defines a gas-permeable window, and wherein the plurality of gas-permeable windows form at least a portion of a surface of the gas-permeable container.
  • the fluid is a cell culture medium, a supplement for a cell culture medium, water, or an aqueous solution.
  • FIG. 1 depicts a schematic design of a water-impermeable barrier 100 comprising a gas-permeable membrane layer 110 and a supportive layer 120, wherein the supportive layer comprises a plurality of openings 1201.
  • FIG. 2 depicts a schematic design of a water-impermeable barrier 200 formed by laminating a gas-permeable membrane layer 210 and a supportive layer 220 comprising a plurality of openings, wherein each of the opening defines a gas-permeable windows 2201 which can correspond to a region in the gas-permeable membrane layer 210.
  • FIGS. 3A-3C depict schematic designs of the plurality of openings defining gas- permeable windows.
  • FIG. 4A shows the top view of a schematic design of a reinforced gas-permeable apparatus 400 comprising one or more ports 403 (e.g., for introducing and/or removing cells and/or one or more agents associated with cell culture) on the side of the apparatus.
  • FIG. 4B shows a cross-sectional side view of a schematic design of the reinforced gas-permeable apparatus.
  • the gas-permeable membrane layer 402 can be inside or outside the supportive layer 404.
  • FIG. 4C shows a working model of the reinforced gas-permeable apparatus.
  • FIG. 5 shows the top view of a schematic design of a reinforced gas-permeable apparatus comprising ports on the side.
  • FIG. 6A shows the top view of a schematic design of a reinforced gas-permeable apparatus 600 comprising one or more ports 603 (e.g., for introducing and/or removing cells and/or one or more agents associated with cell culture) on the top of the apparatus.
  • FIG. 6B shows the side view of a schematic design of a reinforced gas-permeable apparatus 600.
  • FIGS. 6C and 6D show working models of a reinforced gas-permeable apparatus.
  • FIG. 7 illustrates a comparative analysis of the gas permeability among three types of devices: (1) reinforced gas-permeable apparatus 700, (2) a round cell culture dish 710 serving as a first control, and (3) a commercially available bag 720 serving as a second control.
  • the media s color variance in the three types of devices, in the presence of phenol red, serves as an indicator of gas permeability and underlying pH conditions.
  • a sequential series of images for the three types of devices were captured at various time intervals: at the initiation of the experiment at 0 minute (FIG. 7A), at 20 minutes (FIG. 7B), at 40 minutes (FIG. 7C), at 70 minutes (FIG. 7D), at 110 minutes (FIG. 7E), at 150 minutes (FIG. 7F), at 220 minutes (FIG. 7G), and at the end of the experiment 23 hours (FIG. 7H).
  • FIG. 8 illustrates an example of a computing device 1000, in accordance with an embodiment.
  • the computing device is configured to be in the disclosed systems and is configured to perform the operational methods associated with the systems disclosed herein.
  • an apparatus made at least partially of gas-permeable material may be suitable.
  • the reinforced gas-permeable apparatus is useful for culturing cells and tissues in suspension in a liquid nutrient medium with minimum turbulence while at the same time providing the required gas transfer.
  • the apparatus may include ports for easy access to the vessel culture (e.g., for introducing and/or removing cells and/or one or more agents associated with cell culture), allowing the growth substrate to be varied for optimum performance.
  • the gas-permeable membrane and/or apparatus described herein possesses increased mechanical integrity and protection. This is achieved by reinforcement of the gas-permeable material by a supportive layer. As such, the apparatus allows for large scale culture and durability during manipulation, storage and transportation.
  • kits for cell culture as well as methods for using the reinforced gas-permeable apparatus described herein.
  • FIG. 4A and FIG. 4B illustrate perspective views of one embodiment of the reinforced gas-permeable apparatus 400.
  • the reinforced gas-permeable apparatus 400 may comprise a plurality of gas-permeable windows 401 defined by a plurality of openings on a support layer 404 in contact with a gas-permeable membrane 402, wherein the plurality of the gas-permeable windows form at least a portion of the top surface of the reinforced gas- permeable apparatus.
  • the gas-permeable membrane layer 402 can be inside or outside the supportive layer 404.
  • the supportive layer 404 is an outside layer that at least partially encloses the gas-permeable membrane layer 402.
  • the gas-permeable membrane layer can be in the interior of the apparatus (e.g., at least partially enclosed within the apparatus by another layer), or can form part of the layer(s) enclosing the interior of the apparatus.
  • the gas-permeable membrane layer 402 is an outside layer that at least partially encloses the supportive layer 404.
  • the supportive layer can be in the interior of the apparatus (e.g., at least partially enclosed within the apparatus by another layer), or can form part of the layer(s) enclosing the interior of the apparatus.
  • a cell culture media may be loaded into the interior of the apparatus.
  • the reinforced gas-permeable apparatus may comprise one or more ports, such as 403.
  • FIG. 4C shows a working model of the reinforced gas-permeable apparatus 400.
  • the reinforced gas-permeable apparatus may have more than one surface wherein at least a portion of the surface comprises gas-permeable windows.
  • a reinforced gas-permeable apparatus in the shape of a bag comprising two sides may have at least a portion on both sides be gas-permeable, such as shown in FIG. 6D.
  • one of the surfaces of the apparatus comprises one or more gas- permeable windows.
  • some of the surfaces of the apparatus comprises one or more gas-permeable windows.
  • all of the surfaces of the apparatus comprises one or more gas-permeable windows.
  • the gas-permeable apparatus disclosed herein or any one or more of the components thereof can be bio-compatible, e.g., compatible with the cells or tissues cultured in the gas-permeable apparatus.
  • the gas-permeable apparatus disclosed herein or any one or more of the components thereof can be degradable (e.g., biodegradable), for instance, for easy disposal after a single use.
  • the gas- permeable apparatus disclosed herein or any one or more of the components thereof can be sterile or sterilized.
  • the gas-permeable apparatus disclosed herein or any one or more of the components thereof can be flexible.
  • the gas-permeable apparatus disclosed herein or any one or more of the components thereof can be rigid or semirigid. a. Gas-permeable membrane
  • the gas-permeable material of which the bioreactor of this disclosure is constructed by allowing gas (such as O2 and CO2) to diffuse through the apparatus and into the cell culture media.
  • gas such as O2 and CO2
  • the gas-permeable membrane may be on the interior or the exterior of the reinforced gas-permeable apparatus.
  • the gas-permeable material can be any suitable material, such as a porous nonhydrophobic material, a silicone rubber, polyethylene, polyvinyl, polyvinyl chloride, polyurethane, silicone, polyethylene terephthalate glycol, polycarbonate, polystyrene, polypropylene, acrylic, polycarbonate, polyolefin, ethylene vinyl acetate, polysulfone, polyethylene terephthalate, polytetrafluoroethylene (PTFE) or compatible fluoropolymer, and poly (styrene-butadiene- styrene), or a combination thereof.
  • the gas-permeable membrane is a single polymer.
  • the gas-permeable membrane is a copolymer.
  • the gas-permeable membrane comprises commercially available materials including POLYFLEX® (0.002 inch thick polystyrene film manufactured by Plastics Suppliers, Inc., Columbus, Ohio), BREATHE-EASYTM (non-porous polyurethane from Diversified Biotech, Boston, Mass.), 3MTM Medical Film (polyurethane from 3M, MN), ArgotecTM, DelstarTM (polyurethane from Schweitzer-Mauduit International Inc., Georgia), TeflonTM (The Chemours Company, Wilmington, Delaware), TPU film (American polyfilm, BRANFORD, CONNECTICUT) SBS/EVA/SBA, a three-layered co-extruded film of styrene-butadiene-styrene/ethyl vinyl acetate/styrene/butadiene/styrene, non-porous film from BASF, Germany), SBS/PE, a two-layered
  • the gas-permeable membrane may be of any suitable thickness, such as about 0.1 pm to about 5 mm, about 0.2 pm to about 4 mm, about 0.5 pm to about 3 mm, about 1 pm to about 3 mm, about 1.5 pm to about 3 mm, about 1.5 pm to about 2 mm, about 2 pm to about 2 mm, about 2 pm to about 1.5 mm, about 2.5 pm to about 1.5 mm, about 2.5 pm to about 1 mm, about 3 pm to about 1 mm, about 3 pm to about 900 pm, about 3.5 pm to about 900 pm, about 3.5 pm to about 800 pm, about 4 pm to about 800 pm, about 4 pm to about 700 pm, about 4.5 pm to about 700 pm, about 4.5 pm to about 600 pm, about 5 pm to about 600 pm, or about 5 pm to about 500 pm.
  • the thickness of the gas-permeable membrane is about 5 pm to about 500 pm.
  • the reinforced gas-permeable apparatus may comprise a supportive layer 120 in contact with the gas-permeable membrane, and wherein supportive layer 120 comprises a plurality of openings 1201.
  • the supportive layer comprises any suitable material or materials, such as but not limited to plastic, silicon, metal, or a polymer.
  • the support layer is flexible.
  • the support layer is rigid.
  • the supportive layer is gas-permeable.
  • the supportive layer is not gas-permeable.
  • the supportive layer is water-permeable.
  • the supportive layer is not water-permeable.
  • the supportive layer and the gas-permeable membrane may comprise the same materials, while in other variations they may comprise different materials.
  • the supportive layer comprises a porous nonhydrophobic material, a silicone rubber, polyethylene, polyvinyl, polyvinyl chloride, polyurethane, silicone, polyethylene terephthalate glycol, polycarbonate, polystyrene, polypropylene, acrylic, polycarbonate, polyolefin, ethylene vinyl acetate, polysulfone, polyethylene terephthalate, polytetrafluoroethylene (PTFE) or compatible fluoropolymer, and poly (styrene-butadiene- styrene), or a combination thereof.
  • the supportive layer is a single polymer.
  • the supportive layer is a textile.
  • the supportive layer is a foam.
  • the supportive layer is a co-polymer.
  • the supportive layer comprises commercially available materials including POLYFLEX® (0.002 inch thick polystyrene film manufactured by Plastics Suppliers, Inc., Columbus, Ohio), BREATHE-EASYTM (non-porous polyurethane from Diversified Biotech, Boston, Mass.), 3MTM Medical Film (polyurethane from 3M, MN), ArgotecTM, DelstarTM (polyurethane from Schweitzer-Mauduit International Inc., Georgia), TeflonTM (The Chemours Company, Wilmington, Delaware), TPU film (American polyfilm, BRANFORD, CONNECTICUT), SBS/EVA/SBA, a three-layered co-extruded film of styrene-butadiene- styrene/ethyl vinyl acetate/styrene/butadiene/styrene, non-porous film from BASF, Germany), SBS/PE, a two-layered co-extruded film of s
  • the supportive layer may be of any suitable thickness, such as about 1 pm to about 20 mm, about 10 pm to about 19 mm, about 20 pm to about 18 mm, about 30 pm to about 17 mm, about 40 pm to about 16 mm, about 50 pm to about 15 mm, about 55 pm to about 14 mm, about 60 pm to about 13 mm, about 65
  • the thickness of the supportive layer is about 100 pm to about 2 mm.
  • the plurality of openings on the supportive layer may have any suitable shape, including but not limited to circular (see FIG. 3A), square (see FIG. 3B), and hexagonal (see FIG. 3C). It should be appreciated that the plurality of openings on the supportive layer may have any number of configurations or other physical shapes, such as half circles, angled slots, bent or curved slots, triangles, crescents, parallelograms, or the like. In some variations, the plurality of openings are arranged in a two-dimensional array, such a gridlike arrangement.
  • the plurality of openings may have any suitable dimensions.
  • the cross-section of the plurality of openings has a circular shape.
  • the largest cross-sectional diameter of the plurality of openings having a circular shape may be about 1 pm to about 10 pm, about 10 pm to about 100 pm, about 100 pm to about 1 mm, about 1 mm to about 10 mm, about 1 pm to about 1 mm, about 1 pm to about 10 mm, or larger than about 10 mm.
  • the largest cross-sectional diameter of the plurality of openings having a circular shape is about 100 pm to about 500 pm, about 500 pm to about 1 mm, about 1 mm to about 1.5 mm, about 1.5 mm to about 2 mm, about 2 mm to about 2.5 mm, or about 2.5 mm to about 3 mm.
  • the largest cross-sectional length of the plurality of openings having a square shape may be about 1 pm to about 10 pm, about 10 pm to about 100 pm, about 100 pm to about 1 mm, about 1 mm to about 10 mm, larger than about 10 mm, about 1 pm to about 10 mm, about 1 pm to about 1 mm, or about 1 mm to about 10 mm.
  • the length of the edge of the plurality of openings having a hexagonal shape may be about 1 pm to about 10 pm, about 10 pm to about 100 pm, about 100 pm to about 1 mm, about 1 mm to about 10 mm, about 1 pm to about 1 mm, about 1 pm to about 10 mm, or larger than about 10 mm.
  • the total surface area for the cross-section of all openings may be about 1 pm 2 to about 10 pm 2 , about 10 pm 2 to about 100 pm 2 , about 100 pm 2 to about 1 mm 2 , about 1 mm 2 to about 10 mm 2 , about 10 mm 2 to about 100 mm 2 , or larger than 100 mm 2 .
  • the gas-permeable windows have an area of about 1 pm 2 to about 30 cm 2 . It should be appreciated that each one of the plurality of openings need not have the same configuration.
  • the plurality of openings may be closely or sparsely distributed across the supportive layer. In some embodiments, there may be about 1 opening, about 2 openings, about 4 openings, about 8 openings, about 12 openings, about 16 openings, about 20 openings, or more than 20 openings per 10 cm 2 of the supportive layer. In some embodiments, the total surface area for the cross-section of all openings on the supportive layer is less than 1%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, or about 25% relative to the area of the supportive layer.
  • the plurality of openings may be evenly distributed at least partially on a surface of the reinforced gas-permeable apparatus, as shown in FIGS. 4A-4C, wherein the openings evenly distribute as an array.
  • the plurality of openings may not be evenly distributed on a surface of the reinforced gas-permeable apparatus, as exemplifies in FIG. 5.
  • the supportive layer 404 or 604 may be integral to the gas- permeable membrane 402 or 602, while in other variations, the supportive layer may be attached to the gas-permeable membrane in any suitable manner (e.g., using adhesives (glues, adhesive polymers, and the like), welding, laminating, molding, mechanical fasteners, chemical bonding, a combination of these methods, or the like).
  • the supportive layer 404 or 604 may be integral to the reinforced gas-permeable apparatus 400 or 600, while in other variations, the supportive layer may be attached to the reinforced gas-permeable apparatus in any suitable manner (e.g., using adhesives (glues, adhesive polymers, and the like), welding, laminating, molding, mechanical fasteners, chemical bonding, a combination of these methods, or the like).
  • adhesives glues, adhesive polymers, and the like
  • welding laminating, molding, mechanical fasteners, chemical bonding, a combination of these methods, or the like.
  • FIG. 2 show one variation of a water-impermeable barrier 200 formed by a gas- permeable membrane 210 in contact with a supportive layer 220 comprising a plurality of openings 2201, wherein each of the opening define a gas-permeable window 230.
  • the supportive layer 220 may be attached to the gas-permeable membrane 210 in any suitable manner (e.g., using adhesives (glues, adhesive polymers, and the like), welding, laminating, molding, mechanical fasteners, chemical bonding, a combination of these methods, or the like).
  • a thin gas-permeable membrane may be laminated to a supporting layer, such as a supportive scaffold, a mesh, or a grid.
  • thermal-bonding lamination may be used by applying heat and pressure to bond and adhere the gas-permeable membrane and the support layer.
  • adhesive-bonding lamination may be used by applying adhesive to bond and adhere the adhere the gas-permeable membrane and the support layer.
  • Any suitable adhesive may be used, such as a silicone glue, preferably a medical grade silicone glue.
  • the supportive layer and the gas-permeable membrane are not integral.
  • the supportive layer and the gas-permeable membrane are integral.
  • the windows may be formed by molding or pressing on the material to create areas that are thinner than the surroundings.
  • the plurality of windows on the water- impermeable barrier may have any suitable shape, including but not limited to circular (see FIG. 3A), square (see FIG. 3B), and hexagonal (see FIG. 3C). It should be appreciated that the windows on the water- impermeable barrier may have any number of configurations or other physical shapes, such as half circles, angled slots, bent or curved slots, triangles, crescents, parallelograms, or the like.
  • the plurality of openings are arranged in a two-dimensional array, such a gridlike arrangement.
  • the plurality of openings may be evenly distributed at least partially on a surface of the reinforced gas-permeable apparatus, as shown in FIGS. 4A-4C, wherein the openings evenly distribute as an array.
  • the plurality of openings may not be evenly distributed on a surface of the reinforced gas-permeable apparatus, as exemplifies in FIG. 5.
  • the windows may have any suitable dimensions.
  • the crosssection of the windows has a circular shape.
  • the largest cross-sectional diameter of the windows having a circular shape may be about 1 pm to about 10 pm, about 10 pm to about 100 m, about 100 pm to about 1 mm, about 1 mm to about 10 mm, about 1 pm to about 1 mm, about 1 pm to about 10 mm, or larger than about 10 mm.
  • the largest cross-sectional diameter of the windows having a circular shape is about 100 pm to about 500 pm, about 500 pm to about 1 mm, about 1 mm to about 1.5 mm, about 1.5 mm to about 2 mm, about 2 mm to about 2.5 mm, or about 2.5 mm to about 3 mm.
  • the largest cross-sectional length of the windows having a square shape may be about 1 pm to about 10 pm, about 10 pm to about 100 pm, about 100 pm to about 1 mm, about 1 mm to about 10 mm, larger than about 10 mm, about 1 pm to about 10 mm, about 1 pm to about 1 mm, or about 1 mm to about 10 mm.
  • the length of the edge of the windows having a hexagonal shape may be about 1 pm to about 10 pm, about 10 pm to about 100 pm, about 100 pm to about 1 mm, about 1 mm to about 10 mm, about 1 pm to about 1 mm, about 1 pm to about 10 mm, or larger than about 10 mm.
  • the total surface area for the crosssection of all openings may be about 1 pm 2 to about 10 pm 2 , about 10 pm 2 to about 100 pm 2 , about 100 pm 2 to about 1 mm 2 , about 1 mm 2 to about 10 mm 2 , about 10 mm 2 to about 100 mm 2 , or larger than 100 mm 2 .
  • the gas-permeable windows have an area of about 1 pm 2 to about 30 cm 2 . It should be appreciated that each one of the windows need not have the same configuration.
  • the windows may be closely or sparsely distributed across the water-impermeable barrier. In some variations, there may be about 1 window, about 2 windows, about 4 windows, about 8 windows, about 12 windows, about 16 windows, about 20 windows, or more than 20 windows per 10 cm 2 of the water-impermeable barrier.
  • the total surface area for the cross-section of all windows on the water-impermeable barrier is less than 1%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% relative to the area of the water-impermeable barrier.
  • Interior of the apparatus is
  • the reinforced gas-permeable apparatus comprises an interior suitable for containing cell culture media, such as interior 405 and 605 depicted in FIGS. 4B and 6B, respectively.
  • the interior of the reinforced gas-permeable apparatus may be smooth, or the surface may be rough (i.e., having surface irregularities).
  • the interior of the reinforced gas-permeable apparatus may be coated with chemicals that are suitable for the cell growth and maintenance of particular cell types.
  • the interior of the reinforced gas- permeable apparatus may be treated to support cell attachment for adherent cell culture, or be treated to prevent cell attachment for suspension cell culture.
  • the interior of the reinforced gas-permeable apparatus may comprise a porous material, such as a polymer gel or a hydrogel, polymer-based sponge, or mesh.
  • the interior of the reinforced gas-permeable apparatus may comprise a matrix, such as a dried fibrous structure, containing the reagent.
  • the interior of the reinforced gas-permeable apparatus may comprise cellulose (e.g., nitrocellulose, paper-like material), glass fibrous mesh, silk fibrous mesh, and the like.
  • all or a portion of the interior coating may be dissolvable when the interior of the reinforced gas-permeable apparatus (e.g. reinforced gas-permeable apparatus 400 or 600) are in contact with the target cell culture.
  • the dissolvable material may comprise any suitable material, such as but not limited to a salt, micro-particles, nanoparticles, polyglycolide, poly (lactic acid), or poly(lactic-co-glycolic) acid co-polymer, or combinations thereof.
  • all or a portion of the interior of the reinforced gas- permeable apparatus may be meltable when the interior coating of the reinforced gas-permeable apparatus is in contact with the target cell culture.
  • the dissolvable material may comprise any suitable material, such as but not limited to DMSO.
  • the inner surface of the reinforced gas-permeable apparatus may be flat or patterned. For example, various patterns such as microfluidic channels or wells can be created on the inner surface of the reinforced gas-permeable apparatus to support different cell culture purposes.
  • the gas-permeable membrane can be attached to a reinforcing structure that has channels or wells.
  • the interior of the cell culture apparatus comprises one or more microfluidic channels. In some embodiments, the interior of the cell culture apparatus comprises one or more wells.
  • the interior of the reinforced gas-permeable apparatus may have any suitable volume capacity.
  • the volume capacity of the reinforced gas-permeable apparatus may be about 1 cm 3 to about 10 cm 3 , about 10 cm 3 to about 100 cm 3 , about 100 cm 3 to about 1 dm 3 , about 1 dm 3 to about 10 cm 3 , about 10 cm 3 to about 100 dm 3 , or greater than about 100 dm 3 .
  • the reinforced gas-permeable apparatus may have any suitable design.
  • the reinforced gas-permeable apparatus comprises one or more ports suitable for inflow and outflow of the cell culture and/or media.
  • the reinforced gas-permeable apparatus 400 comprises ports 403 on the side of the apparatus. The ports may be further connected to tubing, as shown in FIG. 4A.
  • the reinforced gas-permeable apparatus 600 comprises ports 603 in the center of the top of the apparatus.
  • the one or more ports comprise valves to control the inflow/outflow of the cell culture, gas, and/or media.
  • the reinforced gas-permeable apparatus may be further reinforced, such as at the seams, edges, and/or the ports.
  • the reinforced gas-permeable apparatus may have any suitable shapes, such as having a shape similar to a bag, a pouch, a cuboid, a tetrahedron, a flask, or an ellipsoid.
  • the reinforced gas-permeable apparatus may be flexible or rigid. In some variations, the reinforced gas- permeable apparatus is flexible. In some variations, the reinforced gas-permeable apparatus is rigid. In some embodiments, the reinforced gas-permeable apparatus may further comprise gas impermeable packaging and/or containment element to control accessibility to gas exchange.
  • the apparatus provided herein comprises one or more cell culture media.
  • cell culture media include basal medium eagle (BME), dulbecco's modified eagle medium (DMEM), minimum essential medium (MEM), minimum essential media alpha (NEMA), Glasgow modified minimum essential medium (GMEM), Iscove's modified Dulbecco's medium (IMDM), Leibovits L15 medium, McCoy's 5A medium, F10 nutrient mix, F12 nutrient mix, MCDB 153, media 199, RPMI 1640 medium, Waymouth's MB 752/1 medium, Schneider's Drosophila medium, Grace's insect medium, and Bioinsect-1 serum free medium.
  • the medium may comprise additional ingredients such as ingredients selected from the group comprising fetal bovine serum (FBS), platelet lysate (PL), growth factors, antibiotic such as penicillin or streptomycin, ascorbic acid-2- phosphate (asap), peptides and/or aminoacids, e.g. Glutamax, dexamethasone growth factors such as e.g.
  • FBS fetal bovine serum
  • PL platelet lysate
  • growth factors antibiotic such as penicillin or streptomycin
  • ascorbic acid-2- phosphate (asap) ascorbic acid-2- phosphate
  • peptides and/or aminoacids e.g. Glutamax
  • dexamethasone growth factors such as e.g.
  • basic fibroblast growth factor human recombinant and EGF carbohydrates such as D (+) glucose, sodium hypoxanthine, aminopterin, thymidine, lipids, saturated and unsaturated fatty acids, PUFA, vitamins, phospholipids, minerals, human transferrin, human insulin, progesterone, putrescine, selenite, surfactants such as non-ionic surfactants, antioxidants, 2- mercaptoethanol, cholesterol, hormones, and hydrocortisone.
  • carbohydrates such as D (+) glucose, sodium hypoxanthine, aminopterin, thymidine, lipids, saturated and unsaturated fatty acids, PUFA, vitamins, phospholipids, minerals, human transferrin, human insulin, progesterone, putrescine, selenite, surfactants such as non-ionic surfactants, antioxidants, 2- mercaptoethanol, cholesterol, hormones, and hydrocortisone.
  • the apparatus may be configured to perform cell culture in a solid form, for example, on the surface of a solid culture media, a gel, a membrane, a container, or a slide.
  • the reinforced gas-permeable apparatus described herein may be used to culture a target cell.
  • the target cell culture may be in any suitable form.
  • the target cell culture is in a solid form, for example, on the surface of a solid culture media, a gel, a membrane, a container, or a slide.
  • the target cell may be of any type of interest, including but are not limited to, bacteria, yeast, fungi, or mammalian cells.
  • the target cell culture is a microbiome culture.
  • the target cell culture is a bacteria culture.
  • the target cells are pre-cultured prior to application onto the culture media.
  • the cells to be cultured may be any kind of cells, such as bacterial cells, fungi, yeast cells, plant cells, algae, insect cells, mammalian cells.
  • the cells are mammalian cells, more preferably human cells, more preferably human hematopoietic cells (blood cells).
  • the cells may also be chosen from progenitor cells, embryonic stem cells, induced pluripotent stem cells, skin cells, kidney cells, pancreas cells, liver cells, bone cells, cartilage cells, muscle cells, lung cells, eye cells, ovary cells, omnipotent cells, pluripotent cells, cancer cells.
  • the cells may be primary cells but may also be from a cell-line.
  • the cells may be adherent cells or non-adherent cells.
  • the cells may be primary cells.
  • the cells culture is in the form of blood.
  • cells are cultured using a cell culture apparatus disclosed herein for cell or gene therapy.
  • the cell therapy is a stem cell-based cell therapy.
  • cells cultured using a cell culture apparatus disclosed herein are pluripotent stem cells (PSCs), adult stem cells (ASCs), or cancer stem cells (CSCs).
  • cells cultured using a cell culture apparatus disclosed herein are embryonic stem cells (ESCs), found in the inner blastocyst cell mass of preimplantation embryos; epiblast stem cells (EpiSCs) and embryonic germ cells (EGCs), found in post-implantation embryos; or induced pluripotent stem cells (iPSCs), derived from direct reprogramming of postnatal/adult somatic cells in vitro.
  • ESCs embryonic stem cells
  • EpiSCs epiblast stem cells
  • EECs embryonic germ cells
  • iPSCs induced pluripotent stem cells
  • cells cultured using a cell culture apparatus disclosed herein are hematopoietic stem cells (HSCs), skin stem cells (SSCs), neural stem cells (NSCs), or mesenchymal stem cells (MSCs).
  • HSCs hematopoietic stem cells
  • SSCs skin stem cells
  • NSCs neural stem cells
  • MSCs mesenchymal stem cells
  • the cell therapy is a non-stem cell-based cell therapy.
  • cells cultured using a cell culture apparatus disclosed herein are somatic cells that are isolated from a subject (e.g., the human body), propagated, expanded, selected, and subsequently administered to patients for curative, preventive, or diagnostic purposes.
  • cells cultured using a cell culture apparatus disclosed herein are fibroblasts, chondrocytes, keratinocytes, hepatocytes, pancreatic islet cells, and/or immune cells, such as T cells, dendritic cells (DCs), natural killer (NK) cells, and/or macrophages.
  • DCs dendritic cells
  • NK natural killer
  • cells cultured using a cell culture apparatus disclosed herein are employed as an in vivo source of enzymes, cytokines, and growth factors; as an adoptive cell therapy (ACT) to treat cancers; as transplanted cells, such as hepatocytes or pancreatic islet cells, to correct inborn metabolic errors; or as scaffold-based or -free cellular systems to treat ulcers, bums, or cartilage lesions.
  • cells cultured using a cell culture apparatus disclosed herein are used for tissue regeneration.
  • cells cultured using a cell culture apparatus disclosed herein are modified peripheral or tumor-resident immune cells for transferring into patients to mount an immunologic reaction against tumors.
  • cells cultured using a cell culture apparatus disclosed herein can include tumor- infiltrating lymphocytes (TILs), tumor-specific T- cell receptor (TCR)-modified T cells, chimeric antigen receptor (CAR)-T cells, lymphokine- activated killer (LAK) cells, cytokine-induced killer (CIK) cells, y5 T cells, and/or NK cells.
  • TILs tumor- infiltrating lymphocytes
  • TCR tumor-specific T- cell receptor
  • CAR chimeric antigen receptor
  • LAK lymphokine- activated killer
  • CIK cytokine-induced killer
  • y5 T cells y5 T cells
  • NK cells derived from one or more subjects, including healthy subjects or diseased subjects such as a human patient.
  • At least two stem cell and/or non-stem cell types from isolated cells or tissue extracts are cultured using a cell culture apparatus disclosed herein.
  • the cells are cultured for providing a multicellular therapy.
  • cells cultured using a cell culture apparatus disclosed herein comprise cells of different lymphocyte lineages, such as TILs including T cells and B cells.
  • cells e.g., fibroblasts and keratinocytes
  • a scaffold e.g., 3D biocompatible tissue analogs
  • a cell culture apparatus disclosed herein can comprise (e.g., within the apparatus together with the cultured cells, or as part of the apparatus) biodegradable natural or synthetic polymers (e.g., bovine collagen, hydrogels, sponges) with sophisticated porous networks through which oxygen, nutrients, and metabolites can be exchanged.
  • a cell culture apparatus disclosed herein can be used to culture a heterogeneous mixture of cells, such as a mixture of stromal and vascular cells, e.g., ASCs, granulocytes, monocytes, lymphocytes, pericytes, and/or endothelial progenitor cells (EPCs).
  • ASCs a mixture of stromal and vascular cells
  • EPCs endothelial progenitor cells
  • the reinforced gas-permeable apparatus described herein may be used to condition the cell culture medium before the cell culture medium can be used to support cell growth, including to aerate the cell culture medium to the appropriate O2/CO2 saturation, to ensure appropriate pH of the cell culture medium, and/or to maintain at appropriate temperate the cell culture medium, e.g. 37°C.
  • the reinforced gas-permeable apparatus may be in any suitable shapes.
  • the reinforced gas-permeable apparatus can have a shape similar to a bag, a pouch, a cuboid, a tetrahedron, a flask, or an ellipsoid.
  • the reinforced gas-permeable apparatus can be a vial, a syringe, a can, a bag, a pouch, a flask, a bottle, ajar, a bucket, a drum, a pot, a bin, a keg, a tank, a trunk, a carboy, or a bioreactor.
  • the reinforced gas-permeable apparatus may be fluidly configured in tandems, in parallels, or in any combination thereof, thereby enabling increased storage capacity, improved efficiency, and versatility in managing various types of cell culture media.
  • the reinforced gas-permeable apparatus may be fluidly configured in tandems.
  • the reinforced gas-permeable apparatus may be fluidly configured in parallels.
  • the reinforced gas-permeable apparatus may be in fluid communication with a second reinforced gas-permeable apparatus.
  • the reinforced gas-permeable apparatus may be in fluid communication with a plurality of reinforced gas-permeable apparatus.
  • a plurality of the reinforced gas- permeable apparatus may be in fluid communication with a second set of a plurality of reinforced gas-permeable apparatus.
  • the reinforced gas-permeable apparatus may be stored under different conditions.
  • a reinforced gas-permeable apparatus containing the DMEM may be stored in an incubator, preequilibrated at a temperature of 37 °C, with a 5% CO2 concentration and 85% humidity and another reinforced gas-permeable apparatus containing the L-glutamine may be stored outside the incubator, at ambient temperature of 25°C.
  • the DMEM and the L-glutamine are mixed immediately before they are fluidly delivered to a cell culture.
  • the cell culture medium is a mixture of a freshly prepared cell culture medium and a recycled cell culture medium from a cell culture.
  • the volume percentage of the recycled cell culture medium over the total volume of the cell culture medium may be about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%.
  • the reinforced gas-permeable apparatus may contain the same cell culture medium. In some variations, the reinforced gas-permeable apparatus may contain different cell culture media. In some variations, the reinforced gas-permeable apparatus may contain cell culture medium mixed with an appropriate volume percentage of the recycled cell culture medium. In some variations, the liquid chamber of the reinforced gas-permeable apparatus is in fluid communication with the interior of the cell culture apparatus. In some variations, the liquid chambers of a plurality of the reinforced gas-permeable apparatus are in fluid communication with the interior of the cell culture apparatus.
  • the reinforced gas-permeable apparatus may contain one or plurality of ports.
  • the one or the plurality of ports comprise valves to control the inflow/outflow of the cell culture, gas, and/or media.
  • the present disclosure provides computer systems that are programmed to implement and/or be used in connection with devices and methods of the disclosure.
  • a computer system that is programmed or otherwise configured to implement methods of screening compositions or agents, for instance, by interfacing with a potential user or a user of the reinforced gas-permeable apparatus, kit, or system disclosed herein.
  • the computer system can regulate various aspects of the present disclosure, such as, for example, controlling the installation, operation, maintenance, data collection (such as image collection), data processing (such as image processing), and/or replacement of the reinforced gas-permeable apparatus, device, kit, or system disclosed herein.
  • the computer system can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device.
  • the electronic device can be a mobile electronic device.
  • the computer system comprises a central processing unit (CPU), which can be a single core or multi core processor, or a plurality of processors for parallel processing.
  • the computer system comprises memory or memory location (e.g., random-access memory, read-only memory, flash memory), electronic storage unit (e.g., hard disk), communication interface for communicating with one or more other systems, and peripheral devices, such as cache, other memory, data storage and/or electronic display adapters.
  • the memory, storage unit, interface and peripheral devices are in communication with the CPU through a communication bus such as a motherboard.
  • the storage unit is a data storage unit or data repository for storing data.
  • the computer system is operatively coupled to a computer network with the aid of the communication interface.
  • the network is the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet.
  • the network is a telecommunication and/or data network.
  • the network comprises one or more computer servers, which can enable distributed computing, such as cloud computing.
  • the network in some cases with the aid of the computer system, can implement a peer-to-peer network, which may enable devices coupled to the computer system to behave as a client or a server.
  • the CPU of the computer system can execute a sequence of machine-readable instructions, which can be embodied in a program or software.
  • the instructions may be stored in a memory location, such as the memory.
  • the instructions can be directed to the CPU, which can subsequently program or otherwise configure the CPU to implement methods of the present disclosure.
  • the CPU can be part of a circuit, such as an integrated circuit.
  • One or more other components of the system can be included in the circuit.
  • the storage unit can store files, such as drivers, libraries, and saved programs.
  • the storage unit can store user data, e.g., user preferences and user programs.
  • the computer system in some cases can include one or more additional data storage units that are external to the computer system, such as located on a remote server that is in communication with the computer system through an intranet or the Internet.
  • the computer system communicates with one or more remote computer systems through the network, including a local area network (“LAN”); wide area network (“WAN”), such as the Internet; metropolitan area network (“MAN”); point-to-point or peer-to-peer connection; etc.
  • LAN local area network
  • WAN wide area network
  • MAN metropolitan area network
  • point-to-point or peer-to-peer connection etc.
  • Communication with other devices may be accomplished using any suitable networking protocol.
  • one suitable networking protocol may include the Internet Protocol (“IP”), Transmission Control Protocol (“TCP”), User Datagram Protocol (“UDP”), or combinations thereof, such as TCP/IP or UDP/IP.
  • IP Internet Protocol
  • TCP Transmission Control Protocol
  • UDP User Datagram Protocol
  • the computer system communicates with a remote computer system of a user (e.g., a cloud computing system, a server system).
  • remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants.
  • the user can access the computer system via the network.
  • the computer system comprises a front end.
  • the front end is a native application (app) for a particular operating system or a web app.
  • the native app is a mobile app for e.g., Google, Android, or iOS.
  • the front end uses a JavaScript framework.
  • the front end platform links the app to a cloud service.
  • the front end is based on Angular.
  • the cloud service is based on Azure.
  • the system further comprises a back end.
  • the back end is a cloud service.
  • the cloud back end service comprises data storage, security and/or processing.
  • the app is a preconfigured app.
  • the preconfigured app comprises a user-friendly interface for a computer or mobile device, such as a companion web app.
  • Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system, such as, for example, on the memory or electronic storage unit.
  • the machine executable or machine- readable code can be provided in the form of software.
  • the code can be executed by the processor.
  • the code can be retrieved from the storage unit and stored on the memory for ready access by the processor.
  • machine-executable instructions are stored on memory.
  • the code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code or can be compiled during runtime.
  • the code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.
  • aspects of the systems and methods provided herein can be embodied in programming.
  • Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium.
  • Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk.
  • “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server.
  • another type of media that may bear the software elements includes optical, electrical, and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links.
  • Such processors may comprise, or may be in communication with, media, for example one or more non-transitory computer-readable media, that may store processor-executable instructions that, when executed by the processor, can cause the processor to perform methods according to this disclosure as carried out, or assisted, by a processor.
  • Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications.
  • non-transitory computer-readable medium may include, but are not limited to, an electronic, optical, magnetic, or other storage device capable of providing a processor, such as the processor in a web server, with processor-executable instructions.
  • Other examples of non- transitory computer-readable media include, but are not limited to, a floppy disk, CD-ROM, magnetic disk, memory chip, ROM, RAM, ASIC, configured processor, all optical media, all magnetic tape or other magnetic media, or any other medium from which a computer processor can read. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.
  • the processor, and the processing, described may be in one or more structures, and may be dispersed through one or more structures.
  • the processor may comprise code to carry out methods (or parts of methods) according to this disclosure.
  • Methods and systems of the present disclosure can be implemented by way of one or more algorithms.
  • An algorithm can be implemented by way of software upon execution by the central processing unit.
  • the algorithm can, for example, determine sizes of the zone of inhibition on the target cell culture upon contacting the reinforced gas-permeable loaded with one or more agents.
  • the computer system can be or is in communication with an image capturing device, such as a digital camera, to collect images of the target cell culture upon contacting the reinforced gas-permeable loaded with one or more agents.
  • an image capturing device such as a digital camera
  • the computer system can be or is in communication with an electronic display that comprises a user interface (UI) for providing, for example, an interface to start and/or monitor the progress of image collection of target cell culture at various time points and image processing of the target cell culture.
  • UI user interface
  • Examples of UI’s include, without limitation, a graphical user interface (GUI) and web-based user interface.
  • device 1000 can be a host computer connected to a network.
  • Device 1000 can be a client computer or a server.
  • the device can be any suitable type of microprocessor-based device, such as a dedicated computing device, a personal computer, work station, server, handheld computing device (portable electronic device) such as a phone or tablet, an edge- Al device, or a neural network device.
  • the device can include, for example, one or more of processors 1002, input device 1006, output device 1008, storage 1010, and communication device 1004.
  • Input device 1006 and output device 1008 can generally correspond to those described above and can either be connectable or integrated with the computer.
  • Input device 1006 can be any suitable device that provides input, such as a camera sensor, touchscreen, keyboard or keypad, mouse, or voice-recognition device.
  • Output device 1008 can be any suitable device that provides output, such as an illuminator, a touchscreen, haptics device, or speaker.
  • Storage 1010 can be any suitable device that provides storage, such as an electrical, magnetic, or optical memory including a RAM, cache, hard drive, or removable storage disk.
  • Communication device 1004 can include any suitable device capable of transmitting and receiving signals over a network, such as a network interface chip or device. The components of the computer can be connected in any suitable manner, such as via a physical bus, or wirelessly.
  • Software 1012 which can be stored in storage 1010 and executed by processor 1002, can include, for example, the programming that embodies the functionality of the present disclosure (e.g., as embodied in the devices described above).
  • Software 1012 can also be stored and/or transported within any non-transitory, computer-readable storage medium for use by or in connection with an instruction execution system, apparatus, or device, such as those described above, that can fetch instructions associated with the software from the instruction execution system, apparatus, or device and execute the instructions.
  • a computer-readable storage medium can be any medium, such as storage 1010, that can contain or store programming for use by or in connection with an instruction-execution system, apparatus, or device.
  • Software 1012 can also be propagated within any transport medium for use by or in connection with an instruction-execution system, apparatus, or device, such as those described above, that can fetch instructions associated with the software from the instructionexecution system, apparatus, or device and execute the instructions.
  • a transport medium can be any medium that can communicate, propagate, or transport programming for use by or in connection with an instruction-execution system, apparatus, or device.
  • the transport readable medium can include, but is not limited to, an electronic, magnetic, optical, electromagnetic, or infrared wired or wireless propagation medium.
  • Device 1000 may be connected to a network, which can be any suitable type of interconnected communication system.
  • the network can implement any suitable communications protocol and can be secured by any suitable security protocol.
  • the network can comprise network links of any suitable arrangement that can implement the transmission and reception of network signals, such as wireless network connections, T1 or T3 lines, cable networks, DSL, or telephone lines.
  • Device 1000 can implement any operating system suitable for operating on the network.
  • Software 1012 can be written in any suitable programming language.
  • application software embodying the functionality of the present disclosure can be deployed in different configurations, such as in a client/server arrangement or through a Web browser as a Web-based application or Web service, for example.
  • kits and/or systems for cell culture comprising any of the reinforced gas-permeable apparatus (e.g., the reinforced gas- permeable apparatus 400 or 600 described above).
  • the kits and/or systems may comprise the reinforced gas-permeable apparatus described herein, and one or more cell cultures, such as according to any of the cell cultures described herein.
  • the kit and/or system may optionally comprise a gas chamber for communicating the gas with the surfaces of the cell culture apparatus at sufficient pressure to maintain a gas phase in the gas-permeable apparatus.
  • the reinforced gas-permeable apparatus is placed inside a gas chamber.
  • the gas chamber comprises CO2 and/or O2, and wherein the gas-permeable membrane layer of the apparatus permits exchange of CO2 and/or O2.
  • the kit and/or system may optionally comprising a liquid chamber in fluid communication with the interior of the cell culture apparatus for sending and receiving liquid.
  • the liquid chamber may contain cell culture medium.
  • the liquid chamber is in fluid communication with the interior of the cell culture apparatus wherein the interior comprises one or more microfluidic channels or wells.
  • the kit and/or system may further comprise one or more containers in which the target cell cultures and/or culture media may be prepared. Any suitable containers may be used, such as petri dishes.
  • the kit further comprise one or more culture media pre-packaged in the containers.
  • the kit further comprise one or more target cell cultures pre-packaged in the containers.
  • the system may further optionally comprise one or more libraries of compositions comprising an agent for screening.
  • the kit and/or system may further comprise one or more detectors to monitor the status of the cell culture and/or the medium.
  • detectors include a pH meter, a O2 detector, a CO2 detector, a thermometer, and a photometer.
  • the kit and/or system comprises more than one reinforced gas- permeable apparatus in series and/or parallel connection with each other.
  • the kit and/or system comprises one or more reinforced gas-permeable apparatus in gas and/or fluidic connection with one or more gas-impermeable cell culture apparatus.
  • the connection may be in series and/or parallel.
  • such connected reinforced gas-permeable apparatus and/or gas-impermeable apparatus may form a two dimensional and/or three dimensional network, thereby providing possibility for creating a concentration gradient for gas and/or chemicals suitable for biological assay. This can be achieved by exposing reinforced gas- permeable apparatus at different locations of the network to different conditions.
  • a target cell culture e.g. an eukaryotic cell culture, such as stem cells or blood cells
  • the target cell culture may be introduced to the interior of the reinforced gas-permeable apparatus wherein the cells are maintained and cultured.
  • the cells were incubated in the reinforced gas-permeable apparatus provided herein under conditions suitable to propagate cell growth, wherein the thickness and the area of the gas-permeable window allows for gas exchange between the interior and the exterior of the cell culture apparatus.
  • the cells were incubated in the reinforced gas-permeable apparatus provided herein under conditions suitable to sustain cell viability, wherein the thickness and the area of the gas-permeable window allows for gas exchange between the interior and the exterior of the cell culture apparatus.
  • the cells cultured in the reinforced gas-permeable apparatus may be further analyzed. In some variations, the cells are analyzed while in the reinforced gas- permeable apparatus. In some variations, the cells are analyzed after being removed from the reinforced gas-permeable apparatus.
  • the target cell culture may exhibit instant changes detectible by a desired method.
  • the target cell culture may be incubated for about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours, or more than about 120 hours before analysis.
  • some cells are extracted from the apparatus for analysis. In some variations, the cells are analyzed in the apparatus.
  • the results may be analyzed using any known techniques, such as by determining cell count, identifying differentiation status of the cell, detecting color changes in the target cell culture, detecting microscopic changes in the target cell culture, or detecting fluorescent changes in the target cell culture.
  • a reinforced gas-permeable apparatus may be used as blood transfer containers, media transfer containers, and cell culture container in cell manufacture, blood containment and transport, cell containment and transport, cell-based high throughput screening, and stem cell culture.
  • the reinforced gas-permeable apparatus may be disposable, such that it is configured for a single use. Thus, after completion of the desired processes, it may be discarded. In some variations, the reinforced gas-permeable apparatus may be reprocessed, e.g., by washing, rinsing, and/or sterilizing. In other variations, the reinforced gas-permeable apparatus may be reused by re-loading the interior with cell culture media.
  • Example #1 The Production of a Working Model of the Reinforced Gas-permeable Apparatus 400
  • FIG. 4C a working model of the reinforced gas-permeable apparatus 400 was produced and shown in FIG. 4C.
  • FIG. 4B shows a cross-sectional side view of a schematic design of a reinforced gas-permeable apparatus 400.
  • the reinforced gas-permeable apparatus comprises a plurality of gas-permeable windows 401 that allow gas exchange, a supportive layer 404 in contact with a gas-permeable membrane 402, and an interior 405 suitable for containing cell culture media.
  • the structural strength of the 1 mm gas-permeable membrane 402 is mechanically reinforced by sealing together with the 5 mm supportive layer 404.
  • the reinforced gas-permeable membrane 402 maintains its capacity to exchange of CO2 and/or O2.
  • the working model of the reinforced gas- permeable apparatus 400 was then sterilized following a standard autoclave procedure at 121 °C (250°F) and 20 pounds per square inch (psi) for 30 minutes.
  • Example #2 A Comparative Analysis of the Gas Permeability Among Three Types of Devices [0128]
  • working models of the reinforced gas-permeable apparatus 700 were produced and shown in FIG. 7.
  • the reinforced gas-permeable apparatus comprises a plurality of gas-permeable windows that allow gas exchange, a supportive layer in contact with a gas- permeable membrane, and an interior suitable for containing cell culture media.
  • the structural strength of the gas-permeable membrane is reinforced by sealing together with the supportive layer.
  • the reinforced gas-permeable membrane maintains its capacity to exchange of CO2 and/or O 2 .
  • the round cell culture dish 710 serving as a first control was maintained in an ambient environment with 0.5% CO2 throughout the experiment, and media remained purple, indicating minimal gas permeability.
  • the commercially available bag 720 serving as a second control was maintained in an ambient environment with 5% CO2 throughout the experiment, and media showed a color change to orange/pink at the 23-hour mark (FIG. 7H), indicating a normal gas permeability.
  • the three reinforced gas-permeable apparatus 700 showed a color change to orange/pink before the 110-minute mark (FIG. 7E), a color change occurred over a much shorter period compared to the commercially available bag 720, indicating the establishment of an environment with 5% CO2 and thus, significant gas permeability.
  • FIGS. 6C and 6D Two working models of the reinforced gas-permeable apparatus 600 were produced and shown in FIGS. 6C and 6D.
  • FIG. 6B shows a cross-sectional side view of a schematic design of a reinforced gas-permeable apparatus 600.
  • the reinforced gas-permeable apparatus comprises a plurality of gas-permeable windows 601 that allow gas exchange, a supportive layer 606 in contact with a gas-permeable membrane 602, and an interior 605 suitable for containing cell culture media.
  • FIG. 6C shows a reinforced gas-permeable apparatus with circular gas-permeable windows.
  • FIG. 6D shows a reinforced gas-permeable apparatus with square gas-permeable windows on both sides of the pouch.
  • the structural strength of the 1 mm gas-permeable membrane 602 is reinforced by mechanically sealing together with the 5 mm supportive layer 604.
  • the reinforced gas-permeable membrane 602 maintains its capacity to exchange of CO2 and/or O2.
  • the working models of the reinforced gas-permeable apparatus 600 were then sterilized following a standard autoclave procedure at 121 °C (250°F) and 20 pounds per square inch (psi) for 30 minutes.
  • CHO cells Chinese Hamster Ovary (CHO) cells (CHO-K1, ATCC CCL-61) are used in this study. CHO cells are maintained in a monolayer culture in T-75 cm 2 cell culture flasks (Coming Inc., Corning, NY) using Dulbecco's Modified Eagle Medium (DMEM, Gibco, Life Technologies), supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, Life Technologies), and 1% (v/v) penicillin- streptomycin (Pen-Strep, Gibco, Life Technologies).
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • Pen-Strep penicillin- streptomycin
  • CHO cells are subcultured when they reached 70-80% confluence, usually every 2-3 days.
  • CHO cells are washed twice with Phosphate Buffered Saline (PBS) (Gibco, Life Technologies) and then trypsinized with 0.25% trypsin-EDTA solution (Gibco, Life Technologies).
  • PBS Phosphate Buffered Saline
  • trypsinized with 0.25% trypsin-EDTA solution (Gibco, Life Technologies).
  • Post trypsinization fresh DMEM (10% FBS and 1% Pen- Strep) is added to inactivate trypsin and CHO cells are counted using a hemocytometer.
  • Cell count and viability are assessed using the trypan blue dye exclusion method. Briefly, an aliquot of CHO cells is mixed with 0.4% trypan blue solution (Gibco, Life Technologies) in a 1: 1 ratio and counted using a hemocytometer. Viable cells, appearing clear, and nonviable cells, appearing blue, are counted separately.
  • Example #4 The Condition of Dulbecco's Modified Eagle Medium (DMEM) in the Working Model of the Reinforced Gas-permeable Apparatus 400
  • the reinforced gas-permeable apparatus 400 comprises ports
  • the reinforced gas-permeable apparatus comprises a plurality of gas-permeable windows 401 that allow gas exchange, a supportive layer
  • the structural strength of the 1 mm gas-permeable membrane 402 is reinforced by mechanically sealing together with the 5 mm supportive layer 404.
  • the reinforced gas-permeable membrane 402 maintains its capacity to exchange of CO2 and/or O2.
  • the working model of the reinforced gas-permeable apparatus 400 was then sterilized following a standard autoclave procedure at 121°C (250°F) and 20 pounds per square inch (psi) for 30 minutes.
  • the working model of the reinforced gas-permeable apparatus 400 is aseptically filled with 500 mL DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 1% penicillin-streptomycin.
  • FBS fetal bovine serum
  • the three ports 403 on the side of the apparatus are sealed with sterile caps to prevent contamination.
  • the working model is placed on a benchtop rotator, set at 25 revolutions per minute (RPM), with the gas-permeable windows 401 facing up to ensure appropriate aeration.
  • the benchtop rotator is then placed inside a 37 °C incubator, equilibrated with 5% CO2 to ensure appropriate pH and CO2 saturation. After incubation for 2 hours, the 500 mL DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 1% penicillinstreptomycin in the working model is aerated to grow cells.
  • FBS fetal bovine serum
  • penicillinstreptomycin penicillinstreptomycin

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Abstract

L'invention concerne un appareil renforcé de culture cellulaire perméable aux gaz qui permet un échange de gaz sans perturber la culture cellulaire, y compris des cultures cellulaires dans un système de culture complètement fermé. L'invention concerne également des kits/systèmes le comprenant et des procédés d'utilisation de celui-ci.
PCT/US2023/071528 2022-08-03 2023-08-02 Récipient renforcé perméable aux gaz WO2024030965A2 (fr)

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