Method for manufacturing a container, its manufacturing apparatus, cell culture method, cell culture container, and its manufacturing method and manufacturing apparatus

Abstract

The present invention relates to a method for manufacturing a container, its manufacturing apparatus, a cell culture method, a cell culture container, and its manufacturing method and manufacturing apparatus. The present invention provides a container that reduces the risk of contamination and also enables efficient processing such as culturing and differentiation induction of cells in the same container. In addition, a cell culture container capable of ensuring a wide and flat culture surface, etc. is provided. The method for manufacturing a container includes: placing a bag-shaped membrane container on a placement table formed with a recess, introducing a fluid into the interior of the placed bag-shaped membrane container, heating at least one of the placement table and the pressing member, and pressing the bag-shaped membrane container placed on the placement table with the pressing member. On the other hand, the cell culture container includes: a first container wall having air permeability, serving as a bottom surface, and formed of a planar membrane; and a second container wall having a bulging shape that contacts the peripheral portion of the first container wall and protrudes relative to the first container wall; the first container wall is planar at least in a region other than the region in contact with the port.

Description

[0001] This application is a divisional application of a Chinese patent application with an application number of 201780047758.2, a filing date of July 27, 2017, and an invention title of "Method for Manufacturing a Container, Its Manufacturing Apparatus, Cell Culture Method, Cell Culture Container, and Its Manufacturing Method and Manufacturing Apparatus". Technical Field

[0002] The present invention relates to a method for manufacturing a container capable of storing various liquids and the like and its manufacturing apparatus. In addition, the present invention relates to a technique for culturing various cells, and more specifically, to a cell culture container having air permeability and capable of culturing cells, a cell culture method using the cell culture container, and a manufacturing method of the cell culture container. Background Art

[0003] In the modern medical field represented by gene therapy or regenerative medicine, cells (including tissues, microorganisms, viruses, etc.) as targets are cultured and induced to differentiate in an artificial environment. In recent years, in particular, it has been sought to efficiently culture and induce differentiation of a large number of the above cells in an artificial environment.

[0004] Here, when culturing and inducing differentiation of cells, from the viewpoint of supplying necessary culture medium components to the proliferating cells, it becomes important to maintain the cell density in the culture medium within an appropriate range. The reason is that if the cell density in the culture medium becomes high as the cells proliferate, the proliferation of cells will be hindered due to exhaustion of culture medium components or accumulation of metabolites of the cells themselves. On the other hand, there is also the view that for efficient proliferation of cells, it is important to form cell aggregates to a certain extent; if the cell density in the culture medium is too low, the cells cannot be efficiently proliferated and induced to differentiate.

[0005] Under such circumstances, previously, the following method was used: subculture was repeated to appropriately maintain the cell density in the culture medium.

[0006] As such a method of subculture, a well plate or a flask is sometimes used as a culture container.

[0007] For example, in Patent Document 1, the following technique is disclosed: using a well plate, cells and a culture medium are added to each well to start culturing so as to achieve an appropriate cell density (see paragraph

[0027] etc.). In Patent Document 1, it is proposed that after the cells have sufficiently proliferated in the wells, they are transferred to a flask, a new culture medium is added, and when they have proliferated to a certain amount, they are transferred to a flask with a larger volume and the same treatment is performed to culture a large number of cells.

[0008] In addition, in Patent Document 2, the following technology is disclosed: on one side of a flask-type culture container formed in a polyhedron shape such as a rectangular parallelepiped, a plurality of recesses are formed. In Patent Document 2, first, cell aggregates are formed in the above-mentioned plurality of recesses, and then they are moved to a wide culture surface formed on the opposite side inside the container to form larger aggregates.

[0009] On the other hand, as the cultured cells cultured in the above-mentioned form, etc., according to their existing forms in the culture, they can be classified into adherent culture system cells and suspension culture system cells.

[0010] Adherent culture system cells are cultured cells that proliferate by attaching to, for example, the bottom surface of a culture container for cultured cells. In these adherent culture system cells, in addition to subculture in which existing cultured cells are transferred to a new culture container for proliferation, medium replacement is also appropriately performed.

[0011] Here, regarding the above cell culture, the following culture containers have been proposed.

[0012] As a container preferably used in a laboratory, for example, a culture dish (Petri dish) or a culture flask having a flat bottom surface without deflection is known. Among them, the culture dish can also be sealed inside the container with a lid, and ribs are provided for an exhaust type lid, and an exhaust / non-exhaust position can be selected. In addition, similar to the culture dish, a culture flask with a flat bottom surface also has the advantage of excellent culture surface uniformity and smoothness, so there is also an advantage of obtaining a good field of view during microscopic observation.

[0013] On the other hand, for open system cell culture using a culture dish or a culture flask, closed system cell culture in which cell culture is performed in a tightened space is also known. In such closed system cell culture, for the need to ensure transparency or air permeability and suppress the risk of contamination, a cell culture bag formed of a flexible resin is preferably used.

[0014] However, in a general cell culture bag, there is a problem that the bottom surface that becomes the culture surface becomes uneven when injecting a culture solution, and a tray-shaped container for cell culture such as Patent Document 3 has been proposed. That is, the tray-shaped container for cell culture disclosed in Patent Document 3 has the following configuration: the first container wall has a concave portion having a transparent and single planar bottom surface and a flange-shaped portion formed on the periphery of the concave portion, and the second container wall has air permeability and flexibility that can be deformed.

[0015] Prior Art Documents

[0016] Patent Documents

[0017] Patent Document 1: Japanese Unexamined Patent Application Publication No. 2011-241159

[0018] Patent Document 2: Japanese Patent Application Laid-Open No. 2006-055069

[0019] Patent Document 3: Japanese Patent No. 4780462 Summary of the Invention

[0020] Problems to be Solved by the Invention

[0021] However, when subculturing in this manner, when dispensing cells into each well of a microplate or transferring cells from a microplate to a flask, it is necessary to repeat pipetting operations many times, increasing the cumbersome work. In addition, every time subculturing is performed, the cells must be moved to a new culture container such as a flask. Therefore, not only does the work become cumbersome, but the risk of contamination by accidental bacteria or viruses also increases.

[0022] In addition, in a flask-type culture container such as Patent Document 2, gas replacement can be performed only when the lid blocking the opening is removed and the opening is left open. Therefore, not only is it impossible to supply a sufficient amount of oxygen to the cells in culture, but also the risk of contamination cannot be avoided during gas replacement. Furthermore, when culturing cells in large quantities on a scale other than laboratory level, it is not practical to use a flask-type culture container with a limited capacity.

[0023] Furthermore, regarding cell culture containers, indeed, according to Patent Document 3, it is possible to ensure the flatness of the culture surface to a certain extent, but as described below, there are still many problems to be improved.

[0024] That is, in adherent culture system cells represented by, for example, iPS cells, the degree of cell proliferation depends on the width of the area. Therefore, simply ensuring a flat culture surface is not sufficient, and it is necessary to make the culture surface as flat and extensive as possible. In particular, cells for the purpose of regenerative medicine etc. are extremely precious, and their culture requires a large amount of time and cost. Therefore, high efficiency is also required.

[0025] Furthermore, the culture medium (culture solution) required for cell culture is very expensive. Therefore, there is also a potential need to use a small amount of the culture medium as efficiently as possible. Therefore, it is necessary to make the culture surface required for cell culture as extensive and flat as possible, and to spread the culture solution over every corner of the above-mentioned culture surface using a relatively small amount of the culture medium.

[0026] In addition, it is preferable to make the liquid surface of the culture medium extensive and uniform so that the cells that precipitate to the bottom after seeding are uniformly distributed in density, and the nutrient components of the culture medium uniformly reach all cells.

[0027] However, in prior art cell culture containers including Patent Document 3, there is no recognition or suggestion regarding the above problems.

[0028] The present invention aims to solve the above-mentioned problem as an example. One of its purposes is to provide a method for manufacturing a cell culture container and its manufacturing apparatus that can appropriately maintain the cell density during cultivation, reduce the risk of contamination, and efficiently cultivate and induce differentiation of cells in the same container.

[0029] In addition, one of the purposes of the present invention is to provide a cell culture container, a cell culture method, and a method for manufacturing a cell culture container that can ensure a wide and flat culture surface and can cover every corner of the culture surface with a uniform thickness even with a small amount of culture solution.

[0030] Technical means for solving the problem

[0031] In order to achieve the above object, the method for manufacturing a container in the present embodiment is characterized by including the following steps: placing a bag-shaped membrane container on a placement table formed with a recess; introducing a fluid into the interior of the placed bag-shaped membrane container; and heating at least one of the placement table and a pressing member facing the bag-shaped membrane container placed on the placement table, while pressing the placed bag-shaped membrane container with the pressing member.

[0032] In addition, the manufacturing apparatus for a container in the present embodiment is characterized by including: a placement table having a recess formed on a placement surface for placing a bag-shaped membrane container; a fluid introduction device for introducing a fluid into the interior of the bag-shaped membrane container placed on the placement surface; a pressing member configured to be able to advance and retreat relative to the placement surface and press the bag-shaped membrane container into which the fluid has been introduced; and a heating device for heating at least one of the placement table and the pressing member.

[0033] In order to achieve the above object, the cell culture container in the present embodiment is characterized by including: a first container wall having air permeability, serving as a bottom surface, and composed of a planar membrane; a second container wall having a bulging shape that contacts the peripheral portion of the first container wall and protrudes relative to the first container wall at a position more inside than the peripheral portion; and a port communicating with a culture space surrounded by the bulging shape of the first container wall and the second container wall; at least the region of the first container wall other than the region in contact with the port is planar.

[0034] In addition, the cell culture method in the present embodiment uses the cell culture container in the present embodiment, and is characterized in that: the first container wall is placed in such a manner that it is below the second container wall, and cells and a culture solution are injected through the port.

[0035] In addition, the manufacturing method of the cell culture container in the present embodiment is characterized by including the following steps: placing the first container wall made of a breathable membrane on a placement table in a state where the first container wall and a second container wall arranged opposite to the first container wall overlap; pressing the peripheral portions of the first container wall and the second container wall with a restraining member in a state where the central portion of the second container wall is open; introducing a fluid between the first container wall and the second container wall in a state where the peripheral portion is pressed with the restraining member; and heating at least the pressing member while pressing the central portion of the second container wall with a pressing member.

[0036] In addition, the manufacturing apparatus of the cell culture container in the present embodiment includes a first container wall made of a planar membrane and a second container wall having a bulging shape that contacts the peripheral portion of the first container wall and protrudes relative to the first container wall, and is characterized by including: a placement table for placing the first container wall; a fluid introduction device for introducing a fluid into the space between the first container wall and the second container wall placed on the placement table; a pressing member configured to be able to advance and retreat relative to the placement table to press the second container wall into which the fluid has been introduced into the space; a heating device for heating the pressing member; and a restraining member arranged opposite to the placement table to restrain the periphery of the second container wall placed on the placement table; heating the pressing member with the heating device while restraining the second container wall with the restraining member, and introducing the fluid into the space between the first container wall and the second container wall with the fluid introduction device.

[0037] Effects of the Invention

[0038] According to the present invention, a container having one or more depressions formed on its surface can be efficiently manufactured. Moreover, a container can be efficiently manufactured such that, particularly when applied to cell culture, the cell density during culture can be appropriately maintained, depletion of the culture medium components required for proliferation can be suppressed, and the risk of contamination by foreign substances and the like can also be suppressed.

[0039] In addition, according to the present invention, a wide and flat culture surface can be ensured by the planar first container wall serving as the bottom surface, and even with a small amount of culture solution, the culture solution can reach every corner of the culture surface through the second container wall having a bulging shape. Furthermore, high-quality large-scale cell culture can be carried out in a state where "the cell density during culture is appropriately maintained, depletion of the culture medium components required for proliferation does not occur, and the risk of contamination is suppressed". BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1Schematic explanatory diagram showing the cell culture container 1 of the first embodiment.

[0041] (a) is a top view, (b) is a side view, and (c) is a bottom view.

[0042] Figure 2 Schematic diagram showing the schematic configuration of the manufacturing apparatus 20 for the cell culture container in the first embodiment.

[0043] Figure 3 Schematic diagram for explaining the structure of the mounting table 22 and the pressing member 21 in the manufacturing apparatus 20 for the cell culture container in the first embodiment.

[0044] Figure 4 State transition diagram of the manufacturing apparatus 20 for the cell culture container in the first embodiment.

[0045] Figure 5 Flowchart for explaining the manufacturing method of the cell culture container in the first embodiment.

[0046] Figure 6 Schematic diagram showing the schematic configuration of the manufacturing apparatus 30 for the cell culture container in the second embodiment.

[0047] Figure 7 State transition diagram of the manufacturing apparatus 30 for the cell culture container in the second embodiment.

[0048] Figure 8 Flowchart for explaining the manufacturing method of the cell culture container in the second embodiment.

[0049] Figure 9 Schematic diagram showing the schematic configuration of the manufacturing apparatus 40 for the cell culture container in the third embodiment.

[0050] Figure 10 Schematic diagram showing the schematic configuration of the manufacturing apparatus 50 for the cell culture container in the fourth embodiment.

[0051] Figure 11 Schematic diagram for explaining the positional relationship between the pressing member 21 and the restraining member 29 in the manufacturing apparatus 50 for the cell culture container in the fourth embodiment.

[0052] Figure 12 State transition diagram of the manufacturing apparatus 50 for the cell culture container in the fourth embodiment.

[0053] Figure 13 Flowchart for explaining the manufacturing method of the cell culture container in the fourth embodiment.

[0054] Figure 14Schematic diagram showing the schematic configuration of the manufacturing apparatus 60 for the cell culture container in Modification 1.

[0055] Figure 15 Schematic diagram for explaining the heating device 24 and the restraining member 29 in Modification 2.

[0056] Figure 16 Schematic diagram for explaining the mounting table 22 in Modification 3.

[0057] Figure 17 Exterior perspective view of the cell culture container 10 in the fifth embodiment.

[0058] Figure 18 Side view of the cell culture container 10 in the fifth embodiment.

[0059] Figure 19 Front view of the cell culture container 10 in the fifth embodiment.

[0060] Figure 20 Diagram showing the manufacturing apparatus and manufacturing method of the cell culture container 10 in the fifth embodiment.

[0061] Figure 21 Diagram showing a comparison between the previous type of cell culture container and the cell culture container 10 of the embodiment.

[0062] Figure 22 Diagram showing the cell culture container 10 in Modification 4.

[0063] Figure 23 Diagram showing the cell culture container 10 in Modification 5. Detailed Description of the Invention

[0064] Hereinafter, with appropriate reference to the drawings, the manufacturing method of the container of the present invention and the manufacturing apparatus of the container, etc. when applied to the use of cell culture will be specifically described. In addition, for the convenience of explanation, the X direction, Y direction, and Z direction are respectively defined in the following description, but it is not intended to limit or narrow the scope of the present invention.

[0065] <<First Embodiment>>

[0066] [Cell Culture Container 1]

[0067] Figure 1 The shown cell culture container 1 is a cell culture container having a plurality of depressions on the bottom surface, and includes: a container body 2 made of a known plastic film having air permeability; and an injection / extraction port 3 made of a tubular member through which a culture medium or cells can flow.

[0068] The container body 2 has a bulging shape with its peripheral part sealed and its top surface 2a bulging in a stepped manner, and is formed in such a way that "the edge of the top surface 2a which becomes a flat surface is inclined and connected to the peripheral part". Further, on the bottom surface 2b of the container body 2, a recess 4 serving as a cell culture part is provided. In addition, in this embodiment, a plurality of recesses 4 are provided, but at least one may be provided. Further, the size of the container body 2 is not particularly limited, and for example, it is preferably set to a length of 20 to 1000 mm and a width of 20 to 1000 mm.

[0069] In addition, regarding the air permeability of the plastic film forming the container body 2, according to the air permeability test method of JIS K 7126, the oxygen penetration rate measured at a test temperature of 37°C is preferably 5000 mL / (m 2 ·day·atm) or more.

[0070] As the material of the plastic film for forming such a container body 2, there is no particular limitation as long as it has the required air permeability. For example, thermoplastic resins such as polyethylene, polypropylene, ethylene-vinyl acetate copolymer, polyester, silicone-based elastomer, polystyrene-based elastomer, and tetrafluoroethylene-hexafluoropropylene copolymer (FEP) can be cited. It can be used as a single layer, or the same or different materials can be laminated and used. If the heat meltability when sealing the peripheral part is considered, it is preferably a layer that functions as a sealing layer.

[0071] In addition, the plastic film is preferably partially or entirely transparent so that the progress of cell culture or the state of cells can be confirmed.

[0072] The recess 4 provided on the bottom surface 2b of the container body 2 preferably has an opening diameter (diameter) that can suppress the movement of cells in the container body 2 and make the cells in culture stay in one recess 4. In addition, the opening diameters of all the recesses 4 can be the same, or for example, the bottom surface 2b can be divided into a plurality of regions, and the opening diameters of the recesses 4 can be different for each region, so that the recesses 4 provided on the bottom surface 2b include two or more types of concave parts with different opening diameters. In addition, in Figure 1 the cell culture container 1 shown, the shape of the recess 4 is set to a spherical crown shape so that cells can easily concentrate at the bottom of the recess 4, but the shape of the recess 4 is not limited to this.

[0073] In addition, in order to prevent cells from staying in parts other than the recess 4 on the bottom surface 2b, the area of the recess 4 on the bottom surface 2b is preferably as large as possible within the range that does not impair the formability. Specifically, it is preferably 30 to 90% of the area of the bottom surface 2b. The arrangement of the recesses 4 is preferably set in a staggered shape as shown in the drawing so that the area of the recess 4 on the bottom surface 2b is as large as possible, and it can also be arranged in a grid pattern as needed.

[0074] As described above, the injection / extraction port 3 is composed of a tubular member through which a culture medium or cells can flow. For example, a thermoplastic resin such as polyethylene, polypropylene, vinyl chloride, polystyrene-based elastomer, or FEP can be used as the tubular member forming the injection / extraction port 3, and it is formed into a specific shape by injection molding, extrusion molding, etc.

[0075] When culturing cells using the cell culture container 1 as described above, while maintaining a closed system, the cells to be cultured and the culture medium are injected into the container body 2 via a liquid delivery tube connected to the injection / extraction port 3. Then, the cells injected into the container body 2 precipitate in the culture medium and gather at the bottom of each recess 4.

[0076] [Manufacturing apparatus for cell culture container 1]

[0077] Next, use Figure 2 and Figure 3 , the manufacturing apparatus 20 for the cell culture container in this embodiment will be described.

[0078] As Figure 2 shown, the manufacturing apparatus 20 for the cell culture container includes a pressing member 21, a mounting table T, a heating device 24, and a fluid introduction device 26.

[0079] The pressing member 21 has a function of pressing the bag-shaped film container placed on the mounting surface of the mounting table T. In addition, the bag-shaped film container later becomes the base of the cell culture container 1 and is a container that has not yet formed the recesses 4 or the bulging shape. The pressing member 21 is formed of, for example, a metal such as aluminum or iron or a resin such as plastic. As Figure 3 shown, the outer shape of the pressing member 21 is, for example, rectangular, and is set to be slightly larger than the outer shape of the bag-shaped film container placed on the above-mentioned mounting surface. In addition, regarding the size of the pressing member 21 in the XY plane direction, it is sufficient if it has at least an area capable of pressing the flat top surface of the above-mentioned bulging shape. In the case where, for example, there is a restraining member 29 described in the fourth embodiment, etc., it is restricted inside it.

[0080] Moreover, the pressing member 21 is connected to a drive mechanism 25 and is configured to be able to move forward and backward relative to the mounting surface via the drive mechanism 25. When manufacturing the cell culture container described below, the pressing member 21 presses the bag-shaped film container placed on the mounting surface and into which fluid has been introduced. In addition, there is no particular limitation on the drive mechanism 25, and a known drive mechanism such as a hydraulic cylinder mechanism, a ball screw mechanism, or an electric motor mechanism can be applied.

[0081] The placement table T is a support table with a concave portion formed on the placement surface for placing the bag-shaped film container, and has the function of supporting the bag-shaped film container. In addition, the placement table T of the present embodiment is formed with a plurality of concave portions corresponding to the recess 4 of the cell culture container 1. If there is one recess 4 in the cell culture container 1, the concave portion also becomes one. That is, one or more concave portions are formed on the placement table T of the present embodiment.

[0082] The placement table T of the present embodiment is composed of two materials, namely, a placement table main body portion 22 and a container support portion 23.

[0083] The placement table main body portion 22 is formed of a material with a lower thermal conductivity than the container support portion 23. Moreover, as Figure 2 and Figure 3 shown, a concave portion is provided on the upper surface of the placement table main body portion 22, and the container support portion 23 is accommodated in the concave portion. In addition, the placement table main body portion 22 also has the function of supporting the periphery of the bag-shaped film container when the bag-shaped film container is placed on the placement table T. In other words, the placement table main body portion 22 functions as a placement surface for supporting the periphery of the bag-shaped film container.

[0084] The container support portion 23 is formed of a material with a higher thermal conductivity than the placement table main body portion 22. In the present embodiment, aluminum is used for the container support portion 23, and cast iron is used for the placement table main body portion 22. In addition, as in the example shown below Figure 16 shown, the placement table main body portion 22 and the container support portion 23 may be formed of the same material, and it is not necessary to make the thermal conductivities different.

[0085] As Figure 3 shown, a plurality of concave portions 23a are formed on the upper surface of the container support portion 23 (the placement surface for placing the bag-shaped film container). The concave portions 23a correspond to the recess 4 of the cell culture container 1. Therefore, the opening diameters of all the concave portions 23a can be set to be the same. Or, by dividing the placement surface of the container support portion 23 into a plurality of regions and making the opening diameters of the concave portions 23a different for each region, etc., two or more types of concave portions 23a with different opening diameters can be included.

[0086] In addition, although the shape of the concave portion 23a is set to a spherical crown shape, the shape of the concave portion 23a is not limited thereto, and it may also be a columnar shape or the like.

[0087] In addition, regarding the arrangement of the concave portions 23a, it is preferably arranged in a staggered shape as shown in the figure to make the occupied area of the concave portions 23a as large as possible, but it may also be arranged in a grid shape as needed.

[0088] The heating device 24 has a function of heating at least one of the stage T and the pressing member 21. The heating device 24 of the present embodiment may be, for example, a resistance heating device such as a nichrome wire, and may be buried in the pressing member 21, the stage main body portion 22, or the container support portion 23.

[0089] More specifically, the heating device 24 of the present embodiment is buried inside the stage main body portion 22 (see Figure 2 ), and is buried in the bottom surface side on the pressing side in the pressing member 21 (see Figure 3 ).

[0090] Among them, the heating device 24 buried in the stage main body portion 22 is arranged across the entire surface of the stage main body portion 22 so as to correspond to the bottom surface 2b of the cell culture container 1. Therefore, heat can be transferred without missing the plurality of recesses 4 provided on the bottom surface 2b.

[0091] On the other hand, the heating device 24 buried in the pressing member 21 is not widely arranged on the bottom surface of the pressing member 21, but is arranged corresponding to the position of the edge portion of the top surface 2a of the cell culture container 1. Therefore, meaningless heating can be omitted, and the necessary parts can be heated efficiently.

[0092] The fluid introduction device 26 has a function of introducing a fluid into the inside of the bag-shaped film container placed on the placement surface of the stage T. In the present embodiment, the fluid introduction device 26 introduces a fluid into the inside of the bag-shaped film container through the injection / extraction port 3. In addition, the fluid introduced by the fluid introduction device 26 may be, for example, a liquid or a gas. Among them, as the fluid, pure water or the like can be specifically applied. On the other hand, as the gas, an inert gas such as purified air (clean air) or nitrogen can be applied. From the viewpoint of ease of operation or processing, in the present embodiment, clean air is applied.

[0093] In addition, the fluid introduction device 26 of the present embodiment also has a function of adjusting the supply pressure of the fluid introduced into the inside of the bag-shaped film container. Therefore, the supply pressure can be set to be fixed or variable before and after the bag-shaped film container is pressurized by the pressing member 21.

[0094] In addition, the fluid introduction device 26 may also have a function of controlling the supply flow rate of the fluid, instead of the function of adjusting the supply pressure of the above-mentioned fluid. Therefore, by controlling the flow rate of the fluid supplied to the inside of the bag-shaped film container, it is possible to easily control the protruding amount in the bulging shape of the above-mentioned container body 2. More specifically, for example, when a bulging shape with a smaller protruding amount is formed in the container body 2 (in other words, a container suitable for a thinner liquid thickness (small liquid volume)), the fluid introduction device 26 can also be controlled in such a way that the supply flow rate of the fluid supplied to the bag-shaped film container becomes smaller. On the other hand, when a bulging shape with a larger protruding amount is formed in the container body 2 (in other words, a large container suitable for a thicker liquid thickness (large liquid volume)), the fluid introduction device 26 can also be controlled in such a way that the supply flow rate of the fluid supplied to the bag-shaped film container becomes larger.

[0095] In addition, the manufacturing apparatus 20 for the cell culture container of the present embodiment may further include a control device CP. The control device CP has a function of controlling the operations of the above-mentioned heating device 24, drive mechanism 25, and fluid introduction device 26. Specifically, as the control device CP, a computer having a memory or CPU (not shown) can be exemplified. In addition, the manufacturing apparatus 20 for the cell culture container does not necessarily have to include the control device CP, and it can also be remotely operated from a distant location via a network such as a LAN.

[0096] [Method for manufacturing the cell culture container 1]

[0097] Next, use Figure 4 and Figure 5 to explain the method for manufacturing the cell culture container in the present embodiment. Figure 4 is a state transition diagram of the manufacturing apparatus 20 for the cell culture container in the present embodiment, Figure 5 is a flowchart for explaining the method for manufacturing the cell culture container corresponding to the state transition diagram of Figure 4

[0098] First, as shown in (a) of Figure 4 and step 1 of Figure 5 , place the bag-shaped film container 1' on the placement table T formed with a plurality of recesses 23a. At this time, it is preferably placed in such a way that the periphery of the bag-shaped film container 1' is supported by the placement table body 22.

[0099] After placing the bag-shaped film container 1' in step 1, as shown in (b) of Figure 4 and Figure 5 ​As shown in step 2, a fluid is introduced into the interior of the bag-shaped film container 1'. At this time, in the present embodiment, the fluid introduction device 26 supplies the purified clean air at the supply pressure f1 as described above. In addition, as described above, the fluid introduction device 26 may also supply the clean air based on the supply flow rate instead of the supply pressure.

[0100] Then, as Figure 4 shown in (c) and Figure 5 steps 3 and 4, at least one of the mounting table T and the pressing member 21 facing the bag-shaped film container 1' mounted on the mounting table T is heated, and the bag-shaped film container 1' placed thereon is pressed by the pressing member 21. In other words, after introducing the fluid into the interior of the bag-shaped film container 1', the pressing member 21 is brought close to the mounting table T to press the bag-shaped film container 1'.

[0101] In addition, regarding the heating temperature at this time, it is preferably a temperature that does not cause the bag-shaped film container 1' to melt and soften. For example, it can be set to about 80°C.

[0102] In addition, steps 3 and 4 may not be in this order. For example, after pressing the bag-shaped film container 1' with the pressing member 21, at least one of the mounting table T and the pressing member 21 may be heated by the heating device 24. At this time, in the present embodiment, the fluid introduction device 26 supplies the purified clean air at the supply pressure f3 as described above.

[0103] In the present embodiment, the heating device 24 is provided on both the pressing member 21 and the mounting table T. Therefore, the region of the bag-shaped film container 1' that will later become the recess 4 can be heated by the heating device 24 provided on the mounting table T. Furthermore, the region that will later become the bulging shape can be efficiently heated by the heating device 24 provided on the pressing member 21.

[0104] After starting to heat and press the bag-shaped film container 1' in steps 3 and 4, as Figure 5 shown in step 5, it is determined whether a specific time t1 has elapsed.

[0105] As this specific time t1, there is no particular limitation as long as the above-mentioned recess 4 or bulging shape can be formed. For example, it can be set to about several seconds to several minutes.

[0106] Thus, in the present embodiment, by separating the pressing member 21 from the mounting table T by a specific distance and introducing a fluid into the interior of the bag-shaped film container 1', a bulging shape with a protruding top surface can be formed in the bag-shaped film container 1'.

[0107] In addition, in Steps 3 to 5, the fluid introduction device 26 can also adjust the supply pressure of the fluid before and after the above-described pressurization of the pressing member 21. That is, since the pressure inside the container becomes high due to the pressurization of the pressing member 21 on the bag-shaped film container 1', by adjusting the supply pressure of the fluid with the fluid introduction device 26, excessive change in the pressure inside the container can be suppressed. In other words, the fluid introduction device 26 can also be controlled by the control device CP so that the supply pressure of the fluid becomes a fixed value (assuming f1 = f3) in such a manner that the internal pressure of the bag-shaped film container 1' becomes fixed according to the pressing force applied by the pressing member 21. In addition, the fluid introduction device 26 can also be controlled by the control device CP so that the supply pressure of the fluid becomes variable (assuming f1 ≠ f3) in such a manner that the internal pressure of the bag-shaped film container 1' changes (increases or decreases) according to the pressing force applied by the pressing member 21.

[0108] Moreover, when it is determined in Step 5 that a specific time t1 has elapsed, as shown in (d) of Figure 4 and Step 6 of Figure 5 , after the pressing member 21 is retracted via the drive mechanism 25, the bag-shaped film container 1' is taken out to end the process. The above-described plurality of recesses 4 or bulging shapes are formed in the taken-out bag-shaped film container 1', thereby manufacturing the cell culture container 1 of the present embodiment.

[0109] <<Second Embodiment>>

[0110] Next, with reference to Figures 6 - 8 , the second embodiment of the present invention will be described.

[0111] Here, regarding the differences between the manufacturing apparatus 30 for the cell culture container in the second embodiment and the first embodiment, it can be cited that a suction flow path 23b is provided in the container support portion 23, a suction device 27 is provided, and a temperature control device 28 is further provided, etc.

[0112] Therefore, hereinafter, the differences from the first embodiment will be mainly described. Regarding the components having the same configuration or the same function as those in the first embodiment, the same reference signs as those in the first embodiment are given, and their descriptions are appropriately omitted (the same applies to the following other embodiments or modified embodiments).

[0113] In addition, in the present embodiment, a configuration including both the suction device 27 and the temperature control device 28 is described, but it is not limited to this form, and it is sufficient to include at least one of them.

[0114] As shown in Figure 6 , the manufacturing apparatus 30 for the cell culture container further includes a suction device 27 and is configured to have a temperature control device 28 as an alternative to the heating device 24.

[0115] The suction device 27 has the following function: when a fluid is introduced into the inside of the bag-shaped film container 1' placed on the placement table T, suction is performed through a plurality of recesses 23a formed in the placement table T. In addition, in the container support portion 23 of the present embodiment, a flow path 23b continuous with the recesses 23a is formed, and the flow path 23b penetrates a part of the placement table body 22 and is connected to the suction device 27.

[0116] Moreover, the suction device 27 is connected to a negative pressure source (not shown), and can perform a suction operation through the flow path 23b under the control of the control device CP. Therefore, when the bag-shaped film container 1' is placed, the inside of the recess 23a becomes a negative pressure state, and thus the bottom surface (the surface placed on the container support portion 23) of the bag-shaped film container 1' is sucked.

[0117] Therefore, according to the suction device 27 of the present embodiment, it is possible to assist in "forming the above-mentioned recess 4 on the bottom surface of the bag-shaped film container 1'".

[0118] The temperature control device 28 not only has the function of the heating device 24 described in the first embodiment, but also further has the function of a cooling device that cools at least one of the placement table T and the pressing member 21. As a specific example of the temperature control device 28, various known devices can be applied, such as a Peltier module or the like. In addition, the temperature control device 28 can apply a single component having both a heating function and a cooling function, or can be configured to individually include a heating device such as a nichrome wire and a cooling device such as a fan.

[0119] [Method for manufacturing the cell culture container 1]

[0120] Next, use Figure 7 and Figure 8 to describe the method for manufacturing the cell culture container in the second embodiment. Figure 7 is a state transition diagram of the manufacturing apparatus 30 for the cell culture container in the present embodiment, Figure 8 is a flowchart for explaining the method for manufacturing the cell culture container corresponding to the state transition diagram of Figure 7

[0121] First, as shown in step 1 of (a) in Figure 7 and Figure 8 , place the bag-shaped film container 1' on the placement table T formed with a plurality of recesses 23a. At this time, it is preferably placed in such a manner that the periphery of the bag-shaped film container 1' is supported by the placement table body 22.

[0122] After the bag-shaped film container 1' is placed in step 1, as shown in step 2 of (b) in Figure 7 and Figure 8 , introduce a fluid into the inside of the bag-shaped film container 1'. At this time, the fluid introduction device 26 supplies purified clean air at a supply pressure f1.​

[0123] Subsequently, as shown in steps 3 to 5 of (c) in Figure 7 and Figure 8 at least one of the mounting table T and the pressing member 21 is heated, and the bag-shaped film container 1' is pressed by the pressing member 21. Further, suction is performed through the concave portion 23a of the mounting table T.

[0124] At this time, as the heating temperature using the temperature control device 28 (heating device), a temperature that does not cause the bag-shaped film container 1' to melt and soften is preferably set, for example, to about 80°C.

[0125] In addition, the pressing member 21 presses the bag-shaped film container 1' with a pressing force F, and the suction device 27 performs suction with a suction force f2. That is, in the present embodiment, when a fluid is introduced into the inside of the bag-shaped film container 1' placed on the mounting table T, a suction operation is performed through the plurality of concave portions 23a formed in the mounting table T.

[0126] Therefore, a pressing force F caused by the pressing member 21 and a suction force f2 caused by the suction device 27 are applied to the bag-shaped film container 1', and the control device CP performs the following control: adjusting the supply pressure of the fluid introduction device 26 so that the internal pressure of the bag-shaped film container 1' slightly rises (in this case, the relationship f1 < f3 holds). Therefore, excessive pressure on the inside of the bag-shaped film container 1' is also suppressed. In addition, similarly to the first embodiment, the control device CP can also perform control to make the supply pressure of the fluid introduction device 26 a fixed value (i.e., f1 = f3) or decrease.

[0127] In addition, steps 3 to 5 do not necessarily have to be in this order. As long as these steps are at least parallel for a certain period, they can be performed simultaneously, and the order can also be appropriately changed.

[0128] After steps 3 to 5 start, as shown in step 6 of Figure 8 it is determined whether a specific time t1 has elapsed.

[0129] As this specific time t1, there is no particular limitation as long as the above-mentioned recess 4 or bulging shape can be formed. For example, it can also be about several seconds to several minutes.

[0130] Moreover, when it is determined in step 5 that a specific time t1 has elapsed, as shown in (d) in Figure 7 and Figure 8As shown in step 7, at least one of the mounting table T and the pressing member 21 is cooled. More specifically, the temperature control device 28 functions as a cooling device, and as the above heating device, it performs a cooling operation on the heated area. In addition, there is no particular limitation on the cooling temperature of the temperature control device 28, and it is preferably a temperature that can solidify the film softened during forming. For example, if it is heated to about 80°C, it can be cooled to about 50°C, for example.

[0131] In addition, when the temperature control device 28 performs the above cooling operation, the pressing operation using the pressing member 21, the fluid introduction operation using the fluid introduction device 26, and the suction operation using the suction device 27 are performed in parallel.

[0132] Therefore, the portions that will later become the recesses 4 or bulges in the bag-shaped film container 1' are cooled and solidified during forming. Therefore, according to this embodiment, when demolding is performed after forming is completed, deformation of the container shape can be prevented, and the recesses 4 or bulges can be more surely formed in the cell culture container 1.

[0133] After step 7 starts, as Figure 8 shown in step 8, it is determined whether a specific time t2 has elapsed.

[0134] In addition, the determination in this step 8 is made based on whether the temperature of the bag-shaped film container 1' has reached the reference solidification temperature. Therefore, the specific time t2 can be determined, for example, by "adding a slight margin to the time to reach the above solidification temperature", and there is no particular limitation as long as the above recesses 4 or bulges can be formed, and it can be, for example, about several seconds to several minutes.

[0135] Moreover, when it is determined in step 8 that a specific time t2 has elapsed, as Figure 7 shown in (e) and Figure 8 step 9, after the pressing member 21 is retracted via the drive mechanism 25, the bag-shaped film container 1' is taken out to end. The above-mentioned multiple recesses 4 or bulges are formed in the taken-out bag-shaped film container 1', thereby manufacturing the cell culture container 1 of this embodiment.

[0136] <<Third Embodiment>>

[0137] In addition, with reference to Figure 9 , the third embodiment of the present invention will be described.

[0138] Here, the differences between the manufacturing device 40 of the cell culture container in the third embodiment and the first embodiment can include: the mounting table body 22 without the container support portion 23 is the mounting table T, and the recess formed in the mounting table T becomes a through hole, etc. In addition, in this embodiment, the hole formed in the mounting table T does not necessarily have to be a through hole.

[0139] That is, Figure 9 The manufacturing apparatus 40 of the cell culture container shown includes a mounting table body 22 formed with a plurality of through holes 22a. Among them, the through holes 22a correspond to the concave portions 23a in the first embodiment, and have a shape that penetrates from the mounting surface of the mounting table body 22 (the surface for mounting the bag-shaped film container 1') to the bottom surface on the opposite side. Therefore, in the present invention, the concave portions formed on the mounting surface of the bag-shaped film container 1' are not limited to the shape that does not penetrate downward, and may also be formed as through holes as in this embodiment.

[0140] <<Fourth Embodiment>>

[0141] In addition, with reference to Figures 10 - 13 , the fourth embodiment of the present invention will be described.

[0142] Here, the differences between the manufacturing apparatus 50 of the cell culture container in the fourth embodiment and the first embodiment are as follows: It has a restraining member 29, and while restraining the periphery of the bag-shaped film container 1' with the restraining member 29, fluid is introduced into the interior of the bag-shaped film container 1' with a fluid introduction device 26.

[0143] In addition, the cell culture container 1 in the fourth embodiment is also characterized in the following aspects: The bottom surface on which the depression 4 is formed is flat, and on the other hand, the top surface has a protruding bulge shape. In addition, the "flat shape" in this embodiment means a plane in the X direction and the Y direction, and means a single surface parallel to the XY plane.

[0144] That is, as Figure 10 shown, the manufacturing apparatus 50 of the cell culture container in this embodiment includes a restraining member 29 that restrains the periphery of the bag-shaped film container 1'. The restraining member 29 is disposed opposite to the mounting surface and has a function of restraining the periphery of the bag-shaped film container 1' mounted on the mounting surface. In addition, the restraining member 29 can descend toward the mounting table T independently of the pressing member 21 through a driving mechanism 25.

[0145] In addition, the restraining member 29 and the pressing member 21 do not necessarily descend independently. For example, the restraining member 29 may be connected to the driving mechanism 25 via a spring and descend together with the pressing member 21.

[0146] In addition, a concave portion may be formed in the area of the mounting table body 22 opposite to the restraining member 29, and a heat insulating material such as rock wool or polyurethane resin may be disposed in the concave portion.

[0147] As Figure 11As shown, the restraining member 29 of this embodiment is arranged around the pressing member 21 in a manner opposite to the periphery of the bag-shaped film container 1'. In this embodiment, the restraining member 29 is separated into four parts, one of which ( Figure 11 The restraining member at the left end of the bag-shaped film container 1' has a shape corresponding to the outer shape of the injection and extraction port 3. Therefore, each separate restraining member 29 can restrain the periphery of the bag-shaped film container 1'.

[0148] In addition, the material of the restraining member 29 is not limited, and it can be a metal material such as aluminum or iron. In addition, the restraining member 29 is preferably made of a material with a lower thermal conductivity than the pressing member 21 so as to minimize the heat transfer from the pressing member 21. In addition, the restraining member 29 does not necessarily have to be separated into four parts, and can also be used as Figure 15 Although it is set as an integral structure connected all around as described below, it can also be set as a structure in which some of the four are connected together.

[0149] [Method for Manufacturing Cell Culture Container 1]

[0150] After that, use Figure 12 and Figure 13 , a method for manufacturing a cell culture container in the fourth embodiment is described. Figure 12 : is a state transition diagram of the cell culture container manufacturing device 50 in this embodiment, Figure 13 To illustrate the corresponding Figure 12 A state transition diagram of a flow chart of a method for manufacturing a cell culture vessel.

[0151] First, if Figure 12 (a) and Figure 13 As shown in step 1, the bag-like film container 1' is placed on a mounting table T formed with a plurality of recessed portions 23a. At this time, it is preferable to place the bag-like film container 1' so that the periphery of the bag-like film container 1' is supported by the mounting table body 22.

[0152] After the bag-shaped film container 1' is placed in step 1, Figure 12 (b) and Figure 13 As shown in step 2, the restraining member 29 is lowered toward the mounting table T to restrain the periphery of the bag-like film container 1'.

[0153] Then, if Figure 12 (c) and Figure 13 As shown in step 3, the fluid is introduced into the bag-like film container 1' by the fluid introduction device 26. At this time, the periphery of the bag-like film container 1' is constrained by the constraining member 29, and the fluid is introduced into the bag-like film container 1' placed on the mounting table T. In this embodiment, the fluid introduction device 26 also supplies purified clean air at the supply pressure f1.

[0154] Then, ifFigure 12 In (d) and Figure 13 As shown in Steps 4 to 6, at least one of the mounting table T and the pressing member 21 is heated, the bag-shaped film container 1' is pressed by the pressing member 21, and then suction is performed through the concave portion 23a of the mounting table T.

[0155] At this time, as the heating temperature generated by the heating device 24, a temperature that does not cause the bag-shaped film container 1' to melt and soften is preferable, and for example, it can be set to about 80°C.

[0156] In addition, the pressing member 21 presses the bag-shaped film container 1' with a pressing force F, and the suction device 27 performs suction with a suction force f2.

[0157] Therefore, a pressure F caused by the pressing member 21 and a suction force f2 caused by the suction device 27 are applied to the bag-shaped film container 1', and the control device CP controls in the following manner: the supply pressure of the fluid introduction device 26 is changed from f1 to f3 so that the internal pressure of the bag-shaped film container 1' is slightly reduced (in this case, the relationship f1 > f3 holds). Therefore, it is also possible to suppress excessive pressure being applied to the inside of the bag-shaped film container 1'. In addition, the control device CP can also control the supply pressure of the fluid introduction device 26 so that the internal pressure of the bag-shaped film container 1' is fixed (i.e., f1 = f3) or slightly increased.

[0158] In addition, Steps 4 to 6 do not have to be in this order. As long as these steps are made to be parallel at least for a certain period, they can be performed simultaneously, or the order can be appropriately changed.

[0159] After Steps 4 to 6 start, as Figure 13 shown in Step 7, it is determined whether a specific time t1 has elapsed.

[0160] As this specific time t1, there is no particular limitation as long as the above-mentioned recess 4 or bulging shape can be formed, and for example, it can be set to about several seconds to several minutes.

[0161] Moreover, when it is determined in Step 7 that the specific time t1 has elapsed, as Figure 12 shown in (e) and Figure 13 Step 8, after the pressing member 21 is retracted via the drive mechanism 25, the bag-shaped film container 1' is taken out and the process ends. In the taken-out bag-shaped film container 1', a plurality of recesses 4 are formed on the planar bottom surface, and a bulging shape with a protruding top surface is also formed, thereby completing the cell culture container 1 of this embodiment.

[0162] The above-described First Embodiment to Fourth Embodiment can be variously modified without departing from the gist of the present invention. Hereinafter, modification examples that can be appropriately applied to the above-described First Embodiment to Fourth Embodiment will be described.

[0163] <Example 1 of Modification>

[0164] Figure 14 It is a schematic diagram showing a schematic configuration of a manufacturing apparatus 60 for a cell culture container according to Example 1 of Modification.

[0165] The heating device 24 or the temperature control device 28 of each of the embodiments described above is provided on both the pressing member 21 and the mounting table T. However, the present invention is not limited to this form, and the heating device 24 (or the temperature control device 28) may also be provided on at least one of the pressing member 21 and the mounting table T.

[0166] That is, as Figure 14 shown, the manufacturing apparatus 60 for a cell culture container is in the following form: the heating device 24 is not embedded in the pressing member 21, and the heating device 24 is embedded in the container support portion 23. Of course, in this Example 1 of Modification, it may also be in the following form: the heating device 24 is embedded in the pressing member 21, and the heating device 24 is not embedded in the container support portion 23.

[0167] <Example 2 of Modification>

[0168] Figure 15 It is a schematic diagram showing a schematic configuration of the restraining member 29 and the heating device 24 according to Example 2 of Modification. In the above Figure 11 described embodiment, the restraining member 29 is in a form separated into a plurality of parts, and the heating device 24 embedded in the pressing member 21 is also in a form separated into a plurality of parts.

[0169] However, the present invention is not limited to this form, and as Figure 15 shown, the restraining member 29 may also be in a continuous shape surrounding the periphery of the pressing member 21.

[0170] In addition, regarding the heating device 24 embedded in the pressing member 21, it may also be in a ring shape (circular shape) corresponding to "the position of the edge portion that becomes the top surface 2a of the cell culture container 1 in the bag-shaped film container 1'".

[0171] In addition, as shown in this figure, the gap between the restraining member 29 and the pressing member 21 is preferably as narrow as possible. The reason is that if this gap is large, when a fluid (such as air) is injected into the inside of the bag-shaped film container 1', the film will extend at this gap portion. Therefore, from this point of view, the diameter of the inner hole of the restraining member 29 into which the pressing member 21 can be inserted is preferably as close as possible to the outer edge of the pressing member 21 (that is, the boundary of the bag-shaped film container 1'), and the corners are also R-shaped like the corners of the pressing member 21.

[0172] <Example 3 of Modification>

[0173] Figure 16Schematic diagram showing the schematic configuration of the stage T of Modification 3.

[0174] In the first, second, and fourth embodiments described above, the stage T is composed of the stage body 22 and the container support portion 23. However, the present invention is not limited to this form. For example, a recess 22b corresponding to the above-described recess 23a may be formed on the upper surface of the stage body 22, and a heat insulation groove 22a may be formed.

[0175] That is, the stage T may also be composed of the stage body 22. In addition, the width or depth of the heat insulation groove 22a is not particularly limited. For example, the width may be set to 1 to 5 mm, and the depth may be set to about 5 to 10 mm.

[0176] Moreover, when manufacturing the cell culture container 1, for example, the periphery of the bag-shaped film container 1' may be placed on a region more outside than the heat insulation groove 22a, and further, the remaining portion of the bag-shaped film container 1' (the region that later forms the recess 4) may be placed on a region inside the heat insulation groove 22a.

[0177] Therefore, the heat generated in the region on the recess 22b side is blocked by the heat insulation groove 22a, thereby suppressing it from reaching the periphery of the bag-shaped film container 1'.

[0178] In addition, for example, when the heat influence on the periphery of the bag-shaped film container 1' does not need to be considered much, the heat insulation groove 22a is not necessary and may be appropriately omitted.

[0179] <<Fifth Embodiment>>

[0180] [Cell Culture Container 10]

[0181] Figure 17 An external perspective view showing the cell culture container 10 of the fifth embodiment of the present invention.

[0182] The cell culture container 10 is a flexible cell culture container made of a soft packaging material with a membrane base and formed in a bag shape. The cell culture container 10 has air permeability suitable for cell culture, and preferably a part or all of it has transparency so that the content can be confirmed.

[0183] Such a cell culture container 10 is at least composed of a first container wall 11, a second container wall 12, and a port 13. In addition, the outer shape of the cell culture container 10 is preferably set to a rectangular shape, for example, 20 to 1000 mm in the X direction and 20 to 1000 mm in the Y direction.

[0184] The first container wall 11 is composed of a planar membrane having air permeability and serving as the bottom surface. And as Figure 18As shown, the first container wall 11 of the present embodiment preferably has a thickness z1 of, for example, 30 to 200 μm. Here, the "bottom surface" in the present embodiment refers to the surface that becomes the bottom when the cell culture container 10 is placed on a placement table or the like, and refers to the surface that is lower in the Z direction than the following second container wall 12. In addition, the "planar shape" in the present embodiment means a plane in the X direction and the Y direction, and refers to a single surface parallel to the XY plane.

[0185] Regarding the gas permeability of the first container wall 11, according to the gas permeability test method of JIS K 7126, the oxygen penetration rate measured at a test temperature of 37°C is preferably 5000 mL / (m 2 ·day·atm) or more.

[0186] In addition, the film constituting the first container wall 11 preferably has transparency in part or in whole so that the progress of cell culture or the state of cells can be confirmed. As the material for such a film, there is no particular limitation as long as it has the above gas permeability. For example, thermoplastic resins such as polyethylene, polypropylene, ethylene-vinyl acetate copolymer, polyester, silicone-based elastomer, polystyrene-based elastomer, and tetrafluoroethylene-hexafluoropropylene copolymer (FEP) can be cited. It can be used as a single layer, or the same or different materials can be laminated and used. If the heat meltability at the sealing peripheral portion is considered, it is preferably a layer that functions as a sealing layer.

[0187] In addition, as shown in the figure, the first container wall 11 is composed of a peripheral portion 11a and a central portion 11b. Among them, the peripheral portion 11a is a region facing the peripheral portion 12a of the following second container wall 12. In addition, the central portion 11b is a region closer to the inside than the above peripheral portion 11a, and is a region for forming the following culture space S.

[0188] The second container wall 12 has a bulging shape 12z that contacts the peripheral portion 11a of the first container wall 11 and protrudes relative to the first container wall 11 at a position closer to the inside than the peripheral portion 11a. Similar to the first container wall 11, the second container wall 12 is composed of a film having gas permeability.

[0189] More specifically, regarding the gas permeability of the second container wall 12, according to the gas permeability test method of JIS K 7126, the oxygen penetration rate measured at a test temperature of 37°C is preferably 5000 mL / (m 2 ·day·atm) or more. That is, the gas permeability of the second container wall 12 can also be set to be equal to the gas permeability of the first container wall 11. In addition, the film constituting the second container wall 12 preferably has transparency in part or in whole so that the progress of cell culture or the state of cells can be confirmed. Furthermore, it can also be composed of the same material as the first container wall 11.

[0190] In addition, as Figure 18 shown, the second container wall 12 of the present embodiment preferably has a thickness z2 of, for example, 30 to 200 μm. That is, the thickness z2 of the second container wall 12 may also be set to be equal to the thickness z1 of the first container wall 11. In other words, the thickness ratio of the first container wall 11 to the second container wall 12 may also be made substantially 1.

[0191] The second container wall 12 is composed of a peripheral portion 12a, a rising portion 12b, and a central portion 12c. Among them, the peripheral portion 12a is a region in contact with the peripheral portion 11a of the first container wall 11. In addition, the central portion 12c is a region closer to the inside than the following rising portion 12b, and is a region arranged in the Z direction at a distance corresponding to the desired height for forming the culture space S from the central portion 11b. The rising portion 12b is a region rising from the first container wall 11 so as to separate the central portion 12c from the first container wall 11.

[0192] In addition, in the present embodiment, the peripheral portion 11a of the first container wall 11 and the peripheral portion 12a of the second container wall 12 may also be sealed to each other by heat fusion, and thus, the airtightness of the culture space S can be more surely ensured. However, it is not limited to this form. For example, it may also be a form in which the peripheral portion 11a of the first container wall 11 and the peripheral portion 12a of the second container wall 12 are fixed via a known adhesive.

[0193] In addition, in the idea of ensuring as wide and uniform as possible a liquid thickness and flat culture surface of the culture solution, the width of the rising portion 12b is preferably as narrow as possible, and the width of the sealing region of the peripheral portion is also preferably as narrow as possible.

[0194] In the present embodiment, a bulging shape 12z that bulges in a stepped manner is formed by the above-mentioned rising portion 12b and central portion 12c, and a culture space S is formed inside the bulging shape 12z. In addition, the height of the culture space S in the Z direction is not particularly limited, and may be appropriately set in such a way as to form an appropriate liquid thickness corresponding to the state of the cultured cells. Although it also depends on the size of the culture container, for example, it may be set to several mm to several tens of mm.

[0195] In addition, as Figure 18 or Figure 19As shown, the central portion 12c may also be a plane. In other words, the top surface (central portion 12c) forming the culture space in the second container wall 12 is preferably a plane. In addition, the rising portion 12b of the second container wall 12 is a region where the membrane is deformed into a terraced shape by heating as described below, and the hardness may be higher than the central portion 12c. In other words, the hardness of the rising portion 12b forming the bulging shape 12z in the second container wall 12 may be set to be higher than the hardness of the region (central portion 12c) different from the rising portion 12b in the bulging shape 12z. Furthermore, the hardness of the central portion 12c may also be set to be equal to the hardness of the central portion 11b. In other words, the hardness of the central portion 12c of the second container wall 12 may also be set to be substantially equal to the hardness of the central portion 11b of the first container wall 11 opposite to the central portion 12c.

[0196] like Figure 18 and Figure 19 As shown, the port 13 is a member connected to the culture space S surrounded by the first container wall 11 and the second container wall 12. The port 13 is a tubular member that can flow a culture medium or cells, etc. The port 13 is formed into a specific shape by injection molding or extrusion molding using a thermoplastic resin such as polyethylene, polypropylene, vinyl chloride, polystyrene elastomer, FEP, etc.

[0197] Furthermore, in order to prevent the port 13 from being blocked by the adhesion of the central portion 12c of the second container wall 12 and the central portion 11b of the first container wall 11, a port blocking piece protruding from the base end thereof into the culture space S may be provided at the port 13. When such a port blocking piece is provided, it is preferred that the port blocking piece be provided so as to be located on the central portion 12c side of the culture space S side so as not to interfere with the cells on the surface of the central portion 11b of the first container wall 11.

[0198] In this embodiment, the port 13 may have a semicircular cross section, a flat surface in contact with the first container wall 11 and a curved surface in contact with the second container wall 12.

[0199] Therefore, the generation of a gap between the port 13 and the second container wall 12 is suppressed, and leakage of the culture solution from the culture space S is prevented.

[0200] In this embodiment, it is preferred that at least the surface of the first container wall 11 and the top surface of the second container wall 12 are parallel to each other. Therefore, a large culture space S can be ensured, and even a relatively small amount of culture solution can be efficiently spread to every corner of the culture space S.

[0201] Moreover, more preferably, Figure 19As shown in FIG. 1 , the surface of the first container wall 11, the top surface (central portion 12c) of the second container wall 12, and the bottom surface of the port 13 may be made parallel to each other. In other words, it is preferred that the bottom surface of the port 13 (the contact surface with the first container wall 11) and the surface of the first container wall 11 of this embodiment are flush with each other. Therefore, a relatively small amount of culture solution can be spread more efficiently to every corner of the culture space S.

[0202] [Method for manufacturing cell culture container 10 and device for manufacturing cell culture container 10]

[0203] Second, use Figure 20 , a method and apparatus for manufacturing the cell culture container 10 in this embodiment will be described.

[0204] That is, the cell culture container 10 is manufactured through the following steps using a cell culture container manufacturing device 70 including a pressing member 71 , a restraining member 72 , a mounting table 73 , a heating device 74 , and a fluid introduction device 75 , which are described below.

[0205] First, if Figure 20 As shown in (a), in a state where the "first container wall 11 made of a gas-permeable film" and the "second container wall 12 arranged opposite to the first container wall 11" are overlapped, the first container wall 11 is placed on the placement table 73. At this time, the peripheral edge portion 11a of the first container wall 11 and the peripheral edge portion 12a of the second container wall 12 are preferably sealed, for example, by heat welding.

[0206] In addition, the above-mentioned port 13 is preferably provided at the end of the first container wall 11 and the second container wall 12. Figure 20 The assembly of the first container wall 11 , the second container wall 12 , and the port 13 shown in (a) is referred to as a base body 10 ′ as a cell culture container before completion.

[0207] After the first container wall 11 is placed on the mounting table 73, Figure 20 As shown in (b), in the state where the central portion 12c of the second container wall 12 is open (not pressed), the peripheral portion 12a of the second container wall 12 is pressed and constrained by the constraining member 72. Therefore, the constraining member 72 is arranged opposite to the mounting table 73 and has the function of constraining the periphery of the second container wall 12 mounted on the mounting table 73.

[0208] In addition, as long as the above-mentioned peripheral portion 12a can be constrained, the shape of the constraining member 72 is not particularly limited, and it is preferably a shape that neither excessively nor insufficiently constrains the peripheral edge of the second container wall 12, and more preferably a shape that constrains all four peripheral edges of the region corresponding to R1. In addition, in this embodiment, the constraining member 72 is used for constraint, but as long as the flatness of the first container wall 11 can be ensured, the constraining member 72 can be appropriately omitted.

[0209] After the peripheral edge portion 12a of the second container wall 12 is restrained by the restraining member 72, as shown in FIG. Figure 20 As shown in (c), in a state where the above-mentioned peripheral portion is pressed by the restraining member 72, the fluid is introduced into the space between the first container wall 11 and the second container wall 12 using the fluid introduction device 75. In addition, in this embodiment, the fluid is introduced into the interior of the base body 10' (the space between the first container wall 11 and the second container wall 12) through the port 13. Therefore, in this embodiment, while the second container wall 12 is restrained by the restraining member 72, the fluid is introduced into the space between the first container wall 11 and the second container wall 12 using the fluid introduction device 75.

[0210] In this embodiment, the fluid introduced by the fluid introduction device 75 is a liquid or a gas. As a fluid, it can be, for example, pure water, etc., as a gas, it can be, for example, clean air or an inert gas such as nitrogen. Among them, from the perspective of ease of operation or handling, clean air is used in this embodiment.

[0211] After the fluid is introduced into the interior of the substrate 10', Figure 20 As shown in (d), the pressing member 71 is lowered to a position separated from the mounting table 73 by a specific distance, and the central portion 12c of the second container wall 12 is pressed by the pressing member 71, while at least the pressing member 71 is heated by the heating device 74. At this time, the specific distance is the size (height) of the bulging shape 12z of the cell culture container 10. Therefore, the pressing member 71 is configured to be able to move forward and backward relative to the mounting table 73, and has a function of pressurizing "the second container wall 12 into which the fluid is introduced into the space between the first container wall 11 and the second container wall 12".

[0212] In addition, as the heating device 74 of this embodiment, for example, a well-known resistance heating means such as a nickel-chromium alloy wire can be cited, and typically, the heating device 74 can be provided inside the pressing member 71. In addition, it is also possible to set the heating device 74 inside the mounting table 73, and it is also possible to set the heating device 74 to heat at least one of the mounting table 73 and the pressing member 71.

[0213] In addition, the temperature at which the pressing member 71 is heated by the heating device 74 is determined in consideration of the heat-resistant temperature of the film used for the first container wall, etc., and is preferably heated to a degree that softens the film (for example, about 80° C.). The bulging shape 12z is formed by heating using the pressing member 71. In addition, when heating is performed using the pressing member 71, a heating means (for example, a resistance heating device such as a nichrome wire) may be arranged in a specific area of ​​the pressing member 71 (avoiding the area facing the central portion 12c of the second container wall 12 and facing the peripheral portion 12b of the second container wall 12). Therefore, it is possible to suppress hardening in the area of ​​the second container wall 12 where the bulging shape 12z is not necessarily formed.

[0214] Furthermore, when the second container wall 12 is pressed by the pressing member 71 heated to a desired temperature, it is preferred that the supply pressure of the fluid introduced into the interior of the substrate body 10' (later to become the culture space S) is adjusted by the fluid introduction device 75 so that the internal pressure of the interior of the substrate body 10' (later to become the culture space S) becomes constant. Therefore, it is suppressed that excessive pressure is applied to the interior of the substrate body 10', thereby preventing the extension of the membrane or the destruction of the seal, etc.

[0215] In addition to the pressing member 71 and the like, a cooling device may be further provided, and after the pressing member 71 is heated and while pressing the second container wall 12 , the pressing member 71 may be cooled using the cooling device.

[0216] After a specific time has passed since the second container wall 12 was pressed by the pressing member 71 heated to a desired temperature, Figure 20 As shown in (e), the pressing member 71 and the restraining member 72 are retracted relative to the mounting table 73, and the completed cell culture container 10 is taken out. In addition, it is preferable to seal the port 13 of the cell culture container 10 before retracting the pressing member 71 and the restraining member 72 relative to the mounting table 73. Therefore, foreign matter is prevented from accidentally entering the culture space S.

[0217] <The significance of the flat bottom and bulging shape>

[0218] Previous cell culture containers are often flat in shape with only a portion of the bottom surface being the cell culture surface, but there is no idea of ​​maximizing the cell culture surface, and further, there is no mention of the issue of ensuring the homogeneous diffusion of the culture fluid in the culture space or the teaching of the structure. More specifically, Figure 21 The cell culture container shown in (b) manufactured by the previous method cannot evenly spread the culture solution in the plane direction (XY plane), and the culture solution does not spread to the corners of the container, so it cannot become a good culture space. In addition, there is also warping in the height direction (Z direction), so the density of the culture solution is uneven across the entire surface.

[0219] In contrast, obviously, Figure 21 In the cell culture container of this embodiment shown in (a), the culture solution is uniformly and homogeneously spread in both the above-mentioned plane direction and the height direction. Therefore, even when only a relatively small amount of culture solution can be used, the cell culture container 10 manufactured by this embodiment can make the culture solution uniformly spread to every corner of the central part 11b of the first container wall 11 that serves as the culture surface. Therefore, according to this embodiment, expensive culture solution can be used with maximum efficiency, thereby suppressing the risk of contamination and more reliably culturing precious cells.

[0220] Furthermore, the cell culture method using the cell culture container of this embodiment is a cell culture method using the above-mentioned cell culture container 10, characterized in that the first container wall 11 is placed downward relative to the second container wall 12, and cells and culture fluid are injected through the port 13. At this time, the cell culture container 10 is preferably placed on a placement surface in a cell culture device (CO2 incubator) adjusted to an appropriate temperature (e.g., 37° C.), carbon dioxide concentration (e.g., 5 to 10% CO2 concentration), and humidity (e.g., about 95%).

[0221] Therefore, a wide and flat culture surface can be ensured by the first container wall serving as the bottom surface, and even a small amount of culture solution can be spread to every corner of the culture surface through the second container wall having a bulging shape.

[0222] In addition, in the above embodiment, adherent cells such as iPS cells are used as an example, but the present invention is not limited to this. That is, the present invention can also be applied to a culture container suitable for suspension cells such as hematopoietic cells or ascites cells, or its manufacturing method and manufacturing device, and a cell culture method. The reason is that in fact, in static culture of suspension cells, there is also a need to distribute cells in a wide range and uniformly, and adherent cells also have a form similar to that of suspension cells after seeding until they are precipitated and adhere to the bottom surface.

[0223] The fifth embodiment described above can be modified in various ways without departing from the spirit of the present invention. Modifications that can be appropriately applied to the fifth embodiment will be described below.

[0224] <Variation 4>

[0225] Figure 22 This is a modified example of the port 13 described in the fifth embodiment.

[0226] In the fifth embodiment described above, the surface (upper surface) of the port 13 in contact with the second container wall 12 is a curved surface, but the present invention is not limited to this form, and various port shapes can be adopted.

[0227] For example, Figure 22 The port 14 shown in (a) is formed by making the surface (in this example, the upper surface 14a1 and the side surface 14a2) in contact with the second container wall 12 a flat surface. In other words, the port 14 may also be formed as follows: a rectangular body 14a extending in the Y direction is provided with an injection and extraction port 14b.

[0228] In addition, you can Figure 22 The port 15 shown in (b) is generally configured such that the surface (in this example, the inclined surface 15a1 and the inclined surface 15a2) in contact with the second container wall 12 is a flat surface. In other words, the port 15 may be configured such that a triangular prism-shaped body 15a extending in the Y direction is provided with an injection and extraction port 15b.

[0229] <Variant 5>

[0230] Figure 23 This is another modification of the port 13 described in the fifth embodiment.

[0231] That is, the bottom surface of the port described in the above-mentioned fifth embodiment and variant example 4 is flat, and the outer surface of the area in contact with the port (the peripheral portion 11a in contact with the port) in the first container wall 11 is the same plane as the outer surface other than the area in contact with the port.

[0232] However, the present invention is not limited to this form. Figure 23 As shown, the first container wall 11 only needs to be flat at least in the region other than the region in contact with the port.

[0233] That is, in this modification 5, the port 13 having a general boat shape (almond-shaped cross section) is used, and the bottom surface of the port 13 is not a flat surface but a convex surface facing downward. Figure 23 As shown, the region of the first container wall 11 that contacts the port 13 is formed into a downwardly convex curved surface along the shape of the port 13 .

[0234] In this case, the first container wall 11 becomes flat also in the area other than the area in contact with the port 13, so that the above-mentioned effects of the present invention can be exhibited.

[0235] Furthermore, although the use for cell culture has been described above, the container of the present invention can also be used for uses other than cell culture, for example, when it is desired to store a liquid with a bottom surface that is as flat as possible.

[0236] Furthermore, the ports 13 to 15 described in the above-mentioned modification examples 4 and 5 can also be appropriately applied to the above-mentioned first to fourth embodiments or the first to third modifications.

[0237] <Other Modifications>

[0238] In the first to fourth embodiments or modifications 1 to 3 described above, examples in which the recesses 23a (or recesses 22a) provided on the mounting table T are arranged in a staggered or grid pattern are described, but the present invention is not limited thereto.

[0239] That is, a plurality of recesses may be formed on the mounting table T so that the plurality of depressions 4 in the finally manufactured cell culture container 1 are regularly arranged to form a specific pattern (a decorative pattern or geometric pattern, or characters, figures, symbols, etc.).

[0240] In addition, in each of the above embodiments or variations, the bag-shaped film container 1' is used as a container for culturing cells, but the present invention is not limited thereto. That is, the container with depressions made from the bag-shaped film container 1' of the present invention can also be used for other purposes such as a container for storing food or medicine. Moreover, as long as the depressions are in the above-mentioned specific pattern, a container with high design value can be realized.

[0241] Furthermore, in each of the above embodiments or variations, the cell culture container manufacturing device may include an observation device (not shown) such as a camera (camera assembly). For example, in the third embodiment, an observation device for observing whether a depression of the bag-shaped film container 1' is formed may be provided on the mounting table T.

[0242] Therefore, by placing CCD units and the like in a plurality of holes provided on the mounting table T, it is possible to observe the situation when the bag-like film container 1' is pressurized by the pressing member 21. In this case, the CCD unit only needs to be placed in at least one hole of the mounting table T, and need not be placed in all holes.

[0243] Therefore, based on the observation result by the observation device, the operation of the pressing member 21 or the fluid introduction device 26 can be controlled. Therefore, the bag-like film container 1' can be pressurized neither excessively nor insufficiently by the pressing member 21. Alternatively, the fluid introduction device 26 can introduce the fluid into the bag-like film container 1' at an appropriate supply pressure neither excessively nor insufficiently. Furthermore, the size of the depression formed in the bag-like film container 1' can be adjusted based on the observation result by the observation device.

[0244] The observation device is not necessarily arranged on the mounting table T side, and may be provided on the pressing member 21 side if the pressing member 21 is formed of a transparent material such as glass or heat-resistant plastic.

[0245] Furthermore, if the mounting table T is formed of the transparent material, the interior of the mounting table T (in the case of a recessed portion) can be observed from the outside of the mounting table T, so the observation device can be arranged to the side or obliquely of the mounting table T or the pressing member 21 .

[0246] Furthermore, for example, in the fifth embodiment, an observation device for observing whether the bulging shape 12z of the second container wall 12 is formed may be provided on the pressing member 71. Therefore, the pressing member 71 can form an appropriate bulging shape 12z.

[0247] In addition, the installation position of the observation device in the fifth embodiment is not limited to the pressing member 71 side, and it can also be installed at other positions such as the side or oblique side of the mounting table 73.

[0248] [Industrial Applicability]

[0249] The present invention can be used as a technology for culturing various cells efficiently or a technology for producing a container having good storage properties and high design value.

[0250] Explanation of symbols

[0251] 1: Cell culture container

[0252] 2: Container body

[0253] 3: Injection and extraction port

[0254] 4: Depression

[0255] 10: Cell culture container

[0256] 11: 1st container wall

[0257] 12: Second container wall

[0258] 13, 14, 15: Port

[0259] 21, 71: Pressing member

[0260] 22: Platform body

[0261] 23: Container Support Department

[0262] 24, 74: Heating device

[0263] 25: Driving mechanism

[0264] 26, 75: Fluid introduction device

[0265] 27: Suction device

[0266] 28: Temperature control device

[0267] 29, 72: Constraint components

[0268] 73. T: Mounting table

[0269] CP: Control device

Claims

1. A cell culture container, comprising: A first container wall, which is air-permeable and forms the bottom surface; A second container wall, which has a bulging shape that contacts the peripheral portion of the first container wall and protrudes relative to the first container wall at a position more inward than the peripheral portion; and A port, which communicates with the culture space surrounded by the first container wall and the bulging shape of the second container wall; and The first container wall has the peripheral portion and a central portion that is more inward than the peripheral portion and forms the culture space, The bottom surface of the first container wall is planar in the region in contact with the port, the peripheral portion, and the central portion.

2. The cell culture container according to claim 1, wherein, The top surface of the second container wall that forms the culture space is planar.

3. The cell culture container according to claim 1 or 2, wherein The thickness ratio of the first container wall to the second container wall is substantially 1.

4. The cell culture container according to claim 1 or 2, wherein The second container wall is a membrane with air permeability, and The air permeability of the first container wall is substantially equal to the air permeability of the second container wall.

5. The cell culture container according to claim 4, wherein, The first container wall and the second container wall are made of the same material.

6. The cell culture container according to claim 1 or 2, wherein The surface of the first container wall and the top surface of the second container wall are parallel to each other.

7. The cell culture container according to claim 1 or 2, wherein, The contact surface of the port that contacts the first container wall is in the same plane as the surface of the first container wall.

8. A cell culture method, which uses the cell culture container according to any one of claims 1 to 7, and: places the first container wall in such a way that the first container wall is below the second container wall, and injects cells and culture medium through the port.

9. A manufacturing method of the cell culture container according to any one of claims 1 to 7, which comprises the following steps: Placing the first container wall on a placement table in a state where the first container wall made of a membrane with air permeability and the second container wall arranged opposite to the first container wall overlap; Pressing the peripheral portions of the first container wall and the second container wall with a restraining member in a state where the central portion of the second container wall is open; Introducing a fluid between the first container wall and the second container wall in a state where the peripheral portion is pressed with the restraining member; and While pressing the central portion of the second container wall with a pressing member, heating at least the pressing member.

10. A manufacturing device of the cell culture container according to any one of claims 1 to 7, the cell culture container comprises a first container wall made of a planar membrane, and the manufacturing device is characterized by comprising: A placement table, which places the first container wall; A fluid introduction device, which introduces a fluid into the space between the first container wall and the second container wall placed on the placement table; A pressing member, which is configured to be able to advance and retreat relative to the placement table and presses the second container wall into which the fluid has been introduced in the space; A heating device, which heats the pressing member; and A restraining member, which is arranged opposite to the placement table and restrains the periphery of the second container wall placed on the placement table; and While restraining the second container wall with the above-described restraining member, heating the pressing member with the above-described heating device, and introducing the fluid into the space between the first container wall and the second container wall with the above-described fluid introduction device.

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