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

Abstract

To provide a cell culture container having excellent gas permeability and strength for culturing cells at high density.SOLUTION: Provided is a bag-shaped closed cell culture container 1 having at least one port and formed by planar instruments 11 facing to each other. At lest one of the planar instruments 11 is made of a gas permeable film. The outer surface of at least one gas permeable film is provided with a protruded portion 111 having a shape different from the inner surface shape of the gas permeable film. A culture space S for culturing cells is provided on the inner surface side of the gas permeable film. When the gas permeable film is brought into contact with a flat surface, the protruded portion 111 forms an air-permeable space between the gas permeable film and the flat surface.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a cell culture technique, and more particularly to a cell culture vessel for culturing cells 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 view of this situation, it has been proposed to automatically culture large amounts of cells in a closed system using a bag-shaped cell culture vessel.

[0003] When culturing cells using a bag-shaped cell culture vessel, the vessel may be placed inside an incubator or clamped between a pressing device to suppress the movement of culture fluid within the vessel and stabilize the culture environment. However, bag-shaped cell culture vessels are usually formed by bonding two pieces of film together, and because their outer surface is flat, there is a problem that when they are placed in an incubator, gas permeation through the bottom of the vessel is insufficient.

[0004] In other words, incubator mounting stages are usually made of perforated plates known as punched metal, but the opening rate of these plates is often less than 50%. As a result, except for the perforated areas, the surface of the cell culture vessel and the mounting stage come into close contact with each other, preventing gas from passing into the vessel. As a result, when the cells consume a lot of oxygen during high-density culture, the oxygen concentration around the cells in the vessel decreases, which is a problem. Furthermore, when the container is used while being clamped in a pressing tool, the surface of the container is in close contact with the stand or pressing member of the pressing tool, which also inhibits the gas permeability of the container, lowering the oxygen concentration around the cells and reducing the cell proliferation efficiency.

[0005] Furthermore, when cells are mass-cultured using a bag-shaped cell culture vessel, the strength and gas permeability of the vessel are important. For example, when a 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.

[0006] However, in this case, the density of cultured cells was 500,000 cells / cm 2 At such high 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 a cell culture vessel is manufactured using a gas-permeable film having a thickness of, for example, 50 μm or less, although the gas permeability is sufficient even when the cultured cells are in a high density state, there is a problem that the strength of the vessel is insufficient, there is a risk of the vessel breaking, and handling becomes difficult. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 4806761 Summary of the Invention [Problem to be solved by the invention]

[0008] Here, Patent Document 1 describes that the gas permeability of the bag-shaped container is improved by providing a plurality of ventilation holes at the position of the presser plate of the culture tray that contacts the bag-shaped container. However, since cells typically accumulate on the underside of a bag-shaped container, simply having air 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.

[0009] Therefore, the inventors conducted extensive research and succeeded in making the gas permeability performance of the cell culture vessel sufficient even during high-density culture by providing protrusions on the outer surface of the gas permeable film that constitutes the container, so that when the gas permeable film is brought into contact with a flat surface, the protrusions form a breathable space between the gas permeable film and the flat surface, thereby completing the present invention. Furthermore, such a cell culture vessel can ensure sufficient strength during handling even when the thickness of the gas permeable film is less than 100 μm.

[0010] 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 density, a method for manufacturing the cell culture vessel, a method for manufacturing cells, and a cell culture device. [Means for solving the problem]

[0011] 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 equipment, at least one of the planar equipment being made of a gas-permeable film, at least one of the gas-permeable films having a protrusion on the outer surface thereof, the protrusion having a shape different from the shape of the inner surface of the gas-permeable film, and a culture space for culturing cells on the inner surface side of the gas-permeable film, and configured such that when the gas-permeable film is brought into contact with a flat surface, the protrusion forms a breathable space between the gas-permeable film and the flat surface.

[0012] In addition, it is preferable that the cell culture vessel of the present invention is configured such that, in the outer surface region of the gas permeable film that is outside the culture space, a plurality of approximately triangular prisms are formed in parallel in a mountain range shape as the protrusions. Furthermore, it is also preferable that the cell culture vessel of the present invention is configured such that both of the opposing planar substrates are made of gas-permeable films, and the protrusions are provided on the outer surface of each of the gas-permeable films.

[0013] 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 on at least one of the planar articles, and protrusions having a shape different from the shape of the inner surface of the gas-permeable film are formed on the outer surface of the gas-permeable film so that when the gas-permeable film is brought into contact with a flat surface, a breathable space is formed between the gas-permeable film and the flat surface, 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 gas-permeable film.

[0014] Furthermore, the method for producing cells of the present invention is a method for culturing cells using the above-mentioned cell culture vessel.

[0015] Furthermore, the cell culture device of the present invention is configured to include a culture tool comprising the above-mentioned cell culture vessel, a flat stand on which the cell culture vessel is placed, and a pressing member that presses the cell culture vessel placed on the stand, and in which a breathable space is formed between the gas permeable film and the stand and / or between the gas permeable film and the pressing member by the protrusions formed on the cell culture vessel. Effect of the Invention

[0016] 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 a cell culture vessel, a method for producing cells, and a cell culture device. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1A is a plan view and FIG. 1B is a cross-sectional view taken along line AA of a cell culture vessel according to one embodiment of the present invention. [Diagram 2] FIG. 2A is an enlarged view of a gas-permeable film used in a cell culture vessel according to one embodiment of the present invention, and FIG. 2B is a schematic diagram showing cell culture in the vessel. [Diagram 3]FIG. 2 is a diagram showing a state in which a cell culture vessel according to one embodiment of the present invention is fixed to a culture tool and cultured. [Figure 4] FIG. 1 is a graph showing the measurement results of gas permeability of the cell culture vessels in Test 1 (Example 1, Comparative Example 1). [Diagram 5] FIG. 1 shows the results of cell culture using the cell culture vessels of Test 2 (Example 2, Comparative Example 2). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Hereinafter, embodiments of the cell culture vessel, the cell culture vessel manufacturing method, the cell manufacturing method, and the cell culture device of the present invention will be described in detail. However, the present invention is not limited to the specific contents of the following embodiments and examples.

[0019] The cell culture vessel of this embodiment has at least one port and is a bag-shaped closed cell culture vessel formed by opposing planar equipment, at least one of the planar equipment being made of a gas-permeable film, and the outer surface of at least one of the gas-permeable films is provided with a protrusion having a shape different from the inner surface shape of the gas-permeable film, and a culture space for culturing cells is provided on the inner surface side of the gas-permeable film, and when the gas-permeable film is brought into contact with a flat surface, the protrusion forms a breathable space between the gas-permeable film and the flat surface.

[0020] Specifically, as shown in Fig. 1(a), the cell culture vessel 1 of this embodiment can be obtained by heat-sealing the peripheral parts 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 forming the culture space S in the planar substrate 11 constitutes the culture section in the cell culture vessel 1. Note that the overall shape of the planar substrate 11, the shape of the peripheral parts H, and the shape of the culture space S are not particularly limited, and any shape can be used.

[0021] 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. In addition, the "planar" of the planar article 11 does not mean that the surface of the article is flat and without any irregularities, but means that the overall shape of the article is roughly planar, and also includes planar articles 11 with irregularities formed on the surface.

[0022] As shown in FIG. 1(b), in the cell culture vessel 1 of this embodiment, the outer surface of the planar substrate 11, which is a gas-permeable film, is provided with protrusions 111, and when the gas-permeable film is brought into contact with a flat surface, the protrusions 111 form a breathable space between the gas-permeable film and the flat surface.

[0023] The shape of the protrusion 111 in this embodiment is not particularly limited, but it is preferable that the protrusion 111 has a plurality of approximately triangular prisms arranged in parallel in a mountain range (hereinafter, sometimes referred to as a mountain range pattern), as shown in Figures 1(b) and 2. By forming the protrusions 111 in a mountain-like pattern in this manner, the contact area between the gas-permeable film and the flat surface can be reduced, thereby preventing the gas permeability performance of the cell culture vessel 1 from being impaired.

[0024] Furthermore, such a mountain range pattern can improve the strength of the protrusions in the protruding portion 111, and can prevent the protrusions from being damaged, such as being distorted, by the pressing force of a pressing member, for example. Furthermore, the protrusion 111 may be a plurality of rectangular parallelepipeds arranged in parallel, and may have various other shapes.

[0025] In this embodiment, the protrusions 111 are preferably formed in the outer surface region of the gas permeable film that corresponds to the outside of the culture space S. It is also preferable to use gas-permeable films on both of the opposing planar substrates 11 and provide protrusions 111 on the outer surface of each of the gas-permeable films.

[0026] By providing the cell culture vessel 1 of this embodiment with such protrusions 111, when the cell culture vessel 1 is placed on a flat surface, a breathable space is formed between the gas permeable film facing the flat surface and the flat surface, making it possible to greatly improve the gas permeability performance of the cell culture vessel 1. Furthermore, when the upper surface of the cell culture vessel 1 is pressed with a pressing member, a breathable space is formed between the gas permeable film facing the plane of the pressing member and the plane, making it possible to greatly improve the gas permeability performance of the cell culture vessel 1 in this case as well.

[0027] Furthermore, by blowing air into the space formed by the protrusions 111 between the gas permeable film and the flat surface, it is possible to further improve the gas permeability of the cell culture vessel 1. Moreover, by forming the protrusions 111 on the cell culture vessel 1 of this embodiment in this manner, the strength of the cell culture vessel 1 can also be improved.

[0028] In addition, it is preferable that the shape of the protrusion 111 in this embodiment is different from the shape of the inner surface of the gas permeable film, for example, different from a shape in which multiple wells are formed on the inner surface of a container and the outer surfaces of the wells form hemispheres on the outside of the container. In other words, it is preferable that the protrusion 111 formed on the outer surface of the gas permeable film in the cell culture vessel 1 of this embodiment has a shape that can more effectively achieve ventilation between the gas permeable film and the flat surface, regardless of the inner surface shape of the gas permeable film.

[0029] Therefore, the shape of the inner surface of the gas-permeable film in the cell culture vessel 1 of this embodiment can be set regardless of the shape of the protrusions 111, and it is preferable to make it flat without any irregularities, and it is also preferable to form irregularities on the inner surface of the gas-permeable film regardless of the shape of the protrusions 111 to increase the culture area within the vessel.

[0030] As the flat substrate 11, a resin film or the like can be suitably used, and polyolefin resins such as polyethylene and polypropylene can be used. For example, polyethylene, copolymers of ethylene and α-olefin, copolymers of ethylene and vinyl acetate, ionomers using ethylene, acrylic acid or methacrylic acid copolymers, and metal ions can be mentioned. In addition, polyolefins, styrene-based elastomers, polyester-based thermoplastic elastomers, silicone-based thermoplastic elastomers, silicone resins, etc. can also be used. Furthermore, silicone rubber, soft vinyl chloride resins, polybutadiene resins, ethylene-vinyl acetate copolymers, chlorinated polyethylene resins, 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, fluorine-based resins, etc. may also be used.

[0031] Of the materials for the planar substrate 11, it is preferable to use a thermoplastic resin having particularly excellent gas permeability as the gas permeable film, and for example, a polyolefin resin such as polyethylene or polypropylene, such as linear low density polyethylene (LLDPE), can be suitably used. In addition, in order to make the inside of the cell culture vessel 1 visible, it is preferable that the gas permeable film is made of a transparent material.

[0032] 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 (approximately 10 μm thick), and so the container strength is insufficient, leading to the risk of the bag breaking. However, since the cell culture vessel 1 of this embodiment has protrusions on the outer surface of the gas permeable film, the gas permeable film can be handled even if it is made relatively thin. For this reason, the thickness of the gas permeable film in the cell culture vessel 1 of this embodiment is preferably less than 100 μm, more preferably 70 μm or less and 10 μm or more, and even more preferably 35 μm or less and 20 μm or more.

[0033] FIG. 2(b) shows cell culture in the cell culture vessel 1 of this embodiment. In the figure, a culture medium is filled in the cell culture vessel 1, and the cells 2 are seeded in the culture space S whose volume is expanded by the culture medium, and the culture is performed. A ventilation space V is formed between the protrusion 111 and the mounting surface of the cell culture vessel 1. Thus, the cell culture vessel 1 of this embodiment can have excellent gas permeability when culturing cells, making it possible to culture cells at a high density.

[0034] 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-welded thereto. 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 material such as polyethylene, polypropylene, vinyl chloride, polystyrene elastomer, FEP, or other thermoplastic resin.

[0035] The method for producing cells according to this embodiment is characterized in that the cells are cultured using the cell culture vessel 1 described above. 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 section inside the vessel.

[0036] The manufacturing method for a cell culture vessel of this embodiment is a manufacturing method for a bag-shaped closed cell culture vessel having at least one port and formed by opposing planar articles, characterized in that a gas-permeable film is used on at least one of the planar articles, and protrusions having a shape different from the shape of the inner surface of the gas-permeable film are formed on the outer surface of the gas-permeable film so that when the gas-permeable film is brought into contact with a flat surface, a breathable space is formed between the gas-permeable film and the flat surface, and the peripheral portions of the opposing planar articles are bonded together to form a culture space for culturing cells on the inner side of the gas-permeable film.

[0037] In the method for producing a cell culture vessel of this embodiment, it is preferable to use gas-permeable films on both of the opposing planar substrates and form protrusions on the outer surface of each gas-permeable film. Furthermore, it is also preferable to form the protrusions by thermal transfer, cast film formation, or inflation film formation. According to such a method for producing a cell culture vessel, it is possible to produce a cell culture vessel having excellent gas permeability and strength.

[0038] The cell culture device of this embodiment is characterized by having a culture tool comprising the above-mentioned cell culture vessel, a flat stand on which the cell culture vessel is placed, and a pressing member that presses the cell culture vessel placed on the stand, in which a breathable space is formed between the gas permeable film and the stand and / or between the gas permeable film and the pressing member by a protrusion formed on the cell culture vessel.

[0039] That is, as shown in FIG. 3, the cell culture device 3 of this embodiment includes a cell culture vessel 1, a stand 31, a pressing member 32, and a guide pin 33. The cell culture vessel 1 is placed on a stand 31, and a pressing member 32 presses the upper surface of the cell culture vessel 1. Guide pins 33 are provided at the four corners of the stand 31, and the guide pins 33 pass through guide holes provided in the pressing member 32. The pressing member 32 is supported so as to be movable vertically along these guide pins 33.

[0040] At this time, the pressing member 32 can be fixed so as to press the cell culture vessel 1 with a constant pressure by its own weight. Also, a spring material can be provided on the guide pin 33 to press the pressing member 32 against the cell culture vessel 1 as appropriate. By configuring the cell culture device 3 of this embodiment in this way, it is possible to make the pressing member 32 movable in the vertical direction as the culture medium is injected into or discharged from the cell culture vessel 1.

[0041] In the cell culture device 3 of this embodiment, since the cell culture vessel 1 is provided with the protrusion 111, a breathable space is formed between the gas permeable film facing the upper surface of the stand 31 and the upper surface of the stand 31. In addition, a breathable space is formed between the gas permeable film facing the lower surface of the pressing member 32 and the lower surface of the pressing member 32. Therefore, according to the cell culture device 3 of this embodiment, it is possible to prevent the gas permeability of the cell culture vessel 1 from being impaired by being sandwiched between the stand 31 and the pressing member 32. EXAMPLES

[0042] Tests conducted to confirm the effects of the cell culture vessel according to the embodiment of the present invention will be described below.

[0043] [Test 1] A test was conducted to compare the gas permeability of the cell culture vessel of this embodiment and a conventional cell culture vessel.

[0044] Specifically, a cell culture vessel (Example 1) of this embodiment was prepared with protrusions formed on both surfaces. That is, a film made of linear low-density polyethylene (manufactured by Toyo Seikan Group Holdings Co., Ltd.) with a thickness of 90 μm was prepared, on the entire surface of one side of which protrusions with a height of 0.2 mm, a width of 0.2 mm, and a pitch of 0.42 mm were formed by thermal transfer. At this time, multiple approximately triangular prisms were formed in parallel in a mountain range shape as the protrusions. The other surface of the film was made flat. Then, two sheets of film were stacked so that the protrusions were on the outside, one port was sandwiched between them, and the periphery was formed into a bag shape by heat welding. The external dimensions were 120 mm × 65 mm, and the base area of ​​the culture space was 48 cm 2 It was.

[0045] Furthermore, a conventional cell culture vessel (Comparative Example 1) was prepared in which no protrusions were formed on the surface of a film made of 90 μm-thick linear low-density polyethylene (manufactured by Toyo Seikan Group Holdings, Ltd.), and both surfaces were flat. A port was sandwiched between two sheets of film, and the periphery was formed into a bag shape by heat welding. The external dimensions were 120 mm × 65 mm, and the base area of ​​the culture space was 48 cm. 2 It was.

[0046] Next, a non-contact oxygen concentration meter was placed in each cell culture vessel, and 20 ml of pure water was filled in. The liquid depth in each vessel was 4 mm. Then, the change in the dissolved oxygen concentration in the water near the bottom of each cell culture vessel was measured. At this time, each cell culture vessel was fixed to a culture jig and left in a CO2 incubator at 37°C to adjust the dissolved oxygen in the vessel to approximately 21%, which is the oxygen concentration in the atmosphere. After the oxygen concentration in the vessel stabilized, the oxygen concentration in the CO2 incubator was lowered to 5%.

[0047] In addition, the culture tool used had a flat upper surface on the stand on which the cell culture vessel was placed and a flat lower surface of a pressing member that pressed the cell culture vessel from above. The cell culture vessel was smaller than the upper surface of the stand and the lower surface of the pressing member. The same applies to the following tests.

[0048] Here, the oxygen concentration around the container changes from 21% to 5%, creating an oxygen partial pressure difference between the inside and outside of the container, causing the oxygen inside the container to escape through the gas permeable film. At this time, the rate of change in oxygen concentration correlates with the gas permeability of the film, making it possible to quantitatively measure the gas permeability. The results are shown in Figure 4.

[0049] 4, the oxygen concentration in the cell culture vessel of Example 1 at the start of the measurement was about 20%, while the oxygen concentration in the cell culture vessel of Comparative Example 1 was about 24%. This is because the measurement by the non-contact oxygen concentration meter used in this test is a mechanism in which the change in the element attached to the vessel is measured by the measuring device in a non-contact manner, and the value is accurate when the element and the measuring part are in contact, and the value increases as the element and the measuring part are separated, so although the normal dissolved oxygen at 37°C is 21%, the value increases depending on the thickness of the vessel and the installation state. However, there is no effect on the amount of change, and oxygen permeability is judged based on the amount of change, so this measurement is not problematic in terms of performance evaluation.

[0050] 4, the maximum amount of change in oxygen concentration per minute in the cell culture vessel of Example 1 was 0.6% / min. In contrast, the maximum amount of change in oxygen concentration per minute in the cell culture vessel of Comparative Example 1 was 0.2% / min. In other words, the gas permeability of Example 1 was three times higher than that of Comparative Example 1, and it was found that the cell culture vessel of this embodiment exhibited excellent oxygen permeability.

[0051] [Test 2] Cell culture was carried out using the cell culture vessel of this embodiment and a conventional cell culture vessel, and a test was carried out to compare the culture performances of these vessels. Specifically, a cell culture vessel (Example 2) of this embodiment was prepared with protrusions formed on both surfaces. That is, a film made of linear low density polyethylene (LLDPE, Linear Low Density Polyethylene, manufactured by Toyo Seikan Group Holdings Co., Ltd.) with a thickness of 90 μm was prepared with protrusions with a height of 0.2 mm, a width of 0.2 mm, and a pitch of 0.42 mm formed by thermal transfer on the entire surface. At this time, a number of approximately triangular prisms were formed in parallel in a mountain range shape as the protrusions. The other surface of the film was made flat. Then, two films were stacked with the protrusions facing outward, two ports were sandwiched between them at both ends in the longitudinal direction, and the periphery was formed into a bag shape by heat welding. The external dimensions were 120 mm × 65 mm, and the base area of ​​the culture space was 48 cm 2 It was.

[0052] Furthermore, as a conventional cell culture vessel (Comparative Example 2), a film made of linear low-density polyethylene (manufactured by Toyo Seikan Group Holdings Co., Ltd.) with a thickness of 90 μm was prepared, with no protrusions formed on the surface, and both surfaces were flat. Two ports were sandwiched between the two films at both ends in the longitudinal direction, and the periphery was formed into a bag shape by heat welding. The external dimensions were 120 mm × 65 mm, and the base area of ​​the culture space was 48 cm. 2 It was.

[0053] Furthermore, the culture tool used had a flat upper surface of a stand on which the cell culture vessel was placed and a flat lower surface of a pressing member that pressed the cell culture vessel from above.

[0054] iPS cells (1231A3 strain) were used as the cells to be cultured. StemFit AK02N (Ajinomoto Co., Inc.) was used as the culture medium, and these were filled into each cell culture vessel. The culture medium filled was 10 ml at 37 °C, and the liquid thickness of each vessel was 2 mm. At this time, 10 μM Y-27632 and 24 μl of adhesive substrate (i-Matrix, laminin 511-E8) were added to 10 ml of culture medium in the cell culture vessel, 70,000 cells were injected, and the vessel was set in a cell culture jig to start culture. 1, 4, 5, and 6 days after cell seeding, the entire amount of 10 ml of culture medium (only culture medium without Y-27632 and adhesive substrate) was replaced, and culture was performed for 7 days. After 7 days, the cells were recovered using the detachment solution TrypLE, the cell number was counted, and the proliferation rate was calculated. These were performed twice for each cell culture vessel. The results are shown in Figure 5.

[0055] As shown in the figure, the cell proliferation rate of the cell culture vessel of Example 2 was superior to that of Comparative Example 2, indicating that the cell culture vessel of this embodiment provides appropriate gas permeability performance.

[0056] The present invention is not limited to the above-mentioned embodiments and examples, and various modifications are possible within the scope of the present invention. For example, the shape of the protrusions formed on the cell culture vessel can be appropriately modified to various shapes different from those in the embodiments. [Industrial Applicability]

[0057] The present invention can be suitably used when, for example, mass culturing of cells at high density using a cell culture bag. [Explanation of symbols]

[0058] 1 cell culture vessel 11 Planar equipment 111 Protrusion 12 ports 2 cells 3 Cell culture equipment 31 Mount 32 Pressing member 33 Guide pin S culture space H Periphery V Ventilation space

Claims

1. A bag-shaped 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, and at least one of the gas permeable films has a protrusion on the outer surface thereof that has a shape different from the shape of the inner surface of the gas permeable film, and the inner surface of the gas permeable film has a culture space for culturing cells, and when the gas permeable film is brought into contact with a flat surface, a breathable space is formed between the gas permeable film and the flat surface by the protrusion. In an outer surface region of the gas permeable film that corresponds to the outside of the culture space, a plurality of approximately triangular prisms are formed in parallel in a mountain range shape as the protrusions, The thickness of the region of the gas permeable film where the protrusions are not formed is 10 μm or more and 35 μm or less. A cell culture vessel comprising:

2. 2. The cell culture vessel according to claim 1, wherein both of said opposing planar substrates are made of gas-permeable films, and said protrusions are provided on the outer surface of each of said gas-permeable films.

3. 3. The cell culture vessel according to claim 1, wherein the inner surface of the gas permeable film is flat and free of irregularities.

4. 3. The cell culture vessel according to claim 1, wherein the gas permeable film has an inner surface with projections and recesses.

5. 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 on at least one of the planar articles, and protrusions having a shape different from the shape of the inner surface of the gas permeable film are formed on the outer surface of the gas permeable film so that a breathable space is formed between the gas permeable film and the plane when the gas permeable film is brought into contact with the plane, The peripheral portions of the opposing planar substrates are bonded together to form a culture space for culturing cells on the inner surface side of the gas permeable film; In an outer surface region of the gas permeable film that corresponds to the outside of the culture space, a plurality of approximately triangular prisms are formed in parallel in a mountain range shape as the protrusions, The thickness of the region of the gas permeable film where the protrusions are not formed is 10 μm or more and 35 μm or less. A method for producing a cell culture vessel comprising the steps of:

6. 6. The method for producing a cell culture vessel according to claim 5, wherein a gas permeable film is used for both of the opposing planar substrates, and the protrusions are formed on the outer surface of each of the gas permeable films.

7. 7. The method for producing a cell culture vessel according to claim 5, wherein the protrusions are formed by thermal transfer, cast film formation, or inflation film formation.

8. A method for producing cells, comprising culturing cells using the cell culture vessel according to any one of claims 1 to 4.

9. A cell culture vessel according to any one of claims 1 to 4, and a culture tool including a flat stand on which the cell culture vessel is placed, and a pressing member that presses the cell culture vessel placed on the stand, and in which a breathable space is formed between the gas permeable film and the stand and / or between the gas permeable film and the pressing member by the protrusions formed on the cell culture vessel. A cell culture device characterized by:

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