Cell culture vessel, cell manufacturing method, and cell culture vessel manufacturing method

Abstract

To provide cell culture containers with an excellent gas permeation performance and strength for culturing cells at high density.SOLUTION: Provided is a bag-shaped, closed cell culture container 1 having at least one port 12 and formed by opposing planar members 11, at least one of the planar members 11 consisting of a gas permeable film, a culture space S for culturing cells being provided on the inner surface side of the container in the gas permeable film, a plurality of thin-walled portions 111 and convex portions 112 being formed on the surface of the gas permeable film forming the culture space S, and the thickness of the thin-walled portion 111 being 75 μm or less.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to cell culture technology, and more particularly to a cell culture vessel that enables cells to be cultured at high density. [Background technology]

[0002] In recent years, there has been a demand for efficient mass cultivation of cells and tissues in an artificial environment in the fields of pharmaceutical production, gene therapy, regenerative medicine, immunotherapy, and the like. In this situation, it has been proposed to automatically culture large amounts of cells in a closed system using a bag-like cell culture vessel.

[0003] When cells are cultured using a bag-shaped cell culture vessel, the strength and gas permeability of the vessel are important. For example, when a bag-shaped cell culture vessel is manufactured using a gas-permeable film such as polyethylene or ethylene vinyl acetate, a thickness of 100 μm or more is usually required to ensure the strength of the vessel.

[0004] However, in this case, the density of cultured cells is 500,000 cells / cm 2 At higher densities, the gas permeability of the container becomes insufficient, causing a problem of a decrease in the oxygen concentration around the cells, resulting in a decrease in proliferation efficiency. On the other hand, when cell culture vessels are manufactured using gas-permeable films with a thickness of, for example, 50 μm or less, although the gas permeability is sufficient even when the cultured cells are at a high density, there is a problem that the strength of the vessel is insufficient, which raises the risk of the vessel breaking and makes handling difficult. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2016-77164 [Patent Document 2] Patent No. 4806761 Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, in order to improve the gas permeability and strength of bag-shaped cell culture vessels, multilayer films made by laminating films of different materials and densities are sometimes used as the film that makes up the vessel, but this has not resulted in a significant improvement in performance.

[0007] Silicone is another material with excellent gas permeability. Although silicone has low strength, it can still achieve excellent gas permeability even when made thicker than 300 μm, ensuring strength during handling. However, silicone materials have the problem that it is difficult to weld ports, etc. Furthermore, harmful substances are eluted from silicone materials during radiation sterilization, etc., which may adversely affect subsequent culture performance. Furthermore, silicone materials are difficult to treat with the surface required for culturing adherent cells, so although it is possible to manufacture culture vessels for suspension cells, it is difficult to manufacture culture vessels for adherent cells.

[0008] Therefore, the inventors conducted extensive research and formed multiple thin and convex portions on the surface of the gas-permeable film that constitutes the container, and by making the thickness of the thin portions 75 μm or less, they succeeded in ensuring strength when handled and achieving a gas permeability that allows cells to be cultured at high densities, thereby completing the present invention.

[0009] Patent Document 1 describes a cell culture vessel equipped with a support layer on a gas-permeable film. However, the support layer is intended to prevent bending of the gas-permeable film placed flat inside a vessel such as a flask, and does not improve the strength of the vessel, so it does not solve the problem of improving the gas permeability and strength of a bag-shaped cell culture vessel to enable high-density cell culture.

[0010] Furthermore, Patent Document 2 describes that the gas permeability of the bag-shaped container is improved by providing a plurality of ventilation holes in the position of the presser plate of the culture tray that contacts the bag-shaped container. However, since cells usually accumulate on the underside of a bag-shaped container, simply having ventilation holes in the pressure plate has little effect in improving gas permeability, and this culture tray was not able to fully solve the problem of a decrease in oxygen concentration around the cells in the container.

[0011] In contrast, according to the present invention, the convex portions formed on the surface of the gas-permeable film that constitutes the container ensure strength during handling, and the thin-walled portions of a specified thickness make it possible to improve gas permeability.

[0012] The present invention has been made in consideration of the above circumstances, and aims to provide a cell culture vessel having excellent gas permeability and strength for culturing cells at high densities, a method for producing cells, and a method for producing a cell culture vessel. [Means for solving the problem]

[0013] In order to achieve the above-mentioned object, the cell culture vessel of the present invention is a bag-shaped closed cell culture vessel having at least one port and formed by opposing planar articles, at least one of which is made of a gas-permeable film, and a culture space for culturing cells is provided on the inner surface side of the vessel in the gas-permeable film, and the gas-permeable film that forms this culture space has a plurality of thin-walled portions and convex portions formed therein, and the thickness of the thin-walled portions is 75 μm or less. It is also preferable that the cell culture vessel of the present invention is configured so that the thickness of the thin-walled portion is 20 to 35 μm.

[0014] It is also preferable that the cell culture vessel of the present invention is configured such that the surface of the gas-permeable film on which the convex portions are formed is the outer surface of the vessel in the gas-permeable film, and the inner surface of the vessel in the gas-permeable film is flat and free of irregularities. Furthermore, it is also preferable that the cell culture vessel of the present invention is configured such that the surface of the gas-permeable film on which the convex portions are formed is the inner surface of the vessel in the gas-permeable film, and a plurality of small protrusions are formed on the outer surface of the vessel in the gas-permeable film, and when the outer surface is brought into contact with a flat surface, the small protrusions form a breathable space between the gas-permeable film and the flat surface.

[0015] It is also preferable that the cell culture vessel of the present invention be configured such that both of the opposing planar substrates are made of gas-permeable films, and that the plurality of thin-walled portions and the convex portions are formed on each gas-permeable film.

[0016] In addition, it is also preferable that the cell culture vessel of the present invention has a configuration in which the convex portions are formed in the form of a plurality of approximately triangular prisms arranged in parallel in a mountain range shape. Furthermore, it is also preferable that the cell culture vessel of the present invention has a configuration in which a plurality of approximately triangular prisms are formed in parallel in a mountain range shape as the convex portion, and a plurality of other approximately triangular prisms are formed in parallel in a mountain range shape intersecting the plurality of approximately triangular prisms, and these plurality of approximately triangular prisms are arranged in a lattice shape.

[0017] It is also preferable that the cell culture vessel of the present invention is configured so that adjacent parallel convex portions are formed with an interval of 1 to 5 mm. Furthermore, it is also preferable that the cell culture vessel of the present invention is configured such that the adjacent parallel convex portions are formed without any gap therebetween, and the thin-walled portions are formed in a linear or dotted pattern.

[0018] The method for producing cells of the present invention is a method for culturing cells using the cell culture vessel described above.

[0019] Furthermore, the method for manufacturing a cell culture vessel of the present invention is a method for manufacturing a bag-shaped closed cell culture vessel having at least one port and formed by opposing planar articles, in which a gas-permeable film is used as at least one of the planar articles, a plurality of thin-walled portions with a thickness of 75 μm or less and convex portions are formed on the surface of the gas-permeable film, and the peripheral portions of the opposing planar articles are bonded together to form a culture space for culturing cells on the inner surface side of the vessel in the gas-permeable film. [Effects of the Invention]

[0020] According to the present invention, it is possible to provide a cell culture vessel having excellent gas permeability and strength for culturing cells at high density, a method for producing cells, and a method for producing a cell culture vessel. [Brief explanation of the drawings]

[0021] [Figure 1] 1A is a plan view of a cell culture vessel according to a first embodiment of the present invention, and FIG. 1B is a cross-sectional view taken along line AA. [Figure 2] 1A and 1B are an enlarged view and a BB cross-sectional view, respectively, of a gas-permeable film used in a cell culture vessel according to a first embodiment of the present invention. [Figure 3] FIG. 1 is a diagram showing the oxygen permeability of various gas-permeable films used in the cell culture vessel according to the first embodiment of the present invention. [Figure 4] 1 is a schematic diagram showing a state in which culture is performed using a cell culture vessel according to a first embodiment of the present invention. FIG. [Figure 5] 1A is an enlarged view of a gas-permeable film used in a cell culture vessel according to a second embodiment of the present invention, and FIG. 1B is a cross-sectional view taken along the line CC. [Figure 6] FIG. 1A is a cross-sectional view of a gas-permeable film used in a cell culture vessel according to a third embodiment of the present invention, and FIG. 1B is a cross-sectional view of a gas-permeable film used in a cell culture vessel according to a fourth embodiment of the present invention. [Figure 7]1A is a cross-sectional view of a gas-permeable film used in a cell culture vessel according to a fifth embodiment of the present invention, and FIG. 1B is a cross-sectional view of a gas-permeable film used in a cell culture vessel according to a sixth embodiment of the present invention. [Figure 8] 1A is a cross-sectional view of a gas-permeable film used in a cell culture vessel according to a seventh embodiment of the present invention, and FIG. 1B is a cross-sectional view of a gas-permeable film used in a cell culture vessel according to an eighth embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0022] Hereinafter, embodiments of the cell culture vessel, the cell production method, and the cell culture vessel production method of the present invention will be described in detail. However, the present invention is not limited to the specific contents of the following embodiments.

[0023] [First embodiment] First, a first embodiment of the present invention will be described with reference to FIGS. The cell culture vessel of this embodiment is a bag-shaped, closed cell culture vessel having at least one port and formed by opposing planar substrates, at least one of which is made of a gas-permeable film, a culture space for culturing cells is provided on the inner surface of the container of the gas-permeable film, the gas-permeable film forming this culture space has a plurality of thin-walled portions and protrusions, and the thickness of the thin-walled portions is 75 μm or less. It is also preferable that the thickness of the thin-walled portions is 20 to 35 μm.

[0024] Specifically, as shown in FIG. 1(a), the cell culture vessel 1 of this embodiment can be obtained by heat-sealing the peripheral edges H of, for example, two rectangular planar substrates 11. The space formed between the two planar substrates 11 is used as a culture space S for culturing cells. The area of ​​the planar substrate 11 that forms the culture space S constitutes the culture section of the cell culture vessel 1. There are no particular limitations on the overall shape of the planar substrate 11, the shape of the peripheral edges H, or the shape of the culture space S, and any shape can be used.

[0025] At least one of the planar substrates 11 is made of a gas-permeable film, and it is preferable that both of them are made of gas-permeable films. The "flat" of the flat object 11 does not mean that the surface of the object is flat without any irregularities, but rather that the overall shape of the object is roughly flat This means that the surface of the planar substrate 11 may be uneven.

[0026] As shown in Figure 1(b), in the cell culture vessel 1 of this embodiment, a plurality of thin-walled portions 111 are formed on the planar substrate 11, which is a gas-permeable film. In addition, a plurality of convex portions 112 are formed on the outer surface of the container made of the gas-permeable film. Furthermore, the inner surface of the container made of the gas-permeable film is flat and free of irregularities. The number of convex portions formed on the gas-permeable film is not limited to multiple, and may include convex portions formed by connecting multiple convex portions to form essentially one convex portion, such as the convex portions in the cell culture vessel of the second embodiment.

[0027] In this embodiment, both of the opposing flat substrates 11 are made of gas-permeable film, and each gas-permeable film has a thin portion 111 and a convex portion 112 formed thereon. However, it is also possible to configure only one of the flat substrates 11 to be made of gas-permeable film, with a thin portion 111 and a convex portion 112 formed in the gas-permeable film.

[0028] As shown in FIGS. 2(a) and 2(b), the convex portions 112 are preferably formed by arranging a plurality of approximately triangular prisms in parallel in a mountain range shape (hereinafter, this may be referred to as a mountain range pattern). By forming the convex portions 112 in a mountain range pattern in this manner, the contact area between the gas permeable film and the flat surface can be reduced, preventing the gas permeability performance of the cell culture vessel 1 from being impaired. Furthermore, such a mountain range pattern can improve the strength of the protrusions of the convex portions 112.

[0029] In the cell culture vessel 1 of this embodiment, adjacent parallel convex portions 112 are formed with an interval of 1 to 5 mm. That is, the width (width in the short dimension direction) of the thin-walled portion 111 is formed to be 1 to 5 mm. This allows the cell culture vessel 1 to have sufficient strength and gas permeability when handled. Furthermore, to further improve the strength and gas permeability of the cell culture vessel 1 when handled, the width of the thin-walled portion 111 is preferably 1 to 3 mm, and more preferably 1 to 2 mm.

[0030] The spacing between adjacent parallel convex portions 112 may be less than 1 mm, and even if adjacent parallel convex portions 112 are formed without any spacing between them, as will be explained in the third embodiment described later, not only can strength during handling be ensured, but the gas permeability can be made large enough to allow cells to be cultured at high density.

[0031] Here, with reference to FIG. 3, the oxygen permeability of a gas permeable film having a plurality of thin portions and protrusions formed thereon will be described. First, two types of flat gas-permeable films with no irregularities and seven types of films with thin portions and protrusions were prepared. Specifically, a planar film (1) made of LLDPE (Linear Low Density Polyethylene) and having a thickness of 110 μm and a planar film (2) having a thickness of 30 μm were prepared.

[0032] Furthermore, films (3) to (8) each having a thin portion and a convex portion made of LLDPE were prepared. The thicknesses of the thin portions were 100 μm, 75 μm, 50 μm, 35 μm, 30 μm, and 25 μm, the pitch of the convex portions was 2 mm, the lengths of the bases of the convex portions were 0.15 mm, 0.27 mm, 0.36 mm, 0.4 mm, 0.41 mm, and 0.42 mm, the widths of the thin portions were 1.85 mm, 1.73 mm, 1.64 mm, 1.6 mm, 1.59 mm, and 1.58 mm, the heights of the convex portions were 0.27 mm, 0.51 mm, 0.67 mm, 0.75 mm, 0.77 mm, and 0.82 mm, and the angle of the convex portions (the angle between the base and the oblique side of the convex portion) was 75°.

[0033] Furthermore, a film (9) made of LLDPE and having thin portions and convex portions formed thereon was prepared in which adjacent parallel convex portions were formed continuously without any gaps (thickness of thin portion 25 μm, pitch of convex portions 0.11 mm, length of base of convex portions 0.11 mm, height of convex portions 0.19 mm, angle of convex portions 75°). The films (3) to (9) were all flat films made of LLDPE with a thickness of 110 μm, on which the respective convex portions were formed by thermal transfer.

[0034] Then, oxygen permeability was measured based on the flow method (constant pressure method, JIS K 7126-2) using a gas permeability measuring device (GTR Tech Co., Ltd., flow type gas / water vapor permeability measuring device GTR-20X).

[0035] As shown in Figure 3, the oxygen permeability (ml / m) of the flat films with thicknesses of 110 μm and 30 μm was 2 The oxygen permeabilities of films with thin and convex portions (thin portion thicknesses of 100 μm, 75 μm, 50 μm, 35 μm, 30 μm, 25 μm, and 25 μm (continuous convex portions)) were 8000, 11000, 17000, 25000, 27000, 28000, and 27000, respectively.

[0036] In this way, the film having the thin portions and protrusions not only ensures strength during handling by providing the protrusions, but also enables its oxygen permeability to be higher than that of a 110 μm-thick flat film. That is, as mentioned above, these films are obtained by processing a 110 μm-thick flat film, but by forming the thin portions and protrusions, it is possible to further improve the oxygen permeability.

[0037] In particular, those with a thin-walled portion having a thickness of 75 μm or less can achieve an oxygen permeability of 11,000 or more, and those with a thin-walled portion having a thickness of 35 μm to 25 μm can achieve an excellent oxygen permeability of 25,000 or more. The oxygen permeability is the same whether the convex portions are formed on the outside or inside of the container in the gas-permeable film.

[0038] Resin films and the like are suitable for use as the planar substrate 11, and polyolefin resins such as polyethylene and polypropylene can be used. Examples include polyethylene, copolymers of ethylene and α-olefins, copolymers of ethylene and vinyl acetate, and ionomers using ethylene, acrylic acid, or methacrylic acid copolymers, and metal ions. Polyolefins, styrene-based elastomers, and polyester-based thermoplastic elastomers can also be used. Furthermore, soft vinyl chloride resin, polybutadiene resin, ethylene-vinyl acetate copolymer, chlorinated polyethylene resin, polyurethane-based thermoplastic elastomers, polyester-based thermoplastic elastomers, silicone-based thermoplastic elastomers, styrene-based elastomers such as SBS (styrene-butadiene-styrene), SIS (styrene-isoprene-styrene), SEBS (styrene-ethylene-butylene-styrene), SEPS (styrene-ethylene-propylene-styrene), polyolefin resins, and fluorine-based resins can also be used.

[0039] Of the materials for the planar substrate 11, it is preferable to use a thermoplastic resin with particularly excellent gas permeability as the gas permeable film, and for example, a polyolefin resin such as polyethylene (LLDPE) or polypropylene can be suitably used. Furthermore, in order to make the inside of the cell culture vessel 1 visible, it is preferable that the gas permeable film be made of a transparent material.

[0040] Generally, the thinner the gas-permeable film, the higher its gas permeability. However, the thinner the gas-permeable film, the more difficult it is to handle. In particular, films with thicknesses of 30 μm or 20 μm are similar to food wrap (thickness of approximately 10 μm), and therefore the container strength is insufficient, leading to the risk of the bag breaking. However, the cell culture vessel 1 of this embodiment has the convex portions 112 on the gas permeable film, and therefore can be handled even if the thickness of the thin portion 111 of the gas permeable film is made relatively thin. Therefore, the thickness of the gas-permeable film in the cell culture vessel 1 of this embodiment is preferably less than 100 μm, more preferably 75 μm or less, and even more preferably 35 μm or less and 20 μm or more.

[0041] In the cell culture vessel 1 of this embodiment, at least one port 12 is provided by being sandwiched between the peripheral edges H of two planar substrates 11 and heat-sealed. In FIG. 1, two ports 12 are provided facing each other at both ends in the longitudinal direction of the planar substrate 11, but the number of ports 12 is not limited to this and may be one or three or more. The port 12 may be made of a thermoplastic resin such as polyethylene, polypropylene, vinyl chloride, polystyrene elastomer, or FEP.

[0042] FIG. 4 shows the state of cell culture using the cell culture vessel 1 of this embodiment. In the figure, a cell culture vessel 1 is filled with a culture medium, and cells 2 are seeded in a culture space S whose volume has been expanded by the culture medium, and the cells are then cultured. The cells to be cultured using the cell culture vessel 1 are not particularly limited, and may be floating cells such as lymphocytes and dendritic cells that are cultured while suspended in a culture medium, or adhesive cells such as induced pluripotent stem cells (iPS cells), neural stem cells, embryonic stem cells (ES cells), mesenchymal stem cells, hepatocytes, pancreatic islet cells, cardiac muscle cells, corneal endothelial cells, and lymphocytes in an activation process that are cultured while attached to the culture area within the vessel.

[0043] The cell production method of this embodiment is characterized by culturing cells using the above-described cell culture vessel 1. Similarly, the cell production methods of the embodiments described below are characterized by culturing cells using the cell culture vessel of each embodiment, but the description thereof will be omitted below.

[0044] The method for manufacturing a cell culture vessel of this embodiment is a method for manufacturing a bag-shaped closed cell culture vessel 1 having at least one port 12 and formed by opposing planar articles 11, characterized in that a gas-permeable film is used as at least one of the planar articles 11, multiple thin-walled portions 111 with a thickness of 75 μm or less and convex portions 112 are formed in the gas-permeable film, and the peripheral portions H of the opposing planar articles 11 are bonded together to form a culture space S for culturing cells 2 on the inner surface side of the vessel in the gas-permeable film.

[0045] In the method for producing a cell culture vessel of this embodiment, it is also preferable to form a plurality of thin-walled portions 111 with a thickness of 20 to 35 μm and protrusions 112 in the gas-permeable film. Furthermore, in the method for manufacturing a cell culture vessel of this embodiment, it is also preferable to form convex portions 112 on the outer surface of the vessel in the gas-permeable film, and make the inner surface of the vessel in the gas-permeable film flat and free of irregularities.

[0046] In addition, in the method for manufacturing a cell culture vessel of this embodiment, it is also preferable to use gas-permeable films on both of the opposing planar substrates 11 and form a plurality of thin portions 111 and protrusions 112 on each gas-permeable film. Furthermore, in the method for manufacturing a cell culture vessel of this embodiment, it is also preferable to form a plurality of approximately triangular prisms as the protrusions 112 in a mountain range shape.

[0047] In the method for manufacturing a cell culture vessel of this embodiment, it is also preferable to form adjacent parallel convex portions 112 with an interval of 1 to 5 mm between them. Furthermore, in the method for producing a cell culture vessel of this embodiment, it is also preferable to form the convex portions 112 by thermal transfer or cast film formation.

[0048] Thus, according to the cell culture vessel, cell manufacturing method, and cell culture vessel manufacturing method of this embodiment, it is possible to manufacture a cell culture vessel having excellent gas permeability and strength, in which protrusions are formed on the outside of the vessel at intervals in a mountain-like pattern, and it is possible to efficiently culture large amounts of cells using this cell culture vessel.

[0049] [Second embodiment] Next, a second embodiment of the present invention will be described with reference to FIG. The cell culture vessel of this embodiment differs from the first embodiment in that a gas-permeable film is formed with a plurality of thin-walled portions 111a and convex portions 112a, and as convex portions 112a, a plurality of approximately triangular prisms are formed in parallel in a mountain range shape on the outer surface of the container in the gas-permeable film, and a plurality of other approximately triangular prisms are formed in parallel in a mountain range shape, intersecting the plurality of approximately triangular prisms, and these plurality of approximately triangular prisms are arranged in a lattice shape. The other configurations of the cell culture vessel, the cell manufacturing method, and the method for manufacturing a cell culture vessel of this embodiment are the same as those of the first embodiment, except for the points described below.

[0050] Specifically, as shown in Figures 5(a) and 5(b), in the cell culture vessel of this embodiment, a plurality of thin-walled portions 111a are formed on a planar substrate 11a, which is a gas-permeable film. Furthermore, convex portions 112a are formed on the outer surface of the container made of the gas-permeable film. Furthermore, the inner surface of the container made of the gas-permeable film is flat and free of irregularities.

[0051] In this embodiment, both of the opposing flat substrates 11a are made of gas-permeable film, and each gas-permeable film has a thin portion 111a and a convex portion 112a formed thereon. However, it is also possible to configure only one of the flat substrates 11a to be made of gas-permeable film, with the thin portion 111a and the convex portion 112a formed on that gas-permeable film.

[0052] It is preferable that the convex portion 112a is formed by forming a plurality of approximately triangular prisms in parallel in a mountain range shape, and by intersecting the plurality of approximately triangular prisms with a plurality of other approximately triangular prisms in parallel in a mountain range shape (hereinafter, this may be referred to as a lattice pattern). In FIGS. 5(a) and 5(b), the height of the approximately triangular prism in the horizontal direction is smaller than the height of the approximately triangular prism in the vertical direction.

[0053] This allows a breathable space to be formed between the flat substrate 11a and the plane when the cell culture vessel of this embodiment is placed on a plane with the surface on which the convex portion 112a is formed facing downward. Furthermore, even when the surface of the cell culture vessel of this embodiment on which the convex portion 112a is formed is facing upward and pressed from above using a pressure plate or the like, a breathable space is formed between the flat substrate 11a and the plane. In this way, by forming a convex portion on the cell culture vessel, a breathable space is formed between the cell culture vessel and the flat surface, which also applies to the following embodiments.

[0054] By forming the convex portions 112a in a grid pattern in this way, the contact area between the gas permeable film and the flat surface can be reduced, and the gas permeability of the cell culture vessel can be prevented from being impaired. Furthermore, such a grid pattern can improve the strength of the protrusions of the convex portions 112a. Furthermore, in FIG. 5(a), the protrusions 112a are formed by a plurality of approximately triangular prisms intersecting perpendicularly with a plurality of other approximately triangular prisms, but they may also be formed so as to intersect obliquely.

[0055] In the cell culture vessel of this embodiment, adjacent parallel convex portions 112a are formed with an interval of 1 to 5 mm between them. That is, the width of the thin-walled portion 111a is formed to be 1 to 5 mm, but the width of the thin-walled portion 111a is preferably 1 to 3 mm, and more preferably 1 to 2 mm.

[0056] In the manufacturing method of the cell culture vessel of this embodiment, it is preferable to form a plurality of approximately triangular prisms in a mountain range-like arrangement as the convex portion 112a, and to form a plurality of other approximately triangular prisms in a mountain range-like arrangement intersecting the plurality of approximately triangular prisms, and to form these plurality of approximately triangular prisms in a lattice-like arrangement.

[0057] Thus, according to the cell culture vessel, cell manufacturing method, and cell culture vessel manufacturing method of this embodiment, it is possible to manufacture a cell culture vessel having excellent gas permeability and strength, in which protrusions are formed on the outside of the vessel at intervals in a grid pattern, and it is possible to efficiently culture large amounts of cells using this cell culture vessel.

[0058] [Third embodiment] Next, a third embodiment of the present invention will be described with reference to FIG. 6(a). The cell culture vessel of this embodiment differs from the first embodiment in that a gas-permeable film has a plurality of thin-walled portions 111b and convex portions 112b formed thereon, and the convex portions 112b are formed as a plurality of roughly triangular prisms arranged side by side in a mountain range shape on the outer surface of the gas-permeable film of the vessel, and adjacent parallel convex portions 112b are formed without any gaps between them, so that the thin-walled portions 111b are formed in a linear shape. The other configurations of the cell culture vessel, the cell manufacturing method, and the method for manufacturing a cell culture vessel of this embodiment are the same as those of the first embodiment, except for the points described below.

[0059] Specifically, as shown in Figure 6(a), in the cell culture vessel of this embodiment, a plurality of thin-walled portions 111b are formed in parallel lines (in the depth direction in the figure) on the planar substrate 11b, which is a gas-permeable film. Furthermore, convex portions 112b are formed continuously and parallel to each other in the front-to-back direction (in the depth direction in the figure) on the outer surface of the container made of the gas-permeable film. Furthermore, the inner surface of the container made of the gas-permeable film is flat and free of irregularities.

[0060] In this embodiment, both of the opposing flat substrates 11b are made of gas-permeable film, and each gas-permeable film has a thin portion 111b and a convex portion 112b formed thereon. However, it is also possible to configure only one of the flat substrates 11b to be made of gas-permeable film, with a thin portion 111b and a convex portion 112b formed in the gas-permeable film.

[0061] As shown in FIG. 6(a), the protrusions 112b are preferably formed by arranging a plurality of approximately triangular prisms in parallel in a mountain range shape (mountain range pattern). By forming the convex portions 112b in a mountain range pattern in this way, the contact area between the gas permeable film and the flat surface can be reduced, and the gas permeability of the cell culture vessel can be prevented from being impaired. Furthermore, such a mountain range pattern can improve the strength of the protrusions of the convex portions 112b.

[0062] In the method for manufacturing a cell culture vessel of this embodiment, it is preferable that the convex portions 112b are formed in parallel with each other without any gap between them, and that the thin-walled portions 111b are formed linearly.

[0063] Thus, according to the cell culture vessel, cell manufacturing method, and cell culture vessel manufacturing method of this embodiment, it is possible to manufacture a cell culture vessel having excellent gas permeability and strength, in which the convex portions are formed on the outside of the vessel in a mountain-like pattern without any gaps between them, and it is possible to efficiently culture large amounts of cells using this cell culture vessel.

[0064] [Fourth embodiment] Next, a fourth embodiment of the present invention will be described with reference to FIG. 6(b). The cell culture vessel of this embodiment differs from the first embodiment in that a plurality of thin-walled portions 111c and convex portions 112c are formed in the gas-permeable film, and the convex portions 112c are formed in a lattice pattern on the outer surface of the container in the gas-permeable film, and adjacent parallel convex portions 112c are formed without any gaps between them, so that the thin-walled portions 111c are formed in a dot-like pattern. The other configurations of the cell culture vessel, the cell manufacturing method, and the method for manufacturing a cell culture vessel of this embodiment are the same as those of the first embodiment, except for the points described below.

[0065] Specifically, as shown in Figure 6(b), in the cell culture vessel of this embodiment, a plurality of thin-walled portions 111c are formed in a dotted pattern on a planar substrate 11c, which is a gas-permeable film. Furthermore, on the outer surface of the container made of the gas-permeable film, convex portions 112c are formed continuously and parallel to each other in the front-to-back direction (the depth direction in the figure) and the left-to-right direction without any gaps. Furthermore, the inner surface of the container made of the gas-permeable film is flat and free of irregularities.

[0066] In this embodiment, both of the opposing flat substrates 11c are made of gas-permeable film, and each gas-permeable film has a thin portion 111c and a convex portion 112c formed thereon. However, it is also possible to configure only one of the flat substrates 11c to be made of gas-permeable film, with a thin portion 111c and a convex portion 112c formed in the gas-permeable film.

[0067] It is preferable that the convex portion 112c is formed by forming a plurality of approximately triangular prisms in parallel in a mountain range shape, and by intersecting the plurality of approximately triangular prisms with a plurality of other approximately triangular prisms in parallel in a mountain range shape (a grid pattern). 6(b), the height of the approximately triangular prism in the horizontal direction is shorter than the height of the approximately triangular prism in the vertical direction (depth direction in the figure). This allows a breathable space to be formed between the planar substrate 11c and the flat surface when the cell culture vessel of this embodiment is placed on the flat surface.

[0068] By forming the convex portions 112c in a grid pattern in this way, the contact area between the gas permeable film and the flat surface can be reduced, and the gas permeability of the cell culture vessel can be prevented from being impaired. Furthermore, such a grid pattern can improve the strength of the protrusions of the convex portions 112c. Furthermore, the protrusions 112c can also be formed so that a plurality of approximately triangular prisms intersect obliquely with a plurality of other approximately triangular prisms.

[0069] In the method for manufacturing a cell culture vessel of this embodiment, it is preferable that the protrusions 112c are formed by forming a plurality of approximately triangular prisms in a mountain range shape, and that a plurality of other approximately triangular prisms are formed in a mountain range shape intersecting the plurality of approximately triangular prisms, so that these plurality of approximately triangular prisms are formed in a lattice shape. It is also preferable that adjacent protrusions 112c are formed in parallel with each other without any gaps, and that the thin-walled portions 111c are formed in a dotted shape.

[0070] Thus, according to the cell culture vessel, cell manufacturing method, and cell culture vessel manufacturing method of this embodiment, a cell culture vessel having excellent gas permeability and strength can be manufactured in which the protrusions are formed on the outside of the vessel without any gaps in a grid pattern, and this cell culture vessel can be used to efficiently culture large amounts of cells.

[0071] [Fifth embodiment] Next, a fifth embodiment of the present invention will be described with reference to FIG. The cell culture vessel of this embodiment differs from the first embodiment in that a gas-permeable film has a plurality of thin-walled portions 111d and convex portions 112d formed thereon, and the convex portions 112d are formed as a plurality of roughly triangular prisms arranged side by side in a mountain range shape on the inner surface of the gas-permeable film of the vessel, with adjacent parallel convex portions 112d formed with spaces between them, and a plurality of small protrusions 113d are formed on the outer surface of the gas-permeable film of the vessel, and when the outer surface is brought into contact with a flat surface, the small protrusions 113d form a breathable space between the gas-permeable film and the flat surface. The other configurations of the cell culture vessel, the cell manufacturing method, and the method for manufacturing a cell culture vessel of this embodiment are the same as those of the first embodiment, except for the points described below.

[0072] Specifically, as shown in Figure 7(a), in the cell culture vessel of this embodiment, a plurality of thin-walled portions 111d are formed on a planar substrate 11d, which is a gas-permeable film. Furthermore, convex portions 112d are formed on the inner surface of the gas-permeable film vessel at intervals in parallel in the front-to-rear direction (the depth direction in the figure). Furthermore, a plurality of small protrusions 113d are formed on the outer surface of the gas-permeable film vessel.

[0073] The shape of the small protrusions 113d is not particularly limited as long as they can form a breathable space between the gas-permeable film and the plane, and they can have various shapes such as a cone, a triangular pyramid, a polygonal pyramid, a rectangular parallelepiped, a mountain range pattern, a lattice pattern, etc. The same applies to the small protrusions in the following embodiments.

[0074] In this embodiment, both opposing flat substrates 11d are made of gas-permeable film, and each gas-permeable film has a thin portion 111d, a convex portion 112d, and a small protrusion 113d formed thereon. However, it is also possible to configure only one of the flat substrates 11d to be made of gas-permeable film, with the thin portion 111d, the convex portion 112d, and the small protrusion 113d formed on the gas-permeable film.

[0075] As shown in FIG. 7(a), the protrusions 112d are preferably formed by arranging a plurality of approximately triangular prisms in parallel in a mountain range shape (mountain range pattern). By forming the convex portions 112d in a mountain range pattern in this way, the contact area between the gas permeable film and the flat surface can be reduced, and the gas permeability of the cell culture vessel can be prevented from being impaired. Furthermore, such a mountain range pattern can improve the strength of the protrusions of the convex portions 112d.

[0076] In this embodiment, the thin portion 111d is formed so that its width is 1 to 5 mm, but the width of the thin portion 111d is preferably 1 to 3 mm, and more preferably 1 to 2 mm.

[0077] In the method for manufacturing a cell culture vessel of this embodiment, it is preferable that the convex portions 112d are formed by forming a plurality of approximately triangular prisms in a mountain range shape at intervals on the inner surface of the vessel in the gas-permeable film, and that a plurality of small protrusions 113d are formed on the outer surface of the vessel in the gas-permeable film, so that when the outer surface is brought into contact with a flat surface, the small protrusions 113d form a breathable space between the gas-permeable film and the flat surface.

[0078] Thus, according to the cell culture vessel, cell manufacturing method, and cell culture vessel manufacturing method of this embodiment, it is possible to manufacture a cell culture vessel having excellent gas permeability and strength, in which protrusions are formed on the inside of the vessel at intervals in a mountain-like pattern, and it is possible to efficiently culture large amounts of cells using this cell culture vessel.

[0079] [Sixth embodiment] Next, a sixth embodiment of the present invention will be described with reference to FIG. 7(b). The cell culture vessel of this embodiment differs from the fifth embodiment in that a plurality of thin-walled portions 111e and convex portions 112e are formed in the gas-permeable film, and the convex portions 112e are formed as a plurality of approximately triangular prisms arranged side by side in a mountain range shape on the inner surface of the vessel in the gas-permeable film, and the convex portions 112d are formed parallel to each other with no gap between them. The cell culture vessel, the method for producing cells, and other configurations of the method for producing a cell culture vessel of this embodiment are the same as those of the fifth embodiment, except for the points described below.

[0080] Specifically, as shown in Figure 7(b), in the cell culture vessel of this embodiment, a plurality of thin-walled portions 111e are formed in parallel lines (in the depth direction in the figure) on the planar substrate 11e, which is a gas-permeable film. Furthermore, convex portions 112e are formed on the inner surface of the gas-permeable film vessel, parallel to each other in the front-rear direction (in the depth direction in the figure), without any gaps between them. Furthermore, a plurality of small protrusions 113e are formed on the outer surface of the gas-permeable film vessel.

[0081] In this embodiment, both of the opposing flat substrates 11e are made of gas-permeable film, and each gas-permeable film has a thin portion 111e, a convex portion 112e, and a small protrusion 113e formed thereon. However, it is also possible to configure only one of the flat substrates 11e to be made of gas-permeable film, with the thin portion 111e, the convex portion 112e, and the small protrusion 113e formed on the gas-permeable film.

[0082] As shown in FIG. 7(b), the protrusions 112e are preferably formed by arranging a plurality of approximately triangular prisms in parallel in a mountain range shape (mountain range pattern). By forming the convex portions 112e in a mountain range pattern in this way, the contact area between the gas permeable film and the flat surface can be reduced, and the gas permeability of the cell culture vessel can be prevented from being impaired. Furthermore, such a mountain range pattern can improve the strength of the protrusions of the convex portions 112e.

[0083] In the method for manufacturing a cell culture vessel of this embodiment, it is preferable that the convex portions 112e are formed by forming a plurality of roughly triangular prisms in a mountain range shape on the inner surface of the vessel in the gas-permeable film without any gaps between them, and that a plurality of small protrusions 113e are formed on the outer surface of the vessel in the gas-permeable film, so that when the outer surface is brought into contact with a flat surface, the small protrusions 113e form a breathable space between the gas-permeable film and the flat surface.

[0084] Thus, according to the cell culture vessel, cell manufacturing method, and cell culture vessel manufacturing method of this embodiment, it is possible to manufacture a cell culture vessel having excellent gas permeability and strength, in which protrusions are formed on the inside of the vessel in a mountain-like pattern without any gaps between them, and it is possible to efficiently culture large amounts of cells using this cell culture vessel.

[0085] [Seventh embodiment] Next, a seventh embodiment of the present invention will be described with reference to FIG. 8(a). The cell culture vessel of this embodiment differs from the fifth embodiment in that a plurality of thin-walled portions 111f and convex portions 112f are formed in the gas-permeable film, and the convex portions 112f are formed by providing a plurality of approximately triangular prisms in a lattice pattern on the inner surface of the vessel in the gas-permeable film, and adjacent parallel convex portions 112f are formed with a gap between them. The cell culture vessel, the method for producing cells, and other configurations of the method for producing a cell culture vessel of this embodiment are the same as those of the fifth embodiment, except for the points described below.

[0086] Specifically, as shown in Figure 8(a), in the cell culture vessel of this embodiment, a plurality of thin-walled portions 111f are formed on a planar substrate 11f, which is a gas-permeable film. Furthermore, convex portions 112f are formed on the inner surface of the gas-permeable film vessel at intervals in parallel in the front-rear direction (depth direction in the figure) and left-right direction. Furthermore, a plurality of small protrusions 113f are formed on the outer surface of the gas-permeable film vessel.

[0087] In this embodiment, both opposing flat substrates 11d are made of gas-permeable film, and each gas-permeable film has a thin portion 111d, a convex portion 112d, and a small protrusion 113d formed thereon. However, it is also possible to configure only one of the flat substrates 11d to be made of gas-permeable film, with the thin portion 111d, the convex portion 112d, and the small protrusion 113d formed on the gas-permeable film.

[0088] It is preferable that the convex portion 112f is formed by forming a plurality of approximately triangular prisms in parallel in a mountain range shape, and by intersecting the plurality of approximately triangular prisms with a plurality of other approximately triangular prisms in parallel in a mountain range shape (a grid pattern). 8(a), the height of the approximately triangular prism in the horizontal direction is shorter than the height of the approximately triangular prism in the vertical direction (depth direction in the figure). This allows a breathable space to be formed between the planar substrate 11f and the flat surface when the cell culture vessel of this embodiment is placed on the flat surface.

[0089] By forming the convex portions 112f in a grid pattern in this way, the contact area between the gas permeable film and the flat surface can be reduced, and the gas permeability of the cell culture vessel can be prevented from being impaired. Furthermore, such a grid pattern can improve the strength of the protrusions of the convex portions 112f. Furthermore, the protrusions 112f may be formed so that a plurality of approximately triangular prisms intersect with a plurality of other approximately triangular prisms at an angle.

[0090] In this embodiment, the thin portion 111f is formed so that the width is 1 to 5 mm, but the width of the thin portion 111f is preferably 1 to 3 mm, and more preferably 1 to 2 mm.

[0091] In the method for manufacturing a cell culture vessel of this embodiment, it is preferable that the convex portions 112f are formed by forming a plurality of approximately triangular prisms in a lattice pattern at intervals on the inner surface of the vessel in the gas-permeable film, and that a plurality of small protrusions 113f are formed on the outer surface of the vessel in the gas-permeable film, so that when the outer surface is brought into contact with a flat surface, the small protrusions 113f form a breathable space between the gas-permeable film and the flat surface.

[0092] Thus, according to the cell culture vessel, cell manufacturing method, and cell culture vessel manufacturing method of this embodiment, it is possible to manufacture a cell culture vessel having excellent gas permeability and strength, in which protrusions are formed on the inside of the vessel at intervals in a grid pattern, and it is possible to efficiently culture large amounts of cells using this cell culture vessel.

[0093] [Eighth embodiment] Next, an eighth embodiment of the present invention will be described with reference to FIG. 8(b). The cell culture vessel of this embodiment differs from the fifth embodiment in that a plurality of thin-walled portions 111g and convex portions 112g are formed in the gas-permeable film, and the convex portions 112g are formed by providing a plurality of approximately triangular prisms in a lattice pattern on the inner surface of the vessel in the gas-permeable film, and adjacent parallel convex portions 112g are formed continuously without any gaps. The cell culture vessel, the method for producing cells, and other configurations of the method for producing a cell culture vessel of this embodiment are the same as those of the fifth embodiment, except for the points described below.

[0094] Specifically, as shown in Figure 8(b), in the cell culture vessel of this embodiment, a plurality of thin-walled portions 111g are formed in a dotted pattern on a planar substrate 11g, which is a gas-permeable film. Furthermore, convex portions 112g are formed continuously on the inner surface of the gas-permeable film vessel, parallel to each other in the front-rear direction (the depth direction in the figure) and the left-right direction, without any gaps between them. Furthermore, a plurality of small protrusions 113g are formed on the outer surface of the gas-permeable film vessel.

[0095] In this embodiment, both of the opposing flat substrates 11g are made of gas-permeable film, and each gas-permeable film has a thin portion 111g, a convex portion 112g, and a small protrusion 113g formed thereon. However, it is also possible to configure only one of the flat substrates 11g to be made of gas-permeable film, with the thin portion 111g, the convex portion 112g, and the small protrusion 113g formed on the gas-permeable film.

[0096] It is preferable that the convex portion 112g is formed by forming a plurality of approximately triangular prisms in parallel in a mountain range shape, and by intersecting the plurality of approximately triangular prisms with a plurality of other approximately triangular prisms in parallel in a mountain range shape (a grid pattern). 8(b), the height of the approximately triangular prism in the horizontal direction is shorter than the height of the approximately triangular prism in the vertical direction (the depth direction in the figure). This allows a breathable space to be formed between the planar substrate 11g and the flat surface when the cell culture vessel of this embodiment is placed on the flat surface.

[0097] By forming the convex portions 112g in a grid pattern in this way, the contact area between the gas permeable film and the flat surface can be reduced, and the gas permeability of the cell culture vessel can be prevented from being impaired. Furthermore, such a grid pattern can improve the strength of the protrusions of the convex portions 112g. Furthermore, the protrusions 112g may be formed so that a plurality of approximately triangular prisms intersect with a plurality of other approximately triangular prisms at an angle.

[0098] In the method for manufacturing a cell culture vessel of this embodiment, it is preferable that the convex portions 112g are formed by forming a plurality of approximately triangular prisms in a lattice pattern on the inner surface of the vessel in the gas-permeable film without any gaps, and that a plurality of small protrusions 113g are formed on the outer surface of the vessel in the gas-permeable film, so that when the outer surface is brought into contact with a flat surface, the small protrusions 113g form a breathable space between the gas-permeable film and the flat surface.

[0099] Thus, according to the cell culture vessel, cell manufacturing method, and cell culture vessel manufacturing method of this embodiment, it is possible to manufacture a cell culture vessel having excellent gas permeability and strength, in which the convex portions are formed continuously on the inside of the vessel in a grid pattern without any gaps, and it is possible to efficiently culture large amounts of cells using this cell culture vessel.

[0100] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the present invention. For example, the shape of the convex portion formed on the cell culture vessel may be various shapes different from those of the embodiments, or parts of the first to eighth embodiments may be combined to form a cell culture vessel in which convex portions are formed on both the outside and inside of the vessel. [Industrial Applicability]

[0101] The present invention can be suitably used when, for example, cells are cultured in large quantities at high density using a cell culture bag. [Explanation of symbols]

[0102] 1 cell culture vessel 11,11a~11g Planar equipment 111,111a~111g Thin wall part 112, 112a~112g convex part 113d~113g Small protrusion 12 ports 2 cells S culture space H Periphery

Claims

1. A bag-like closed cell culture vessel having at least one port and formed by opposing planar substrates, At least one of the planar substrates is made of a gas-permeable film, a culture space for culturing cells is provided on the inner surface side of the container in the gas-permeable film, a plurality of thin portions and convex portions are formed on the gas-permeable film that forms this culture space, and the thickness of the thin portions is 25 μm or more and 75 μm or less, The surface of the gas-permeable film on which the convex portions are formed is the outer surface of the container of the gas-permeable film, and the inner surface of the container of the gas-permeable film is flat and free of irregularities. A cell culture vessel characterized by:

2. 2. The cell culture vessel according to claim 1, wherein the thickness of the thin-walled portion is 25 μm or more and 35 μm or less.

3. 3. The cell culture vessel according to claim 1, wherein both of the opposing planar substrates are made of gas-permeable films, and the plurality of thin portions and the convex portions are formed on each of the gas-permeable films.

4. 4. The cell culture vessel according to claim 1, wherein the convex portions are formed by arranging a plurality of substantially triangular prisms in a mountain range shape.

5. The cell culture vessel according to any one of claims 1 to 3, characterized in that the convex portion is formed by a plurality of approximately triangular prisms arranged in parallel in a mountain range shape, and a plurality of other approximately triangular prisms arranged in parallel in a mountain range shape, intersecting the plurality of approximately triangular prisms, and these plurality of approximately triangular prisms are arranged in a lattice shape.

6. 6. The cell culture vessel according to claim 1, wherein the adjacent parallel convex portions are formed at intervals of 1 to 5 mm.

7. 6. The cell culture vessel according to claim 1, wherein the adjacent parallel convex portions are formed without any gaps therebetween, and the thin-walled portions are formed in a linear or dotted shape.

8. A method for producing cells, comprising culturing cells using the cell culture vessel according to any one of claims 1 to 7.

9. A method for producing a bag-shaped closed cell culture vessel having at least one port and formed by opposing planar substrates, comprising: A gas-permeable film is used as at least one of the planar substrates, and a plurality of thin-walled portions and convex portions each having a thickness of 25 μm or more and 75 μm or less are formed on the gas-permeable film; the surface of the gas-permeable film on which the convex portions are formed is an outer surface of a container in the gas-permeable film, and the inner surface of the container in the gas-permeable film is flat and free of irregularities; The peripheral edges of the opposing planar substrates are bonded together to form a culture space for culturing cells on the inner surface side of the container in the gas-permeable film. A method for manufacturing a cell culture vessel, comprising:

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