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

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 facing to each other. At lest one of the planar instruments 11 is made of a gas permeable film. A gas-permeable reinforcing member 13 is provided on the outer surface side of at least one gas permeable film with respect to the container. On the inner surface side of the gas permeable film with respect to the container, a culture space S for culturing cells is provided. The gas permeable film has a thickness of less than 100 μm.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a cell culture technique, and in particular 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 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 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 was 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 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 at a high density, 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]

[0005] [Patent Document 1] JP 2016-77164 A 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 constitutes the vessel, but this has not led to a significant improvement in performance.

[0007] Silicone materials are also an example of materials with excellent gas permeability. Although silicone materials have low strength, they can 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. In addition, harmful substances are eluted from silicone materials by radiation sterilization, etc., which may adversely affect the subsequent culture performance. Furthermore, silicone materials are difficult to perform the surface treatment required for culturing adhesive cells, so although it is possible to manufacture culture vessels for non-adherent cells, there is also the problem that it is difficult to manufacture culture vessels for adhesive cells.

[0008] Therefore, the inventors conducted extensive research and succeeded in completing the present invention by providing a breathable reinforcing member on the outer surface of the gas-permeable film that constitutes the container, thereby ensuring strength when handled even when the gas-permeable film is made thinner than 100 μm, and by making the gas permeability large enough to culture cells at high density.

[0009] Here, 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 is not intended to improve the strength of the vessel, and is not capable of resolving the problem of improving the gas permeability and strength of a bag-shaped cell culture vessel to enable high-density cell culture.

[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, and a method for manufacturing cells. [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 articles, at least one of the planar articles being made of a gas-permeable film, at least one of the gas-permeable films being provided with a breathable reinforcing member on the outer surface side of the vessel, and at least one of the gas-permeable films being provided with a culture space for culturing cells on the inner surface side of the vessel, and the gas-permeable film being configured to have a thickness of less than 100 μm.

[0012] In the cell culture vessel of the present invention, the breathable reinforcing member is preferably made of a mesh material or a porous material. It is also preferable that the cell culture vessel of the present invention is configured such that the breathable reinforcing member is made of a film with an uneven surface treatment, and when the gas-permeable film and the breathable reinforcing member are brought into close contact with each other, the uneven surface treatment forms gaps through which air can pass.

[0013] In addition, the cell culture vessel of the present invention is preferably configured such that the gas-permeable reinforcing member is fixed to the gas-permeable film by heat welding. In addition, it is preferable that the cell culture vessel of the present invention is configured so that the thickness of the gas-permeable film having the breathable reinforcing member on the outer surface side is 70 μm or less and 10 μm or more, and it is more preferable that the thickness of the gas-permeable film is 35 μm or less and 20 μm or more.

[0014] In addition, the manufacturing method of the cell culture vessel of the present invention is a manufacturing method of a bag-shaped cell culture vessel having at least one port and formed by opposing planar equipment, in which a gas-permeable film having a thickness of less than 100 μm is used as the planar equipment, the port is sandwiched between the opposing planar equipment to form a bag shape, a culture space for culturing cells is formed on the inner surface side of the vessel in the gas-permeable film, and an air-permeable reinforcing member is fixed to the outer peripheral edge or the entire outer surface of the gas-permeable film.

[0015] In addition, the manufacturing method of the cell culture vessel of the present invention is a manufacturing method of a bag-shaped cell culture vessel having at least one port and formed by opposing planar equipment, in which a gas-permeable film having a thickness of less than 100 μm is used as the planar equipment, a breathable reinforcing member is fixed to the outer peripheral edge or the entire outer surface of the gas-permeable film, the port is sandwiched between the opposing planar equipment to form a bag shape, and a culture space for culturing cells is formed on the inner surface side of the vessel in the gas-permeable film.

[0016] Furthermore, the method for producing cells of the present invention is a method for culturing cells using the above-mentioned cell culture vessel. Effect of the Invention

[0017] 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 the cell culture vessel, and a method for producing cells. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1A is a plan view and FIG. 1B is a front view of a cell culture vessel according to one embodiment of the present invention. [Diagram 2] FIG. 1 is a diagram showing the oxygen permeability of a gas-permeable film that can be used in a cell culture vessel according to one embodiment of the present invention. [Diagram 3]1A is an enlarged view of a breathable reinforcing member provided in a cell culture vessel according to one embodiment of the present invention, FIG. 1B is a partial cross-sectional view taken along the line AA in FIG. 1A, and FIG. 1C is a partial cross-sectional view taken along the line BB in FIG. 1A. [Figure 4] FIG. 1 is a schematic diagram showing cell culture in a cell culture vessel according to one embodiment of the present invention. [Diagram 5] FIG. 13 is a plan cross-sectional view (between the gas-permeable film and the breathable reinforcing member) of a modified example of the cell culture vessel according to one embodiment of the present invention. [Figure 6] 1A is a partial cross-sectional view taken along line CC of a plan view (not shown) corresponding to a plan cross-sectional view of a modified example of a cell culture vessel according to one embodiment of the present invention, and FIG. 1B is a partial cross-sectional view taken along line DD of the plan view (not shown). [Figure 7] FIG. 2 is a plan cross-sectional view (between two gas-permeable films) of a modified example of a cell culture vessel according to one embodiment of the present invention. [Figure 8] FIG. 2 is a photograph of the cell culture vessel (prototype 1) of Example 1. [Figure 9] FIG. 2 is a diagram showing a partial cross-sectional photograph (a) taken along the EE cross section of the cell culture vessel of Example 1, and a partial cross-sectional photograph (b) taken along the FF cross section of the same vessel. [Figure 10] FIG. 2 is a photograph showing a state in which the cell culture vessel of Example 1 is filled with liquid and its strength is checked. [Figure 11] FIG. 1 is a photograph of the cell culture vessel (prototype 2) of Example 2. [Figure 12] FIG. 13 is a photograph of a partial cross section of the cell culture vessel of Example 2 taken along the line GG. [Figure 13] FIG. 1 is a graph showing the measurement results of gas permeability of the cell culture vessel in Test 1 (Example 3: film thickness 33 μm+mesh welding, Example 4: film thickness 50 μm+mesh welding, Comparative Example 1: film thickness 110 μm). [Figure 14] FIG. 13 is a graph showing the measurement results of gas permeability of the cell culture vessel in Test 2 (Example 5: film thickness 50 μm+welded textured film, Comparative Example 2: film thickness 110 μm). [Figure 15]FIG. 13 is a graph showing the results of cell culture using the cell culture vessel of Test 3 (Example 6: film thickness 33 μm+mesh welding, Comparative Example 3: film thickness 110 μm). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Hereinafter, embodiments of the cell culture vessel, the method for producing the cell culture vessel, and the method for producing cells 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.

[0020] 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 at least one of the gas-permeable films is provided with a breathable reinforcing member on the outer surface side of the vessel, and a culture space for culturing cells on the inner surface side of the vessel of the gas-permeable film, characterized in that the thickness of the gas-permeable film is less than 100 μm.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] In general, the thinner the gas-permeable film, the higher its gas permeability. For example, as shown in Figure 2, in a film made of linear low density polyethylene (LLDPE), the oxygen permeability (ml / m2) at a thickness of 110 μm is 2The oxygen permeability (H2O2 / 24Hr / atm) is approximately 9,000 when the thickness is 75 μm, approximately 13,000 when the thickness is 50 μm, approximately 19,000 when the thickness is 30 μm, approximately 31,000 when the thickness is 20 μm, and approximately 48,000 when the thickness is 20 μm.

[0026] On the other hand, 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), so the container strength is insufficient and there is a risk of the bag breaking. Therefore, in order to provide the cell culture vessel 1 of this embodiment with excellent gas permeability and strength, as described below, the gas permeable film is provided with an air permeable reinforcing member 13 on the outer surface thereof.

[0027] At least one port 12 is provided by being sandwiched and heat-sealed between two planar substrates 11 at the periphery H of the planar substrate 11. In Fig. 1(a) and Fig. 1(b), two ports 12 are provided facing each other at both ends 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.

[0028] A breathable reinforcing member 13 is fixed to the outer surface of the gas-permeable film, and in the example of Figures 1(a) and 1(b), a breathable reinforcing member 13 is fixed to each of the two gas-permeable films. The cell culture vessel 1 of this embodiment is provided with the breathable reinforcing member 13, so that it is possible to ensure strength during handling even if the gas permeable film has a thickness of less than 100 μm.

[0029] As described above, the thinner the gas-permeable film, the higher its gas permeability, but the thinner the gas-permeable film, the more difficult it is to handle. For this reason, the cell culture vessel 1 of this embodiment uses the breathable reinforcing member 13 to improve both gas permeability and strength. From this viewpoint, the thickness of the gas-permeable film provided with the breathable reinforcing member 13 on the outer surface side is more preferably 70 μm or less and 10 μm or more, and further preferably 35 μm or less and 20 μm or more.

[0030] In addition, in the cell culture vessel 1 of this embodiment, when a gas-permeable film is used on only one side of the planar substrate 11, the breathable reinforcing member 13 can be provided only on the outer surface of the gas-permeable film.

[0031] In this embodiment, the breathable reinforcing member 13 can be roughly divided into two types: a first reinforcing member and a second reinforcing member. The first reinforcing member may be a mesh material as shown in Fig. 3(a) or a porous material such as a sponge.

[0032] Specific mesh and porous materials are not particularly limited, but are preferably those having a melting point close to that of the planar material 11 and made of similar material, such as polyethylene mesh material (NBC Meshtec Co., Ltd., PE 200 mesh, etc.) or polyethylene porous material (Teijin Limited, Miraim, large aperture ≦3 μm).

[0033] In the cell culture vessel 1 of this embodiment, a mesh material shown in Fig. 3(a) can be suitably used as the breathable reinforcing member 13. Fig. 3(b) shows a partial cross-sectional view (region including the culture space) in cross section AA of Fig. 1(a) when a mesh material is used as the breathable reinforcing member 13, and Fig. 3(c) shows a partial cross-sectional view (welded region of the peripheral portion H) in cross section BB of the same Fig. 1(a).

[0034] When a mesh material or a porous material is used as the breathable reinforcing member 13, it is preferable that the breathable reinforcing member 13 is fixed to the peripheral edge part H on the outer surface side of the gas permeable film. The breathable reinforcing member 13 may also be fixed to the entire surface of the outer surface side of the gas permeable film. The fixing of such breathable reinforcing member 13 to the outer surface side of the gas permeable film can be suitably carried out by heat welding.

[0035] FIG. 4 shows how a cell culture vessel 1 formed using a mesh material as a breathable reinforcing member 13 is filled with culture medium and cells 2 are seeded into the culture space S whose volume has been expanded by the culture medium, thereby carrying out culture.

[0036] The cells 2 cultured in the cell culture vessel 1 of this embodiment 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 a culture section in the vessel.

[0037] Such a cell culture vessel 1 provided with the gas-permeable reinforcing member 13 can have excellent gas permeability and strength, and therefore allows cells to be cultured at a high density. It should be noted that, when a second reinforcing member described below is used as the breathable reinforcing member 13, it is also possible to culture cells at a high density in the same manner.

[0038] Next, a modified example of the cell culture vessel of this embodiment in which a second reinforcing member is used as the breathable reinforcing member will be described with reference to FIGS. In this modification, a film having projections and recesses formed on the inner surface side of the container (hereinafter, sometimes referred to as a projection-and-recess processed film) is used as the second reinforcing member. The material for the textured film is not particularly limited, but is preferably one that has a melting point close to that of the gas permeable film and is made of a similar material, and in particular, linear low density polyethylene or the like can be suitably used.

[0039] A planar cross-sectional view (between the gas-permeable film and the breathable reinforcing member) of a modified example of the cell culture vessel of this embodiment is shown in Fig. 5. A partial cross-sectional view of the plan view (not shown) corresponding to the planar cross-sectional view at CC section is shown in Fig. 6(a), and a partial cross-sectional view of the plan view at DD section is shown in Fig. 6(b). Furthermore, a planar cross-sectional view (between two gas-permeable films) of a modified example of the cell culture vessel of this embodiment is shown in Fig. 7.

[0040] As shown in these figures, in the cell culture vessel 1a, a resin film with a concave-convex processing is used as the breathable reinforcing member 13a, and a plurality of protrusions 131a are formed on the breathable reinforcing member 13a by the concave-convex processing. The inner surface side with the concave-convex processing (the side with the protrusions 131a) is laminated on the outer surface of the gas permeable film.

[0041] The breathable reinforcing member 13a is fixed to the peripheral portion Ha on the outer surface side of the gas-permeable film, and a plurality of ventilation holes T are formed in the peripheral portion Ha where the breathable reinforcing member 13a and the gas-permeable film are not in close contact with each other. The shape and number of the ventilation holes T are not limited to those shown in FIG. 5, and it is also preferable to form a large number of fine ventilation holes with an opening diameter of about several tens of μm.

[0042] By forming such an air vent T in the cell culture vessel 1a, it is possible to form a passage through which air can escape from the outer surface of the gas permeable film to the outside of the cell culture vessel 1a through the gaps in the uneven processing on the inner surface of the breathable reinforcing member 13a and the air vent T. In addition, by blowing air through the gaps in the uneven processing and the ventilation holes T, it is possible to further improve the gas permeability of the cell culture vessel 1a.

[0043] In order to form the ventilation holes T in the cell culture vessel 1a in this manner, it is preferable to fix the gas permeable film of the breathable reinforcing member 13a to the outer peripheral edge portion Ha so that the ventilation holes T are formed parallel to the wall surface of the rectangular protrusion portion 131a, as shown in Figure 5, for example. Furthermore, the breathable reinforcing member 13a can be preferably fixed to the outer surface side of the gas permeable film by heat welding.

[0044] In the cell culture vessel 1a, the planar substrate 11a and the port 12a can have the same configurations as the planar substrate 11 and the port 12 in the cell culture vessel 1 described above, respectively. Moreover, the thickness of the gas-permeable film on the outer surface of which the breathable reinforcing member 13a is provided is more preferably 70 μm or less and 10 μm or more, and further preferably 35 μm or less and 20 μm or more.

[0045] In this way, by providing the cell culture container 1a with the breathable reinforcing member 13a, the container can be made to have excellent gas permeability and strength, and therefore it is possible to culture cells at high density by filling the culture space Sa with culture medium and seeding the cells.

[0046] The method for producing cells according to this embodiment is characterized in that cells are cultured using the cell culture vessel according to this embodiment described above.

[0047] Furthermore, the manufacturing method for a cell culture vessel of this embodiment is a manufacturing method for a bag-shaped cell culture vessel having at least one port and formed by opposing planar articles, characterized in that a gas-permeable film having a thickness of less than 100 μm is used as the planar article, the port is sandwiched between the opposing planar articles to form a bag shape, a culture space for culturing cells is formed on the inner surface side of the vessel in the gas-permeable film, and an air-permeable reinforcing member is fixed to the outer peripheral edge or the entire outer surface of the gas-permeable film.

[0048] According to this method for manufacturing a cell culture vessel, it is possible to easily manufacture a cell culture vessel having excellent gas permeability and strength by, for example, molding a bag-shaped vessel and then heat-welding a breathable reinforcing member to the outer surface of a gas-permeable film.

[0049] Furthermore, a preferred method for producing a cell culture vessel of this embodiment is a method for producing a bag-shaped cell culture vessel having at least one port and formed by opposing planar articles, in which a gas-permeable film having a thickness of less than 100 μm is used as the planar article, an air-permeable reinforcing member is fixed to the outer peripheral edge or the entire outer surface of the gas-permeable film, the port is sandwiched between the opposing planar articles to form a bag shape, and a culture space for culturing cells is formed on the inner surface side of the vessel in the gas-permeable film.

[0050] According to this method for manufacturing a cell culture vessel, the container material is produced by heat welding the breathable reinforcing member to the outer surface of the gas permeable film, and then this material is molded into a bag shape, making it possible to easily manufacture a cell culture vessel with excellent gas permeability and strength.

[0051] As described above, according to the cell culture vessel, the method for manufacturing the cell culture vessel, and the method for manufacturing cells of this embodiment, it is possible to manufacture a cell culture vessel having excellent gas permeability and strength for culturing cells at high density, thereby making it possible to mass-cultivate cells. EXAMPLES

[0052] Hereinafter, examples and tests carried out to confirm the effects of the cell culture vessel according to the embodiment of the present invention will be specifically described.

[0053] [Example 1] A prototype 1 of the cell culture vessel of this embodiment was produced. Specifically, two sheets of 30 μm-thick linear low-density polyethylene (manufactured by Toyo Seikan Group Holdings Co., Ltd.) were used as the planar equipment, and two sheets of polyethylene mesh material (NBC Meshtec Co., Ltd., PE200 mesh) were used as the breathable reinforcement material, and one port was sandwiched around the periphery and 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 A photograph of the prototype of this cell culture vessel is shown in Figure 8. Also, a partial cross-sectional photograph of the prototype in the EE section of Figure 8 is shown in Figure 9(a), and a partial cross-sectional photograph of the prototype in the FF section of Figure 9(b).

[0054] As these figures show, even when an extremely thin planar article with a thickness of 30 μm is used, by providing an air permeability reinforcing member made of a mesh material on its outer surface, a bag-shaped cell culture vessel can be formed. In Fig. 9(a), the two planar articles are positioned near the lower air permeability reinforcing member, with the upper air permeability reinforcing member raised. Also, in Fig. 9(b), it can be seen that the thin polyethylene film and the polyethylene mesh material are firmly welded together.

[0055] In addition, when 20 ml of ink-containing liquid was poured into the culture space of the cell culture vessel prototype 1, the liquid thickness was 4 mm. The strength of the cell culture vessel was then confirmed by tapping it with a hand. As a result, as shown in Figure 10, it was found that the cell culture vessel had sufficient strength without breaking.

[0056] [Example 2] A second prototype of the cell culture vessel of this embodiment was produced. Specifically, two sheets of 30μm-thick linear low-density polyethylene (manufactured by Toyo Seikan Group Holdings Co., Ltd.) were used as the planar material, and two sheets of textured film (with protrusions measuring 110μm in thickness, 0.1mm in width, 1mm in length, 0.1mm in height, and spaced 1mm apart) were used as the breathable reinforcement material, and the periphery was heat-sealed to form a bag shape. The external dimensions were 120mm x 65mm. A photograph of prototype 2 of this cell culture vessel is shown in Figure 11. A partial cross-sectional photograph of the GG cross section of this cell culture vessel in Figure 11 is shown in Figure 12.

[0057] As shown in these figures, even when an extremely thin, planar substrate with a thickness of 30 μm is used, a bag-shaped cell culture vessel can be formed by providing an air-permeable reinforcing member made of an unevenly textured film on its outer surface.

[0058] [Test 1] As the cell culture vessel of this embodiment, vessels with different film thicknesses were prepared, and a conventional cell culture vessel was also prepared, and a test was performed to compare the gas permeability of each.

[0059] Specifically, the following two types of cell culture vessels of this embodiment (Example 3: 33 μm+mesh welding, Example 4: 50 μm+mesh welding) were prepared. Specifically, two sheets of linear low-density polyethylene (Toyo Seikan Group Holdings Co., Ltd.) with a thickness of 33 μm and two sheets of linear low-density polyethylene (Toyo Seikan Group Holdings Co., Ltd.) with a thickness of 50 μm and four sheets of polyethylene mesh material (NBC Meshtec Co., Ltd., PE200 mesh) as a breathable reinforcement member were used, and one port was sandwiched around the periphery to form 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.

[0060] As a conventional cell culture vessel (Comparative Example 1), two sheets of linear low-density polyethylene (manufactured by Toyo Seikan Group Holdings Co., Ltd.) with a thickness of 110 μm were used, and one port was sandwiched at the periphery to form a bag shape by heat welding. The external dimensions were 120 mm × 65 mm, and the bottom area of ​​the culture space was 48 cm. 2It was.

[0061] Then, the change in oxygen gas concentration near the bottom of each cell culture vessel was measured. Specifically, a non-contact oxygen concentration meter was placed in each cell culture vessel, which was then filled with 30 ml of pure water, and 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%.

[0062] 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.

[0063] 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, so gas permeability can be measured quantitatively. The results are shown in Figure 13.

[0064] As shown in the figure, the cell culture vessels of Examples 3 and 4 exhibited a large change in oxygen concentration compared to the cell culture vessel of Comparative Example 1, demonstrating superior oxygen permeability. Specifically, the maximum amount of change in oxygen concentration per minute in the cell culture vessel of Example 3 was 1.1% / min, and the maximum amount of change in oxygen concentration per minute in the cell culture vessel of Example 4 was 0.8% / min.

[0065] In contrast, the maximum change in oxygen concentration per minute was 0.4% / min in the cell culture vessel of Comparative Example 1. That is, the gas permeability of Example 3 was 2.75 times that of Comparative Example 1, and the gas permeability of Example 4 was twice that of Comparative Example 1. Thus, it was found that the cell culture vessel of this embodiment has strength suitable for handling and exhibits excellent oxygen permeability.

[0066] [Test 2] A test was conducted to compare the gas permeability of the cell culture vessel of this embodiment and a conventional cell culture vessel.

[0067] Specifically, the cell culture vessel of this embodiment (Example 5) was made of two sheets of 50 μm-thick linear low-density polyethylene (manufactured by Toyo Seikan Group Holdings Co., Ltd.) and two sheets of textured film (with protrusions of 110 μm thickness, width 0.1 mm, length 1 mm, height 0.1 mm, and intervals of 1 mm) as a breathable reinforcing member, and was formed into a bag shape by heat welding with one port sandwiched on the periphery. The external dimensions were 120 mm × 65 mm, and the base area of ​​the culture space was 48 cm. 2 It was.

[0068] Furthermore, as a conventional cell culture vessel (Comparative Example 2), a vessel similar to that in Test 1 was prepared. Then, 30 ml of pure water was placed in each cell culture vessel, and the change in oxygen gas concentration near the bottom of the cell culture vessel was measured in the same manner as in Test 1. The results are shown in FIG.

[0069] 14, the oxygen concentration in the cell culture vessel of Example 5 at the start of the measurement was about 22%, while the oxygen concentration in the cell culture vessel of Comparative Example 2 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.

[0070] As shown in FIG. 14, the cell culture vessel of Example 5 exhibited a large change in oxygen concentration compared to the cell culture vessel of Comparative Example 2, and was found to have excellent oxygen permeability. Specifically, the maximum change in oxygen concentration per minute in the cell culture vessel of Example 5 was 0.5% / min.

[0071] In contrast, the maximum change in oxygen concentration per minute was 0.2% / min in the cell culture vessel of Comparative Example 2. That is, the gas permeability of Example 5 was 2.5 times that of Comparative Example 2. Thus, it was found that the cell culture vessel of this embodiment has strength suitable for handling and exhibits excellent oxygen permeability.

[0072] [Test 3] 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.

[0073] Specifically, two sheets of linear low-density polyethylene (manufactured by Toyo Seikan Group Holdings Co., Ltd.) with a thickness of 33 μm were used as the planar equipment in the cell culture vessel of this embodiment (Example 6), and two sheets of polyethylene mesh material (NBC Meshtec Co., Ltd., PE200 mesh) were used as the breathable reinforcement member, and two ports were sandwiched at both ends of the longitudinal direction of the periphery to form 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.

[0074] In addition, two sheets of linear low-density polyethylene (manufactured by Toyo Seikan Group Holdings Co., Ltd.) with a thickness of 110 μm were used as the planar material in the conventional cell culture vessel (Comparative Example 3), and two ports were sandwiched at both ends of the longitudinal direction of the periphery to form 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.

[0075] Furthermore, the culture tool used was one in which the entire upper surface of a stand on which the cell culture vessels were placed and the entire lower surface of a pressing member that pressed the cell culture vessels from above were flat.

[0076] Jurkat (a cell line derived from human leukemia T cells) was used as the cells to be cultured. The culture medium used was ALyS505N-0 manufactured by Cell Science Institute, supplemented with 2% FBS (fetal bovine serum), and these were filled into each cell culture vessel. At the start of culture, cells were seeded to give 1 million cells / ml, and the number of cells was counted on the first and second days, followed by centrifugation, the supernatant was discharged, and the culture medium was replaced. Culture was continued until the third day, the number of cells was counted, and the cell density was calculated. The results are shown in Figure 15.

[0077] As shown in the figure, the cell growth performance of the cell culture vessel of Example 6 is greater than that of the cell culture vessel of Comparative Example 3. Specifically, on the third day, the cell density in Example 6 was 23% higher than that in Comparative Example 3. Thus, it was found that the cell culture vessel of this embodiment ensures sufficient strength for handling even when the thickness of the gas-permeable film is thinned to about 30 μm, and provides appropriate gas permeability when culturing cells at high density.

[0078] The present invention is not limited to the above-mentioned embodiments and examples, and various modifications can be made within the scope of the present invention. For example, when a textured film is used as the breathable reinforcing member, the shape of the protrusions can be appropriately changed to various other shapes. [Industrial Applicability]

[0079] The present invention can be suitably used in cases where cells are cultured in large quantities at high density using a cell culture bag. [Explanation of symbols]

[0080] 1,1a,1b,1c Cell culture vessel 11,11a,11b,11c Planar equipment 12,12a port 13, 13a, 13b, 13c Breathable reinforcement member 131a Protrusion 2 cells S,Sa culture space H,Ha Periphery T Vent

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 the gas-permeable film is provided with a gas-permeable reinforcing member on the outer surface side of the container, and the gas-permeable film is provided with a culture space for culturing cells on the inner surface side of the container, and the thickness of the gas-permeable film is less than 100 μm; The breathable reinforcing member is made of a mesh material or a film with a textured surface, When the gas-permeable film and the breathability reinforcing member are brought into close contact with each other, a gap through which air can pass is formed, The gas-permeable film has a plurality of ventilation holes at the outer peripheral edge portion thereof where the breathable reinforcing member and the gas-permeable film are not in close contact with each other. A cell culture vessel comprising:

2. 2. The cell culture vessel according to claim 1, wherein the air-permeable reinforcing member is fixed to the peripheral edge of the outer surface of the gas-permeable film.

3. 2. The cell culture vessel according to claim 1, wherein the air-permeable reinforcing member is fixed to the entire outer surface of the gas-permeable film.

4. 4. The cell culture vessel according to claim 1, wherein the gas-permeable reinforcing member is fixed to the gas-permeable film by heat welding.

5. 5. The cell culture vessel according to claim 1, wherein the gas-permeable film on the outer surface of which the breathable reinforcing member is provided has a thickness of 70 μm or less and 10 μm or more.

6. 6. The cell culture vessel according to claim 1, wherein the gas-permeable film on the outer surface of which the breathable reinforcing member is provided has a thickness of 35 μm or less and 20 μm or more.

7. A method for producing a bag-shaped cell culture vessel having at least one port and formed by opposing planar substrates, comprising: A gas-permeable film having a thickness of less than 100 μm is used as the planar substrate, and the port is sandwiched between the opposing planar substrates to form a bag shape, forming a culture space for culturing cells on the inner surface side of the container in the gas-permeable film, A breathable reinforcing member is fixed to the outer peripheral edge portion or the entire outer surface of the gas permeable film, The breathable reinforcing member is made of a mesh material or a film with a textured surface, When the gas-permeable film and the breathable reinforcing member are brought into close contact with each other, a gap through which air can pass is formed, A plurality of ventilation holes are formed in the peripheral portion of the outer surface of the gas permeable film, where the breathable reinforcing member and the gas permeable film are not in close contact with each other. A method for producing a cell culture vessel comprising the steps of:

8. A method for producing a bag-shaped cell culture vessel having at least one port and formed by opposing planar substrates, comprising: A gas-permeable film having a thickness of less than 100 μm is used as the planar substrate, and a breathable reinforcing member is fixed to the outer peripheral edge or the entire outer surface of the gas-permeable film; The port is sandwiched between the opposing planar substrates to form a bag shape, and a culture space for culturing cells is formed on the inner surface side of the container in the gas permeable film; The breathable reinforcing member is made of a mesh material or a film with a textured surface, When the gas-permeable film and the breathable reinforcing member are brought into close contact with each other, a gap through which air can pass is formed, A plurality of ventilation holes are formed in the peripheral portion of the outer surface of the gas permeable film, where the breathable reinforcing member and the gas permeable film are not in close contact with each other. A method for producing a cell culture vessel comprising the steps of:

9. A method for producing cells, comprising culturing cells using the cell culture vessel according to any one of claims 1 to 6.

Images