Cell culture device and cell manufacturing method

Abstract

To provide a cell culture jig capable of culturing cells at a high density while suppressing the inhibition of the gas permeation performance of the cell culture container.SOLUTION: Provided is a cell culture jig 1 for holding a bag-shaped closed cell culture container 2 formed by planar instruments 21, 22 facing to each other in which at lest one thereof is made of a gas permeable film. The cell culture jig 1 comprises a mount 11 on which the cell culture container 2 is placed, and a pressing member 12 that presses the cell culture container 2 against the mount 11. In the mount 11, a gas permeable film is formed of an instrument that allows air to permeate to and from the mount 11. The gas permeable film side of the cell culture container 2 is placed on the mount 11.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to cell culture technology, and more particularly to a cell culture device 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 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 culturing cells using a bag-shaped cell culture vessel, the vessel may be clamped between a pressing jig to suppress the movement of the culture medium within the vessel and stabilize the culture environment. However, since bag-shaped cell culture vessels are usually formed by bonding two sheets of film together and have a flat outer surface, when they are used while being clamped in a pressing jig, the surface of the vessel comes into close contact with the base and pressing member of the pressing jig, which impairs the vessel's gas permeability, lowers the oxygen concentration around the cells, and reduces cell growth efficiency. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 4806761 Summary of the Invention [Problem to be solved by the invention]

[0005] Here, Patent Document 1 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.

[0006] Therefore, the inventors conducted extensive research and completed the present invention, which has succeeded in preventing the gas permeability of the cell culture vessel from being impaired during culture by forming at least the stand from a material that allows air to pass between the surface of the cell culture vessel and the stand in a cell culture device that is equipped with a stand on which a bag-shaped cell culture vessel is placed and a pressing member that presses the cell culture vessel against the stand.

[0007] The present invention has been made in consideration of the above circumstances, and aims to provide a cell culture device that can culture cells at high density while preventing the gas permeability performance of a cell culture vessel from being impaired, and a method for producing cells. [Means for solving the problem]

[0008] In order to achieve the above-mentioned object, the cell culture device of the present invention is a cell culture device that holds a bag-shaped closed cell culture vessel formed by opposing planar materials, at least one of which is made of a gas-permeable film, and is equipped with a stand on which the cell culture vessel is placed and a pressing member that presses the cell culture vessel against the stand, and the stand is formed of a material that allows air to pass through between the gas-permeable film and the stand, and the gas-permeable film side of the cell culture vessel is placed on the stand.

[0009] Furthermore, it is preferable that the cell culture device of the present invention is configured such that the pressing member is formed of a material that allows ventilation between the gas-permeable film and the pressing member, and the gas-permeable film side of the cell culture vessel is pressed against the pressing member.

[0010] Furthermore, it is preferable that the cell culture device of the present invention is configured such that guide pins are provided at the four corners of the stand, the guide pins pass through guide holes provided in the pressing member, and the pressing member is provided so as to be movable vertically along the guide pins.

[0011] Furthermore, it is preferable that the cell culture device of the present invention is configured such that support columns are erected at the four corners of the stand, a top plate is fixed to the support columns, guide pins are provided on the top plate, the guide pins pass through guide holes provided in the pressing member, and the pressing member is provided so as to be movable vertically along the guide pins.

[0012] The cell production method of the present invention is a method for culturing cells using the above-mentioned cell culture device. [Effects of the Invention]

[0013] According to the present invention, it is possible to provide a cell culture device and a method for producing cells that can culture cells at high density while preventing the gas permeability of the cell culture vessel from being impaired. [Brief explanation of the drawings]

[0014] [Figure 1] 1A is a perspective view showing a cell culture device according to one embodiment of the present invention and a cell culture vessel placed thereon, and FIG. 1B is an enlarged view of the surface of a stand and a pressing member of the cell culture device. [Figure 2] 1 is a front view showing a cell culture device according to one embodiment of the present invention and a cell culture vessel placed thereon. [Figure 3] 1 is a side view showing a cell culture device according to one embodiment of the present invention and a cell culture vessel placed thereon. [Figure 4] FIG. 10 is a schematic diagram showing a modified example of a cell culture device according to one embodiment of the present invention and a cell culture vessel placed thereon. [Figure 5] FIG. 1 is a schematic diagram showing cell culture using a cell culture device according to one embodiment of the present invention. [Figure 6]FIG. 1 is a graph showing the measurement results of the gas permeability of cell culture vessels fixed to cell culture jigs in Test 1 (Example 1, Comparative Example 1). [Figure 7] FIG. 10 is a graph showing the measurement results of the gas permeability of the cell culture vessel fixed to the cell culture jig (Example 2, Example 3) in Test 2. [Figure 8] FIG. 10 shows the results of cell culture using the cell culture device of Test 3 (Example 4, Comparative Example 2). [Figure 9] FIG. 10 shows the results of cell culture using the cell culture device of Test 4 (Example 5, Comparative Example 3). DETAILED DESCRIPTION OF THE INVENTION

[0015] Hereinafter, embodiments of the cell culture device and the cell production method of the present invention will be described in detail, but the present invention is not limited to the specific contents of the following embodiments and examples.

[0016] The cell culture device of this embodiment is a cell culture device that holds a bag-shaped closed cell culture vessel formed by opposing planar materials, at least one of which is made of a gas-permeable film, and is characterized in that it comprises a stand on which the cell culture vessel is placed and a pressing member that presses the cell culture vessel against the stand, and the stand is formed of a material that allows air to pass through between the gas-permeable film and the stand, and the gas-permeable film side of the cell culture vessel is placed on the stand. Furthermore, in the cell culture device of this embodiment, it is preferable that the pressing member is formed of a material that allows ventilation between the gas-permeable film and the pressing member, and that the gas-permeable film side of the cell culture vessel is pressed against the pressing member.

[0017] Specifically, as shown in Figures 1 to 3, the cell culture device 1 of this embodiment includes a stand 11 on which a cell culture vessel 2 is placed, and a pressing member 12 that presses the cell culture vessel 2 against the stand 11. Support columns 13 are erected at the four corners of the stand 11, and a top plate 14 is fixed to the support columns 13. In addition, the top plate 14 is provided with guide pins 15, which pass through guide holes provided in the pressing member 12, and the pressing member 12 is provided so as to be movable vertically along the guide pins 15. It is also preferable to provide an opening in the top plate 14 so that the inside of the cell culture device 1 can be seen through this opening.

[0018] The stand 11 is formed of a material that allows air to pass between the gas-permeable film and the stand 11, and is preferably formed by, for example, processing the surface of the material to have an uneven surface, and more preferably, protrusions 111 are formed on the surface of the material by processing the surface to have an uneven surface.

[0019] Furthermore, the pressing member 12 is formed of a material that allows air to pass between the gas-permeable film and the pressing member 12, and is preferably formed by, for example, processing the surface of the material to have an uneven surface, and more preferably, protrusions 121 are formed on the surface of the material by processing the surface to have an uneven surface.

[0020] As the protrusions 111 and 121, it is preferable that a plurality of approximately triangular prisms are formed in parallel in a mountain range shape (hereinafter, this may be referred to as a mountain range pattern), as shown in Figures 1(b), 2, and 3. By forming the protrusions 111 and 121 in a mountain-like pattern in this manner, the contact area between the gas-permeable film and the stand 11 and pressing member 12 can be reduced, thereby preventing the gas permeability performance of the cell culture vessel 2 from being impaired.

[0021] Furthermore, such a mountain range pattern can improve the strength of the protrusions in the protrusions 111 and 121, and can prevent the protrusions from being damaged, such as being distorted, by the pressing force of the pressing member 12. Furthermore, by providing flat portions between the plurality of approximately triangular prisms in the mountain range pattern, it is possible to ensure visibility of the inside of the cell culture vessel 2 when the gas permeable film is made of a transparent material.

[0022] It is also preferable to use a porous material as the breathable material.Furthermore, it is also preferable to use a perforated plate as the breathable material. In the cell culture device 1 of this embodiment, even if a porous material or a perforated plate is used as the breathable material, it is possible to prevent the gas permeability of the cell culture vessel from being impaired.

[0023] Furthermore, the cell culture jig 1 of this embodiment is preferably provided with permanent magnets (not shown) at the four corners of the top plate 14 as a biasing means for biasing the pressing member 12 downward, and also with permanent magnets at positions corresponding to the permanent magnets of the pressing member 12 with their same polarities facing each other as a biasing means. By using such a biasing means, the pressing member 12 can be made movable in the vertical direction while pressing vertically against the planar substrate 21 in the cell culture vessel 2 due to the repulsive force acting between the permanent magnets. The biasing means is not limited to a permanent magnet, and the biasing means may be omitted and the pressing member 12 may be moved up and down by its own weight.

[0024] The base 11 can be made of, for example, a synthetic resin such as polycarbonate. The pressing member 12 may be made of a synthetic resin such as polycarbonate, or may be made of glass. It is preferable that the pressing member 12 is partially or entirely transparent so that the progress of the culture and the state of the culture object can be checked.

[0025] Next, a modified example of the cell culture device of this embodiment will be described with reference to FIG. As shown in the figure, a cell culture vessel 2 is placed on a stand 11a of a cell culture jig 1a, and a pressing member 12a presses the upper surface of the cell culture vessel 2. Guide pins 15a are provided at the four corners of the base 11a, and the guide pins 15a pass through guide holes provided in the pressing member 12a. The pressing member 12a is provided so as to be movable in the vertical direction along the guide pins 15a.

[0026] At this time, the pressing member 12a can be fixed so as to press the cell culture vessel 2 with a constant pressure due to its own weight. Also, a spring material can be attached to the guide pin 15a to appropriately press the pressing member 12a against the cell culture vessel 2. This allows the pressing member 12a to be movable in the vertical direction as the culture medium is injected into or discharged from the cell culture vessel 2.

[0027] In this embodiment, the cell culture vessel 2 is not particularly limited, but can be formed, for example, by heat-sealing the peripheral edges of two rectangular planar substrates 21, 22. The space formed between the two planar substrates is used as a culture space for culturing cells, and the area of ​​the planar substrates 21, 22 that forms the culture space constitutes the culture section of the cell culture vessel 2.

[0028] At least one of the planar substrates 21, 22 is preferably made of a gas-permeable film, and both are preferably made of a gas-permeable film. Suitable gas-permeable films include polyolefin resins such as polyethylene (linear low density polyethylene, LLDPE) and polypropylene. Furthermore, to allow the interior of the cell culture vessel 2 to be visible, the gas-permeable film is preferably made of a transparent material. It should be noted that the "flat" of the flat articles 21 and 22 does not mean that the surface of the article is flat and without any irregularities, but rather that the overall shape of the article is roughly the same, and includes articles with irregularities formed on the surface of the article.

[0029] When the cell culture vessel 2 is filled with a culture medium, the pressing member 12 is pushed up by the cell culture vessel 2 as shown in FIGS. On the other hand, when the culture medium is discharged from the cell culture vessel 2 through the port 23, the thickness of the culture medium in the cell culture vessel 2 decreases accordingly, and the pressing member 12 moves down accordingly.

[0030] 2, two ports 23 are provided facing each other at both ends in the longitudinal direction of the cell culture vessel 2, but the number of ports is not limited to this and may be one or three or more. Examples of materials that can be used for the ports include thermoplastic resins such as polyethylene, polypropylene, vinyl chloride, polystyrene elastomers, and FEP.

[0031] FIG. 5 shows the state of cell culture using the cell culture device 1 (1a) of this embodiment. In the figure, a cell culture vessel 2 is filled with a culture medium, and cells 3 are seeded in the culture space whose volume has been expanded by the culture medium, and the culture is then performed. An air vent space V is formed between the protrusion 111 (111a) of the stand 11 (11a) of the cell culture tool 1 (1a) and the planar substrate 22 of the cell culture vessel 2 on the stand side.

[0032] Furthermore, a ventilation space V is also formed between the protrusion 121 (121a) of the pressing member 12 (12a) of the cell culture tool 1 (1a) and the planar substrate 21 on the top plate side of the cell culture vessel 2. As described above, the cell culture jig 1 (1a) of this embodiment can prevent the gas permeability of the cell culture vessel 2 from being impaired when culturing cells, making it possible to culture cells at high densities.

[0033] The cell production method of this embodiment is characterized by culturing cells using the cell culture device 1 described above. The cells to be cultured using the cell culture device 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 a culture section in a container.

[0034] As described above, the cell culture device of this embodiment can prevent the gas permeability of the cell culture vessel from being impaired, making it possible to culture cells at high density. [Example]

[0035] Tests conducted to confirm the effects of the cell culture device according to the embodiment of the present invention will be described below.

[0036] [Test 1] The cell culture device of this embodiment and a conventional cell culture device were prepared, and the same cell culture vessel was fixed to each to conduct a test to compare gas permeability.

[0037] Specifically, as a cell culture jig of this embodiment (Example 1), a device was prepared in which protrusions were formed on the entire upper surface of the stand on which the cell culture vessel was placed and on the entire lower surface of the pressing member that pressed the cell culture vessel from above. Polycarbonate was used as the material for the stand and the pressing member, and a mountain range pattern, as shown in the above-described embodiment, was formed as protrusions on the entire upper surface of the stand and the entire lower surface of the pressing member. The height of the protrusions in the mountain range pattern formed was 200 μm, the width was 400 μm, and the pitch was 800 μm. The size of the entire upper surface of the stand and the entire lower surface of the pressing member was larger than the size of the cell culture vessel (described below) to be fixed thereto. This also applies to the following tests.

[0038] A film with flat surfaces made of 110 μm-thick linear low-density polyethylene (manufactured by Toyo Seikan Group Holdings Co., Ltd.) was prepared as the cell culture vessel material to be fixed to this cell culture jig. One port was sandwiched between two pieces of film, and the periphery was heat-sealed to form a bag shape. The external dimensions were 120 mm × 65 mm, and the base area of ​​the culture space was 48 cm. 2 It was.

[0039] Furthermore, as a conventional cell culture jig (Comparative Example 1), a jig was prepared in which the entire upper surface of a stand on which a cell culture vessel is placed and the entire lower surface of a pressing member that presses the cell culture vessel from above are flat. The material of the stand and the pressing member was a flat plate made of polycarbonate. Furthermore, the same cell culture vessel as in Example 1 was prepared as the cell culture vessel to be fixed to this cell culture jig.

[0040] Next, a non-contact oxygen concentration meter was placed in each cell culture vessel, and 20 ml of pure water was filled. The liquid depth in each vessel was 4 mm. The change in the dissolved oxygen concentration in the water near the bottom of each cell culture vessel was then measured. At this time, each cell culture vessel was fixed to its respective cell culture jig and placed in a CO2 incubator at 37°C, where the dissolved oxygen in the vessel was adjusted to approximately 21%, the atmospheric oxygen concentration. After the oxygen concentration in the vessel stabilized, the oxygen concentration in the CO2 incubator was lowered to 10%, and then raised to 21%.

[0041] Here, when the oxygen concentration around the container changes from 21% to 10%, a difference in oxygen partial pressure occurs between the inside and outside of the container, causing the oxygen inside the container to escape through the gas-permeable film. Furthermore, when the oxygen concentration around the container changes from 10% to 21%, oxygen outside the container enters the container through the gas-permeable film. The rate of change in oxygen concentration correlates with the gas permeability of the film, allowing quantitative measurement of gas permeability. The results are shown in Figure 6.

[0042] In Figure 6, the oxygen concentration in the cell culture vessel of Example 1 decreased to about 10%, while the oxygen concentration in the cell culture vessel of Comparative Example 1 decreased only to about 12%. This is because the rate of decrease in the oxygen concentration in Comparative Example 1 was slow, and it was expected that it would take a considerable amount of time to decrease to 10%, as in Example 1. Therefore, Comparative Example 1 was stopped at about 12%, and the measurement was resumed after returning it to 21%.

[0043] As shown in Figure 6, when the oxygen concentration around the container was changed from 21% to 10%, the maximum change in oxygen concentration per minute in the cell culture container of Example 1 was 0.75 / min. In contrast, the maximum change in oxygen concentration per minute in the cell culture container of Comparative Example 1 was 0.25% / min. Furthermore, when the oxygen concentration around the container was changed from 10% to 21%, the maximum change in oxygen concentration per minute in the cell culture container of Example 1 was 0.6% / min. In contrast, the maximum change in oxygen concentration per minute in the cell culture container of Comparative Example 1 was 0.3% / min.

[0044] That is, when the oxygen concentration around the container was changed from 21% to 10%, the gas permeability of Example 1 was about three times greater than that of Comparative Example 1, and when the oxygen concentration around the container was changed from 10% to 21%, the gas permeability of Example 1 was about twice greater than that of Comparative Example 1. Thus, it was found that the cell culture device of this embodiment exhibits superior oxygen permeability compared to conventional cell culture devices.

[0045] [Test 2] Different configurations (Example 2 and Example 3) were prepared as cell culture jigs of this embodiment, and the same cell culture vessel was fixed to each to conduct a test to compare gas permeability.

[0046] Specifically, as a cell culture vessel of this embodiment (Example 2), a pressing member that presses the cell culture vessel from above was prepared, in which protrusions were formed on the entire lower surface thereof, and no protrusions were formed on the upper surface of the stand on which the cell culture vessel was placed. Polycarbonate was used as the material for the pressing member, and a mountain range pattern was formed as the protrusions on the entire lower surface of the pressing member, similar to Example 1. A flat plate made of polycarbonate was used as the material for the stand. Furthermore, the same cell culture vessel as in Example 1 was prepared as the cell culture vessel to be fixed to this cell culture jig.

[0047] Next, as a cell culture vessel of this embodiment (Example 3), a vessel in which protrusions were formed on the entire upper surface of a stand on which the cell culture vessel was placed was prepared. No protrusions were formed on the lower surface of the pressing member that presses the cell culture vessel from above. Polycarbonate was used as the material of the stand, and a mountain range pattern was formed as the protrusions on the entire top surface of the stand, similar to Example 1. A flat plate made of polycarbonate was used as the material of the pressing member. Furthermore, the same cell culture vessel as in Example 1 was prepared as the cell culture vessel to be fixed to this cell culture jig.

[0048] Next, a non-contact oxygen concentration meter was placed in each cell culture vessel, and 20 ml of pure water was filled. The liquid depth in each vessel was 4 mm. The change in the dissolved oxygen concentration in the water near the bottom of each cell culture vessel was then measured. At this time, each cell culture vessel was fixed to its respective cell culture jig and placed in a CO2 incubator at 37°C, where it was left for a while until the dissolved oxygen in the vessel reached approximately 21%, the atmospheric oxygen concentration. After the oxygen concentration in the vessel stabilized, the oxygen concentration in the CO2 incubator was lowered to 10%, and then raised to 21%. The results are shown in Figure 7.

[0049] 7, the oxygen concentration in the cell culture vessel of Example 2 at the start of measurement was about 20%, while the oxygen concentration in the cell culture vessel of Example 3 was about 24%. This is because the measurement using the non-contact oxygen concentration meter used in this test is a system in which the change in an element attached to the vessel is measured non-contact with the measuring device, and the value is accurate when the element and the measuring unit are in contact, and the value increases as the element and the measuring unit become more distant from each other. Therefore, although the dissolved oxygen at 37°C is normally 21%, the value increases depending on the thickness of the vessel and the installation state. However, this does not affect the amount of change, and oxygen permeability is determined based on the amount of change, so this measurement does not pose a problem in terms of performance evaluation.

[0050] As shown in Figure 7, when the oxygen concentration around the container was changed from 21% to 10%, the maximum change in oxygen concentration per minute in the cell culture container of Example 2 was 0.4% / min. In contrast, the maximum change in oxygen concentration per minute in the cell culture container of Example 3 was 0.8% / min. Furthermore, when the oxygen concentration around the container was changed from 10% to 21%, the maximum change in oxygen concentration per minute in the cell culture container of Example 2 was 0.25% / min. In contrast, the maximum change in oxygen concentration per minute in the cell culture container of Example 3 was 1.1% / min.

[0051] That is, when the oxygen concentration around the container was changed from 21% to 10%, the gas permeability of Example 3 was about twice as high as that of Example 2, and when the oxygen concentration around the container was changed from 10% to 21%, the gas permeability of Example 3 was about four times as high as that of Example 2. In this way, it was found that forming the protrusions on the cell culture jig on the upper surface of the stand on which the cell culture vessel is placed is more effective in improving the oxygen permeability of the cell culture vessel fixed to the cell culture jig and used for cell culture than forming them on the lower surface of the pressing member that presses the cell culture vessel from above.

[0052] [Test 3] The cell culture device of this embodiment and a conventional cell culture device were prepared, and the same cell culture vessel was fixed to each to carry out cell culture, and a test was conducted to compare the culture performance of these devices.

[0053] Specifically, a cell culture jig of this embodiment (Example 4) was prepared that was the same as Example 1. That is, a cell culture jig was prepared in which protrusions were formed on the entire upper surface of a stand on which a cell culture vessel was placed and on the entire lower surface of a pressing member that pressed the cell culture vessel from above. The protrusions formed a mountain range pattern over the entire upper surface of the base and the entire lower surface of the pressing member. Furthermore, the same cell culture vessel as in Example 1 was prepared as the cell culture vessel to be fixed to this cell culture jig.

[0054] Furthermore, a conventional cell culture jig (Comparative Example 2) was prepared that was the same as Comparative Example 1. That is, the entire upper surface of the stand on which the cell culture vessels were placed and the entire lower surface of the pressing member that pressed the cell culture vessels from above were flat. Furthermore, the same cell culture vessel as in Example 1 was prepared as the cell culture vessel to be fixed to this cell culture jig.

[0055] iPS cells (1231A3 strain) were used for culture. StemFit AK02N (Ajinomoto Co., Inc.) was used as the culture medium, and these were filled into each cell culture vessel. The culture medium was 10 ml at 37°C, with a liquid thickness of 2 mm in each vessel. 10 ml of culture medium was added to the cell culture vessel, along with 10 μM Y-27632 and 24 μl of adhesive substrate (i-Matrix, laminin 511-E8). 70,000 cells were then added to the cell culture device and culture was initiated. 10 ml of culture medium (only culture medium without Y-27632 or adhesive substrate) was replaced on days 1, 4, 5, and 6 after cell seeding, and the cells were cultured for 7 days. After 7 days, cells were harvested using the detachment solution TrypLE, counted, and the proliferation fold was calculated. This procedure was repeated twice for each cell culture device. The results are shown in Figure 8.

[0056] As shown in the figure, the cell proliferation rate of the cell culture vessel of Example 4 was superior to that of Comparative Example 2, and it was found that the cell culture device of this embodiment can suppress the inhibition of the gas permeability performance of the cell culture vessel and enable cells to be cultured at high density.

[0057] [Test 4] The cell culture device of this embodiment and a conventional cell culture device were prepared, and the same cell culture vessel was fixed to each to carry out cell culture, and a test was conducted to compare the culture performance of these devices.

[0058] Specifically, as a cell culture device (Example 5) of this embodiment, a stand on which a cell culture vessel is placed has an upper surface entirely made of a porous material. The lower surface of a pressing member that presses the cell culture vessel from above is not made of a porous material but is flat. The stand was made of a porous polypropylene filter plate (pore diameter 400 μm, thickness 3 mm, AS ONE Corporation 3-2531-02), and the pressing member was made of a flat polycarbonate plate. Furthermore, the same cell culture vessel as in Example 1 was prepared as the cell culture vessel to be fixed to this cell culture jig.

[0059] Furthermore, a conventional cell culture jig (Comparative Example 3) was prepared that was the same as Comparative Example 1. That is, the entire upper surface of the stand on which the cell culture vessels were placed and the entire lower surface of the pressing member that pressed the cell culture vessels from above were flat. Furthermore, the same cell culture vessel as in Example 1 was prepared as the cell culture vessel to be fixed to this cell culture jig.

[0060] iPS cells (1231A3 strain) were used for culture. StemFit AK02N (Ajinomoto Co., Inc.) was used as the culture medium, and these were filled into each cell culture vessel. The culture medium was 10 ml at 37°C, with a liquid thickness of 2 mm in each vessel. 10 μM Y-27632 and 24 μl of adhesive substrate (i-Matrix, laminin 511-E8) were added to 10 ml of culture medium, and 70,000 cells were added. The vessel was then placed in a cell culture jig and cultured. 1, 4, 5, and 6 days after cell seeding, the entire volume of the culture medium was replaced with 10 ml (only culture medium without Y-27632 or adhesive substrate), and the vessel was cultured for 7 days. After 7 days, cells were harvested using the detachment solution TrypLE, counted, and the proliferation fold was calculated. The results are shown in Figure 9.

[0061] As shown in the figure, the cell proliferation rate of the cell culture vessel of Example 5 was superior to that of Comparative Example 3, and it was found that the cell culture device of this embodiment can suppress the inhibition of the gas permeability performance of the cell culture vessel and enable cells to be cultured at high density.

[0062] The present invention is not limited to the above-described 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 device may be modified as needed, such as to have various shapes different from those in the embodiments. [Industrial Applicability]

[0063] The present invention can be suitably used when a cell culture bag is fixed to a cell culture jig and used to culture cells in large quantities at high density. [Explanation of symbols]

[0064] 1,1a Cell culture jig 11,11a Mounting stand 111,111a Protrusion 12, 12a Pressing member 121,121a Protrusion 13 Support pillar 14 Top plate 15,15a Guide pin 2 Cell culture vessel 21,22 Planar equipment 23 ports V Ventilation space

Claims

1. A cell culture device for holding a bag-shaped closed cell culture vessel formed by opposing planar substrates, at least one of which is made of a gas-permeable film, a stand on which the cell culture vessel is placed; a pressing member that presses the cell culture vessel against the stand, The stand is formed of a material that allows air to pass between the gas permeable film and the stand, A protrusion is provided on the surface of the ventilable device, When the gas-permeable film side of the cell culture vessel is placed on the stand, The protrusions form a ventilation space between the gas permeable film and the surface of the stand. A cell culture device characterized by:

2. The pressing member is formed of a material that allows air to pass between the gas permeable film and the pressing member, and the gas permeable film side of the cell culture vessel is pressed against the pressing member.

2. The cell culture device according to claim 1.

3. Guide pins are provided at the four corners of the frame, and the guide pins pass through guide holes provided in the pressing member, and the pressing member is provided so as to be movable in the vertical direction along the guide pins.

3. The cell culture device according to claim 1 or 2.

4. Support columns are erected at the four corners of the stand, a top plate is fixed to the support columns, guide pins are provided on the top plate, the guide pins pass through guide holes provided in the pressing member, and the pressing member is provided so as to be movable in the vertical direction along the guide pins.

3. The cell culture device according to claim 1 or 2.

5. 3. The cell culture device according to claim 2, wherein the breathable material of the pressing member is a porous material.

6. 3. The cell culture device according to claim 2, wherein the breathable material of the pressing member is a perforated plate.

7. 3. The cell culture device according to claim 2, wherein the breathable material of the pressing member is formed by processing the surface of the material into irregularities.

8. 8. The cell culture device according to claim 7, wherein protrusions are formed on the surface of the breathable substrate by the uneven processing.

9. 9. The cell culture device according to claim 1, wherein the protrusions are formed in the form of a plurality of substantially triangular prisms arranged in parallel in a mountain range shape.

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

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