WO2018066287A1 - Cell culture container, cell culture system, cell culture method and method for manufacturing cell culture container - Google Patents

Cell culture container, cell culture system, cell culture method and method for manufacturing cell culture container Download PDF

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Publication number
WO2018066287A1
WO2018066287A1 PCT/JP2017/031831 JP2017031831W WO2018066287A1 WO 2018066287 A1 WO2018066287 A1 WO 2018066287A1 JP 2017031831 W JP2017031831 W JP 2017031831W WO 2018066287 A1 WO2018066287 A1 WO 2018066287A1
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Prior art keywords
cell culture
tubular container
container
coating
tubular
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PCT/JP2017/031831
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French (fr)
Japanese (ja)
Inventor
郷史 田中
貴彦 戸谷
亮 末永
直己 高橋
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東洋製罐グループホールディングス株式会社
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Publication of WO2018066287A1 publication Critical patent/WO2018066287A1/en

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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

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  • the present invention relates to a cell culture technique for efficiently culturing a large amount of cells in a closed container.
  • cells In cell culture, cells generally have a property of sedimenting to the bottom surface of the culture vessel because the specific gravity thereof is larger than that of the culture solution. For this reason, in order to obtain an optimal culture environment, the cell density per cell (cells / cm 3 , volume cell density) and the cell density at the bottom (cells / cm 2 , bottom area cell density) Therefore, there are two parameters as culture conditions: volume cell density and bottom area cell density.
  • proteins and polymers may be coated on the culture surface and cultured.
  • cells may not be present in most of the coated part. In such a case, expensive proteins are wasted, which is a cause of increased culture costs. become.
  • the present inventors have intensively studied, formed a thin tubular container (tubular container) using a flexible member, and sealed the cells and culture solution in the sealed space in the tubular container. It was found that the above-mentioned problems can be solved by culturing the yeast. That is, with such a tubular container, when the cells settle, the tube gathers around the lowest point of the tube. Therefore, the bottom surface of the tubular container can be changed by deforming the tubular container by pressing the container. it can. That is, according to the number of cells, it is possible to control the bottom area cell density to a suitable range while keeping the volume cell density constant. Further, by flowing a coating reagent (coating solution) on the bottom surface in the tubular container, it is possible to easily coat only the culture section, and it is possible to reduce the amount of expensive coating reagent used.
  • a coating reagent coating solution
  • Examples of the technique for coating the bottom surface in the culture vessel with the coating reagent include the carrier for culturing T lymphocytes described in Patent Document 1. However, this technique did not enable partial coating in the culture vessel. In cell culture, there has been no technique that enables control of the bottom area cell density by changing the bottom surface of the tubular container formed by using a flexible member.
  • An object of the present invention is to provide a cell culture container, a cell culture system, a cell culture method, and a method for producing the cell culture container.
  • the cell culture container of the present invention is a cell culture container comprising a tubular container, wherein a sealed space for culturing cells is formed in the tubular container, and is formed on a part of the inner surface of the tubular container.
  • the coating reagent is coated.
  • the cell culture system of the present invention includes the cell culture container and a pressing device that changes the bottom area in the tubular container by pressing and deforming the tubular container during cell culture.
  • the cell culture method of the present invention is a cell culture method using the above-described cell culture container, wherein the tubular container is pressed and deformed during cell culture to change the cell culture area in the tubular container. It is as.
  • the method for producing a cell culture container of the present invention is the method for producing a cell culture container described above, wherein the coating reagent is injected into the tubular container, and the coating reagent is disposed in the longitudinal direction in the tubular container. The coating reagent is applied to the lower region in the tubular container to form a coating layer.
  • the present invention in the cell culture container, it is possible to easily coat only the culture part with the coating reagent, reduce the amount of expensive coating reagent used, and make the culture area variable.
  • FIG. 1 It is a schematic diagram which shows schematic structure of the cell culture container which concerns on embodiment of this invention. It is a schematic diagram which shows the cell culture container which consists of a cyclic
  • coating two types of coating reagents which concern on embodiment of this invention.
  • FIG. 6 is a schematic diagram showing an embodiment in which cell culture is performed on a second coating reagent. It is a schematic diagram which shows the cell culture container formed by apply
  • the cell culture container of the present embodiment is a cell culture container composed of a tubular container, wherein a sealed space for culturing cells is formed in the tubular container, and a coating reagent is coated on a part of the inner surface of the tubular container. It is characterized by that.
  • the cell culture container of the present embodiment is composed of, for example, a closed tubular container 1 formed in a cylindrical shape using a flexible member. Can do.
  • ports 11 for taking in and out substances from the outside are provided at both ends in the longitudinal direction of the tubular container 1.
  • This substance includes a liquid such as a culture solution, a gas such as air, and a solid matter such as a cell.
  • the arrangement of the ports 11 in the tubular container 1 is not limited to that shown in FIG. 1, and may be provided on one end in the longitudinal direction or on the side surface (on the curved surface) of the cylinder, and the number thereof is not particularly limited.
  • a cross-sectional view perpendicular to the central axis of the tubular container 1 is shown on the right side of FIG. That is, the central axis of the tubular container 1 is a line that passes through the center of a cross section that is parallel to the longitudinal direction of the tubular container 1 and perpendicular to the longitudinal direction of the tubular container 1.
  • a coating reagent is applied to the lower region (first coating region 12) in the tubular container 1 to form a coating layer.
  • the lower region in the tubular container 1 is a region within the lower half of the inner surface of the tubular container 1 in a state where the tubular container 1 is arranged so that the central axis of the tubular container 1 is horizontal. It can be a region in the tubular container 1 shown as a 1/6 arc of the circumference including the lower end of 1 as the center. In addition, the lower region in the tubular container 1 is the inside of the tubular container 1 shown as an arc of 1/2, 1/4, 1/8, or 1/12 of the circumference including the lower end of the tubular container 1 as the center. The region may be a region, and may be a region within the lower half of the inner surface of the tubular container 1, and is not particularly limited.
  • the coating reagent is not particularly limited as long as it is used by coating a container in cell culture.
  • cells of growth stimulating factors such as anti-CD3 antibody, laminin, collagen, gelatin, fibronectin and the like Adhesive factors, MPC (2-Methacryloylethyl Phosphoryl Choline) polymer, polyvinyl alcohol, polyhydroxyethyl methacrylate, polyethylene glycol, hydrogel, and other low cell adhesion treatment agents can be suitably used.
  • growth stimulating factor such as anti-CD3 antibody, laminin, collagen, gelatin, fibronectin and the like
  • Adhesive factors MPC (2-Methacryloylethyl Phosphoryl Choline) polymer
  • polyvinyl alcohol polyhydroxyethyl methacrylate
  • polyethylene glycol polyethylene glycol
  • hydrogel and other low cell adhesion treatment agents
  • the inner surface of the tubular container 1 can be subjected to a low cell adhesion treatment, and adherent cells that can be cultured by forming aggregates can be suitably cultured.
  • adherent cells such as fibronectin and laminin
  • adhesion to culture vessels by adherent cells such as fibroblasts, hepatocytes, neurons, pluripotent stem cells, and mesenchymal stem cells Can also be promoted.
  • the sealed space in the tubular container 1 is filled with the cells 2 and the culture solution 3, and cell culture is performed.
  • the cells 2 are not particularly limited, and adherent cells or suspension cells can be suitably used. Specifically, for example, adherent cells such as iPS cells, mesenchymal stem cells, neural stem cells, embryonic stem cells, hepatocytes, pancreatic islet cells, cardiomyocytes, corneal endothelial cells, and floating cells such as lymphocytes, erythrocytes and megakaryocytes Can be mentioned.
  • adherent cells such as iPS cells, mesenchymal stem cells, neural stem cells, embryonic stem cells, hepatocytes, pancreatic islet cells, cardiomyocytes, corneal endothelial cells, and floating cells such as lymphocytes, erythrocytes and megakaryocytes Can be mentioned.
  • lymphocytes When lymphocytes are cultured as the cells 2, it is preferable to coat the tubular container 1 with an anti-CD3 antibody and / or an anti-CD28 antibody as a coating reagent to form the cell culture container of this embodiment.
  • an anti-CD3 antibody and / or an anti-CD28 antibody As a coating reagent to form the cell culture container of this embodiment.
  • lymphocytes When lymphocytes are cultured using such a cell culture container, when the lymphocytes settle, they gather around the lowest point in the tubular container 1, so that the anti-CD3 antibody or the like is partially coated in the region. Collected naturally. For this reason, by partially coating the first coating region 12 in the tubular container 1 with a small amount of antibody, it is possible to appropriately stimulate lymphocytes, thereby reducing the amount of antibody and reducing costs. It becomes possible.
  • iPS cells When iPS cells are cultured as the cells 2, it is preferable to coat the tubular container 1 with, for example, MPC polymer as a coating reagent to form the cell culture container of this embodiment.
  • MPC polymer As a coating reagent to form the cell culture container of this embodiment.
  • iPS cells When iPS cells are cultured using such a cell culture container, iPS cells accumulate on the bottom surface without adhering to the inner surface of the tubular container 1, and then form aggregates. This makes it possible to form and aggregate iPS cell aggregates.
  • the cell culture container of this embodiment is composed of an annular tubular container 1 as shown in FIG.
  • an annular tubular container 1 can be formed by connecting the ends of the tubular container 1 with one or more connecting members 16.
  • the flow of the cells 2 and the culture solution 3 in the tubular container 1 can be performed by driving a pump.
  • a peristaltic pump that can send the cells 2 and the culture solution 3 in the tubular container 1 by squeezing the tubular container 1 can be suitably used.
  • the connecting member 16 forms an annular sealed space in the tubular container 1 by connecting the ends of the tubular container 1.
  • a T-shaped tube connector is used as the connection member 16, and the end portion of the tubular container 1 is inserted and connected to the opposed opening.
  • vertical to the tubular container 1 is used as the port 11, and can be connected to an external bag etc. via another tube.
  • the connection member 16 is not limited to such a T-shaped tube connector, and may be a tube connector such as a Y-type, an I-type, or an X-type, or other tube connection device.
  • Such a cell culture container can be easily manufactured by connecting the elongated tubular container 1 with the connecting member 16, and it is easy to form a long channel. For this reason, it is possible to easily manufacture a culture container having a large capacity compared to a bag-shaped culture container, and it is possible to make the culture container more suitable for mass culture of cells.
  • the tubular container 1 can be formed by sealing a thermoplastic resin sheet by means such as heat sealing, and can also be formed by various molding methods such as blow molding. Moreover, the tubular container 1 can also be formed by connecting with the connecting member 16 as described above. Furthermore, the tubular container 1 can also be formed by connecting a plurality of tubular containers 1 having different materials and rigidity using the connecting member 16.
  • the tubular container 1 is preferably partly or entirely made of a member having elastic deformability. Since at least a part of the tubular container 1 is elastically deformable, the culture solution and cells in the tubular container 1 can be efficiently transferred using a peristaltic pump (also referred to as a tube pump, a roller pump, or a squeezing pump). Can be sent. Further, since the tubular container 1 can be bent freely, the cell culture container can be arranged with excellent space efficiency.
  • a peristaltic pump also referred to as a tube pump, a roller pump, or a squeezing pump.
  • the tubular container 1 consists of a member which has gas permeability.
  • the oxygen permeability coefficient is 400 ml ⁇ mm / m 2 ⁇ day ⁇ atm (37 ° C-80% RH) or more
  • the carbon dioxide permeability coefficient is 1200 ml ⁇ mm / m 2 ⁇ day ⁇ atm (37 ° C-80%).
  • RH or more
  • the oxygen permeability coefficient is 1000 ml ⁇ mm / m 2 ⁇ day ⁇ atm (37 ° C.-80% RH) or more
  • the carbon dioxide permeability coefficient is 3000 ml ⁇ mm / m 2 ⁇ day ⁇ atm. More preferably (37 ° C.-80% RH) or higher. If the tubular container 1 has such gas permeability, excellent cell growth efficiency can be obtained.
  • the tubular container 1 has transparency so that the contents can be confirmed.
  • the material of the tubular container 1 include silicone rubber, soft vinyl chloride resin, polybutadiene resin, ethylene-vinyl acetate copolymer, chlorinated polyethylene resin, polyurethane-based thermoplastic elastomer, polyester-based thermoplastic elastomer, silicone-based thermoplastic.
  • Elastomers eg, SBS (styrene / butadiene / styrene), SIS (styrene / isoprene / styrene), SEBS (styrene / ethylene / butylene / styrene), SEPS (styrene / ethylene / propylene / styrene)), polyolefin Resin, fluorine resin, or the like can be used. That is, it is preferable to use these as the material of the tubular container 1 and to have the above-described elastic deformability, gas permeability, and transparency.
  • SBS styrene / butadiene / styrene
  • SIS styrene / isoprene / styrene
  • SEBS styrene / ethylene / butylene / styrene
  • SEPS styrene
  • tubular container 1 a rigid container such as polystyrene or polyethylene terephthalate may be used as the tubular container 1.
  • the cell culturing method of the present embodiment is a cell culturing method using the above cell culturing container, wherein the tubular container 1 is pressed and deformed during cell culturing to change the cell culturing area in the tubular container 1.
  • the tubular container 1 is crushed in the vertical direction and becomes a flat shape (see FIG. 3).
  • the cross section perpendicular to the central axis of the tubular container 1 has an elliptical shape, a substantially elliptical shape, a track shape for track and field, a shape obtained by extending the track shape in the longitudinal direction, and the like.
  • cell culture for example, in the initial stage of culture, cell culture is performed in the lowermost region in the tubular container 1, and after the number of cells grows to a certain level or more, the tubular container 1 is
  • the cell culture can be performed on the bottom surface in the tubular container 1 having an increased bottom area by pressing and deforming to increase the culture area.
  • the cell culture container 3 of this embodiment when the cell culture container 3 of this embodiment is filled with the cells 2 and the culture solution 3, the cells 2 gather in the lowest region in the tubular container 1, so that the bottom area cell density can be increased. For this reason, the culture efficiency of the cell in the initial stage of culture
  • the tubular container 1 after the number of cells grows to a certain level or more, the tubular container 1 can be pressed and deformed to expand the culture area on the bottom surface in the tubular container 1, thereby easily improving the cell culture efficiency.
  • the tubular container 1 it is preferable to culture lymphocytes using such a cell culture method and cell culture vessel.
  • the tubular container 1 can be pressed and deformed to increase the cell culture area in the tubular container 1.
  • the bottom area cell density can be controlled within an appropriate range, and the culture environment can be improved. That is, by deforming the tubular container 1 according to the number of cells in the tubular container 1, it becomes possible to give an activation stimulus to the lymphocytes by the anti-CD3 antibody in an environment having an optimal bottom area cell density.
  • the cell culture method of the present embodiment is a cell culture method using the above cell culture container, and the cell culture surface is changed by rotating the tubular container 1 around its central axis during cell culture.
  • a method is also preferred.
  • the cell culture container of the present embodiment is coated with a coating reagent in the lower region (first coating region 12) and the upper region (second coating region 13) in the tubular container 1. Can be applied to form a coating layer.
  • the upper region is a region within the upper half of the inner surface of the tubular container 1 when the tubular container 1 is arranged so that the central axis of the tubular container 1 is horizontal, and is defined by the same method as the lower region.
  • the tubular container 1 can be a region in the tubular container 1 shown as an arc of 1/6 of the circumference including the upper end of the tubular container 1 as the center.
  • a side region described later can also be defined by a similar method. That is, the side region is a region within the left and right halves on the inner surface of the tubular container 1 in a state where the tubular container 1 is arranged so that the central axis of the tubular container 1 is horizontal. Or it can be set as the area
  • the coating reagents for the first coating region 12 and the second coating region 13 may be the same or different.
  • the reagent that coats the first coating region 12 is referred to as a first coating reagent
  • the reagent that coats the second coating region 13 is referred to as a second coating reagent.
  • the tubular container 1 is rotated by 180 ° (vertically inverted) about its central axis, so that the second Cells can subsequently be cultured in the coating region 13.
  • the tubular container 1 is rotated by 180 ° (vertically inverted) about its central axis, so that the second Cells can subsequently be cultured in the coating region 13.
  • laminin 511 is used as the first coating reagent
  • MPC polymer is used as the second coating reagent
  • iPS cells are cultured in the first coating region 12
  • the second coating reagent is used. Cells can subsequently be cultured in the coating region 13.
  • iPS cells are adhered and cultured in the tubular container 1 in the first coating region 12, and after the number of cells increases to a certain level or more, the cells are peeled off from the bottom surface using a predetermined drug or the like. Is rotated to form an agglomerate of iPS cells in the second coating region 13 and culture can be performed efficiently.
  • the cell culture method of the present embodiment includes a method in which the tubular container 1 is pressed and deformed during cell culture to change the cell culture area in the tubular container 1, and the tubular container 1 is centered on its central axis during cell culture. It is also preferable to combine the methods of rotating and changing the cell culture surface. Specifically, for example, as shown in FIG. 5, laminin 511 is coated on the first coating region 12, and MPC polymer is coated on the second coating region 13 to form a cell culture container.
  • This cell culture container is composed of an annular tubular container 1.
  • the tubular container 1 is pressed and deformed to enlarge the bottom area.
  • the iPS cells adhere to the bottom surface and are cultured.
  • iPS cells are cultured by static culture (culture in a static state). After the iPS cells have grown to a certain level or more, the pressure on the tubular container 1 is released, the tubular container 1 is returned to a cylindrical shape, and the cells are peeled off from the bottom using a predetermined drug or the like.
  • the tubular container 1 is rotated around its central axis, and the second coating region 13 coated with the MPC polymer is turned downward to form a cell culture surface.
  • iPS cells are cultured with the culture solution 3 to form aggregates, and then circulated in one direction in the tubular container 1 to perform cell culture.
  • the iPS cells are sent in the tubular container 1 while maintaining the aggregates without adhering to the bottom surface in the tubular container 1, thereby obtaining uniform aggregates.
  • the cell culture container which consists of such an annular tubular container 1 can be used suitably also for culture
  • the tubular container 1 is pressed and deformed to enlarge the bottom area, and lymphocytes are activated by the anti-CD3 antibody in stationary culture. Make it. After the lymphocytes are activated, the pressure on the tubular container 1 is released, the tubular container 1 is returned to the cylindrical shape, the lymphocytes and the culture solution 3 are circulated by a pump, and are circulated in one direction in the tubular container 1. Perform cell culture. Thereby, lymphocytes proliferate in a floating state. For this reason, it is possible to prevent the lymphocytes from being excessively activated by contacting the anti-CD3 antibody for an unnecessarily long time, and to improve the proliferation efficiency of lymphocytes.
  • the tubular container 1 In the case of culturing lymphocytes, after activating the lymphocytes, the tubular container 1 is rotated around its central axis, and then pressed and deformed to culture on the bottom surface in the tubular container 1. The area can be expanded so that the lymphocytes do not contact the first coating region 12 to prevent the lymphocytes from being activated excessively, and the proliferation efficiency of the lymphocytes can be improved.
  • the cell culture container of this embodiment includes a lower region (first coating region 12), an upper region (second coating region 13), a side region ( It is also preferable that the third coating region 14 and the fourth coating region 15) are coated with a coating reagent and dried to form a coating layer.
  • the coating reagents for the first coating region 12, the second coating region 13, the third coating region 14, and the fourth coating region 15 may be the same or different.
  • the cell culture system of the present embodiment includes the cell culture container described above and a pressing device that presses and deforms the tubular container during cell culture to change the bottom area in the tubular container. . According to such a cell culture system, it is possible to increase the culture area according to the cell density by pressing the cell culture container.
  • the cell culture system of the present embodiment includes a rotation drive device that rotates the tubular container around its central axis during cell culture and moves the bottom surface in the tubular container. According to such a cell culture system, it is possible to select a culture surface by rotating the cell culture container, and for example, it is possible to change from a coated surface to an uncoated surface.
  • the cell culture system of the present embodiment preferably includes a tubular container 1, a pressing device 4, and a rotation driving device 5.
  • a tubular container 1 is placed on a loading table and can be pressed by a pressing device 4 from above.
  • the tubular container 1 is deformed into a flat shape by being pressed by the pressing device 4, and the bottom area in the tubular container 1 can be increased.
  • Ports 11 provided at both ends of the tubular container 1 are respectively attached to the rotational drive device 5 and are rotationally driven around the central axis of the tubular container 1 by the rotational drive device 5, thereby rotating the tubular container 1. It has become.
  • the tubular container 1 is preferably in a state of being floated from the loading table by a support table or the like.
  • the rotation of the tubular container 1 by the rotation drive device 5 can be rotated to 180 ° clockwise and counterclockwise around the central axis, for example.
  • the rotation drive device 5 is not limited to the one attached to the port 11.
  • the rotation drive device 5 is attached to the connection member 16 in the annular tubular container 1 and rotates the tubular container 1 together with the connection member 16, or the tubular container 1.
  • the tubular container 1 may be directly attached and rotated.
  • the cell culture system of the present embodiment includes a cell culture container in which an end of the tubular container is connected by one or more connecting members to form an annular sealed space, and a pump. It is also preferable that the culture medium and the cells in the tubular container are cultured while being fed in one direction in the tubular container by driving the pump. According to such a cell culture system, cells can be circulated in one direction in a tubular container together with a culture solution, and a cell aggregate of uniform size can be obtained.
  • the method for producing a cell culture container according to the present embodiment is the above-described method for producing a cell culture container, injecting a coating reagent into a tubular container, and moving the coating reagent in the longitudinal direction in the tubular container, A coating reagent is applied to a lower region in the tubular container, and the coating reagent is dried to form a coating layer. Specifically, as shown in FIG.
  • the coating reagent 6 is injected into the tubular container 1, the coating reagent 6 is moved in the longitudinal direction in the tubular container 1, and, if necessary, the tubular container By moving the coating reagent 6 in the circumferential direction in 1, the coating reagent 6 is applied to the lower region (first coating region 12) in the tubular container 1 to form a coating layer. Thereby, in the lower region including the lowermost region in the tubular container 1, the coating reagent 6 is solid-phased to form a coating layer.
  • the tubular container 1 is crushed to bring the upper surface in the tubular container 1 into contact with the lower surface in the tubular container 1, thereby The coating reagent 6 applied to the lower region is also applied to the upper region in the tubular container 1, and the same coating reagent 6 is solidified in both the upper region and the lower region in the tubular container 1 to form a coating layer. It is also preferable to form. Furthermore, after forming a coating layer in the lower region including the lowermost region in the tubular container 1, the lower region including the new lowermost region in the tubular container 1 is obtained by rotating the tubular container 1 around its central axis. It is also preferable to form a coating layer.
  • the coating reagent can be easily coated only on the culture part in the cell culture container.
  • the amount of expensive coating reagent used can be reduced, and the culture area can be made variable.
  • the culture solution and the cells in the tubular container can be circulated in one direction, thereby preventing the aggregates of cells from colliding with each other and uniform aggregates. It is also possible to obtain
  • the present invention can be suitably used for culturing a large amount of cells to be cultured by applying a coating reagent to a culture vessel.

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Abstract

A cell culture container comprising a tubular container 1, wherein a closed space for culturing cells is formed in the tubular container 1 and a coating reagent is coated on a part of the inner surface of the tubular container 1; a cell culture system provided with the cell culture container; a cell culture method wherein the cell culture container is used; and a method for manufacturing the cell culture container, whereby the coating reagent can be easily coated exclusively on a culture section so that the usage of the expensive coating reagent can be reduced and the culture area can be varied.

Description

細胞培養容器、細胞培養システム、細胞培養方法、及び細胞培養容器の製造方法Cell culture container, cell culture system, cell culture method, and method for manufacturing cell culture container
　本発明は、閉鎖系容器において細胞を効率良く大量に培養するための細胞培養技術に関する。 The present invention relates to a cell culture technique for efficiently culturing a large amount of cells in a closed container.
　近年、医薬品の生産や、遺伝子治療、再生医療、免疫療法等の分野において、細胞や組織、微生物などを人工的な環境下で効率良く大量に培養することが求められている。
　このような状況において、培養容器（培養バッグ）に細胞と培養液を充填して、閉鎖系で自動的に細胞を大量培養することが行われている。 In recent years, in the fields of pharmaceutical production, gene therapy, regenerative medicine, immunotherapy, and the like, it has been required to efficiently culture a large amount of cells, tissues, microorganisms, and the like in an artificial environment.
Under such circumstances, it has been practiced to fill a culture vessel (culture bag) with cells and a culture solution and automatically culture a large number of cells in a closed system.
　細胞培養においては、一般に細胞は培養液よりもその比重が大きいことから、培養容器の底面へ沈降するという性質がある。このため、最適な培養環境を得るためには、体積当たりの細胞の培養密度（cells/cm３，体積細胞密度）と、底面での細胞の培養密度（cells/cm2，底面積細胞密度）を適切にコントロールすることが必要であり、培養条件として体積細胞密度と底面積細胞密度の２つのパラメータが存在する。 In cell culture, cells generally have a property of sedimenting to the bottom surface of the culture vessel because the specific gravity thereof is larger than that of the culture solution. For this reason, in order to obtain an optimal culture environment, the cell density per cell (cells / cm 3 , volume cell density) and the cell density at the bottom (cells / cm 2 , bottom area cell density) Therefore, there are two parameters as culture conditions: volume cell density and bottom area cell density.
　また、細胞培養において、細胞への刺激、あるいは培養容器への細胞の接着又は非接着などを制御するために、タンパク質やポリマー等を培養面にコーティングして培養する場合がある。しかしながら、底面積細胞密度が低い培養条件では、コーティングした部分のほとんどに細胞が存在しない場合があり、このような場合においては、高価なタンパク質等が無駄に消費され、培養コストが上昇する一因になる。 In cell culture, in order to control stimulation of cells or adhesion or non-adhesion of cells to a culture container, proteins and polymers may be coated on the culture surface and cultured. However, under the culture conditions with a low bottom area cell density, cells may not be present in most of the coated part. In such a case, expensive proteins are wasted, which is a cause of increased culture costs. become.
　一方、閉鎖系の細胞培養では、可撓性部材からなるバッグ形状の培養容器などが多用されているところ、このような培養バッグ内において、部分的にタンパク質等をコーティングすることは難しく、一方で全面に亘ってコーティングを行うと余分なコストがかかるという問題があった。また、培養バッグを用いる場合には、細胞を局所的に集めるなどといった底面積細胞密度のコントロールが難しいという問題もあった。 On the other hand, in closed cell culture, a bag-shaped culture container made of a flexible member is often used. In such a culture bag, it is difficult to partially coat protein or the like. If coating is performed over the entire surface, there is a problem that extra costs are required. In addition, when a culture bag is used, there is a problem that it is difficult to control the cell density of the bottom area, such as collecting cells locally.
特開２０００－２１２２００号公報JP 2000-212200 A
　そこで、本発明者らは鋭意研究し、可撓性部材を用いて細長の管状容器（チューブ状容器）を形成して、この管状容器内の密閉空間に細胞及び培養液を封入した状態で細胞を培養することによって、上記問題を解消できることを見いだした。
　すなわち、このような管状容器であれば、細胞が沈降した際に管の最下点を中心に集まるため、容器を押圧することなどにより管状容器を変形させることで、その底面を変化させることができる。すなわち、細胞数に応じて、体積細胞密度を一定にしつつ、底面積細胞密度を変化させて好適な範囲にコントロールすることが可能となる。
　また、管状容器内の底面にコーティング用試薬（コーティング液）を流すことによって、培養部のみに簡単にコーティングすることができ、高価なコーティング用試薬の使用量を低減することも可能となる。 Accordingly, the present inventors have intensively studied, formed a thin tubular container (tubular container) using a flexible member, and sealed the cells and culture solution in the sealed space in the tubular container. It was found that the above-mentioned problems can be solved by culturing the yeast.
That is, with such a tubular container, when the cells settle, the tube gathers around the lowest point of the tube. Therefore, the bottom surface of the tubular container can be changed by deforming the tubular container by pressing the container. it can. That is, according to the number of cells, it is possible to control the bottom area cell density to a suitable range while keeping the volume cell density constant.
Further, by flowing a coating reagent (coating solution) on the bottom surface in the tubular container, it is possible to easily coat only the culture section, and it is possible to reduce the amount of expensive coating reagent used.
　培養容器内の底面にコーティング用試薬のコーティングが行われる技術としては、特許文献１に記載のＴリンパ球培養用担体などを挙げることができる。しかしながら、当該技術は、培養容器内において部分的にコーティングを行うことを可能にしたものではなかった。
　また、細胞培養において、可撓性部材を用いて形成された管状容器を押圧することなどにより、その底面を変化させることで、底面積細胞密度のコントロールを可能とする技術は、見当たらなかった。 Examples of the technique for coating the bottom surface in the culture vessel with the coating reagent include the carrier for culturing T lymphocytes described in Patent Document 1. However, this technique did not enable partial coating in the culture vessel.
In cell culture, there has been no technique that enables control of the bottom area cell density by changing the bottom surface of the tubular container formed by using a flexible member.
　本発明は、上記事情に鑑みなされたものであり、細胞培養容器において、培養部のみにコーティング用試薬を簡単にコーティングできると共に、高価なコーティング用試薬の使用量を低減でき、培養面積を可変にすることが可能な細胞培養容器、細胞培養システム、細胞培養方法、及び細胞培養容器の製造方法の提供を目的とする。 The present invention has been made in view of the above circumstances, and in a cell culture container, it is possible to easily coat a coating reagent only on the culture part, reduce the amount of expensive coating reagent used, and make the culture area variable. An object of the present invention is to provide a cell culture container, a cell culture system, a cell culture method, and a method for producing the cell culture container.
　上記目的を達成するため、本発明の細胞培養容器は、管状容器からなる細胞培養容器であって、細胞を培養するための密閉空間が管状容器内に形成され、管状容器の内面の一部にコーティング用試薬がコーティングされた構成としてある。
　また、本発明の細胞培養システムは、上記の細胞培養容器と、細胞培養中に前記管状容器を押圧して変形させ、前記管状容器内の底面積を変化させる押圧装置とを備えた構成としてある。
　また、本発明の細胞培養方法は、上記の細胞培養容器を用いた細胞培養方法であって、細胞培養中に前記管状容器を押圧変形させて、前記管状容器内における細胞培養面積を変化させる方法としてある。
　また、本発明の細胞培養容器の製造方法は、上記の細胞培養容器の製造方法であって、前記管状容器内に前記コーティング用試薬を注入し、前記管状容器内の長手方向に前記コーティング用試薬を移動させて、前記管状容器内の下方領域に前記コーティング用試薬を塗布し、コーティング層を形成する方法としてある。 In order to achieve the above object, the cell culture container of the present invention is a cell culture container comprising a tubular container, wherein a sealed space for culturing cells is formed in the tubular container, and is formed on a part of the inner surface of the tubular container. The coating reagent is coated.
The cell culture system of the present invention includes the cell culture container and a pressing device that changes the bottom area in the tubular container by pressing and deforming the tubular container during cell culture. .
Further, the cell culture method of the present invention is a cell culture method using the above-described cell culture container, wherein the tubular container is pressed and deformed during cell culture to change the cell culture area in the tubular container. It is as.
The method for producing a cell culture container of the present invention is the method for producing a cell culture container described above, wherein the coating reagent is injected into the tubular container, and the coating reagent is disposed in the longitudinal direction in the tubular container. The coating reagent is applied to the lower region in the tubular container to form a coating layer.
　本発明によれば、細胞培養容器において、培養部のみにコーティング用試薬を簡単にコーティングできると共に、高価なコーティング用試薬の使用量を低減でき、培養面積を可変にすることが可能となる。 According to the present invention, in the cell culture container, it is possible to easily coat only the culture part with the coating reagent, reduce the amount of expensive coating reagent used, and make the culture area variable.
本発明の実施形態に係る細胞培養容器の概略構成を示す模式図である。It is a schematic diagram which shows schematic structure of the cell culture container which concerns on embodiment of this invention. 本発明の実施形態に係る環状の管状容器からなる細胞培養容器を示す模式図である。It is a schematic diagram which shows the cell culture container which consists of a cyclic | annular tubular container which concerns on embodiment of this invention. 本発明の実施形態に係る細胞培養容器における管状容器を押圧変形させて使用する態様を示す模式図である。It is a schematic diagram which shows the aspect which press-deforms and uses the tubular container in the cell culture container which concerns on embodiment of this invention. 本発明の実施形態に係る二種類のコーティング用試薬を塗布して形成された細胞培養容器を示す模式図である。It is a schematic diagram which shows the cell culture container formed by apply | coating two types of coating reagents which concern on embodiment of this invention. 本発明の実施形態に係る二種類のコーティング用試薬を塗布して形成された細胞培養容器における管状容器を押圧変形させて第一のコーティング用試薬上で細胞培養を行った後、培養面を変更して第二のコーティング用試薬上で細胞培養を行う態様を示す模式図である。After culturing the cell on the first coating reagent by pressing and deforming the tubular container in the cell culture container formed by applying two types of coating reagents according to the embodiment of the present invention, the culture surface is changed. FIG. 6 is a schematic diagram showing an embodiment in which cell culture is performed on a second coating reagent. 本発明の実施形態に係る四種類のコーティング用試薬を塗布して形成された細胞培養容器を示す模式図である。It is a schematic diagram which shows the cell culture container formed by apply | coating four types of coating reagents which concern on embodiment of this invention. 本発明の実施形態に係る細胞培養システムの概略構成を示す模式図である。It is a mimetic diagram showing a schematic structure of a cell culture system concerning an embodiment of the present invention. 本発明の実施形態に係る細胞培養容器の製造方法において、管状容器の内面にコーティング用試薬をコーティングする様子を示す模式図である。In the manufacturing method of the cell culture container which concerns on embodiment of this invention, it is a schematic diagram which shows a mode that the reagent for a coating is coated on the inner surface of a tubular container.
　以下、本発明の細胞培養容器、細胞培養システム、細胞培養方法、及び細胞培養容器の製造方法の実施形態について詳細に説明する。ただし、本発明は、以下の実施形態の具体的な内容に限定されるものではない。 Hereinafter, embodiments of the cell culture container, the cell culture system, the cell culture method, and the method for producing the cell culture container of the present invention will be described in detail. However, the present invention is not limited to the specific contents of the following embodiments.
　本実施形態の細胞培養容器は、管状容器からなる細胞培養容器であって、細胞を培養するための密閉空間が管状容器内に形成され、管状容器の内面の一部にコーティング用試薬がコーティングされたことを特徴とする。
　具体的には、本実施形態の細胞培養容器は、図１の左側に示すように、例えば、可撓性部材を用いて円筒状に形成された閉鎖系の管状容器１からなるものとすることができる。管状容器１の長手方向の両端には、外部と物質の出し入れをするためのポート１１が備えられている。この物質には、培養液などの液体の他、空気などの気体や、細胞などの固形物等が含まれる。
　管状容器１におけるポート１１の配置は、図１に限定されず、長手方向の一方の端部や円筒の側面（曲面上）に設けても良く、その個数も特に限定されない。 The cell culture container of the present embodiment is a cell culture container composed of a tubular container, wherein a sealed space for culturing cells is formed in the tubular container, and a coating reagent is coated on a part of the inner surface of the tubular container. It is characterized by that.
Specifically, as shown on the left side of FIG. 1, the cell culture container of the present embodiment is composed of, for example, a closed tubular container 1 formed in a cylindrical shape using a flexible member. Can do. At both ends in the longitudinal direction of the tubular container 1, ports 11 for taking in and out substances from the outside are provided. This substance includes a liquid such as a culture solution, a gas such as air, and a solid matter such as a cell.
The arrangement of the ports 11 in the tubular container 1 is not limited to that shown in FIG. 1, and may be provided on one end in the longitudinal direction or on the side surface (on the curved surface) of the cylinder, and the number thereof is not particularly limited.
　図１の右側には、管状容器１の中心軸に対して垂直な断面図が示されている。すなわち、管状容器１の中心軸は、管状容器１の長手方向に平行で、管状容器１の長手方向に垂直な断面の中心を通る線である。
　また、管状容器１内の下方領域（第一のコーティング領域１２）には、コーティング用試薬が塗布され、コーティング層が形成されている。 A cross-sectional view perpendicular to the central axis of the tubular container 1 is shown on the right side of FIG. That is, the central axis of the tubular container 1 is a line that passes through the center of a cross section that is parallel to the longitudinal direction of the tubular container 1 and perpendicular to the longitudinal direction of the tubular container 1.
In addition, a coating reagent is applied to the lower region (first coating region 12) in the tubular container 1 to form a coating layer.
　管状容器１内の下方領域とは、管状容器１の中心軸が水平になるように管状容器１が配置された状態において、管状容器１の内面における下半分以内の領域であり、例えば、管状容器１の下端を中心として含む円周の１／６の円弧として示される管状容器１内の領域とすることができる。また、管状容器１内の下方領域は、管状容器１の下端を中心として含む円周の１／２、１／４、１／８、又は１／１２等の円弧として示される管状容器１内の領域とすることもでき、管状容器１の内面の下半分以内の領域であれば良く、特に限定されない。 The lower region in the tubular container 1 is a region within the lower half of the inner surface of the tubular container 1 in a state where the tubular container 1 is arranged so that the central axis of the tubular container 1 is horizontal. It can be a region in the tubular container 1 shown as a 1/6 arc of the circumference including the lower end of 1 as the center. In addition, the lower region in the tubular container 1 is the inside of the tubular container 1 shown as an arc of 1/2, 1/4, 1/8, or 1/12 of the circumference including the lower end of the tubular container 1 as the center. The region may be a region, and may be a region within the lower half of the inner surface of the tubular container 1, and is not particularly limited.
　コーティング用試薬は、細胞培養において容器にコーティングして使用されるものであれば良く、特に限定されないが、例えば、抗ＣＤ３抗体などの増殖刺激因子のタンパク質、ラミニン、コラーゲン、ゼラチン、フィブロネクチンなどの細胞接着因子、ＭＰＣ（2-Methacryloylethyl　Phosphoryl　Choline）ポリマー、ポリビニルアルコール、ポリヒドロキシエチルメタクリレート、ポリエチレングリコール及びハイドロゲル等の細胞低接着処理剤を好適に用いることができる。
　本実施形態の細胞培養容器における管状容器１の内面に増殖刺激因子をコーティングすることで、リンパ球などの細胞を活性化させることができる。また、ＭＰＣポリマーやハイドロゲルをコーティングすることで、管状容器１の内面に細胞低接着処理を施すことができ、凝集塊を形成させて培養できる接着細胞などを好適に培養することが可能となる。
　また、コーティング用試薬としてフィブロネクチン、ラミニンなどの細胞接着促進剤をコーティングすることで、繊維芽細胞、肝細胞、神経細胞や多能性幹細胞、間葉系幹細胞などの接着細胞による培養容器への接着を促進させることもできる。 The coating reagent is not particularly limited as long as it is used by coating a container in cell culture. For example, cells of growth stimulating factors such as anti-CD3 antibody, laminin, collagen, gelatin, fibronectin and the like Adhesive factors, MPC (2-Methacryloylethyl Phosphoryl Choline) polymer, polyvinyl alcohol, polyhydroxyethyl methacrylate, polyethylene glycol, hydrogel, and other low cell adhesion treatment agents can be suitably used.
By coating the growth stimulating factor on the inner surface of the tubular container 1 in the cell culture container of this embodiment, cells such as lymphocytes can be activated. In addition, by coating with MPC polymer or hydrogel, the inner surface of the tubular container 1 can be subjected to a low cell adhesion treatment, and adherent cells that can be cultured by forming aggregates can be suitably cultured. .
In addition, by coating cell adhesion promoters such as fibronectin and laminin as a coating reagent, adhesion to culture vessels by adherent cells such as fibroblasts, hepatocytes, neurons, pluripotent stem cells, and mesenchymal stem cells Can also be promoted.
　管状容器１内の密閉空間には細胞２と培養液３が充填され、細胞培養が行われる。
　細胞２としては、特に限定されず、接着細胞又は浮遊細胞を好適に用いることができる。具体的には、例えばｉＰＳ細胞、間葉系幹細胞、神経幹細胞、胚性幹細胞、肝細胞、膵島細胞、心筋細胞、角膜内皮細胞等の接着細胞や、リンパ球、赤血球、巨核球などの浮遊細胞を挙げることができる。
　本実施形態の細胞培養容器を用いることにより、凝集塊（スフェロイド）を形成させて培養できる接着細胞や浮遊細胞を培養することができ、また接着細胞を管状容器１内に接着させて培養することもできる。 The sealed space in the tubular container 1 is filled with the cells 2 and the culture solution 3, and cell culture is performed.
The cells 2 are not particularly limited, and adherent cells or suspension cells can be suitably used. Specifically, for example, adherent cells such as iPS cells, mesenchymal stem cells, neural stem cells, embryonic stem cells, hepatocytes, pancreatic islet cells, cardiomyocytes, corneal endothelial cells, and floating cells such as lymphocytes, erythrocytes and megakaryocytes Can be mentioned.
By using the cell culture vessel of the present embodiment, it is possible to culture adherent cells and floating cells that can be cultured by forming aggregates (spheroids), and to adhere and adhere the adherent cells in the tubular vessel 1 You can also.
　細胞２として、リンパ球を培養する場合は、コーティング用試薬として抗ＣＤ３抗体及び／又は抗ＣＤ２８抗体を管状容器１にコーティングして、本実施形態の細胞培養容器を形成することが好ましい。
　このような細胞培養容器を用いてリンパ球を培養すれば、リンパ球が沈降した際に、管状容器１における最下点を中心に集まるため、抗ＣＤ３抗体等が部分的にコーティングされた領域に自然に集められる。このため、管状容器１における第一のコーティング領域１２に、少ない抗体量で部分的にコーティングをすることで、リンパ球に適切に刺激を与えることができ、抗体量を削減してコストダウンを図ることが可能となる。 When lymphocytes are cultured as the cells 2, it is preferable to coat the tubular container 1 with an anti-CD3 antibody and / or an anti-CD28 antibody as a coating reagent to form the cell culture container of this embodiment.
When lymphocytes are cultured using such a cell culture container, when the lymphocytes settle, they gather around the lowest point in the tubular container 1, so that the anti-CD3 antibody or the like is partially coated in the region. Collected naturally. For this reason, by partially coating the first coating region 12 in the tubular container 1 with a small amount of antibody, it is possible to appropriately stimulate lymphocytes, thereby reducing the amount of antibody and reducing costs. It becomes possible.
　また、細胞２として、ｉＰＳ細胞を培養する場合は、コーティング用試薬として例えばＭＰＣポリマーを管状容器１にコーティングして、本実施形態の細胞培養容器を形成することが好ましい。
　このような細胞培養容器を用いてｉＰＳ細胞を培養すれば、ｉＰＳ細胞は管状容器１の内面に接着することなく、底面上に溜まり、その後凝集塊を形成する。これにより、ｉＰＳ細胞の凝集塊を形成させて培養することが可能となる。 When iPS cells are cultured as the cells 2, it is preferable to coat the tubular container 1 with, for example, MPC polymer as a coating reagent to form the cell culture container of this embodiment.
When iPS cells are cultured using such a cell culture container, iPS cells accumulate on the bottom surface without adhering to the inner surface of the tubular container 1, and then form aggregates. This makes it possible to form and aggregate iPS cell aggregates.
　また、このように細胞の凝集塊を形成させて培養する場合には、本実施形態の細胞培養容器を、図２に示すような環状の管状容器１からなるものとして、培養液３を細胞２と共に満注となるように充填し、管状容器１内で一方向に送流させながら、細胞培養を行うことが好ましい。これにより、細胞の凝集塊が互いにぶつかり合うことを抑止でき、より均一なサイズの凝集塊を得ることが可能となる。このような環状の管状容器１は、管状容器１の端部を一又は二以上の接続部材１６で接続することにより形成することができる。 Further, in the case of culturing with the formation of cell agglomerates in this way, the cell culture container of this embodiment is composed of an annular tubular container 1 as shown in FIG. At the same time, it is preferable to perform cell culture while filling the tube so as to be filled and sending it in one direction in the tubular container 1. Thereby, it is possible to prevent the cell aggregates from colliding with each other, and it is possible to obtain aggregates of a more uniform size. Such an annular tubular container 1 can be formed by connecting the ends of the tubular container 1 with one or more connecting members 16.
　本実施形態の細胞培養容器を環状の管状容器１からなるものとする場合、管状容器１内における細胞２と培養液３の送流は、ポンプを駆動させることで行うことができる。ポンプとしては、管状容器１をしごくことによって管状容器１内の細胞２と培養液３を送流できるペリスタルティック方式のポンプを好適に用いることができる。 When the cell culture container according to the present embodiment is composed of the annular tubular container 1, the flow of the cells 2 and the culture solution 3 in the tubular container 1 can be performed by driving a pump. As the pump, a peristaltic pump that can send the cells 2 and the culture solution 3 in the tubular container 1 by squeezing the tubular container 1 can be suitably used.
　接続部材１６は、管状容器１の端部を接続することで、管状容器１内に環状の密閉空間を形成させる。図２では、接続部材１６としてＴ型のチューブコネクタを用い、対向する開口部に管状容器１の端部を挿入して接続している。また、管状容器１に垂直な開口部はポート１１として用いられ、他のチューブを介して外部のバッグなどに接続可能にされている。なお、接続部材１６としては、このようなＴ型のチューブコネクタに限定されず、Ｙ型、Ｉ型、Ｘ型等のチューブコネクタや、その他のチューブ接続器具を用いても良い。 The connecting member 16 forms an annular sealed space in the tubular container 1 by connecting the ends of the tubular container 1. In FIG. 2, a T-shaped tube connector is used as the connection member 16, and the end portion of the tubular container 1 is inserted and connected to the opposed opening. Moreover, the opening part perpendicular | vertical to the tubular container 1 is used as the port 11, and can be connected to an external bag etc. via another tube. Note that the connection member 16 is not limited to such a T-shaped tube connector, and may be a tube connector such as a Y-type, an I-type, or an X-type, or other tube connection device.
　このような細胞培養容器は、細長の管状容器１を接続部材１６で接続することによって簡単に製造でき、また流路を長く形成することが容易である。このため、バッグ形状の培養容器に比較して容量の大きい培養容器を容易に製造することができ、より細胞の大量培養に適したものとすることが可能である。 Such a cell culture container can be easily manufactured by connecting the elongated tubular container 1 with the connecting member 16, and it is easy to form a long channel. For this reason, it is possible to easily manufacture a culture container having a large capacity compared to a bag-shaped culture container, and it is possible to make the culture container more suitable for mass culture of cells.
　管状容器1は、熱可塑性樹脂シートをヒートシールなどの手段でシールすることで形成することができ、またブロー成形等の種々の成形方法によって形成することもできる。また、管状容器1は、上記のように、接続部材１６で接続して形成することもできる。さらに、管状容器1は、接続部材１６を用いて、材質や剛性などが異なる管状容器１を複数組み合わせて接続して形成することもできる。 The tubular container 1 can be formed by sealing a thermoplastic resin sheet by means such as heat sealing, and can also be formed by various molding methods such as blow molding. Moreover, the tubular container 1 can also be formed by connecting with the connecting member 16 as described above. Furthermore, the tubular container 1 can also be formed by connecting a plurality of tubular containers 1 having different materials and rigidity using the connecting member 16.
　管状容器１は、一部又は全部が弾性変形性を有する部材からなるものであることが好ましい。管状容器１の少なくとも一部が弾性変形性を有することで、ペリスタルティック方式のポンプ（チューブポンプ、ローラーポンプ、しごきポンプとも称する）を用いて、管状容器１内の培養液と細胞を効率的に送流することができる。また、管状容器１を自由に曲げることができるため、細胞培養容器を優れたスペース効率で配置することが可能となる。 The tubular container 1 is preferably partly or entirely made of a member having elastic deformability. Since at least a part of the tubular container 1 is elastically deformable, the culture solution and cells in the tubular container 1 can be efficiently transferred using a peristaltic pump (also referred to as a tube pump, a roller pump, or a squeezing pump). Can be sent. Further, since the tubular container 1 can be bent freely, the cell culture container can be arranged with excellent space efficiency.
　また、管状容器１は、ガス透過性を有する部材からなるものであることが好ましい。具体的には、酸素透過係数が４００ｍｌ・ｍｍ／ｍ２・ｄａｙ・ａｔｍ（３７℃－８０％ＲＨ）以上、二酸化炭素透過係数が１２００ｍｌ・ｍｍ／ｍ２・ｄａｙ・ａｔｍ（３７℃－８０％ＲＨ）以上のものであることが好ましく、酸素透過係数が１０００ｍｌ・ｍｍ／ｍ２・ｄａｙ・ａｔｍ（３７℃－８０％ＲＨ）以上、二酸化炭素透過係数が３０００ｍｌ・ｍｍ／ｍ２・ｄａｙ・ａｔｍ（３７℃－８０％ＲＨ）以上のものであることがより好ましい。管状容器１がこのようなガス透過性を有すれば、優れた細胞増殖効率を得ることができる。 Moreover, it is preferable that the tubular container 1 consists of a member which has gas permeability. Specifically, the oxygen permeability coefficient is 400 ml · mm / m 2 · day · atm (37 ° C-80% RH) or more, and the carbon dioxide permeability coefficient is 1200 ml · mm / m 2 · day · atm (37 ° C-80%). RH) or more is preferable, the oxygen permeability coefficient is 1000 ml · mm / m 2 · day · atm (37 ° C.-80% RH) or more, and the carbon dioxide permeability coefficient is 3000 ml · mm / m 2 · day · atm. More preferably (37 ° C.-80% RH) or higher. If the tubular container 1 has such gas permeability, excellent cell growth efficiency can be obtained.
　さらに、管状容器１は、内容物を確認できるように、一部又は全部が透明性を有することが好ましい。
　管状容器１の材料としては、例えば、シリコーンゴム、軟質塩化ビニル樹脂、ポリブタジエン樹脂、エチレン－酢酸ビニル共重合体、塩素化ポリエチレン樹脂、ポリウレタン系熱可塑性エラストマー、ポリエステル系熱可塑性エラストマー、シリコーン系熱可塑性エラストマー、スチレン系エラストマー（例えば、ＳＢＳ（スチレン・ブタジエン・スチレン）、ＳＩＳ（スチレン・イソプレン・スチレン）、ＳＥＢＳ（スチレン・エチレン・ブチレン・スチレン）、ＳＥＰＳ（スチレン・エチレン・プロピレン・スチレン））、ポリオレフィン樹脂、フッ素系樹脂等を用いることができる。
　すなわち、管状容器１の材料としてこれらを用いると共に、上記のような弾性変形性、ガス透過性、及び透明性を有するものとすることが好ましい。 Furthermore, it is preferable that part or all of the tubular container 1 has transparency so that the contents can be confirmed.
Examples of the material of the tubular container 1 include silicone rubber, soft vinyl chloride resin, polybutadiene resin, ethylene-vinyl acetate copolymer, chlorinated polyethylene resin, polyurethane-based thermoplastic elastomer, polyester-based thermoplastic elastomer, silicone-based thermoplastic. Elastomers, Styrenic elastomers (eg, SBS (styrene / butadiene / styrene), SIS (styrene / isoprene / styrene), SEBS (styrene / ethylene / butylene / styrene), SEPS (styrene / ethylene / propylene / styrene)), polyolefin Resin, fluorine resin, or the like can be used.
That is, it is preferable to use these as the material of the tubular container 1 and to have the above-described elastic deformability, gas permeability, and transparency.
　なお、管状容器１として、ポリスチレンやポリエチレンテレフタレートなどのリジッドなものを用いても良い。 Note that a rigid container such as polystyrene or polyethylene terephthalate may be used as the tubular container 1.
　本実施形態の細胞培養方法は、上記の細胞培養容器を用いた細胞培養方法であって、細胞培養中に管状容器１を押圧変形させて、管状容器１内における細胞培養面積を変化させることを特徴とする。
　具体的には、管状容器１を固定して、上方向及び／又は下方向から押圧することにより、管状容器１は、上下方向に押しつぶされて扁平な形状となる（図３参照）。このとき、管状容器１の中心軸に対して垂直な断面は、楕円形、略楕円形、陸上競技のトラック形状、トラック形状を長手方向に引き延ばした形状等となる。
　このような細胞培養方法及び細胞培養容器によれば、例えば、培養の初期段階では、管状容器１内の最下領域で細胞培養を行い、細胞数が一定以上に増殖した後、管状容器１を押圧変形させて培養面積を増加させ、底面積の増加した管状容器１内の底面で細胞培養を行うことができる。 The cell culturing method of the present embodiment is a cell culturing method using the above cell culturing container, wherein the tubular container 1 is pressed and deformed during cell culturing to change the cell culturing area in the tubular container 1. Features.
Specifically, by fixing the tubular container 1 and pressing from above and / or from below, the tubular container 1 is crushed in the vertical direction and becomes a flat shape (see FIG. 3). At this time, the cross section perpendicular to the central axis of the tubular container 1 has an elliptical shape, a substantially elliptical shape, a track shape for track and field, a shape obtained by extending the track shape in the longitudinal direction, and the like.
According to such a cell culture method and cell culture container, for example, in the initial stage of culture, cell culture is performed in the lowermost region in the tubular container 1, and after the number of cells grows to a certain level or more, the tubular container 1 is The cell culture can be performed on the bottom surface in the tubular container 1 having an increased bottom area by pressing and deforming to increase the culture area.
　すなわち、本実施形態の細胞培養容器に細胞２と培養液３を充填すると、細胞２は管状容器１内の最下領域に集まるため、底面積細胞密度を高めることができる。このため、培養の初期段階における細胞の培養効率を容易に向上させることができる。
　また、細胞数が一定以上に増殖した後に、管状容器１を押圧変形させて、管状容器１内の底面における培養面積を拡張して、細胞の培養効率を簡単に向上させることができる。 That is, when the cell culture container 3 of this embodiment is filled with the cells 2 and the culture solution 3, the cells 2 gather in the lowest region in the tubular container 1, so that the bottom area cell density can be increased. For this reason, the culture efficiency of the cell in the initial stage of culture | cultivation can be improved easily.
In addition, after the number of cells grows to a certain level or more, the tubular container 1 can be pressed and deformed to expand the culture area on the bottom surface in the tubular container 1, thereby easily improving the cell culture efficiency.
　また、このような細胞培養方法及び細胞培養容器を用いてリンパ球を培養することも好ましい。このとき、管状容器１における第一のコーティング領域１２を比較的広く（例えば管状容器１の内面の下半分に）設けて、第一のコーティング薬剤として、抗ＣＤ３抗体を用いることが好ましい。
　そして、例えば管状容器１における底面積細胞密度が大きく、培養条件が不適切である場合に、管状容器１を押圧変形させて、管状容器１内における細胞培養面積を増加させることができる。このため、底面積細胞密度を適切な範囲に制御でき、培養環境を向上させることが可能となる。
　すなわち、管状容器１における細胞数に応じて管状容器１を変形させることにより、最適な底面積細胞密度の環境下で、リンパ球に抗ＣＤ３抗体による活性化刺激を与えることが可能となる。 It is also preferable to culture lymphocytes using such a cell culture method and cell culture vessel. At this time, it is preferable to provide the first coating region 12 in the tubular container 1 relatively wide (for example, in the lower half of the inner surface of the tubular container 1) and use an anti-CD3 antibody as the first coating agent.
For example, when the bottom area cell density in the tubular container 1 is large and the culture conditions are inappropriate, the tubular container 1 can be pressed and deformed to increase the cell culture area in the tubular container 1. For this reason, the bottom area cell density can be controlled within an appropriate range, and the culture environment can be improved.
That is, by deforming the tubular container 1 according to the number of cells in the tubular container 1, it becomes possible to give an activation stimulus to the lymphocytes by the anti-CD3 antibody in an environment having an optimal bottom area cell density.
　また、本実施形態の細胞培養方法を、上記の細胞培養容器を用いた細胞培養方法であって、細胞培養中に管状容器１をその中心軸を中心として回転させて、細胞培養面を変更させる方法とすることも好ましい。
　このとき、本実施形態の細胞培養容器を、例えば図４に示すように、管状容器１内の下方領域（第一のコーティング領域１２）及び上方領域（第二のコーティング領域１３）にコーティング用試薬を塗布して、コーティング層を形成したものとすることができる。上方領域は、管状容器１の中心軸が水平になるように管状容器１が配置された状態において、管状容器１の内面における上半分以内の領域であり、下方領域と同様の方法により定義することができ、例えば、管状容器１の上端を中心として含む円周の１／６の円弧として示される管状容器１内の領域とすることができる。なお、後述する側方領域についても、同様の方法により定義することができる。すなわち、側方領域は、管状容器１の中心軸が水平になるように管状容器１が配置された状態において、管状容器１の内面における左右半分以内の領域であり、例えば、管状容器１の左端又は右端を中心として含む円周の１／６の円弧として示される管状容器１内の領域とすることができる。 Further, the cell culture method of the present embodiment is a cell culture method using the above cell culture container, and the cell culture surface is changed by rotating the tubular container 1 around its central axis during cell culture. A method is also preferred.
At this time, as shown in FIG. 4, for example, the cell culture container of the present embodiment is coated with a coating reagent in the lower region (first coating region 12) and the upper region (second coating region 13) in the tubular container 1. Can be applied to form a coating layer. The upper region is a region within the upper half of the inner surface of the tubular container 1 when the tubular container 1 is arranged so that the central axis of the tubular container 1 is horizontal, and is defined by the same method as the lower region. For example, it can be a region in the tubular container 1 shown as an arc of 1/6 of the circumference including the upper end of the tubular container 1 as the center. Note that a side region described later can also be defined by a similar method. That is, the side region is a region within the left and right halves on the inner surface of the tubular container 1 in a state where the tubular container 1 is arranged so that the central axis of the tubular container 1 is horizontal. Or it can be set as the area | region in the tubular container 1 shown as 1/6 circular arc of the circumference including a right end as a center.
　第一のコーティング領域１２と第二のコーティング領域１３のコーティング用試薬は同一のものであっても異なるものであっても良い。便宜上、第一のコーティング領域１２にコーティングする試薬を第一のコーティング用試薬と称し、第二のコーティング領域１３にコーティングする試薬を第二のコーティング用試薬と称する。 The coating reagents for the first coating region 12 and the second coating region 13 may be the same or different. For convenience, the reagent that coats the first coating region 12 is referred to as a first coating reagent, and the reagent that coats the second coating region 13 is referred to as a second coating reagent.
　このような細胞培養方法及び細胞培養容器を用いれば、第一のコーティング領域１２で細胞を培養した後、管状容器１をその中心軸を中心として１８０°回転（上下反転）させて、第二のコーティング領域１３で細胞を引き続き培養することができる。
　具体的には、例えば、第一のコーティング用試薬としてラミニン５１１を用いると共に、第二のコーティング用試薬としてＭＰＣポリマーを用いて、第一のコーティング領域１２でｉＰＳ細胞を培養した後、第二のコーティング領域１３で細胞を引き続き培養することができる。これにより、第一のコーティング領域１２においてｉＰＳ細胞を管状容器１内に接着させて培養させ、細胞数が一定以上に増加した後に、所定の薬剤などを用いて底面から細胞を剥がし、管状容器１を回転させて、第二のコーティング領域１３においてｉＰＳ細胞の凝集塊を形成させて、培養を効率的に行うことが可能となる。 If such a cell culture method and a cell culture container are used, after culturing cells in the first coating region 12, the tubular container 1 is rotated by 180 ° (vertically inverted) about its central axis, so that the second Cells can subsequently be cultured in the coating region 13.
Specifically, for example, after laminin 511 is used as the first coating reagent and MPC polymer is used as the second coating reagent, iPS cells are cultured in the first coating region 12, and then the second coating reagent is used. Cells can subsequently be cultured in the coating region 13. As a result, iPS cells are adhered and cultured in the tubular container 1 in the first coating region 12, and after the number of cells increases to a certain level or more, the cells are peeled off from the bottom surface using a predetermined drug or the like. Is rotated to form an agglomerate of iPS cells in the second coating region 13 and culture can be performed efficiently.
　さらに、本実施形態の細胞培養方法を、細胞培養中に管状容器１を押圧変形させて管状容器１内における細胞培養面積を変化させる方法と、細胞培養中に管状容器１をその中心軸を中心として回転させて細胞培養面を変更させる方法を組み合わせたものとすることも好ましい。
　具体的には、例えば図５に示すように、第一のコーティング領域１２にラミニン５１１をコーティングし、第二のコーティング領域１３にＭＰＣポリマーをコーティングして、細胞培養容器を形成する。この細胞培養容器は、環状の管状容器１からなるものとする。 Furthermore, the cell culture method of the present embodiment includes a method in which the tubular container 1 is pressed and deformed during cell culture to change the cell culture area in the tubular container 1, and the tubular container 1 is centered on its central axis during cell culture. It is also preferable to combine the methods of rotating and changing the cell culture surface.
Specifically, for example, as shown in FIG. 5, laminin 511 is coated on the first coating region 12, and MPC polymer is coated on the second coating region 13 to form a cell culture container. This cell culture container is composed of an annular tubular container 1.
　そして、ラミニン５１１をコーティングした第一のコーティング領域１２を下にして、管状容器１を押圧変形させて底面積を拡大させる。このようにして管状容器１内の底面にｉＰＳ細胞を播種すると、ｉＰＳ細胞は底面に接着して培養される。このとき、ｉＰＳ細胞は静置培養（静置させた状態での培養）により培養される。
　ｉＰＳ細胞が一定以上に増殖した後、管状容器１の押圧を解除して、管状容器１を円筒形状に戻し、所定の薬剤などを用いて底面から細胞を剥がす。次いで、管状容器１をその中心軸を中心として回転させて、ＭＰＣポリマーをコーティングした第二のコーティング領域１３を下にして細胞培養面とする。そして、ｉＰＳ細胞を培養液３と共に培養し凝集塊を形成後、管状容器１内で一方向に環流させて、細胞培養を行う。これにより、ｉＰＳ細胞は管状容器１内の底面に接着することなく凝集塊を維持したまま、管状容器１内で送流されることで、均一なサイズの凝集塊を得ることが可能となる。 Then, with the first coating region 12 coated with laminin 511 facing down, the tubular container 1 is pressed and deformed to enlarge the bottom area. When iPS cells are seeded on the bottom surface in the tubular container 1 in this way, the iPS cells adhere to the bottom surface and are cultured. At this time, iPS cells are cultured by static culture (culture in a static state).
After the iPS cells have grown to a certain level or more, the pressure on the tubular container 1 is released, the tubular container 1 is returned to a cylindrical shape, and the cells are peeled off from the bottom using a predetermined drug or the like. Next, the tubular container 1 is rotated around its central axis, and the second coating region 13 coated with the MPC polymer is turned downward to form a cell culture surface. Then, iPS cells are cultured with the culture solution 3 to form aggregates, and then circulated in one direction in the tubular container 1 to perform cell culture. As a result, the iPS cells are sent in the tubular container 1 while maintaining the aggregates without adhering to the bottom surface in the tubular container 1, thereby obtaining uniform aggregates.
　また、このような環状の管状容器１からなる細胞培養容器は、リンパ球の培養にも好適に用いることができる。
　具体的には、例えば、抗ＣＤ３抗体をコーティングした第一のコーティング領域１２を下にして、管状容器１を押圧変形させて底面積を拡大させ、静置培養でリンパ球を抗ＣＤ３抗体により活性化させる。
　リンパ球が活性化した後、管状容器１の押圧を解除して、管状容器１を円筒形状に戻し、リンパ球と培養液３をポンプで循環させ、管状容器１内で一方向に環流させて、細胞培養を行う。これにより、リンパ球は、浮遊した状態で増殖していく。このため、リンパ球が抗ＣＤ３抗体に必要以上の長時間に亘って接触して過度に活性化することを防止し、リンパ球の増殖効率を向上させることが可能となる。 Moreover, the cell culture container which consists of such an annular tubular container 1 can be used suitably also for culture | cultivation of a lymphocyte.
Specifically, for example, with the first coating region 12 coated with an anti-CD3 antibody facing down, the tubular container 1 is pressed and deformed to enlarge the bottom area, and lymphocytes are activated by the anti-CD3 antibody in stationary culture. Make it.
After the lymphocytes are activated, the pressure on the tubular container 1 is released, the tubular container 1 is returned to the cylindrical shape, the lymphocytes and the culture solution 3 are circulated by a pump, and are circulated in one direction in the tubular container 1. Perform cell culture. Thereby, lymphocytes proliferate in a floating state. For this reason, it is possible to prevent the lymphocytes from being excessively activated by contacting the anti-CD3 antibody for an unnecessarily long time, and to improve the proliferation efficiency of lymphocytes.
　また、リンパ球を培養する場合には、リンパ球を活性化させた後、管状容器１をその中心軸を中心として回転させて、その後に押圧して変形させて管状容器１内の底面における培養面積を拡張し、リンパ球が第一のコーティング領域１２に接しないようにして、リンパ球が過度に活性化することを防止し、リンパ球の増殖効率を向上させることもできる。 In the case of culturing lymphocytes, after activating the lymphocytes, the tubular container 1 is rotated around its central axis, and then pressed and deformed to culture on the bottom surface in the tubular container 1. The area can be expanded so that the lymphocytes do not contact the first coating region 12 to prevent the lymphocytes from being activated excessively, and the proliferation efficiency of the lymphocytes can be improved.
　さらに、本実施形態の細胞培養容器を、例えば図６に示すように、管状容器１内の下方領域（第一のコーティング領域１２）、上方領域（第二のコーティング領域１３）、側方領域（第三のコーティング領域１４、第四のコーティング領域１５）にコーティング用試薬をコーティングして乾燥させ、コーティング層を形成したものとすることも好ましい。このとき、第一のコーティング領域１２、第二のコーティング領域１３、第三のコーティング領域１４、及び第四のコーティング領域１５のコーティング用試薬は同一のものであっても異なるものであっても良い。また、本実施形態の細胞培養容器において、コーティング領域として五種類以上の領域を形成させても良い。 Furthermore, as shown in FIG. 6, for example, the cell culture container of this embodiment includes a lower region (first coating region 12), an upper region (second coating region 13), a side region ( It is also preferable that the third coating region 14 and the fourth coating region 15) are coated with a coating reagent and dried to form a coating layer. At this time, the coating reagents for the first coating region 12, the second coating region 13, the third coating region 14, and the fourth coating region 15 may be the same or different. . Moreover, in the cell culture container of this embodiment, you may form five or more types of area | regions as a coating area | region.
　本実施形態の細胞培養システムは、上記の細胞培養容器と、細胞培養中に前記管状容器を押圧して変形させ、管状容器内の底面積を変化させる押圧装置とを備えたことを特徴とする。このような細胞培養システムによれば、細胞培養容器を押圧することで、細胞密度に応じて培養面積を増加させることが可能となる。
　また、本実施形態の細胞培養システムは、細胞培養中に前記管状容器をその中心軸を中心として回転させ、前記管状容器内の底面を移動させる回転駆動装置を備えたことを特徴とする。このような細胞培養システムによれば、細胞培養容器を回転させることで、培養面を選択することができ、例えばコーティング面から未コーティング面へ変更することが可能となる。 The cell culture system of the present embodiment includes the cell culture container described above and a pressing device that presses and deforms the tubular container during cell culture to change the bottom area in the tubular container. . According to such a cell culture system, it is possible to increase the culture area according to the cell density by pressing the cell culture container.
In addition, the cell culture system of the present embodiment includes a rotation drive device that rotates the tubular container around its central axis during cell culture and moves the bottom surface in the tubular container. According to such a cell culture system, it is possible to select a culture surface by rotating the cell culture container, and for example, it is possible to change from a coated surface to an uncoated surface.
　具体的には、例えば図７に示すように、本実施形態の細胞培養システムは、管状容器１と、押圧装置４と、回転駆動装置５とを備えたものとすることが好ましい。
　同図において、管状容器１は積載台上に載置され、上方から押圧装置４により押圧可能な構成となっている。このように管状容器１は、押圧装置４により押圧されることで、扁平な形状に変形し、管状容器１内の底面積を増加させることが可能となっている。 Specifically, for example, as shown in FIG. 7, the cell culture system of the present embodiment preferably includes a tubular container 1, a pressing device 4, and a rotation driving device 5.
In the figure, a tubular container 1 is placed on a loading table and can be pressed by a pressing device 4 from above. Thus, the tubular container 1 is deformed into a flat shape by being pressed by the pressing device 4, and the bottom area in the tubular container 1 can be increased.
　管状容器１の両端に備えられたポート１１は、それぞれ回転駆動装置５に取り付けられ、回転駆動装置５によって、管状容器１の中心軸を中心として回転駆動され、これによって管状容器１を回転させる構成となっている。管状容器１を回転させるに際しては、管状容器１は支持台などにより、積載台から浮かせた状態とすることが好ましい。回転駆動装置５による管状容器１の回転は、例えばその中心軸を中心として右回り及び左回りに１８０°まで回転可能なものとすることができる。
　また、回転駆動装置５は、ポート１１に取り付けられるものに限定されず、例えば環状の管状容器１における接続部材１６に取り付けて、接続部材１６と共に管状容器１を回転させるものや、管状容器１に直接取り付けて、管状容器１を回転させるものなどとすることもできる。 Ports 11 provided at both ends of the tubular container 1 are respectively attached to the rotational drive device 5 and are rotationally driven around the central axis of the tubular container 1 by the rotational drive device 5, thereby rotating the tubular container 1. It has become. When rotating the tubular container 1, the tubular container 1 is preferably in a state of being floated from the loading table by a support table or the like. The rotation of the tubular container 1 by the rotation drive device 5 can be rotated to 180 ° clockwise and counterclockwise around the central axis, for example.
Further, the rotation drive device 5 is not limited to the one attached to the port 11. For example, the rotation drive device 5 is attached to the connection member 16 in the annular tubular container 1 and rotates the tubular container 1 together with the connection member 16, or the tubular container 1. The tubular container 1 may be directly attached and rotated.
　また、本実施形態の細胞培養システムは、管状容器の端部が一又は二以上の接続部材で接続されて環状の密閉空間が形成された細胞培養容器と、ポンプとを備え、管状容器にポンプが取り付けられ、ポンプを駆動させることにより、管状容器内の培養液と細胞が、管状容器内において一方向に送流されながら培養される構成とすることも好ましい。このような細胞培養システムによれば、細胞を培養液と共に管状容器内で一方向に環流させることができ、均一なサイズの細胞凝集塊を得ることが可能となる。 In addition, the cell culture system of the present embodiment includes a cell culture container in which an end of the tubular container is connected by one or more connecting members to form an annular sealed space, and a pump. It is also preferable that the culture medium and the cells in the tubular container are cultured while being fed in one direction in the tubular container by driving the pump. According to such a cell culture system, cells can be circulated in one direction in a tubular container together with a culture solution, and a cell aggregate of uniform size can be obtained.
　本実施形態の細胞培養容器の製造方法は、上記の細胞培養容器の製造方法であって、管状容器内にコーティング用試薬を注入し、管状容器内の長手方向にコーティング用試薬を移動させて、管状容器内の下方領域にコーティング用試薬を塗布し、コーティング用試薬を乾燥させて、コーティング層を形成することを特徴とする。
　具体的には、図８に示すように、管状容器１内にコーティング用試薬６を注入し、管状容器１内の長手方向にコーティング用試薬６を移動させて、また必要に応じて、管状容器１内の円周方向にコーティング用試薬６を移動させることにより、管状容器１内の下方領域（第一のコーティング領域１２）にコーティング用試薬６を塗布し、コーティング層を形成する。
　これにより、管状容器１内の最下領域を含む下方領域において、コーティング用試薬６が固相化されて、コーティング層が形成される。 The method for producing a cell culture container according to the present embodiment is the above-described method for producing a cell culture container, injecting a coating reagent into a tubular container, and moving the coating reagent in the longitudinal direction in the tubular container, A coating reagent is applied to a lower region in the tubular container, and the coating reagent is dried to form a coating layer.
Specifically, as shown in FIG. 8, the coating reagent 6 is injected into the tubular container 1, the coating reagent 6 is moved in the longitudinal direction in the tubular container 1, and, if necessary, the tubular container By moving the coating reagent 6 in the circumferential direction in 1, the coating reagent 6 is applied to the lower region (first coating region 12) in the tubular container 1 to form a coating layer.
Thereby, in the lower region including the lowermost region in the tubular container 1, the coating reagent 6 is solid-phased to form a coating layer.
　また、管状容器１内の下方領域にコーティング用試薬６を塗布した後に、管状容器１を押し潰して管状容器１内の上面を管状容器１内の下面に接触させることにより、管状容器１内の下方領域に塗布されたコーティング用試薬６を管状容器１内の上方領域にも塗布し、同一のコーティング用試薬６を管状容器１内の上方領域及び下方領域の両方に固相化して、コーティング層を形成させることも好ましい。
　さらに、管状容器１内の最下領域を含む下方領域にコーティング層を形成した後、管状容器１をその中心軸を中心として回転させて、管状容器１内の新たな最下領域を含む下方領域にコーティング層を形成することも好ましい。 In addition, after applying the coating reagent 6 to the lower region in the tubular container 1, the tubular container 1 is crushed to bring the upper surface in the tubular container 1 into contact with the lower surface in the tubular container 1, thereby The coating reagent 6 applied to the lower region is also applied to the upper region in the tubular container 1, and the same coating reagent 6 is solidified in both the upper region and the lower region in the tubular container 1 to form a coating layer. It is also preferable to form.
Furthermore, after forming a coating layer in the lower region including the lowermost region in the tubular container 1, the lower region including the new lowermost region in the tubular container 1 is obtained by rotating the tubular container 1 around its central axis. It is also preferable to form a coating layer.
　以上説明したように、本実施形態の細胞培養容器、細胞培養システム、細胞培養方法、及び細胞培養容器の製造方法によれば、細胞培養容器において、培養部のみにコーティング用試薬を簡単にコーティングできると共に、高価なコーティング用試薬の使用量を低減でき、培養面積を可変にすることが可能となる。
　また、容量の大きい細胞培養容器を容易に製造することも可能となる。
　さらに、環状の管状容器からなるものとすれば、管状容器内の培養液と細胞を一方向に環流することができ、これによって細胞の凝集塊が互いにぶつかり合うことを抑止して均一な凝集塊を得ることも可能である。 As described above, according to the cell culture container, the cell culture system, the cell culture method, and the cell culture container manufacturing method of the present embodiment, the coating reagent can be easily coated only on the culture part in the cell culture container. At the same time, the amount of expensive coating reagent used can be reduced, and the culture area can be made variable.
In addition, it is possible to easily manufacture a cell culture container having a large capacity.
Furthermore, if it consists of an annular tubular container, the culture solution and the cells in the tubular container can be circulated in one direction, thereby preventing the aggregates of cells from colliding with each other and uniform aggregates. It is also possible to obtain
　本発明は、以上の実施形態に限定されるものではなく、本発明の範囲内において、種々の変更実施が可能であることは言うまでもない。
　例えば、複数の環状の管状容器が接続部材により接続されたものとしたり、管状容器に一又は二以上の培養液バッグや細胞回収バッグを接続したものとするなど適宜変更することが可能である。
　この明細書に記載の文献及び本願のパリ優先の基礎となる日本出願明細書の内容を全てここに援用する。 It goes without saying that the present invention is not limited to the above embodiment, and that various modifications can be made within the scope of the present invention.
For example, it is possible to appropriately change such that a plurality of annular tubular containers are connected by a connecting member, or one or two or more culture solution bags or cell collection bags are connected to the tubular container.
The contents of the documents described in this specification and the specification of the Japanese application that is the basis of Paris priority of the present application are all incorporated herein.
　本発明は、コーティング用試薬を培養容器に塗布して培養が行われる細胞を、大量に培養する場合に好適に利用することが可能である。 The present invention can be suitably used for culturing a large amount of cells to be cultured by applying a coating reagent to a culture vessel.
　１　管状容器
　１１　ポート
　１２　第一のコーティング領域
　１３　第二のコーティング領域
　１４　第三のコーティング領域
　１５　第四のコーティング領域
　１６　接続部材
　２　細胞
　３　培養液
　４　押圧装置
　５　回転駆動装置
　６　コーティング用試薬 DESCRIPTION OF SYMBOLS 1 Tubular container 11 Port 12 1st coating area | region 13 2nd coating area | region 14 3rd coating area | region 15 4th coating area | region 16 Connection member 2 Cell 3 Culture solution 4 Pressing device 5 Rotation drive device 6 Reagent for coating

Claims (13)

  1. 　管状容器からなる細胞培養容器であって、細胞を培養するための密閉空間が前記管状容器内に形成され、前記管状容器の内面の一部にコーティング用試薬がコーティングされたことを特徴とする細胞培養容器。 A cell culture container comprising a tubular container, wherein a sealed space for culturing cells is formed in the tubular container, and a coating reagent is coated on a part of the inner surface of the tubular container Culture container.
  2. 　前記管状容器の中心軸が水平になるように前記管状容器が配置された状態において、前記管状容器の内面における下半分以内の領域にコーティング用試薬がコーティングされたことを特徴とする請求項１記載の細胞培養容器。 The coating reagent is coated in a region within the lower half of the inner surface of the tubular container in a state where the tubular container is arranged so that the central axis of the tubular container is horizontal. Cell culture container.
  3. 　前記管状容器の中心軸が水平になるように前記管状容器が配置された状態において、前記管状容器の内面における下半分以内の領域、及び、上半分以内の領域にコーティング用試薬がコーティングされたことを特徴とする請求項１又は２記載の細胞培養容器。 In the state where the tubular container is arranged so that the central axis of the tubular container is horizontal, a coating reagent is coated on the area within the lower half and the area within the upper half of the inner surface of the tubular container. The cell culture container according to claim 1 or 2.
  4. 　前記管状容器内に二以上の種類のコーティング用試薬がコーティングされたことを特徴とする請求項１～３のいずれかに記載の細胞培養容器。 The cell culture container according to any one of claims 1 to 3, wherein two or more kinds of coating reagents are coated in the tubular container.
  5. 　前記コーティング用試薬が、タンパク質、ＭＰＣポリマー、ハイドロゲル、フィブロネクチン、ゼラチン、又はラミニンであることを特徴とする請求項１～４のいずれかに記載の細胞培養容器。 The cell culture container according to any one of claims 1 to 4, wherein the coating reagent is protein, MPC polymer, hydrogel, fibronectin, gelatin, or laminin.
  6. 　前記管状容器が、当該細胞培養容器の外部と物質の出し入れをするためのポートを備えたことを特徴とする請求項１～５のいずれかに記載の細胞培養容器。 The cell culture container according to any one of claims 1 to 5, wherein the tubular container is provided with a port for taking in and out substances from the outside of the cell culture container.
  7. 　前記管状容器が、ガス透過性を有することを特徴とする請求項１～６のいずれかに記載の細胞培養容器。 The cell culture container according to any one of claims 1 to 6, wherein the tubular container has gas permeability.
  8. 　前記管状容器の端部が一又は二以上の接続部材で接続されて環状の密閉空間が形成されたことを特徴とする請求項１～７のいずれかに記載の細胞培養容器。 The cell culture container according to any one of claims 1 to 7, wherein an end of the tubular container is connected by one or more connecting members to form an annular sealed space.
  9. 　請求項１～８のいずれかに記載の細胞培養容器と、
    　細胞培養中に前記管状容器を押圧して変形させ、前記管状容器内の底面積を変化させる押圧装置と、を備えた
    　ことを特徴とする細胞培養システム。     A cell culture container according to any one of claims 1 to 8,
    And a pressing device that changes the bottom area of the tubular container by pressing and deforming the tubular container during cell culture.
  10. 　細胞培養中に前記管状容器をその中心軸を中心として回転させ、前記管状容器内の底面を移動させる回転駆動装置を備えた
    　ことを特徴とする請求項９記載の細胞培養システム。     The cell culture system according to claim 9, further comprising: a rotation driving device that rotates the tubular container around its central axis during cell culture and moves a bottom surface of the tubular container.
  11. 　請求項１～８のいずれかに記載の細胞培養容器を用いた細胞培養方法であって、
    　細胞培養中に前記管状容器を押圧変形させて、前記管状容器内における細胞培養面積を変化させることを特徴とする細胞培養方法。     A cell culture method using the cell culture container according to any one of claims 1 to 8,
    A cell culture method, wherein the tubular container is pressed and deformed during cell culture to change a cell culture area in the tubular container.
  12. 　細胞培養中に前記管状容器をその中心軸を中心として回転させて、細胞培養面を変更させることを特徴とする請求項１１記載の細胞培養方法。 The cell culture method according to claim 11, wherein the cell culture surface is changed by rotating the tubular container about its central axis during cell culture.
  13. 　請求項１～８のいずれかに記載の細胞培養容器の製造方法であって、
    　前記管状容器内に前記コーティング用試薬を注入し、
    　前記管状容器内の長手方向に前記コーティング用試薬を移動させて、前記管状容器の中心軸が水平になるように前記管状容器が配置された状態において、前記管状容器の内面における下半分以内の領域に前記コーティング用試薬を塗布し、
    　コーティング層を形成する
    　ことを特徴とする細胞培養容器の製造方法。     A method for producing a cell culture container according to any one of claims 1 to 8,
    Injecting the coating reagent into the tubular container,
    A region within the lower half of the inner surface of the tubular container in a state where the coating reagent is moved in the longitudinal direction in the tubular container and the tubular container is arranged so that the central axis of the tubular container is horizontal. Apply the coating reagent to
    A method for producing a cell culture container, comprising forming a coating layer.
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