WO2016140213A1 - Procédé de culture cellulaire mettant en œuvre un module à fibres creuses - Google Patents

Procédé de culture cellulaire mettant en œuvre un module à fibres creuses Download PDF

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Publication number
WO2016140213A1
WO2016140213A1 PCT/JP2016/056218 JP2016056218W WO2016140213A1 WO 2016140213 A1 WO2016140213 A1 WO 2016140213A1 JP 2016056218 W JP2016056218 W JP 2016056218W WO 2016140213 A1 WO2016140213 A1 WO 2016140213A1
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Prior art keywords
cell culture
hollow fiber
cells
cell
container
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PCT/JP2016/056218
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English (en)
Japanese (ja)
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真希子 平岡
達哉 山口
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東洋紡株式会社
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Priority to JP2017503658A priority Critical patent/JPWO2016140213A1/ja
Publication of WO2016140213A1 publication Critical patent/WO2016140213A1/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
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor

Definitions

  • the present invention relates to a cell culture method using a hollow fiber module for culturing cells.
  • Stem cells are cells that can form organs and tissues, and are considered to exist in most organs and tissues even in adults.
  • stem cells embryonic cells (ES cells) are all-purpose cells and have the ability to differentiate into all tissues and organs.
  • somatic stem cells cannot differentiate into all organs and tissues, but differentiate into specific tissues and organs. Somatic stem cells that can be collected from human tissues can be collected from patients themselves and have no interest in rejection.
  • somatic stem cells in humans are known to be mesenchymal stem cells, hematopoietic stem cells, neural stem cells, cardiac muscle stem cells, pancreatic stem cells, skin stem cells, bone marrow stem cells, retinal stem cells, corneal endothelial stem cells, and the like.
  • mesenchymal stem cells hematopoietic stem cells
  • neural stem cells hematopoietic stem cells
  • cardiac muscle stem cells pancreatic stem cells
  • skin stem cells skin stem cells
  • bone marrow stem cells retinal stem cells
  • corneal endothelial stem cells corneal endothelial stem cells
  • Regenerative medicine has been developed in which somatic stem cells cultured outside the body are used for the treatment of the same person or others, and some have begun to be put into practical use. In such regenerative medicine, it is necessary to increase a small amount of cells collected from the human body to the number of cells necessary for transplantation outside the body. In addition, when culturing outside the body, it is important to prevent contamination of cultured cells.
  • Non-patent Document 1 Culture of cells used for regenerative medicine is currently performed in accordance with GMP (Good Manufacturing Practice) in a cell culture clean room called a cell processing center (CPC) (Non-patent Document 1). ).
  • the problems in this case are that stem cells are cultivated by an engineer, and that labor and cost are very high for the preparation of cells for one patient, and biology by manual operation. There is a risk of global contamination.
  • this invention consists of the following structures. 1.
  • the following [1] to [4] configuration [1] A module using a porous membrane hollow fiber, two openings connected to the inner space of the hollow fiber, and two openings connected to a space formed inside the module and outside the hollow fiber
  • Cell culture container [2] having at least one liquid permeable liquid medium storage container [3] channel network connecting the cell culture container and the liquid medium storage container [4] provided in the channel network
  • a method of culturing using a cell culturing apparatus comprising means for controlling the supply of liquid to the cell culture container and / or the discharge of liquid from the cell culture container, wherein the inside of the hollow fiber of the cell culture container
  • the linear velocity of the liquid medium flowing through the space is controlled to 0.001 mm / min to 5 mm / min
  • the linear velocity of the liquid medium flowing through the hollow fiber outer space is controlled to 0.001 mm / min to 5 mm / min, respectively.
  • Cell culture method characterized by. 2. 2. The cell culture method according to 1, wherein the cell culture device further includes the following configuration [5]. [5] CO 2 incubator that covers at least a part of [2] and / or [3] 3. The cell culture method according to 1 or 2, wherein a gas permeable channel tube is used for the channel network. 4). 4. The cell culture method according to any one of 1 to 3, wherein in the cell culture apparatus, the flow of the medium from the liquid medium storage container to the cell culture container is unidirectional. 5. 5. The cell culture method according to any one of 1 to 4, wherein the cell culture container is detachable. 6). 6. The cell culture method according to any one of 1 to 5, wherein the hollow fiber is coated with a cell adhesive protein.
  • the module using the porous membrane hollow fiber used in the cell culture method of the present invention is configured to allow a liquid medium to flow in the space inside the hollow fiber and the space outside the hollow fiber, respectively.
  • various cells such as mesenchymal stem cells that are present in a very small number in a diverse cell population can be cultured in a safe, simple and highly efficient manner compared to conventional methods. Make it possible.
  • the structural example of the hollow fiber module used for the cell culture apparatus of this invention is shown.
  • the SEM image which observed the surface form of the hollow fiber made from CTA used for the automatic cell culture apparatus which concerns on one Embodiment of this invention is shown.
  • 1 is a hollow fiber sectional view observation result.
  • 2 is the observation result of the surface form inside the hollow fiber.
  • 3 is the observation result of the surface form of the outside of the hollow fiber.
  • the structure of the cell culture apparatus used in Example 1 and Comparative Example 2 of the present invention is shown in a simplified manner.
  • the observation result of the cell cultured on the hollow fiber inner surface used for the automatic cell culture device concerning one embodiment of the present invention is shown.
  • Example 1 The structure of the cell culture apparatus used in the comparative example 1 is simplified and shown.
  • One embodiment of the present invention includes the following [1] to [4]: [1] A module using a porous membrane hollow fiber, two openings connected to the inner space of the hollow fiber, and two openings connected to a space formed inside the module and outside the hollow fiber
  • Cell culture container [2] having at least one liquid permeable liquid medium storage container [3] channel network connecting the cell culture container and the liquid medium storage container [4] provided in the channel network
  • a method of culturing using a cell culturing apparatus comprising means for controlling the supply of liquid to the cell culture container and / or the discharge of liquid from the cell culture container, wherein the inside of the hollow fiber of the cell culture container
  • the linear velocity of the liquid medium flowing through the space is controlled to 0.001 mm / min to 5 mm / min
  • the linear velocity of the liquid medium flowing through the hollow fiber outer space is controlled to 0.001 mm / min to 5 mm / min, respectively. It is a cell culture method, characterized by.
  • the overall size of the cell culture apparatus used in the cell culture method of the present invention is not particularly limited, but is preferably a small one that can be installed in a CO 2 incubator. Since the culture apparatus is small, a plurality of culture apparatuses can be installed in the CO 2 incubator at the same time.
  • the weight of the entire culture apparatus is not particularly limited, but is preferably light enough to be installed in a CO 2 incubator.
  • the cell culture container used for the cell culture method of the present invention is a module using a porous membrane hollow fiber, and has two openings connected to the inner space (lumen) of the hollow fiber, and an outer space (outer lumen). If it has two opening parts connected to), it will not specifically limit.
  • a hollow fiber having a large specific surface area porous membrane hollow fiber
  • the module can be miniaturized, and a small culture apparatus that can be installed in a commercially available CO 2 incubator can be realized.
  • the hollow fiber module used for the cell culture apparatus of this invention can also be made removable.
  • the structure of a module using such a porous membrane hollow fiber is not particularly limited.
  • a hollow fiber module is not particularly limited.
  • the module case 31 having a shape in which a necessary number of porous membrane hollow fibers 32 are bundled and filled is included.
  • the two end conduits 33 are connected to each other by a suitable sealing material (for example, a polyurethane potting agent) so as not to block the hollow portion of the hollow fiber at both ends of the hollow fiber bundle.
  • a liquid or the like introduced from one of the conduits 33 is configured to be led out from one end conduit 33 (that is, to flow in one direction) through the hollow fiber lumen.
  • the other two side conduits 34 are a space inside the module case 31 and an outer space of the hollow fiber (hereinafter, also simply referred to as “external space side”). Connected and configured such that liquid or the like introduced from one of the side conduits 34 is led out from one side conduit 34 that passes through the outer lumen side of the module 17 (ie, flows in one direction). Has been.
  • the size of the hollow fiber module as a cell culture container used in the cell culture method of the present invention can be selected according to the required number of cells. In general, it is preferable to use a larger cell culture container as the required number of cells increases. In addition, when the difference between the amount of cells held and the required amount is large, it may be scaled up as appropriate. For example, when culturing cells on the hollow fiber lumen side, the number of cells is proportional to the membrane area based on the inner diameter of the hollow fiber, so if the initial number of cells is small, such as somatic stem cells, the initial culture is small. A large amount of somatic stem cells can be obtained in the latter half of the culture by scaling up the module step by step using the module.
  • the material of the porous membrane hollow fiber used as the culture substrate of the cell culture vessel is not particularly limited as long as it can take a structure that allows a solution or a low-molecular substance to permeate.
  • Cellulose materials such as cellulose acetate, cellulose triacetate (CTA), regenerated cellulose, polyethylene, polypropylene, polysulfone, polystyrene, polyvinyl alcohol, polymethyl methacrylate, polyacrylonitrile, fluorine resin, polyamide, polyethersulfone, polyvinylidene fluoride (
  • An insoluble carrier having a skeleton structure of a thermoplastic polymer such as PVDF can be preferably used. These derivatives may be the main component.
  • the porous membrane hollow fiber used in the present invention may be one obtained by chemically modifying these materials.
  • the hollow fiber to be used is preferably subjected to a hydrophilic treatment.
  • a hydrophilic treatment By hydrophilizing the hollow fiber, it becomes easy to supply a liquid component such as a culture solution to the cultured cells.
  • the method for hydrophilizing the hollow fiber include a method of treating the hollow fiber with a hydrophilic polymer such as ethylene-vinyl alcohol copolymer, glycerin, and ethanol.
  • proteins such as collagen, laminin, fibronectin, vitronectin and the like may be coated to improve adhesion to the hollow fiber.
  • the size of the porous membrane hollow fiber used in the present invention is not particularly limited, but the inner diameter is preferably 20 to 1000 ⁇ m, more preferably about 50 to 500 ⁇ m.
  • the film thickness is not particularly limited, but the hollow fiber may have an appropriate strength and the permeability of the substance may be set within a favorable range, and is preferably about 10 to 200 ⁇ m, for example.
  • the pore diameter of the porous membrane hollow fiber used in the present invention is not limited as long as it has a through-hole that allows cells to pass through but does not allow cells to pass through but allows culture medium components such as water, salts, and proteins to pass through.
  • it is desirable to have a large pore size with good material exchange efficiency. For example, it is preferably about 0.001 to 5 ⁇ m, and if it has a through hole of about 0.002 to 3 ⁇ m, preferable.
  • the hollow fiber module used in the cell culture method of the present invention can be produced, for example, by storing tens to tens of thousands of hollow fiber membranes in a cylindrical container.
  • the cell culture is preferably performed on the lumen side.
  • seeding is performed by injecting a cell suspension from the end conduit, and after seeding, the cell suspension is switched to the medium and perfused. Incubate for a certain period of time. During this time, it is preferable that the culture medium is also injected into the outer space side from the side conduit and perfused.
  • the liquid medium has a role of supplying nutrients necessary for cells and gases such as oxygen and carbon dioxide.
  • the hollow fiber module is sterilized by an appropriate method and supplied.
  • the sterilization method is not particularly limited, and examples thereof include autoclaving sterilization, gamma ray sterilization, and ethylene oxide gas sterilization.
  • the material used for the liquid culture medium storage container used in the cell culture method of the present invention is not particularly limited, but industrially has excellent moldability, can withstand gamma sterilization, and observes the state of the internal culture medium. It is preferable that the material be transparent.
  • the gas permeability of the liquid medium storage container is such that the oxygen permeability at 37 ° C. is 200 ⁇ 10 ⁇ 10 cm 3 / (cm 2 ⁇ sec ⁇ cmHg) or more, and the carbon dioxide permeability is 1200 ⁇ 10 ⁇ 10. It is preferably cm 3 / (cm 2 ⁇ sec ⁇ cmHg) or more.
  • Such materials include low density polyethylene (oxygen permeability coefficient: 0.4 ⁇ 10 ⁇ 10 to 6 ⁇ 10 ⁇ 10 cm 3 (STP) ⁇ cm / (cm 2 ⁇ sec ⁇ cmHg), carbon dioxide permeability coefficient: 2 ⁇ 10 ⁇ 10 to 30 ⁇ 10 ⁇ 10 cm 3 (STP) ⁇ cm / (cm 2 ⁇ sec ⁇ cmHg)), polyurethane (oxygen transmission coefficient: 1 ⁇ 10 ⁇ 10 to 5 ⁇ 10 ⁇ 10 cm 3 (STP ) ⁇ Cm / (cm 2 ⁇ sec ⁇ cmHg), carbon dioxide permeability coefficient: 5 ⁇ 10 ⁇ 10 to 40 ⁇ 10 ⁇ 10 cm 3 (STP) ⁇ cm / (cm 2 ⁇ sec ⁇ cmHg)), polypropylene (oxygen) Permeability coefficient: 2 ⁇ 10 ⁇ 10 to 50 ⁇ 10 ⁇ 10 cm 3 (STP) ⁇ cm / (cm 2 ⁇ sec ⁇ cmHg), carbon dioxide
  • the culture medium storage container can be prepared by adjusting the size and thickness so that the oxygen permeability and the carbon dioxide permeability become the desired values, but both strength and gas permeability are compatible.
  • the thickness of the container is preferably 100 to 1000 ⁇ m, more preferably 100 to 500 ⁇ m.
  • the form of the liquid medium storage container used in the present invention is not particularly limited, but it is necessary to have at least one opening that can supply the liquid medium to the outside. It is preferable that the opening is configured to be connectable to a flow path to be described later so that the culture medium can be supplied to the hollow fiber module. In addition, the opening can be sealed when not in use and connected to the flow path when the medium is not in contact with the outside so as to prevent evaporation of the liquid medium and reduce the risk of contamination. It is preferable to be configured to be capable of (for example, interconnecting with a sterile connector). Moreover, it is preferable that parts other than the said opening part are comprised so that the culture medium in a container cannot contact the exterior. Examples of such liquid medium storage containers include commercially available culture bags (available from Nipro, Toyo Seikan, etc.), plastic bottles, and the like.
  • the configuration is not particularly limited, but includes at least a connection portion between the hollow fiber module and the liquid medium storage container so that the medium can be supplied from the liquid medium storage container to the liquid medium storage container. It needs to be piped.
  • the cell culture container has a pipe for supplying the hollow fiber module for seeding cells, a pipe for collecting the cells cultured in the hollow fiber module, and the hollow fiber module. A pipe or the like for discarding the cultured medium may be connected.
  • “connection” may be either directly connected or connected through a flow path such as a pipe.
  • the material of the channel network used in the cell culture method of the present invention is not particularly limited.
  • the gas permeability of the channel network is such that the oxygen permeability at 37 ° C. is 200 ⁇ 10 ⁇ 10 cm 3 / (cm 2 ⁇ sec ⁇ cmHg) or more, and the carbon dioxide permeability is 1200 ⁇ 10 ⁇ 10 cm. 3 / (cm 2 ⁇ sec ⁇ cmHg) or more is preferable.
  • Such materials include low density polyethylene (oxygen permeability coefficient: 0.4 ⁇ 10 ⁇ 10 to 6 ⁇ 10 ⁇ 10 cm 3 (STP) ⁇ cm / (cm 2 ⁇ sec ⁇ cmHg), carbon dioxide permeability coefficient: 2 ⁇ 10 ⁇ 10 to 30 ⁇ 10 ⁇ 10 cm 3 (STP) ⁇ cm / (cm 2 ⁇ sec ⁇ cmHg)), polypropylene (oxygen permeability coefficient: 2 ⁇ 10 ⁇ 10 to 50 ⁇ 10 ⁇ 10 cm 3 (STP ) ⁇ Cm / (cm 2 ⁇ sec ⁇ cmHg), carbon dioxide permeability coefficient: 5 ⁇ 10 ⁇ 10 to 20 ⁇ 10 ⁇ 10 cm 3 (STP) ⁇ cm / (cm 2 ⁇ sec ⁇ cmHg)), poly-4-methyl Penten-1 (oxygen permeability coefficient: 2 ⁇ 10 ⁇ 10 to 32 ⁇ 10 ⁇ 10 cm 3 (STP) ⁇ cm / (cm 2 ⁇ sec ⁇ cmH
  • the laminated body using these may be sufficient, it is not limited to these.
  • the size and thickness of the channel network (tubes) may be adjusted so that the oxygen permeability and carbon dioxide permeability are as desired, but the strength and gas permeability can be improved.
  • the thickness of the tube is preferably 0.5 mm to 3 mm. If the gas permeability is too high, bubbles may be generated in the tube, which may impede the flow of the culture medium (medium). It is preferable.
  • the form of a method for supplying a liquid such as a cell suspension or a medium is not particularly limited. However, if the liquid medium storage container is a soft one such as a culture bag, the container may be transferred to the container. A method of squeezing the medium by applying a roller is suitable.
  • the installation location of the pump is not particularly limited, but it is preferable to install the pump on the discharge side in order to avoid the risk of contamination of liquid when the liquid passes through the pump portion.
  • the cell culturing apparatus may be an apparatus further including the following configuration [5]. [5] A CO 2 incubator covering at least a part of [2] and / or [3]
  • a liquid medium has a role of supplying nutrients necessary for cells and gases such as oxygen and carbon dioxide.
  • gases such as oxygen and carbon dioxide.
  • a gas permeable container is used as the liquid medium storage container, and / or a flow channel tube at a portion connecting the liquid medium storage container and the hollow fiber module.
  • the CO 2 incubator used in the present invention may cover all the configurations [1] to [4].
  • the gas permeable component for example, [2] and / or ([[ 3] or a part other than gas permeable part)
  • the gas permeable component for example, a component having no gas permeability in [1] or [3] above, supply and / or discharge of liquid medium, etc. It is not always necessary to cover the inside of the CO 2 incubator with respect to the means for controlling the above.
  • the CO 2 incubator used in the present invention is not particularly limited as long as the CO 2 concentration of both the hollow fiber module and the liquid medium storage container can be kept constant, but is preferably sealed.
  • the CO 2 incubator used in the present invention has a role as a thermostat as well as maintaining a constant concentration of CO 2 .
  • the setting of the CO 2 concentration and temperature is not particularly limited, but it is preferable to set the CO 2 concentration to 5% and the temperature to 37 ° C., respectively. Further, considering this, the CO 2 incubator used in the present invention preferably further covers the configuration of [1].
  • CO 2 incubator that can take the means as described above, for example, commercially available products such as those manufactured by Panasonic, Thermo Fisher Scientific, and Yamato Scientific can be used.
  • Adhesive animal cell is suitable.
  • the origin of the cell is not particularly limited, and those derived from any animal such as human, mouse, rat and the like can be used.
  • Adhesive animal cells can target both primary cultured cells and established cells. It does not ask
  • it may be a cell line or a primary cell. Examples include cell lines such as NIH3T3 cells, Hela cells, COS cells, HEK cells, L929 cells, Daudi cells, Jurkat cells, KG-1a cells, and various hybridoma cell lines.
  • primary cells such as nerve cells, keratinocytes, fibroblasts, lung epithelial cells, hepatocytes, blood cells and the like can be mentioned.
  • stem cells and progenitor cells such as ES cells, hematopoietic stem cells, hepatic stem cells, and mesenchymal stem cells may be used. These cells may be cells into which a foreign gene has been introduced before culturing, or may be cells that have been stimulated and processed in advance with stimulating factors such as antibodies and ligands.
  • means for seeding cells is not particularly limited.
  • a method using the liquid medium storage container can be mentioned. This method is a method in which a solution containing cells to be seeded is placed in the liquid medium storage container, and poured into the hollow fiber module as a cell culture container via a connecting flow path for perfusion. After seeding, the liquid medium storage container may be switched to a medium containing no medium and perfused for culturing.
  • a culture bag is used for a liquid medium storage container
  • a solution containing cells and medium to be seeded is placed in the culture bag and set in a cell culture device, and then the solution is transferred to the hollow fiber module to start seeding.
  • the culture may be performed by switching to a culture bag containing only the medium.
  • This method is preferably used when the amount of the solution containing cells to be seeded is large.
  • a pipe that allows the solution to flow into the hollow fiber module by injection using a syringe or the like may be separately configured.
  • Cell culture in the cell culture device used in the cell culture method of the present invention can be performed by continuing to send the medium to the cell culture container (preferably in a CO 2 incubator).
  • the liquid medium has a role of supplying the cells with nutrients necessary for the cells and gases such as oxygen and carbon dioxide.
  • the hollow fiber module is used as a cell culture container of a cell culture device.
  • the supply of the medium to the hollow fiber module requires two routes on the lumen side and the lumen side.
  • liquid medium storage containers may be used separately on the lumen side and the outer lumen side in supplying the medium, or the medium is supplied from one container to both the lumen side and the outer lumen side. Also good.
  • the medium composition on the lumen side and the medium composition on the outer lumen side may be the same or different, but are preferably the same composition.
  • the medium used for the culture is determined according to the type of the cultured cell, and may be any medium that is usually used as the medium for the cell.
  • the culture method of the present invention can also be used for serum-free culture.
  • the flow rate on the lumen side and the flow rate on the outer lumen side may be the same or different, but in the method of the present invention, the linear velocity of the liquid medium flowing through the hollow fiber lumen side is set to 0.001 mm / It is necessary to control the linear velocity of the liquid medium flowing on the outer side of the hollow fiber to 0.001 mm / min to 5 mm / min.
  • the flow rate of the medium during the culture is not particularly limited as long as it is within the above range, but a flow rate suitable for cell growth is preferable. From this viewpoint, it is preferable that the inner cavity side is 0.002 to 5 mm / min and the outer cavity side is 0.002 to 5 mm / min.
  • the inner cavity side is 0.002 to 4 mm / min and the outer cavity side is 0.002 to 4 mm / min.
  • the flow rate of a culture medium is preferably adjusted based on measurement results such as the concentration of glucose and lactate in the medium.
  • the medium flow rate is adjusted according to the size of the module.
  • the flow of the medium from the liquid medium storage container to the hollow fiber module is preferably unidirectional.
  • one direction means that the route from introduction of the culture medium to the hollow fiber module is always in the same direction.
  • a so-called circulation type in which the medium once derived from the hollow fiber module is introduced again from the introduction port may be used.
  • at least one of the inner side and the outer side of the hollow fiber may be a circulation type, or both of them may be a circulation type.
  • the hollow fiber module which is a cell culture vessel, may be shaken or rolled. It is preferable to perform such an operation in that the cells can be uniformly seeded in the hollow fiber module, the cells can be removed when bubbles are generated, and the nutrients of the medium are evenly distributed to the cultured cells. .
  • the cell culturing apparatus used in the cell culturing method of the present invention may include means for monitoring the components and concentrations of cellular metabolites such as pH, glucose, lactate, oxygen, etc. over time, and analysis means.
  • a sensor for detection can be included.
  • the sensor can be attached at any position on the inner tube of the hollow fiber or the tube of the outer port of the hollow fiber.
  • the culture medium can be collected periodically by attaching a fraction collector to the inside of the hollow fiber or the outlet of the outside of the hollow fiber.
  • the concentration of glucose or lactate in the medium can be used as an index for adjusting the flow rate of the medium because it depends on cell proliferation. For example, when the glucose concentration is decreased, the cells are growing, so that nutrients are supplied to the cells. Therefore, the flow rate of the medium can be increased to provide an environment in which the cells are more likely to grow.
  • the cell culture device used in the cell culture method of the present invention includes a work controller (work panel) for controlling the above operations and processes and for the convenience of monitoring and the like. May be.
  • the means for collecting the cells in the cell culture device used in the cell culture method of the present invention is not particularly limited.
  • divalent cation-free phosphate buffered saline PBS
  • PBS divalent cation-free phosphate buffered saline
  • the PBS is further perfused for a fixed time, and then the PBS is removed and a protease such as trypsin is filled inside and outside the hollow fiber.
  • the cultured cells are detached from the hollow fiber lumen by trypsin treatment and collected from the hollow fiber module.
  • the cell culture device used in the cell culture method of the present invention may further have a cell collection container and a waste liquid collection container as necessary.
  • the form of the cell collection container is not particularly limited.
  • a glass bottle, a plastic bottle, a culture bag, etc. are mentioned.
  • the form of the waste liquid collection container is not particularly limited.
  • a glass bottle, a plastic bottle, a culture bag, etc. are mentioned.
  • FIG. 1 shows one configuration example of a cell culture apparatus used in the cell culture method of the present invention.
  • reference numerals 1 and 2 denote gas-permeable liquid medium storage containers (such as culture bags), respectively.
  • the liquid medium storage container 1 via the sterile connection connector 13 and the valve 4, to the end conduit 33a of the cell culture container 17 (in the embodiment, a hollow fiber module.
  • FIG. 2 shows a configuration example thereof)
  • the flow path is connected so that the medium contained in the liquid medium storage container 1 can be transferred to the lumen side of the cell culture container 17.
  • a flow path is connected to the side conduit 34 a of the cell culture container (hollow fiber module in the embodiment) 17 via the aseptic connection connector 14 and the valve 5.
  • the medium contained in the container 2 can be transferred to the outer cavity side of the cell culture container 17. From the lumen side of the cell culture container 17, a flow path is further connected to the waste liquid collection container 18 via the valve 7, the valve 11, the liquid feeding pump 20, the valve 12, and the aseptic connection connector 16. The medium that has passed through can be discarded. In addition, supply of liquid (medium etc.) from the liquid medium storage container 1 to the cell culture container 17 and / or discharge of the liquid from the cell culture container by the liquid feed pump 20 provided in the flow path, Furthermore, it is possible to control the flow rate or the like in discarding the liquid.
  • a flow path is further connected to the waste liquid collection container 18 via the valve 6, the valve 9, the liquid feeding pump 19, the valve 10, and the aseptic connection connector 16.
  • the medium that has passed through the container 17 can be discarded.
  • supply of liquid (medium etc.) from the liquid medium storage container 2 to the cell culture container 17 and / or discharge of the liquid from the cell culture container by the liquid feeding pump 19 provided in the flow path Furthermore, it is possible to control the flow rate or the like in discarding the liquid.
  • the cell collection container 3 includes a valve 8, a sterile connection connector that branches from the lumen side of the cell culture container 17 via the valve 7 and further branches off from the waste system flow path on the lumen side.
  • the flow path is connected via 15 so that the cells cultured in the cell culture vessel 17 can be collected.
  • the flow path network may be further provided with a pump or the like to collect cells.
  • the cell may be collected by removing the cell culture container 17 from the cell culture device. In this case, there is no configuration from the branch of the waste-side flow path on the lumen side to the cell collection container 3. Good.
  • 41 is a CO 2 incubator. In FIG. 1, most of the above-described configuration is covered.
  • reference numeral 21 denotes a work controller (work panel), which may be set for convenience such as performing the above-described operations and processes and monitoring.
  • the hollow fiber module for culture used in each example described later was prepared as follows.
  • the material of the hollow fiber to be used is shown in each example.
  • both ends are fixed to the module case with a polyurethane potting agent so as not to block the hollow part of the hollow fiber.
  • FIG. 4 shows a simplified configuration of the cell culture apparatus used in this example.
  • a hollow fiber module (17 in FIG. 4) (hollow fiber: manufactured by CTA, manufactured by Toyobo Co., Ltd.) coated with fibronectin (manufactured by Corning) on the day before cell seeding was used.
  • the hollow fiber had an inner diameter of 200 ⁇ m, an outer diameter of 250 ⁇ m, and a film thickness of 25 ⁇ m.
  • SEM image surface form observation result of the hollow fiber made from CTA is shown.
  • 1 is a hollow fiber cross-sectional view observation result
  • 2 is a hollow fiber inner surface form observation result
  • 3 is a hollow fiber outer surface form observation result.
  • the mesenchymal stem cells used were primary cells purchased from Takara Bio Inc.
  • the cell seeding density was 1900 cells / cm 2 .
  • the flow rate of the medium was 0.33 mm / min on the hollow fiber lumen side and 3.46 mm / min on the hollow fiber outer lumen side.
  • a gas-permeable culture bag 1L (manufactured by Nipro Co., Ltd.) (1 (luminal side) and 2 (external side) in FIG. 4) is provided in the liquid medium storage container, and a gas-permeable silicone is provided in the channel network.
  • a tube was used.
  • GlutaMAX manufactured by Life Technologies
  • a total of two Perista Biomini pumps (manufactured by Ato) (19 (luminal side) and 20 (external side) in FIG. 4) were used for hollow fiber inner perfusion and outer perfusion. .
  • the cells were cultured at 37 ° C. for 7 days in a CO 2 incubator.
  • the medium supply method was unidirectional on both the hollow fiber lumen side and the hollow fiber outer lumen side.
  • Mesenchymal stem cells were seeded inside the hollow fiber (the number of seeded cells was 3.0 ⁇ 10 5 cells / module) and allowed to stand for 2 days, and then perfusion inside the hollow fiber was started. After 7 days of culture, the medium perfusion was stopped and the cells grown in the hollow fiber module were collected.
  • FIG. 5 shows the observation results of the cells cultured on the hollow fiber lumen side. It was confirmed that cells adhered and proliferated on the hollow fiber lumen side.
  • FIG. 6 shows a simplified configuration of the cell culture device used in this comparative example.
  • Mesenchymal stem cells were seeded inside the hollow fiber similar to that used in Example 1, and after standing for 2 days, perfusion inside the hollow fiber was started.
  • the mesenchymal stem cells used were primary cells purchased from Takara Bio Inc.
  • the cell seeding density was 1900 cells / cm 2 .
  • the flow rate of the medium was 0.33 mm / min on the hollow fiber lumen side and 3.46 mm / min on the hollow fiber outer lumen side.
  • a gas-impermeable glass bottle manufactured by Asahi Glass Co., Ltd.
  • 51 luminal side
  • 52 exitternal lumen side
  • a gas-permeable glass bottle is used for the channel network.
  • Tube made of silicone
  • Reference numeral 53 denotes a gas exchange tube (silicone tube in this comparative example).
  • the length of the silicone tube was 1.5 m.
  • ⁇ -MEM manufactured by Life Technologies
  • Perto Biominipumps (56 (inner lumen side) and 57 (external lumen side) in FIG. 6) manufactured by ATTO Corporation were used.
  • the cells were cultured at 37 ° C. for 7 days in a CO 2 incubator.
  • the medium supply method was unidirectional on both the hollow fiber lumen side and the hollow fiber outer lumen side.
  • Mesenchymal stem cells were seeded inside the hollow fiber and allowed to stand for 2 days, and then perfusion inside the hollow fiber was started. After 7 days of culture, the medium perfusion was stopped and the cells grown in the hollow fiber module were collected. At the time of cell recovery, a cell dissociation reagent, 0.25% trypsin solution (manufactured by Life Technologies) was used.
  • FIG. 4 shows a simplified configuration of the cell culture device used in this comparative example.
  • a hollow fiber module (17 in FIG. 4) (hollow fiber: manufactured by CTA, manufactured by Toyobo Co., Ltd.) coated with fibronectin (manufactured by Corning) on the day before cell seeding was used.
  • the hollow fiber had an inner diameter of 200 ⁇ m, an outer diameter of 250 ⁇ m, and a film thickness of 25 ⁇ m.
  • SEM image surface form observation result of the hollow fiber made from CTA is shown.
  • the mesenchymal stem cells used were primary cells purchased from Takara Bio Inc.
  • the cell seeding density was 1900 cells / cm 2 .
  • the flow rate of the medium was 20 mm / min on the hollow fiber lumen side and 30 mm / min on the hollow fiber outer lumen side.
  • a gas-permeable culture bag 1L (manufactured by Nipro Co., Ltd.) (1 (lumen side) and 2 (external side) in FIG. 4) is used for the liquid medium storage container, and a silicone tube is used for the channel network.
  • GlutaMAX (manufactured by Life Technologies) containing 10% serum was used as the medium.
  • a total of two Perista Biomini pumps manufactured by Ato
  • the cells were cultured at 37 ° C. for 7 days in a CO 2 incubator.
  • the medium supply method was unidirectional on both the hollow fiber lumen side and the hollow fiber outer lumen side.
  • Mesenchymal stem cells were seeded inside the hollow fiber (the number of seeded cells was 3.0 ⁇ 10 5 cells / module) and allowed to stand for 2 days, and then perfusion inside the hollow fiber was started. After 7 days of culture, the medium perfusion was stopped and the cells grown in the hollow fiber module were collected.
  • a cell dissociation reagent, 0.25% trypsin solution manufactured by Life Technologies
  • Table 1 summarizes the results of the cell culture experiments of Example 1 and Comparative Examples 1 and 2.
  • a gas-permeable culture bag for the liquid medium storage container (medium supply container)
  • the cell growth rate was improved as compared with the case where a glass bottle without gas permeability was used.
  • the cell growth rate was significantly lower than that of the present invention even when a gas exchange tube was connected.
  • As a gas exchange method in the method using a gas permeable silicone tube, even if the length of the tube is 1.5 m is not sufficient in order to sufficiently perform the gas exchange.
  • the linear velocity of the liquid medium flowing through the hollow fiber lumen side is 0.001 mm / min to 5 mm / min
  • the linear velocity of the liquid medium flowing through the hollow fiber lumen side is 0.001 mm / min to 5 mm / min.
  • the cell proliferation rate improved by controlling each.
  • the present invention it is possible to provide a cell culture apparatus that can culture various cells including stem cells in a safe, simple and highly efficient manner as compared with conventional methods.
  • Liquid medium storage container 3 having gas permeability 3 Cell collection container 4 to 12 Valve 13 to 16 Aseptic connection connector 17 Cell culture container (hollow fiber module in the embodiment) 18 Waste liquid collection containers 19 and 20 Liquid feed pump 21 Work controller (work panel) 31 Module Case 32 Porous Membrane Hollow Fiber 33 End Conduit 34 Side Conduit 41 CO 2 Incubator 51, 52 Liquid Medium Storage Container (Glass Bottle) Not Gas Permeable 53 Gas exchange tube 54 Hollow fiber module 55 Waste liquid collection container 56, 57 Liquid feed pump

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Abstract

L'invention a pour objet de fournir un dispositif de culture cellulaire qui permet de cultiver, de manière plus sûre, plus simple et selon un rendement plus élevé que dans l'art antérieur, diverses cellules telles que des cellules souches mésenchymateuses, ou similaire, présentes seulement en petit nombre dans une population cellulaire désordonnée. Ce dispositif de culture cellulaire contient ［１］ un réceptacle de culture cellulaire, ［２］ un réceptacle de stockage de milieu de culture liquide présentant une perméabilité au gaz, ［３］ un réseau de trajet d'écoulement connectant ledit réceptacle de culture cellulaire et ledit réceptacle de stockage de milieu de culture liquide, et ［４］ un moyen qui est agencé dans ledit réseau de trajet d'écoulement, et qui commande l'alimentation et/ou la décharge d'un liquide.
PCT/JP2016/056218 2015-03-05 2016-03-01 Procédé de culture cellulaire mettant en œuvre un module à fibres creuses WO2016140213A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017143775A (ja) * 2016-02-17 2017-08-24 東洋紡株式会社 ガス不透過性管を用いた細胞培養装置および細胞培養方法
JP2018050502A (ja) * 2016-09-27 2018-04-05 米満　吉和 懸濁液を無菌的に処理するための器具
CN110117527A (zh) * 2019-05-15 2019-08-13 刘宝全 一种干细胞代谢废物的强化排出方法
CN110283716A (zh) * 2019-06-20 2019-09-27 清华大学 一种用于无细胞蛋白质连续合成的装置和方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03292884A (ja) * 1990-04-12 1991-12-24 Mitsubishi Rayon Co Ltd 細胞培養方法
JPH04148676A (ja) * 1990-10-09 1992-05-21 Tabai Espec Corp 灌流培養方法
JPH10108673A (ja) * 1996-10-04 1998-04-28 Asahi Medical Co Ltd 中空糸型培養器を用いた動物細胞の培養方法
JP2007222063A (ja) * 2006-02-23 2007-09-06 Scimedia Ltd 培養装置及び培養方法
JP2012044908A (ja) * 2010-08-25 2012-03-08 Nomura Unison Co Ltd 細胞培養用中空糸モジュールおよび細胞培養方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03292884A (ja) * 1990-04-12 1991-12-24 Mitsubishi Rayon Co Ltd 細胞培養方法
JPH04148676A (ja) * 1990-10-09 1992-05-21 Tabai Espec Corp 灌流培養方法
JPH10108673A (ja) * 1996-10-04 1998-04-28 Asahi Medical Co Ltd 中空糸型培養器を用いた動物細胞の培養方法
JP2007222063A (ja) * 2006-02-23 2007-09-06 Scimedia Ltd 培養装置及び培養方法
JP2012044908A (ja) * 2010-08-25 2012-03-08 Nomura Unison Co Ltd 細胞培養用中空糸モジュールおよび細胞培養方法

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017143775A (ja) * 2016-02-17 2017-08-24 東洋紡株式会社 ガス不透過性管を用いた細胞培養装置および細胞培養方法
JP2018050502A (ja) * 2016-09-27 2018-04-05 米満　吉和 懸濁液を無菌的に処理するための器具
JP6989907B2 (ja) 2016-09-27 2022-02-15 吉和 米満 懸濁液を無菌的に処理するための器具
CN110117527A (zh) * 2019-05-15 2019-08-13 刘宝全 一种干细胞代谢废物的强化排出方法
CN110283716A (zh) * 2019-06-20 2019-09-27 清华大学 一种用于无细胞蛋白质连续合成的装置和方法

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