WO2020090799A1 - 細胞培養装置、および細胞培養方法 - Google Patents

細胞培養装置、および細胞培養方法 Download PDF

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Publication number
WO2020090799A1
WO2020090799A1 PCT/JP2019/042323 JP2019042323W WO2020090799A1 WO 2020090799 A1 WO2020090799 A1 WO 2020090799A1 JP 2019042323 W JP2019042323 W JP 2019042323W WO 2020090799 A1 WO2020090799 A1 WO 2020090799A1
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cell culture
medium
gas
cells
circulation line
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English (en)
French (fr)
Japanese (ja)
Inventor
孝之 江村
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Astec Co Ltd
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Astec Co Ltd
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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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/04—Apparatus for enzymology or microbiology with gas introduction means
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/04—Tissue, human, animal or plant cell, or virus culture apparatus with means providing thin layers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor

Definitions

  • the present invention relates to a cell culture device and a cell culture method.
  • Cell culture bag made of flexible sheet made of organic polymer with gas permeability for mass production of proteins used for antibody drugs and mass production of cells used for evaluation of drugs, cosmetics, foods, etc. Have been developed (for example, Patent Documents 1 and 2).
  • the cell culture bag as described above has been conventionally used for culturing floating cells, but in recent years, it has been cultivated for adhesive cells (also called anchorage-dependent cells) by subjecting the inner surface to hydrophilic treatment or an adhesive coat. Is also used.
  • a cell culture bag is used, other bags and containers, various processing devices (for example, external devices for injecting reagents, culture medium, cells, etc.) via tubes connected to the inlet and outlet, cells after culture Or an external device for collecting waste liquid) or the like to form a closed cell culture system. Since such a cell culture system can perform cell seeding, subculture, medium exchange, cell recovery, etc. without exposure to the outside world, it is a preferable system with a further reduced risk of contamination. ..
  • T-flasks are commonly used.
  • the T-flask is a thick container, which is used to contain a culture medium at the bottom and an air layer at the top.
  • the gas concentration in the culture medium fluctuates greatly as cells grow due to cell culture. Typically, as cells grow, they consume oxygen and reduce the oxygen concentration in the medium.
  • Patent Documents 1 and 2 it is possible to supply gas to some extent by adopting a film having gas permeability as a film forming the bag. Further, in the T-flask, the culture is performed while exchanging gas between the upper air layer and the medium.
  • Patent Document 3 discloses that a cell culture container having a gas permeable portion in a part of the main wall and / or a part or all of the side wall. Is disclosed. Further, in Patent Document 4, a gas permeable portion is provided at one or more portions selected from the two outer plate-shaped portions and the respective side wall portions, and one selected from the two outer plate-shaped portions and the respective side wall portions is provided. The above part discloses a cell culture container provided with an inlet / outlet for a culture solution.
  • the gas concentration gradient was likely to occur because the medium in the cell culture vessel did not flow easily.
  • the gas concentration in the culture medium near the outer periphery of the cell culture container near the gas permeation part is controlled by exchanging gas with the atmospheric gas, but the gas inside the cell culture container is separated from the gas permeation part by cell culture.
  • the effect of gas consumed and produced was large.
  • the dissolved oxygen concentration is high near the outer periphery near the gas permeation part and cell culture can be stably continued, but the dissolved oxygen concentration is low in the inner part far from the gas permeation part and cell growth is stopped or slowed down. There were cases.
  • an object of the present invention is to provide a cell culture device and a cell culture method suitable for controlling the gas concentration of the medium in the cell culture container.
  • the present inventor has found that the following invention meets the above object, and has reached the present invention. That is, the present invention relates to the following inventions.
  • a cell culture container having a hollow inner layer for filling a medium and cells, A circulation line attached to the cell culture vessel for circulating the medium, A liquid feed pump that is provided in the circulation line and feeds the medium to the cell culture container, A cell culture device comprising: a gas exchange unit having a hollow fiber membrane, which is provided in the circulation line and exchanges a gas contained in the culture medium circulated in the circulation line with a regulating gas.
  • a cell culture device comprising: a gas exchange unit having a hollow fiber membrane, which is provided in the circulation line and exchanges a gas contained in the culture medium circulated in the circulation line with a regulating gas.
  • ⁇ 3> The cell according to ⁇ 1> or ⁇ 2>, wherein the adjusting gas is a gas in which at least one selected from the group consisting of oxygen concentration, nitrogen concentration, carbon dioxide concentration, and humidity is adjusted. Incubator.
  • ⁇ 4> The cell culture device according to any one of ⁇ 1> to ⁇ 3>, wherein the hollow fiber membrane is a silicone membrane.
  • ⁇ 5> The cell culture device according to any one of ⁇ 1> to ⁇ 4>, wherein the cell culture container has two plate-shaped portions that are arranged to face each other at a predetermined interval.
  • ⁇ 6> The cell culture device according to any one of ⁇ 1> to ⁇ 5>, in which the cell culture container has stacked plate-shaped portions.
  • a filling step of filling a medium and cells into a cell culture container having a hollow inner layer A culturing step of culturing the cells filled in the cell culture container, A circulation in which the medium in the circulation line is circulated by feeding the medium in the circulation line by a liquid feeding pump provided in a circulation line attached to the cell culture container for circulating the medium.
  • a gas exchange step of performing gas exchange by a gas exchange means having a hollow fiber membrane for performing gas exchange between the gas contained in the medium and the adjusting gas provided in the circulation line and circulated in the circulation line A method for culturing cells.
  • the culture can be performed while controlling the gas concentration of the medium in the cell culture container. Furthermore, according to the present invention, in the state where the medium is filled in the cell culture container, the filled medium can be cultured while exchanging the gas, so that the cell culture container can be cultured without requiring an air layer. Therefore, a place where the adhesive cells can be cultured in a wide range such as the upper and lower surfaces in the cell culture container can be obtained. Furthermore, closed-system culture with limited gas exchange is possible. Therefore, a large amount of cells can be cultured with a relatively small culture device or culture equipment.
  • the cell culture device of the present invention is provided with a cell culture container having a hollow inner layer for filling a medium and cells, a circulation line attached to the cell culture container for circulating the medium, and provided in the circulation line.
  • a gas exchange means having a hollow fiber membrane.
  • the cell culture method of the present invention comprises a filling step of filling a medium and cells into a cell culture container having a hollow inner layer, a culture step of culturing the cells filled in the cell culture container, and a cell culture container A circulating step of circulating the medium in the circulation line by feeding the medium in the circulation line by a liquid feed pump provided in a circulation line for circulating the attached medium; A gas exchange step of performing gas exchange by a gas exchange means provided in a line and having a hollow fiber membrane for performing a gas exchange between the gas contained in the culture medium and the adjusting gas circulated in the circulation line.
  • the cell culture method of the present invention By culturing cells by the cell culturing method of the present invention, it is possible to perform culturing while controlling the gas concentration of the medium in the cell culture container. Further, the cell culture method of the present invention is suitable for culture in which an air layer does not occur in the cell culture container, is a closed system, and is suitable for large-scale cell culture. In the present application, the cell culture method of the present invention can be carried out by the cell culture device of the present invention, and configurations corresponding to each other in the present application can be mutually utilized.
  • FIG. 1 is a schematic diagram showing a first embodiment of the cell culture device of the present invention.
  • FIG. 1 shows a cell culture device 10.
  • the cell culture device 10 includes a cell culture container 21, circulation lines 311, 312, 313, a liquid feed pump 41, and a gas exchange means 51.
  • the cell culture container 21 has openings 211 and 212, the opening 211 is connected to the circulation line 311, and the opening 212 is connected to the circulation line 313.
  • the circulation line 311 is connected to the opening 511 of the gas exchange means 51.
  • the opening 512 of the gas exchange means is connected to the circulation line 312.
  • the circulation line 312 is connected to the liquid feed pump 41, and the liquid feed pump 41 is also connected to the circulation line 313.
  • the circulation lines 311, 312, 313 are tubes that can transfer the medium in the cell culture vessel 21.
  • the liquid feed pump 41 can feed the medium, and may feed the medium in the F11 direction from the circulation line 312 side to the circulation line 313 side, or may feed the medium in the opposite F12 direction.
  • the culture medium in the circulation lines 311, 312, 313, the cell culture vessel 21 appropriately connected to these, or the gas exchange means 51 is sequentially transferred, and the medium is in these systems.
  • the gas exchange means 51 has openings 513 and 514 through which the culture medium does not leak, in addition to the portions where the culture medium circulates, such as the openings 511 and 512. Gas can pass through the openings 513 and 514.
  • the medium introduced / derived from the openings 511, 512 exchanges gas with the gas introduced / derived from the openings 513, 514 to adjust the gas concentration in the medium. Since this medium circulates in the system as described above, it is also transferred to the cell culture vessel 21 in a state where the gas concentration is adjusted in the gas exchange means 51. As a result, the cells cultured in the cell culture vessel 21 are cultured in the medium whose gas concentration is adjusted. Further, the cell culture device 10 can operate a portion of the cell culture container 21 or the like in which the medium can exchange gas as a closed system only inside the substantial gas exchange means 51, and is introduced / extracted from the opening 513 and the opening 514. The gas exchange of the culture medium can be controlled only by controlling the humidity and the gas concentration of the gas to be used.
  • the cell culture vessel 21 has a hollow inner layer that is filled with a medium and cells.
  • 2 to 4 are views for explaining the detailed structure of the cell culture container 21.
  • FIG. 2 is a plan view of the cell culture container 21.
  • 3 (a) is a right side view of the cell culture container 21, and
  • FIG. 3 (b) is a front view.
  • the cell culture container 21 can be made into a substantially rectangular parallelepiped shape. When viewed in a plan view, the shape may be a square shape or a rectangular shape, and may be any size capable of culturing cells. For example, the size may be about 20 mm to 500 mm on the vertical side and about 20 mm to 500 mm on the horizontal side. The area may be about 400 mm 2 to 2.5 ⁇ 10 5 mm 2 .
  • the cell culture container is not limited to a substantially rectangular parallelepiped shape, and may have any other shape such as a columnar shape or a polygonal pillar shape that allows cell culture therein.
  • the cell culture container 21 has openings 211 and 212.
  • the openings 211 and 212 are provided on a diagonal line and are arranged so that replacement can be performed more efficiently when the medium in the cell culture container 21 is circulated. Although two openings are shown here, three or more openings may be provided, and it may be used by connecting the circulation lines of the medium or by replacing other components. Good. Further, it is preferable that the opening not used during the culture is closed by providing a lid. It is preferable that the cell culture container 21 has a high hermeticity so that the liquid does not leak from other than the opening.
  • various materials that can be used for the cell culture container can be used.
  • resin, glass, metal or the like can be used. It is preferable to use a resin such as polystyrene, polycarbonate, or polyethylene terephthalate, which makes it easy to observe the culture condition from the outside and has a low necessity for conducting a material evaluation test.
  • the shape of the cell culture vessel 21 is preferably one that does not easily deform.
  • a plate material having a sufficient thickness of, for example, 1 mm or more or 1.5 mm or more using the above-mentioned materials, it may be used as a cell culture container or not deformed by an external force when handling it. it can. When such a material is hard to be deformed, the cells can be stably cultured and easily proliferate and grow in a state where the cells are adhered to the inner wall of the cell culture container 21.
  • FIG. 4 is a sectional view of the cell culture container 21 taken along the line AA (see FIG. 3A).
  • the cell culture container 21 has a hollow inner layer between the upper and lower plates 213 and the side walls 214, and serves as a place for cell culture.
  • the hollow inner layer is opened to the outside by the hollow portion 2111.
  • the opening 212 is opened.
  • the cells 216 adhere to the inner surfaces 2131 of the upper and lower plates 213 on the flat plate, and the medium 215 is circulated inside thereof.
  • the medium 215 is a fluid medium, and is a liquid medium or a jelly-like medium.
  • the inner surface 2131 which is a place where cells adhere, is appropriately subjected to surface treatment such as flame treatment (frame treatment), corona treatment, plasma treatment, VUV (vacuum ultraviolet light) according to the type of cells to be cultured and adhered. It is preferable to make it easy to do.
  • surface treatment such as flame treatment (frame treatment), corona treatment, plasma treatment, VUV (vacuum ultraviolet light) according to the type of cells to be cultured and adhered. It is preferable to make it easy to do.
  • the cell culture container 21 has two plate-shaped portions (upper and lower plates 213) arranged so as to face each other at a predetermined interval.
  • the two plate-shaped portions are closed so as to hermetically seal the periphery except for the portion where the opening is provided. Since the openings 211 and 212 are connected to the circulation line, the inner layer of the cell culture container 21 becomes a sealed space.
  • the inner layer is filled with a medium and cells in a container, and further, the medium is circulated by a circulation line during culturing, so that the medium and the cells can be used in a high filling rate.
  • the filling rate of the medium and cells in the cell culture vessel 21 (total amount of medium and cells / volume of inner layer) when performing cell culture is 80% by volume or more, 90% by volume or more, 95% by volume or more, 99% by volume or more. can do. That is, it is possible to perform the culture substantially without containing the air layer in the cell culture container 21. As a result, the respective surfaces (upper and lower surfaces) on the inner layer side of the two plate-shaped portions serve as a place for cells to adhere, and a large amount of cells can be efficiently cultured.
  • the thickness d1 of the inner layer of the cell culture vessel 21 can be set to an arbitrary thickness at which cells can be cultured.
  • the thickness d1 corresponds to a predetermined distance between the two plate-shaped portions arranged so as to face each other.
  • the thickness d1 is preferably, for example, about 2 mm to 20 mm.
  • the lower limit of the thickness may be 3 mm or more or 4 mm or more.
  • adherent cells that have undergone culturing may have a thickness of about 2 mm, and may be thickened so that they can be easily cultured on the upper and lower surfaces of the cell culture container.
  • the upper limit of the thickness may be 15 mm or less, 12 mm or less, and 10 mm or less because the cells adhere to the upper and lower surfaces even if the thickness is thick and the amount of the medium for circulation may be excessive. With such a thickness, even if the medium 215 circulated in the cell culture vessel 21 is relatively small, cells are easily adhered to and cultivated above and below the inner layer.
  • the cell culture vessel 21 may be laminated. Since the cell culture container 21 has a thin rectangular parallelepiped shape, the cell culture container 21 can be used by being arranged so as to be stacked above and below it. A pipe for connecting the openings in parallel may be provided to connect with the circulation line, and circulation and gas exchange may be performed by one liquid feed pump or gas exchange means. Since the cell culture container can be a thin container, it is possible to perform high-density culture with a small volume as a whole even if they are stacked. As such a cell culture container 21, with reference to the cell culture containers disclosed in JP-A-2018-33387 and JP-A-2018-143210, those equivalent thereto can be used.
  • the cell culture device 10 is attached to the cell culture container 21 and has circulation lines 311, 312, and 313 for circulating the medium in the cell culture container.
  • a resin tube or the like can be used for the circulation lines 311 to 313.
  • the cell culture device 10 has a liquid feed pump 41 provided between the circulation lines 312 and 313 for feeding the medium to the cell culture vessel 21.
  • the liquid feed pump 41 any pump that can feed the medium can be used.
  • the size of a normal cell culture container 21 for culturing with the cell culture device of the present invention depends on the inner volume of the cell culture device 10, particularly the cell culture container 21, the type of cells to be cultured, the amount of culture, and the like. On the other hand, it is possible to use a material capable of sending a liquid of about 0.01 mL / min to 100 mL / min.
  • the liquid may be continuously fed during the culture or may be intermittently fed.
  • the amount of liquid fed is reduced to suppress the amount of medium to be gas-exchanged by the gas exchange means 51, and in the latter stage of culture, the amount of liquid fed is increased and the amount of medium to be gas exchanged by the gas exchange means 51. It is preferable that the amount of liquid to be sent can be appropriately changed or that the device can be programmed and operated so that the amount can be increased.
  • the liquid feed direction of the liquid feed pump 41 may be the F11 direction which is the suction direction from the gas exchange means 51 or the F12 direction which is the feed direction from the gas exchange means 51. When the medium is difficult to be delivered from the gas exchange means 51, it may be positively delivered as the F12 direction to be delivered to the gas exchange means 51 side.
  • the F11 direction which is the suction direction from the gas exchanging means 51, may be set so that a negative pressure for sucking the adjustment gas introduced from is easily generated.
  • the liquid supply pump may be one, but a plurality of liquid supply pumps may be appropriately provided in consideration of the liquid supply amount, gas exchange efficiency, and the like.
  • the cell culture device 10 has a gas exchange means 51 provided in the circulation lines 311, 312 for exchanging the gas contained in the circulated culture medium with the adjustment gas.
  • the gas exchanging means 51 has a hollow fiber membrane, and the medium or the adjusting gas is passed through the inside and the outside of the hollow fiber membrane to exchange gas between the medium and the adjusting gas through the hollow fiber membrane. ..
  • This gas exchange means is, for example, a hollow fiber membrane cartridge (so-called hollow fiber cartridge) in which both ends of a hollow fiber bundle having a plurality of hollow fiber membranes are fixed in a hollow member while holding the hollow portions in the open state. Can be used.
  • the medium circulated by the circulation line is circulated in one of the inner layer and the outer layer of the hollow fiber membrane, and the adjusting gas is circulated in the other side, and gas exchange between the medium and the adjusting gas is performed through the hollow fiber membrane.
  • the openings 511, 512 of the gas exchange means 51 are communicated with the flow path of the fluid flowing in the outer layer of the hollow fiber membrane, and the openings 513, 514 of the fluid flowing in the inner layer of the hollow fiber membrane. It is designed to communicate with the flow path. Therefore, the medium flows through the outer layer of the hollow fiber membrane, and the adjusting gas flows through the inner layer.
  • FIG. 5 shows a modification of the first embodiment of the cell culture device.
  • the cell culture device 101 is a device in which the connection state of the circulation line to the gas exchange means 51 is changed, the circulation line 311 is connected to the opening 513, and the circulation line 312 is connected to the opening 514. In this way, the inside or outside of the hollow fiber membrane through which the medium or the preparation gas is passed can be appropriately selected and cultured.
  • the hollow fiber membrane has a very low or no liquid permeability, and it is preferable that only the gas is selectively permeated.
  • a material having such gas exchangeability for example, a hollow fiber membrane made of silicone can be used.
  • FIG. 6 is a diagram showing a second embodiment of the cell culture device of the present invention.
  • the cell culture device 11 has, in addition to the first cell culture device 10, a medium transfer line capable of changing the medium and the like.
  • a three-way joint or the like is provided in the circulation line 311 to connect the pipe 611.
  • the circulation line 313 is also provided with a three-way joint or the like to connect the pipe 612.
  • the circulation line 311 is provided with an opening / closing valve 321, the pipe 611 is provided with an opening / closing valve 621, the circulation line 313 is provided with an opening / closing valve 322, and the pipe 612 is provided with an opening / closing valve 622.
  • the circulation through the gas exchange means 51 can be stopped and the cell culture vessel 21 can be connected to the outside of the system via the pipe 611 and the pipe 612.
  • the medium or the like can be circulated in the F211 direction and the F222 direction, or the medium or the like can be circulated in the F212 direction and the F221 direction by using an external liquid feed pump or the like.
  • the medium is replaced during the culture at the time of starting operation, when the cell culture container 21 is filled with the medium or the like, or according to the type of cells or the culture time according to the purpose of the experiment. At times, it can also be used when the culture is completed and the cells are collected.
  • FIG. 7 is a diagram showing a third embodiment of the cell culture device of the present invention.
  • the cell culture device 12 has a gas pump 71 and a filter 72 in addition to the second cell culture device 11.
  • the openings 513 and 514 of the gas exchange means 51 may be simply opened so that air or the like in the atmosphere can circulate, and oxygen or nitrogen in the air can be used. It is also possible to perform gas exchange between the medium and the medium that is circulated in the gas exchange means 51, and adjust the gas concentration to correspond to the oxygen concentration in the air.
  • the cell culture device 12 is configured to more positively control the amount of gas exchange and the like by using the gas pump 71 and the filter 72.
  • the gas pump 71 may increase the flow rate of the air in the atmosphere to improve the gas exchange amount, or the gas delivered by the gas pump 71 may have the gas concentration adjusted.
  • the gas exchange means 51 performs gas exchange between the adjusting gas and the medium.
  • This adjusting gas is for adjusting the gas concentration of the medium.
  • the conditioning gas may be air to supply the oxygen consumed in the medium.
  • the gas pump 71 as described above may be used to supply the gas in the tank or the like in which the adjusting gas is stored.
  • the gas that is introduced into and discharged from the gas exchange means via the filter 72 is appropriately used. It can also be adjusted.
  • the medium substantially contained in the cell culture container or the like is in contact with the outside air only in the gas exchange means, and therefore the gas in the gas exchange means is It is excellent in that the culture system can be managed simply by controlling and controlling the culture.
  • FIG. 8 shows another embodiment of the cell culture container used in the cell culture device of the present invention.
  • the cell culture container has laminated plate-shaped portions.
  • Each of the cell culture vessels 21 has a plate-shaped portion, and the upper and lower surfaces in the vessel serve as adhesion surfaces to which cells adhere during cell culture.
  • Such a cell culture vessel 21 may be used by stacking a plurality of layers. In FIG. 8, 6 layers are laminated.
  • the openings 211 and 212 of each cell culture vessel 21 can be connected at the opening 211 side by a manifold 217.
  • the manifold 217 has a branch on the side of the opening 211 of the cell culture vessel 21, and the side of the circulation line 311 is integrated and connected at one place of the connecting portion 2171. Similarly, the opening 212 side can be connected to the circulation line 313 via the manifold 218.
  • the number of hollow inner layers of the cell culture container to be stacked is not particularly limited and may be, for example, 2 or more, 3 or more, 5 or more, and 20 or less, 15 or less, 12 or less. be able to. At this time, it may be one that can be separated for each part constituting each layer, or may be common so that one plate serves as an upper and lower partition.
  • the present invention relates to a culture method using such a cell culture device.
  • the culturing method of the present invention comprises, as described above in part, a filling step of filling a medium and cells into a cell culture container having a hollow inner layer, and a culturing step of culturing the cells filled in the cell culture container.
  • Circulating the medium in the circulation line by feeding the medium in the circulation line with a liquid feed pump provided in a circulation line attached to the cell culture container for circulating the medium.
  • Gas for gas exchange by a circulation step and a gas exchange means having a hollow fiber membrane for gas exchange between the gas contained in the medium and the adjustment gas provided in the circulation line and circulated in the circulation line And a replacement process.
  • the culturing method of the present invention has a filling step of filling a cell culture container having a hollow inner layer with a medium and cells.
  • the cell culture container and the air layer such as the circulation line are filled with the cell suspension and the medium.
  • the cells to be cultured and the medium are filled in the cell culture container.
  • the cell suspension may be made into a cell suspension by first filling the medium with cells and then seeding the cells, or in a state where the cells are previously seeded into the medium to give a cell suspension, and then placed in a cell culture container or the like. It may be filled. Further, in order to remove the air layer from the cell culture container or the like, it may be appropriately deaerated and filled, or an exhaust passage may be provided to remove the air layer.
  • the culturing method of the present invention has a culturing step of culturing cells filled in a cell culture container.
  • the cells need not be cultivated in the state of cell suspension, but are cultivated in the state of being adhered to the cell culture container.
  • the cells to be cultured may not be particularly limited. Since it is preferable that the cells are adhered and cultured in the cell culture container, cells having adhesiveness and the like are suitable culture targets. Examples thereof include ES cells, iPS cells, stem cells, fibroblasts, CHO (Chinese Hamster Ovary) cells, HEK 293 (Human Embryonic Kidney cells 293) cells, and adipocytes.
  • the medium used for cell culture of the present invention is preferably a medium having high fluidity such as a liquid medium for circulation.
  • the specific composition of the medium may be appropriately prepared according to the subject of culture and the purpose of culture.
  • DMEM medium Dulbecco's modified Eagle medium
  • MEM medium Minimum Essential. Medium
  • StemFit registered trademark
  • a cell suspension or the like is prepared and a medium and cells are filled in a cell culture container. At this time, it is preferable to stop the circulation of the medium and culture until the cells to be cultured adhere to the inner wall of the cell culture container.
  • the amount of cells to be seeded is appropriately set in consideration of the type of cells, the compatibility with the medium, the culture period, and the like.
  • the temperature at which the cells are cultured can be controlled by the optimum temperature of the cells and the experimental conditions. Since the medium in which the cell culture is in contact with the outside air is limited to the gas exchange means, there is little need to control the conditions of the gas such as humidity, and management may be limited to the atmosphere of the gas exchange means.
  • the cell culture method of the present invention circulates the medium in the circulation line by feeding the medium in the circulation line with a liquid feed pump provided in the circulation line for circulating the medium attached to the cell culture vessel. It has a circulation process to. This circulation step is carried out in parallel with the culture step while appropriately or continuously circulating the cells after the cells are adhered and stabilized in the cell culture container.
  • the cell culture method of the present invention comprises a gas exchange means having a hollow fiber membrane for performing gas exchange between a gas contained in the medium and a regulating gas provided in the circulation line and circulated in the circulation line. And a gas exchange step of exchanging gas.
  • This gas exchange is a gas exchange in a limited place, and it is easy to manage the culture conditions.
  • the contact area between the culture medium and the adjusting gas can be widened according to the surface area of the hollow fiber membrane, so that controlling the adjusting gas allows gas exchange with excellent responsiveness. It is also possible to control the gas concentration of the medium.
  • the medium since the medium is circulated, it is cultured while being continuously supplied to the cell culture container. Therefore, even if air is temporarily contained in the cell culture container, the air can be cultured so as to be pushed out of the container.
  • the extruded air can be supplemented by the hollow fiber membrane of the gas exchanging means, or can be cultured while exchanging the gas and being exhaled to the adjusting gas side. Therefore, the culture can be performed in the system such as the cell culture container without the air layer substantially.
  • the cell culture device and cell culture method of the present invention are suitable for controlling the gas concentration of the medium in the cell culture vessel. Furthermore, by appropriately adjusting the culture conditions and the configuration of the culture device, it becomes possible to perform culture with various advantages. For example, it is possible to perform a culture with a low degree of freedom in the surrounding environment or a device by limiting the place where humidity is controlled. Also, the medium exchange during the culture can be easily performed. It is also possible to perform a closed system culture with reduced contamination. Further, it is suitable for downsizing, and it is possible to carry out mass culture in a space-saving manner. Furthermore, it is suitable for automation because it enables stable large-scale culture in a closed system.
  • Cell culture device A polystyrene plate having a plate thickness of about 3 mm was used as the upper and lower plates, and used as a thin rectangular cell culture container having a width of 260 mm, a length of 210 mm, and an upper and lower plate interval of 8 mm.
  • the inner surface of the plate used for the lower side was treated with plasma to improve the adhesiveness.
  • a gas permeable silicone film was arranged in four directions, and polystyrene columns having high rigidity were provided in the four corners to maintain the height (see JP-A-2018-33387). Further, two through holes were provided in the upper plate near both ends in the width direction to serve as a connecting portion for the circulation line tube.
  • -Circulation line A circulation line was provided using a silicone tube.
  • -Liquid-feeding pump A liquid-feeding pump (manufactured by Welco) capable of feeding up to a liquid-feeding amount of about 20 mL / min was connected to the tube of the circulation line to feed the liquid.
  • the solution was fed at about 1 to 5 mL / min, and at the latter stage of culture, the solution was fed at 5 to 10 mL / min while observing the culture state.
  • a silicone hollow fiber gas separation membrane containing a hollow fiber membrane made of silicone (“M60 (number of membranes: 4500)" manufactured by Nagayanagi Industry Co., Ltd.) was used as a gas exchange means.
  • Example 1 With the configuration according to the first embodiment, a cell line was cultivated by attaching a circulation line, a liquid feed pump, and a silicone hollow fiber gas separation membrane column (gas exchange means) to the cell culture container.
  • the culture temperature was 37 ° C., and the culture was performed in a CO 2 incubator with a CO 2 concentration of 5%.
  • Example 1 Since the cell culture vessels used in Example 1 and Comparative Example 1 have a gas exchange section made of silicone on the side wall, from the start of the culture to the third day, both cultures had a high dissolved oxygen concentration in both the center and the end. Maintained culture was possible. However, when the oxygen consumption increased with the progress of culturing and the increase in the number of cells, it was considered that the gas-exchanged oxygen could not reach the center in Comparative Example 1, and the dissolved oxygen concentration in the center decreased. On the other hand, in Example 1, since the medium was circulated while exchanging gas, the state where the dissolved oxygen concentration was high was maintained in the center as well. The number of cells on day 7 of culture was significantly higher in Example 1. From the microscopic observation image, it is confirmed that the cell density of Example 1 is higher.
  • the cell culture device and cell culture method of the present invention can be used for cell culture of various cells, can perform culture without requiring an air layer in the cell culture container, and can be used for large-scale cell culture in a closed system. Suitable and industrially useful.

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