JP6926410B2 - Cell culture container, cell culture method, cell culture container manufacturing method and cell culture container manufacturing equipment - Google Patents

Description

The present invention relates to a technique for culturing various cells, more specifically, a cell culture vessel having gas permeability and capable of culturing cells, a cell culture method using this cell culture vessel, and this cell culture vessel. Regarding the manufacturing method of.

In the modern medical field represented by gene therapy, cell therapy, and regenerative medicine, target cells (including tissues, microorganisms, viruses, etc.) are cultured and induced to differentiate in an artificial environment. Such cultured cells can be classified into adhesive culture cells and suspension culture cells according to their presence in the culture.

Adhesive culture cells are cultured cells that adhere to, for example, the bottom surface of a culture vessel for culturing cells and proliferate. In these adhesive culture cells, in addition to subculture in which existing cultured cells are transferred to a new culture vessel and proliferated, medium exchange is also appropriately performed.

Here, regarding the above-mentioned cell culture, the following culture vessels have been conventionally proposed.
For example, as a container preferably used in a laboratory, a petri dish (Petri dish) having a flat bottom surface without distortion and a culture flask are known. Of these, the Petri dish can be sealed inside the container with a lid, and the vent type lid is provided with ribs so that the degassing / non-gas venting position can be selected. Further, the culture flask having a flat bottom surface as in the Petri dish also has an advantage that a good field of view can be obtained at the time of microscopic observation because the culture surface is excellent in uniformity and smoothness.

On the other hand, in contrast to open cell culture using Petri dishes and culture flasks, closed cell culture in which cells are cultured in a tightly closed space is also known. In such a closed cell culture, a cell culture bag made of a flexible resin is preferably used because of the need to suppress the risk of contamination while ensuring transparency and gas permeability.
However, in a general cell culture bag, there is a problem that the bottom surface, which is the culture surface, becomes uneven when the culture solution is injected. Therefore, for example, a tray-shaped container for cell culture as in Patent Document 1 has been proposed. That is, the tray-shaped container for cell culture disclosed in Patent Document 1 has a recess in which the first container wall is transparent and has a single flat bottom surface, and a flange-shaped portion formed on the periphery of the recess. The second container wall has a gas permeable structure and is deformable and flexible.

Patent No. 4780462

It is true that according to Patent Document 1, the flatness of the culture surface can be ensured to some extent, but as described below, there are still many problems to be improved.
That is, in adhesive culture cells such as iPS cells, the degree of cell proliferation depends on the size of the area, so it is not sufficient to simply secure a flat culture surface and culture as much as possible. The surface needs to be flat and wide. In particular, cells intended for use in regenerative medicine are extremely valuable, and their culture requires a great deal of time and cost, so high efficiency is also required.

Furthermore, since the medium (culture solution) required for cell culture is very expensive, there is a potential need to use a small amount of medium as efficiently as possible. Therefore, the culture surface required for culturing the cells must be flat as wide as possible, and the culture solution must be spread to every corner of the culture surface using a relatively small amount of medium.
In addition, it is desirable that the liquid thickness of the medium is wide and uniform so that the cells that settle on the bottom surface after seeding are uniformly distributed in density and the nutrients in the medium are evenly distributed to all cells.
However, the conventional type cell culture vessels including Patent Document 1 have no recognition or suggestion regarding the above-mentioned problems.

In view of solving the above-mentioned problems as an example, the present invention can secure a wide and flat culture surface and can spread even a small amount of culture solution to every corner of the culture surface with a uniform thickness. It is an object of the present invention to provide a cell culture vessel, a cell culture method, and a method for producing a cell culture vessel.

In order to achieve the above object, the cell culture container of the present invention comes into contact with the first container wall made of a flat film having gas permeability and a bottom surface at the peripheral edge of the first container wall, and at the same time. A port leading to a second container wall having a bulging shape protruding from the first container wall inside the peripheral edge portion, and a culture space surrounded by the bulging shape of the first container wall and the second container wall. The first container wall is flat except for a region in contact with the port.

Further, the cell culture method of the present invention is the cell culture method using the cell culture container of the present invention described above, and the first container wall is downward with respect to the second container wall. It is characterized in that cells and a culture medium are injected through the port while placing the container wall.

Further, in the method for producing a cell culture container of the present invention, a first container wall made of a gas-permeable film and a second container wall arranged to face the first container wall are placed on top of each other. A step of placing the first container wall on a pedestal and a step of pressing the first container wall and the peripheral edge of the second container wall with a restraining member while the central portion of the second container wall is open. And the step of introducing the fluid between the first container wall and the second container wall in a state where the peripheral portion is pressed by the restraining member, and the pressing member against the central portion of the second container wall. It is characterized by having at least a step of heating the pressing member while pressing with.

Further, the apparatus for manufacturing a cell culture container of the present invention has a bulging shape that comes into contact with the first container wall made of a flat film at the peripheral edge of the first container wall and projects from the first container wall. A device for manufacturing a cell culture container including a second container wall having the first container wall, a mounting table on which the first container wall is placed, the first container wall mounted on the above-mentioned table, and the second container wall. A fluid introduction device that introduces fluid into the space between them, a pressing member that is arranged so as to be able to advance and retreat with respect to the above-mentioned stand and pressurizes the second container wall into which the fluid is introduced into the space, and the pressing. The restraining member includes a heating device for heating the member and a restraining member which is arranged to face the above-mentioned stand and restrains the periphery of the second container wall mounted on the mounting stand. 2 The pressing member is heated by the heating device while restraining the container wall, and the fluid is introduced into the space between the first container wall and the second container wall by the fluid introduction device. ..

According to the present invention, a wide and flat culture surface can be secured by the flat first container wall as the bottom surface, and the culture surface is secured by the second container wall having a bulging shape even if the amount of the culture solution is small. It is possible to spread to every corner of. Furthermore, the cell density at the time of culturing is maintained appropriately, and high-quality cells can be mass-cultured in a state where the risk of contamination is suppressed without depletion of the medium components required for proliferation.

It is an external perspective view of the cell culture container 10 in an embodiment. It is a side view of the cell culture container 10 in an embodiment. It is a front view of the cell culture container 10 in an embodiment. It is a figure which shows the manufacturing apparatus and manufacturing method of the cell culture container 10 in an embodiment. It is a figure which shows the comparison between the cell culture container of the conventional type and the cell culture container 10 of an embodiment. It is a figure which shows the cell culture container 10 in the modification 1. It is a figure which shows the cell culture container 10 in the modification 2.

Hereinafter, the cell culture vessel, the cell culture method, and the method for producing the cell culture vessel of the present invention will be specifically described with reference to the drawings as appropriate. For convenience of explanation, the X direction, the Y direction, and the Z direction are defined respectively in the following description, but the scope of rights of the present invention is not intentionally limited or reduced.

[Cell culture container]
FIG. 1 shows an external perspective view of the cell culture vessel 10 according to the embodiment of the present invention.
The cell culture container 10 is a flexible cell culture container formed in a bag shape using a film-based flexible packaging material as a material. It is preferable that the cell culture vessel 10 has gas permeability suitable for culturing cells, and a part or all of the cell culture container 10 is transparent so that the contents can be confirmed.

Such a cell culture vessel 10 is configured to include at least a first vessel wall 1, a second vessel wall 2, and a port 3. Further, the outer shape of the cell culture vessel 10 is preferably rectangular, for example, 20 to 1000 mm in the X direction and 20 to 1000 mm in the Y direction.

The first container wall 1 is made of a flat film having gas permeability and serving as a bottom surface. As shown in FIG. 2, the first container wall 1 of the present embodiment preferably has a thickness t1 of, for example, 30 to 200 μm. Here, the "bottom surface" in the present embodiment means a surface that becomes the bottom when the cell culture container 10 is placed on a mounting table or the like, and is below the second container wall 2 described later in the Z direction. The surface that becomes. Further, the "planar shape" in the present embodiment means a flat surface in the X direction and the Y direction, and means a single plane parallel to the XY plane.

This for the first gas permeable container wall 1 comprises, in compliance with the gas permeability test method of JIS K 7126, permeability of oxygen was measured at a test temperature 37 ° C. ^{is, 5000mL / (m 2 · day} · Atm) or more is preferable.

Further, it is preferable that the film constituting the first container wall 1 is partially or wholly transparent so that the progress of cell culture and the state of cells can be confirmed. The material used for such a film is not particularly limited as long as it has the above-mentioned gas permeability. Examples thereof include thermoplastic resins such as polyethylene, polypropylene, ethylene-vinyl acetate copolymer, polyester, silicone-based elastomer, polystyrene-based elastomer, and tetrafluoroethylene-hexafluoropropylene copolymer (FEP). These may be used as a single layer, or may be used by laminating the same or different materials, but in consideration of heat-sealing property when sealing the peripheral portion, they have a layer that functions as a sealant layer. Is preferable.

Further, as shown in the figure, the first container wall 1 is configured to have a peripheral portion 1a and a central portion 1b. Of these, the peripheral edge portion 1a is a region facing the peripheral edge portion 2a of the second container wall 2 described later. Further, the central portion 1b is a region inside the peripheral portion 1a described above, and is a region forming a culture space S described later.

The second container wall 2 has a bulging shape 2z that is in contact with the peripheral edge portion 1a of the first container wall 1 and projects from the first container wall 1 inside the peripheral edge portion 1a. Like the first container wall 1, the second container wall 2 is made of a gas-permeable film.
More specifically, regarding the gas permeability of the second container wall 2, the oxygen permeability measured at a test temperature of 37 ° C. is 5000 mL / (in accordance with the gas permeability test method of JIS K 7126). m ² · day · atm) or higher than it is preferable. That is, the gas permeability of the second container wall 2 may be set to be equal to the gas permeability of the first container wall 1. Further, the film constituting the second container wall 2 is preferably partially or wholly transparent so that the progress of cell culture and the state of cells can be confirmed, and further, the first container wall 2 is formed. It may be composed of the same material as 1.

Further, as shown in FIG. 2, the second container wall 2 of the present embodiment preferably has a thickness t2 of, for example, 30 to 200 μm. That is, the thickness t2 of the second container wall 2 may be set to be equal to the thickness t1 of the first container wall 1. In other words, the thickness ratio of the first container wall 1 and the second container wall 2 may be substantially 1.

The second container wall 2 is configured to have a peripheral portion 2a, a rising portion 2b, and a central portion 2c. Of these, the peripheral edge portion 2a is a region that comes into contact with the peripheral edge portion 1a of the first container wall 1. Further, the central portion 2c is a region inside the rising portion 2b described later, and is a region arranged apart from the central portion 1b in the Z direction by a desired height for forming the culture space S. The rising portion 2b is a region where the central portion 2c rises from the first container wall 1 so as to be separated from the first container wall 1.

In the present embodiment, the peripheral edge portion 1a of the first container wall 1 and the peripheral edge portion 2a of the second container wall 2 may be sealed to each other by heat welding, whereby the airtightness of the culture space S is more reliably sealed. Be secured. However, the present invention is not limited to this embodiment, and an embodiment in which the peripheral edge portion 1a of the first container wall 1 and the peripheral edge portion 2a of the second container wall 2 are fixed via, for example, a known adhesive may be used.
Further, under the idea of securing a flat culture surface having a uniform thickness of the culture solution as much as possible, the narrower the width of the rising portion 2b, the better, and the narrower the width of the sealing region at the peripheral portion. The narrower the better.

In the present embodiment, the above-mentioned rising portion 2b and the central portion 2c form a bulging shape 2z that bulges like a plateau, and a culture space S is formed inside the bulging shape 2z. The height of the culture space S in the Z direction is not particularly limited and may be appropriately set so as to have an appropriate liquid thickness according to the state of the cells to be cultured. It may be mm to several tens of mm.

As shown in FIG. 2 or 3, the central portion 2c may be a flat surface. In other words, it is preferable that the top surface (central portion 2c) of the second container wall 2 forming the culture space is flat. Further, the rising portion 2b of the second container wall 2 is a region where the film is deformed into a part of the plateau shape by heating as described later, and the hardness may be higher than that of the central portion 2c. In other words, the hardness of the rising portion 2b of the second container wall 2 forming the bulging shape 2z is set higher than the hardness of the region (central portion 2c) different from the rising portion 2b of the bulging shape 2z. You may. Further, the hardness of the central portion 2c may be set equal to the hardness of the central portion 1b. In other words, the hardness of the central portion 2c of the second container wall 2 may be set to be substantially equal to the hardness of the central portion 1b of the first container wall 1 facing the central portion 2c.

As shown in FIGS. 2 and 3, the port 3 is a member leading to the culture space S surrounded by the first container wall 1 and the second container wall 2. The port 3 is a tubular member through which a medium, cells, or the like can flow. The port 3 is formed into a predetermined shape by injection molding, extrusion molding, or the like using a thermoplastic resin such as polyethylene, polypropylene, vinyl chloride, polystyrene elastomer, or FEP.

In addition, in order to prevent the port 3 from being blocked by the central portion 2c of the second container wall 2 and the central portion 1b of the first container wall 1 sticking to the port 3, the culture space is formed from the base end thereof. A port blockage prevention piece protruding into S may be provided. When such a port blockage prevention piece is provided, the port blockage prevention piece is provided on the central portion 2c side of the culture space S side so as not to interfere with the cells existing on the surface of the central portion 1b on the first container wall 1. It is preferable to provide it so that it is located.

Further, in the present embodiment, the cross section of the port 3 is semicircular, and the surface of the port 3 in contact with the first container wall 1 is made flat while the surface in contact with the second container wall 2 is formed. It may be configured to have a curved surface.
As a result, the formation of a gap between the port 3 and the second container wall 2 is suppressed, and the culture solution is prevented from leaking from the culture space S.

Further, in the present embodiment, it is preferable that at least the surface of the first container wall 1 and the top surface of the second container wall 2 are parallel to each other. As a result, it is possible to secure a wide culture space S, and even if a relatively small amount of the culture solution is used, the culture solution can be efficiently distributed to every corner of the culture space S.
More preferably, as shown in FIG. 3, the surface of the first container wall 1, the top surface of the second container wall 2 (central portion 2c), and the bottom surface of the port 3 may be parallel to each other. In other words, it is preferable that the bottom surface of the port 3 of the present embodiment (contact surface in contact with the first container wall 1) is flush with the surface of the first container wall 1. This makes it possible to more efficiently distribute a relatively small amount of the culture solution to every corner of the culture space S.

[Method for manufacturing cell culture container and device for manufacturing cell culture container]
Next, the manufacturing method and the manufacturing apparatus of the cell culture vessel 10 in the present embodiment will be described with reference to FIG.
That is, the cell culture container 10 uses the cell culture container manufacturing apparatus including the pressing member 21, the restraining member 22, the mounting table 23, the heating device 24, and the fluid introduction device 25, which are described below, respectively, and performs each of the following steps. Manufactured after.

First, as shown in FIG. 4A, the first container wall 1 made of a gas-permeable film and the second container wall 2 arranged to face the first container wall 1 are overlapped with each other. The first container wall 1 is placed on the mounting table 23. At this time, it is preferable that the peripheral edge portion 1a of the first container wall 1 and the peripheral edge portion 2a of the second container wall 2 are sealed by, for example, heat welding.

Further, it is preferable that the above-mentioned port 3 is installed at the end of the first container wall 1 and the second container wall 2. The aggregate of the first container wall 1, the second container wall 2 and the port 3 shown in FIG. 4A is referred to as a base body 10'as a cell culture container before completion.

After the first container wall 1 is placed on the mounting table 23, as shown in FIG. 4 (b), the central portion 2c of the second container wall 2 is open (not pressed). 2 The peripheral edge portion 2a of the container wall 2 is pressed by the restraining member 22 to be restrained. In this way, the restraining member 22 is arranged so as to face the mounting table 23, and has a function of restraining the periphery of the second container wall 2 mounted on the mounting table 23.
Although no particular limitation on the shape of the restraining member 22 the rim portion 2a as described above as long as possible constraint, the shape for restraining the second peripheral edge of the container wall 2 without excess or deficiency is preferable, and corresponds to the R ₁ region It is desirable to have a shape that constrains all four sides of the peripheral edge including the one. Further, in the present embodiment, the restraint member 22 is used for restraint, but the restraint member 22 may be omitted as appropriate as long as the flatness of the first container wall 1 is ensured.

After restraining the peripheral edge portion 2a of the second container wall 2 with the restraining member 22, as shown in FIG. 4C, the fluid introducing device 25 is used in a state where the peripheral edge portion described above is pressed by the restraining member 22. A fluid is introduced between the first container wall 1 and the second container wall 2. In the present embodiment, the fluid is introduced into the inside of the base body 10'(the space between the first container wall 1 and the second container wall 2) via the port 3. As described above, in the present embodiment, the fluid is introduced into the space between the first container wall 1 and the second container wall 2 by the fluid introduction device 25 while restraining the second container wall 2 by the restraining member 22.

The fluid introduced by the fluid introduction device 25 in this embodiment is a liquid or a gas. Examples of the fluid include pure water, and examples of the gas include purified air (clean air) and an inert gas such as nitrogen. Of these, clean air is applied in this embodiment from the viewpoint of ease of handling and processing.

After the fluid is introduced into the base body 10', as shown in FIG. 4D, the pressing member 21 is lowered to a position separated from the mounting table 23 by a predetermined distance, and the second container wall 2 At least the pressing member 21 is heated by the heating device 24 while being pressed against the central portion 2c by the pressing member 21. At this time, the above-mentioned predetermined distance becomes the size (height) of the bulging shape 2z of the cell culture vessel 10. In this way, the pressing member 21 is arranged so as to be able to advance and retreat with respect to the mounting table 23, and the second container wall 2 in which the fluid is introduced into the space between the first container wall 1 and the second container wall 2 is provided. It has a function to pressurize.
Examples of the heating device 24 of the present embodiment include known resistance heating means such as nichrome wire, and the heating device 24 can be typically installed inside the pressing member 21. Further, the heating device 24 may be installed inside the mounting table 23, and the heating device 24 may heat at least one of the mounting table 23 and the pressing member 21.

The temperature at which the heating device 24 heats the pressing member 21 is determined in consideration of the heat resistant temperature of the film used for the first container wall or the like, but the heating is such that the film softens (for example, about 80 ° C.). Is preferable. By heating using the pressing member 21, the above-mentioned bulging shape 2z is formed. When heating by the pressing member 21, a specific region of the pressing member 21 (a region facing the peripheral portion 2b of the second container wall 2 while avoiding a region facing the central portion 2c of the second container wall 2). A heating means (for example, a resistance heating device such as a nichrome wire) may be arranged in the container. As a result, it is possible to prevent the second container wall 2 from being hardened to a region that is not necessarily necessary for forming the bulging shape 2z.

Further, when the second container wall 2 is pressed by the pressing member 21 heated to a desired temperature, the base body is set so that the internal pressure inside the base body 10'(which later becomes the culture space S) becomes constant. It is preferable that the supply pressure of the fluid introduced into the inside of the 10'is adjusted by the fluid introduction device 25. As a result, excessive pressure is suppressed from being applied to the inside of the base body 10', and stretching of the film and breakage of the seal are prevented.
The pressing member 21 or the like is further provided with a cooling device, and the pressing member 21 is cooled by using this cooling device while the pressing member 21 is being heated and the second container wall 2 is being pressed. You may.

After the second container wall 2 is pressed by the pressing member 21 heated to a desired temperature and a predetermined time elapses, the pressing member 21 and the restraining member 22 are pressed against the mounting table 23 as shown in FIG. 4 (e). Is evacuated and the completed cell culture vessel 10 is taken out to complete the process. It is preferable to seal the port 3 of the cell culture container 10 before retracting the pressing member 21 and the restraining member 22 with respect to the mounting table 23. As a result, it is possible to prevent foreign substances and the like from inadvertently invading the culture space S.

<Significance of flat bottom surface and bulging shape>
In conventional cell culture vessels, there are some shapes in which only a part of the bottom surface, which is the culture surface of cells, is flat, but there is no idea of ensuring the maximum culture surface of cells, and further, the culture solution in the culture space There was no presentation of the problem or even suggestion of its composition in ensuring homogeneous diffusivity. More specifically, in the cell culture vessel manufactured by the conventional method as shown in FIG. 5 (b), the culture solution cannot spread uniformly in the plane direction (XY plane), and the corner portion of the container is cultured. Since the liquid does not spread, it cannot be a good culture space. Further, since the culture solution has a warp also in the height direction (Z direction), the density of the culture solution is totally generated.

On the other hand, in the cell culture vessel of the present embodiment shown in FIG. 5A, it is clear that the culture solution spreads uniformly and uniformly in both the plane direction and the height direction described above. In this way, the cell culture vessel 10 produced by the present embodiment has a reference to the central portion 1b of the first container wall 10 that serves as the culture surface, even when only a relatively small amount of the culture solution can be used. It is possible to evenly distribute the culture solution to every corner. Therefore, according to the present embodiment, it is possible to use an expensive culture solution as efficiently as possible, and it is possible to more reliably cultivate valuable cells while suppressing the risk of contamination.

The cell culture method using the cell culture container of the present embodiment is the cell culture method using the cell culture container 10 described above, and the first container wall 1 is opposed to the second container wall 2. It is characterized in that cells and a culture solution are injected through the port 3 while placing the first container wall so as to be downward. At this time, the cell culture vessel 10, a suitable temperature (e.g. 37 ° C.), carbon dioxide concentration (e.g. 5 to 10% CO ₂ concentration) and humidity (e.g., about 95%) the cell culture apparatus is adjusted to (CO ₂ It is preferably placed on the mounting surface in the incubator).

As a result, a wide and flat culture surface can be secured by the first container wall which is the bottom surface, and even if a small amount of the culture solution is used, the second container wall having a bulging shape can spread to every corner of the culture surface. It becomes possible.
In the above-described embodiment, adherent cells such as iPS cells have been described as an example, but the present invention is not limited to this example. That is, the present invention may be applied to a culture vessel for floating cells such as hematopoietic cells and ascites cells, a method and an apparatus for producing the same, and a cell culture method. In fact, even in the static culture of floating cells, there is a need to uniformly distribute the cells over a wide range, and the adherent cells take almost the same mode as the suspended cells until they settle on the bottom surface and adhere to each other after seeding.

The above-described embodiment can be modified in various ways without departing from the spirit of the present invention. Hereinafter, a modified example that can be appropriately applied to the above-described embodiment will be described.

<Modification example 1>
FIG. 6 is a modified example of the port 3 described in the present embodiment.
The port 3 of the embodiment described above has a curved surface (upper surface) in contact with the second container wall 2, but the present invention is not limited to this aspect, and various port shapes can be adopted.

For example, as in the port 4 shown in FIG. 6A, the surfaces in contact with the second container wall 2 (in this example, the upper surface 4a ₁ and the side _{surface 4a 2} ) may be flat. In other words, the port 4 may have a structure in which an injection outlet 4b is added to a rectangular parallelepiped main body 4a extending in the Y direction.

Further, as in the port 5 shown in FIG. 6B, the surfaces in contact with the second container wall 2 (in this example, the slope 5a ₁ and the slope 5a ₂ ) may be flat. In other words, the port 5 may have a structure in which an injection outlet 5b is added to a triangular columnar main body 5a extending in the Y direction.

<Modification 2>
FIG. 7 is another modification of the port 3 described in this embodiment.
That is, the bottom surface of the port 3 described in the above embodiment and the first modification is flat, and is a region of the first container wall 1 in contact with the port 3 (peripheral portion 1a in contact with the port 3). The outer surface was coplanar with the outer surface except for the area in contact with the port 3.
However, the present invention is not limited to this aspect, and as shown in FIG. 7, the first container wall 1 may be flat at least except for the region in contact with the port 3.

That is, in this modification, the port 3 having a general boat-shaped shape (the cross section is shaped like an almond) is used, and the bottom surface of the port 3 is not a flat surface but a curved surface that is convex downward. .. When the port 3 having such a shape is used, as shown in FIG. 7, the region of the first container wall 1 in contact with the port 3 has a downwardly convex curved surface along the shape of the port 3.
Even in such a case, since the first container wall 1 is flat except for the region in contact with the port 3, the above-mentioned effect of the present invention can be obtained.
Although the above description has been given for use in cell culture, the container of the present invention may be used for purposes other than cell culture, for example, when it is desired to store a liquid or the like with a flat surface as the bottom surface. ..

The present invention can be used, for example, as a technique for efficiently culturing various cells or a technique for storing a liquid in a desired state.

1 1st container wall 2 2nd container wall 3, 4, 5 Port 10 Cell culture container 21 Pressing member 22 Restraining member 23 Mounting stand 24 Heating device 25 Fluid introduction device

Claims (2)

A step of placing the first container wall on a mounting table in a state where a first container wall made of a gas-permeable film and a second container wall arranged so as to face the first container wall are overlapped with each other. ,
A step of pressing the first container wall and the peripheral edge of the second container wall with a restraining member while the central portion of the second container wall is open.
A step of introducing a fluid between the first container wall and the second container wall while the peripheral portion is pressed by the restraining member.
A step of heating at least the pressing member while pressing the central portion of the second container wall with the pressing member.
A method for producing a cell culture container, which comprises. Manufacture of a cell culture vessel including a first container wall made of a flat film and a second container wall having a bulging shape that is in contact with the peripheral edge of the first container wall and protrudes from the first container wall. It ’s a device,
A mounting table on which the first container wall is mounted and
A fluid introduction device that introduces a fluid into the space between the first container wall and the second container wall mounted on the above-mentioned stand, and
A pressing member that is arranged so as to be able to advance and retreat with respect to the above-mentioned stand and pressurizes the second container wall into which the fluid is introduced into the space.
A heating device that heats the pressing member and
Includes a restraining member that is arranged to face the above-mentioned pedestal and restrains the periphery of the second container wall mounted on the pedestal.
While restraining the second container wall with the restraining member and heating the pressing member with the heating device, the fluid introduction device introduces the fluid into the space between the first container wall and the second container wall. An apparatus for manufacturing a cell culture container.

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