Method for manufacturing a cell culture vessel, and cell culture vessel

Abstract

To provide methods for manufacturing a cell culture vessel having no protrusions or thin-walled portions on the culture surface.SOLUTION: Provided is a method for manufacturing a cell culture vessel having a cell culture surface, in which a primary mold 10 provided with a convex portion 11 for forming a concave-convex pattern structure 31 on the surface of a container material 30, and a secondary mold 20 provided with convex portions 21 and 22 for flattening a part of the surface of the container material 30 are used; the primary mold 10 is pressed against the container material 30 to form the concave-convex pattern structure 31 on the container material 30; the secondary mold 20 is pressed against a thin-walled portion 33 formed in the container material 30 to change the thin-walled portion 33 into a flattened portion 35; and the cell culture vessel is formed using, as a culture surface, the surface of the container material 30 comprising at least a portion of the modified flattened portion 35 and the formed concave-convex structure 31.SELECTED DRAWING: Figure 1

Description

Technical Field

[0005]

[0001] The present invention relates to cell culture technology, and particularly to a method for manufacturing a cell culture container having a fine concavo-convex pattern structure on a culture surface.

Background Art

[0002] In recent years, in the fields of pharmaceutical production, gene therapy, regenerative medicine, immunotherapy, etc., it has been required to efficiently and massively culture cells and tissues in an artificial environment. <000001七杀ツ>In such a situation, cells are automatically and massively cultured using a bag-shaped cell culture container made of a thermoplastic resin. In addition, it has also been proposed to improve the culture efficiency by forming a fine concavo-convex pattern structure on the culture surface of the cell culture container.

[0003] As a method for forming a fine concavo-convex pattern structure on the culture surface, generally, a thermal transfer method in which a flat or belt-shaped mold for forming a concavo-convex pattern structure is pressed against the surface of a thermoplastic film and heated and pressurized, or a method in which a molten resin material is brought into contact with the surface of a cooling roll for forming a concavo-convex pattern structure and molded, etc. are available. Although these methods can be used when manufacturing a cell culture container having a culture surface of a normal size, there is a problem that they cannot be easily applied when manufacturing a cell culture container having a large-sized culture surface.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] In other words, forming a textured pattern structure on a large-sized culture surface requires a large-sized mold, which poses a problem as the manufacturing cost of the mold becomes enormous. Furthermore, when using a large-sized mold, simply increasing the size of the mold is not sufficient to properly form a textured pattern structure on the culture surface; a large-scale transfer device is required to uniformly pressurize a large area, which also presents a problem.

[0006] On the other hand, instead of using a single large mold, methods can be considered in which the culture surface is divided and processed by using multiple smaller molds side by side, or by using the small molds in multiple stages to process the culture surface. However, when the culture surface is processed in this way, softened resin flows into the mold boundaries, causing large protrusions. Additionally, because the resin flows out at the periphery of the mold, the resin in the areas between molds used multiple times and at the periphery of the mold becomes thinner, making it prone to cracking.

[0007] The risk of such cracks occurring is a significant problem in the manufacturing of cell culture vessels, and large protrusions on the culture surface can obstruct the flow of culture medium. Furthermore, the presence of protrusions on the culture surface prevents the culture medium thickness in the cell culture vessel from being reduced to below the height of the protrusions when draining the medium, resulting in increased consumption of culture medium during medium changes.

[0008] In particular, the problem of thin resin is not an issue if a certain thickness is acceptable, as cracks can be prevented by making the equipment thicker. However, in the case of bag-shaped cell culture vessels, for example, it is necessary to increase the gas permeability of the container wall, so there is a demand to make the equipment as thin as possible. For this reason, the risk of cracking has been a serious problem in the manufacturing of cell culture vessels.

[0009] Here, Patent Document 1 discloses an endless belt-shaped metal mold capable of forming an uneven pattern structure on a large-sized culture surface. However, the production of such a belt-shaped mold is expensive, and there is a risk of the resin tearing when peeling the film with the uneven pattern structure from the mold.

[0010] Furthermore, Patent Document 2 discloses a technique for arranging multiple molds side by side and transferring a fine pattern formed on the surface of the molds. However, this technique relates to nano-order display elements for manufacturing large-screen liquid crystal displays, and it involves pattern formation on a photocurable resin rather than thermal transfer. When this mold is applied to thermal transfer, resin flows into the gaps between the molds, creating protrusions, and therefore it is not possible to solve the problem of processing the culture surface in sections as described above.

[0011] Therefore, the inventors diligently conducted research and succeeded in solving the above problem by using a primary mold equipped with protrusions to form an uneven pattern structure on the surface of the container material, and a secondary mold equipped with protrusions to flatten a part of the surface of the container material, thereby completing the present invention. This invention has been made in view of the above circumstances, and aims to provide a method for manufacturing a cell culture vessel that does not have protrusions or thin-walled parts on the culture surface, and a cell culture vessel. [Means for solving the problem]

[0012] To achieve the above objective, the present invention provides a method for manufacturing a cell culture vessel having a cell culture surface, comprising: using a primary mold equipped with protrusions for forming an uneven pattern structure on the surface of a container material; and a secondary mold equipped with protrusions for flattening a part of the surface of the container material; pressing the primary mold against the container material to form the uneven pattern structure on the container material; pressing the secondary mold against a thin-walled portion formed on the container material to modify the thin-walled portion into a flattened portion; and using the surface of the container material, including at least a part of the modified flattened portion and the formed uneven pattern structure, as the culture surface to form the cell culture vessel.

[0013] Furthermore, the method for manufacturing a cell culture vessel of the present invention preferably involves arranging a plurality of primary molds adjacent to each other along a boundary line and pressing them against the container material to form the uneven pattern structure on the container material, and then pressing the secondary mold against the protruding portions and thin-walled portions formed in the portions of the container material corresponding to the boundary lines of the plurality of primary molds to modify the protruding portions and thin-walled portions into flattened portions.

[0014] Furthermore, the method for manufacturing a cell culture vessel of the present invention also preferably involves pressing a single primary mold against the container material multiple times to form the uneven pattern structure on the container material, and then pressing the secondary mold against the thin-walled portion formed in the portion of the container material corresponding to the periphery of the primary mold to modify the thin-walled portion into a flattened portion.

[0015] Furthermore, the method for manufacturing the cell culture vessel of the present invention may also involve pressing the thin-walled portion and a predetermined area inside the thin-walled portion with the secondary mold. Furthermore, the method for manufacturing a cell culture vessel of the present invention may also involve bonding the container material to a reinforcing material and pressing the primary mold and the secondary mold against the opposite side of the bonded surface of the reinforcing material on the container material.

[0016] Also, the method for manufacturing the cell culture container of the present invention is preferably a method of pressing the primary mold and the secondary mold to form the concavo-convex pattern structure and the flattening portion on the container material by thermal transfer or melt extrusion molding. Also, the method for manufacturing the cell culture container of the present invention is preferably a method of forming a plurality of substantially triangular prisms arranged in a mountain range shape or forming a plurality of concave portions with or without fine holes as the concavo-convex pattern structure. Also, the method for manufacturing the cell culture container of the present invention is preferably a method in which the cell culture container is a bag-shaped container having a cell culture surface inside.

[0017] The cell culture container of the present invention is a bag-shaped cell culture container having a cell culture surface inside, and is provided with a plurality of regions in which a concavo-convex pattern structure is formed on the culture surface, and a flattening portion is provided between the plurality of regions in which the concavo-convex pattern structure is formed, and a flattening portion is provided on the entire peripheral edge of the culture surface. Also, the cell culture container of the present invention preferably has a configuration in which the concavo-convex pattern structure is a plurality of substantially triangular prisms arranged in a mountain range shape or a plurality of concave portions with or without fine holes.

Advantages of the Invention

[0018] According to the present invention, it is possible to provide a method for manufacturing a cell culture container having no protrusions or thin portions on the culture surface, and a cell culture container.

Brief Description of the Drawings

[0019] [Figure 1] It is a schematic diagram showing the steps of the method for manufacturing a cell culture container according to an embodiment of the present invention. [Figure 2] It is a schematic diagram showing the state of the first step in the method for manufacturing a cell culture container according to an embodiment of the present invention. [Figure 3] It is a schematic diagram showing the state of performing the first step and the second step of the method for manufacturing a cell culture container according to an embodiment of the present invention by thermal transfer. [Figure 4]It is a schematic diagram showing a state where a primary mold is arranged on a container material in a method for manufacturing a cell culture container according to an embodiment of the present invention, and a secondary mold. [Figure 5] It is a schematic diagram showing an arrangement of a primary mold and a modification example of a secondary mold in a method for manufacturing a cell culture container according to an embodiment of the present invention. [Figure 6] It is a schematic diagram showing a primary mold and a reference example thereof in a method for manufacturing a cell culture container according to an embodiment of the present invention. [Figure 7] It is a schematic diagram showing a reference example of a secondary mold in a method for manufacturing a cell culture container according to an embodiment of the present invention. [Figure 8] It is a diagram showing the result of Test 1 regarding the method for manufacturing a cell culture container according to an embodiment of the present invention. [Figure 9] It is a diagram showing the result of Test 2 regarding the method for manufacturing a cell culture container according to an embodiment of the present invention. [Figure 10] It is a diagram showing a photograph of a protruding portion and a thin portion of a container material obtained by Comparative Example 3 in Test 3 regarding the method for manufacturing a cell culture container according to an embodiment of the present invention. [Figure 11] It is a diagram showing a photograph of a flattened portion where the protruding portion obtained by Example 3 in Test 3 regarding the method for manufacturing a cell culture container according to an embodiment of the present invention is modified and a flattened portion where the thin portion is modified. [Figure 12] It is a diagram showing the result of Test 3 regarding the method for manufacturing a cell culture container according to an embodiment of the present invention.

Embodiments for Carrying Out the Invention

[0020] Hereinafter, the method for manufacturing a cell culture container of the present invention and embodiments of the cell culture container will be described in detail with reference to the drawings. However, the present invention is not limited to the specific contents of the following embodiments and examples described later.

[0021] The method for manufacturing a cell culture vessel according to this embodiment is a method for manufacturing a cell culture vessel having a cell culture surface, and as shown in Figure 1, it uses a primary mold 10 equipped with protrusions 11 that form an uneven pattern structure 31 on the surface of the container material 30, and a secondary mold 20 equipped with protrusions (21, 22) for flattening a part of the surface of the container material 30. Furthermore, the method for manufacturing a cell culture vessel according to this embodiment includes a first step of pressing a primary mold 10 against the container material 30 to form an uneven pattern structure 31 on the container material 30.

[0022] Furthermore, the method for manufacturing a cell culture vessel according to this embodiment includes a second step in which a secondary mold 20 is pressed against the thin-walled portion 33 formed on the container material 30 in the first step, thereby transforming the thin-walled portion 33 into a flattened portion 35. Furthermore, in the method for manufacturing a cell culture vessel of this embodiment, the cell culture vessel is formed by using the surface of a container material 30, which includes at least a portion of the modified flattened portion 35 and the formed uneven pattern structure 31, as the culture surface.

[0023] In this specification and in the claims, the flattened portion means a surface that does not have an uneven pattern structure, and does not mean that it is completely flat, but includes cases where it has a curve. Furthermore, the protrusions 11 of the primary mold 10 only need to be capable of forming an uneven pattern structure on the container material 30, and may be a configuration in which multiple triangular prisms are arranged in a mountain range-like pattern, as shown in Figure 1 (hereinafter sometimes referred to as a V-pattern). In the same figure, the primary mold 10 is shown as a cross-sectional view of the center in the depth direction as seen from the front, and the protrusions 11 are formed in a triangular prism shape in the depth direction.

[0024] In the method for manufacturing a cell culture vessel according to this embodiment, it is preferable that in the first step, a plurality of primary molds 10 are placed adjacent to each other along a boundary line and pressed against the container material 30 to form an uneven pattern structure 31 on the container material 30. Furthermore, in the method for manufacturing the cell culture vessel of this embodiment, it is preferable to press the secondary mold 20 against the protrusions 32 and thin-walled portions 33 formed in the container material 30 at the boundaries of the multiple primary molds 10, thereby modifying the protrusions 32 and thin-walled portions 33 into flattened portions (34, 35).

[0025] Furthermore, in the method for manufacturing the cell culture vessel of this embodiment, it is also preferable that in the first step, a single primary mold 10 is used to press against the container material 30 multiple times to form an uneven pattern structure 31 on the container material 30. Furthermore, in the method for manufacturing the cell culture vessel of this embodiment, it is also preferable to press the secondary mold 20 against the thin-walled portion (including the thin-walled portion formed in the portion corresponding to the boundary line) formed in the portion of the primary mold 10 of the container material 30, thereby modifying the thin-walled portion into a flattened portion (34, 35).

[0026] The primary mold 10 and the secondary mold 20 are molds made of, for example, metal or silicon. In the embodiments described later, the primary mold 10 was made using silicon material and the secondary mold 20 was made using aluminum material. In this specification and in the claims, molds made of materials other than metal are also collectively referred to as molds.

[0027] The container material 30 can be, for example, a polyethylene sheet with a thickness of 0.1 mm. Specifically, the material for the container equipment 30 can preferably be a resin film, and polyolefin resins such as polyethylene and polypropylene can be used. For example, polyethylene, copolymers of ethylene and α-olefin, copolymers of ethylene and vinyl acetate, ionomers using copolymers of ethylene and acrylic acid or methacrylic acid and metal ions can be used. Polyolefins, styrene elastomers, polyester thermoplastic elastomers can also be used. Furthermore, flexible polyvinyl chloride resin, polybutadiene resin, ethylene-vinyl acetate copolymer, chlorinated polyethylene resin, polyurethane thermoplastic elastomer, polyester thermoplastic elastomer, silicone thermoplastic elastomer, styrene elastomer, for example, SBS (styrene-butadiene-styrene), SIS (styrene-isoprene-styrene), SEBS (styrene-ethylene-butylene-styrene), SEPS (styrene-ethylene-propylene-styrene), polyolefin resin, fluororesin, etc. may also be used.

[0028] Figure 2 shows an example of the arrangement of the primary mold 10 and container equipment 30 in the first process. In Figure 2, two primary molds 10 are placed on the container material 30, and by pressing the primary molds 10 against the container material 30, an uneven pattern structure 31 is formed on the surface of the container material 30. In this case, in the container material 30, the portion corresponding to the boundary line between the two primary molds 10 often forms a protruding portion 32 as a result of resin flowing into the small gap between the primary molds 10. In addition, in the peripheral portion of the primary mold 10 excluding the boundary line, a thin-walled portion 33 is often formed as a result of resin flowing out to the outside of the primary mold 10, resulting in a thinner container material 30.

[0029] When a cell culture vessel is manufactured using a container or equipment 30 with such protrusions 32 formed on it, there is a problem that the delivery of the culture medium is obstructed by the protrusions 32. In addition, when the protrusions 32 are present, it is not possible to reduce the thickness of the culture medium in the cell culture vessel to less than the height of the protrusions 32 when discharging the medium, which leads to a problem that the amount of culture medium consumed during medium exchange increases. Furthermore, when manufacturing cell culture vessels using container equipment 30 with a thin-walled section 33, cracks are prone to occur in the thin-walled section 33, leading to a problem of leakage of the culture medium.

[0030] According to the method for manufacturing cell culture vessels of this embodiment, by modifying these protruding portions 32 and thin-walled portions 33 into flattened portions (boundary flattened portions 34, peripheral flattened portions 35) using a secondary mold 20, it is possible to manufacture cell culture vessels without protruding portions 32 and thin-walled portions 33, thereby resolving these problems.

[0031] In addition, according to the method for manufacturing a cell culture vessel of this embodiment, a flattened portion is formed inside the cell culture vessel, and a part of the uneven pattern structure 31 is crushed. However, this is a minor loss compared to the problems of the present invention and does not pose a particular problem in cell culture.

[0032] Furthermore, in the method for manufacturing cell culture vessels according to this embodiment, it is preferable to use the container equipment 30 by bonding it to the reinforcing material. That is, it is preferable to bond the container equipment 30 to the reinforcing material and then press the primary mold 10 and the secondary mold 20 against the opposite side of the bonded surface of the container equipment 30 to the reinforcing material. By using the container equipment 30 bonded to the reinforcing material in this way, the protruding portion 32 and thin-walled portion 33 formed on the container equipment 30 by the primary mold 10 can be stably converted into a flattened portion by the secondary mold 20.

[0033] Furthermore, it is preferable that this reinforcing material has an adhesive layer, and that the container equipment 30 is adhered to and fixed to this adhesive layer. Here, simply stacking the container material 30 on the reinforcing material would have little effect in stably transforming the protruding portion 32 and thin-walled portion 33 formed on the container material 30 into a flattened portion by the secondary mold 20. On the other hand, by firmly attaching it to a reinforcing material that is harder than the container material 30 (such as polyethylene) and has an adhesive layer, such an effect can be fully obtained.

[0034] In other words, by attaching the reinforcing material to the container equipment 30, thermal shrinkage during the cooling of the container equipment 30 during the heat transfer process in the first and second steps is suppressed, thereby reducing deformation such as waviness of the container equipment 30. Furthermore, when peeling the container equipment 30 off the reinforcing material after the heat transfer is complete, the presence of the adhesive layer makes it easy to peel off. While there are no particular limitations on the material used for the reinforcing material, polyester-based materials are suitable, and polyethylene terephthalate (PET), which is harder than polyethylene, readily available, and inexpensive, is preferable. Furthermore, as the material for the adhesive layer, for example, an acrylic adhesive can be suitably used.

[0035] Furthermore, in this embodiment, it is preferable to use a thermoplastic resin as described above as the material for the container material 30, and it is preferable to form the uneven pattern structure 31 on the container material 30 by pressing with the primary mold 10 by heat transfer or melt extrusion molding.

[0036] When forming the uneven pattern structure 31 on the container equipment 30 by thermal transfer, for example, a thermal transfer apparatus 40 as shown in Figure 3 can be used. That is, a cushioning material 50 (such as silicone rubber) is placed on a base plate 41, and a container equipment 30 with a reinforcing material 60 having an adhesive layer (such as polyethylene terephthalate) bonded to its lower surface is placed on the cushioning material 50. Then, a plurality of primary molds 10 are placed on the upper surface of the container equipment 30, and the first step can be performed by heating the primary molds 10 while pressing a heating plate 43 against the primary molds 10 using a pressure cylinder 42.

[0037] Next, the primary mold 10 is removed from the upper surface of the container equipment 30, and the secondary mold 20 is attached to the lower surface of the heating plate 43. At this time, the secondary mold 20 is positioned such that the boundary protrusion 21 of the secondary mold 20 is located above the protrusion 32 formed on the container equipment 30, and the peripheral protrusion 22 of the secondary mold 20 is located above the thin-walled portion 33 formed on the container equipment 30. Then, the second step can be performed by using the pressurizing cylinder 42 to press the secondary mold 20, which is placed on the heating plate 43, against the container equipment 30 while heating the container equipment 30.

[0038] In the method for manufacturing a cell culture vessel according to this embodiment, the shape of the uneven pattern structure 31 formed on the container material 30 is not particularly limited as long as it is an uneven pattern, but for example, it can be a V-pattern in which multiple triangular prisms are arranged in a mountain range-like manner, as shown in the schematic diagram of Figure 1. In the same figure, the container material 30 is shown as a cross-sectional view of the center in the depth direction when viewed from the front, and the uneven pattern structure 31 is formed in a triangular prism shape in the depth direction.

[0039] The uneven pattern structure 31 formed on the container material 30 may be a shape other than the V pattern described above. For example, it may be a configuration in which multiple triangular prisms are spaced apart and arranged in a mountain range-like pattern. It may also be a configuration in which multiple recesses (wells) are formed. These recesses may be hemispherical, conical, or pyramidal, and the shape of the recesses is not particularly limited. Furthermore, the recesses may or may not have fine holes at their bottoms.

[0040] Next, with reference to Figure 4, the primary mold 10 and secondary mold 20 used in the method for manufacturing cell culture vessels of this embodiment will be described in more detail. Figure 4(a) shows a state in which two primary molds 10 are arranged in the container equipment 30, and Figure 4(b) is a schematic diagram showing the structure of the secondary mold 20 used in this case, with the upper side showing a plan view and the lower side showing a front view.

[0041] As shown in Figure 4(a), when two primary molds 10 are placed on the container material 30 and pressed against the container material 30, a recessed pattern structure 31 is formed on the container material 30. As a result, protrusions 32 are formed in the parts of the container material 30 corresponding to the boundaries of the primary molds 10. In addition, thin-walled portions 33 are formed in the parts of the container material 30 corresponding to the peripheral edges excluding the boundaries of the primary molds 10.

[0042] Therefore, in this embodiment, a container material 30 without the protruding portion 32 and the thin-walled portion 33 is obtained by pressing a secondary mold 20 equipped with a boundary protrusion 21 and a peripheral protrusion 22, as shown in Figure 4(b). In other words, the secondary mold 20 includes a boundary projection 21 for pressing the protruding portion 32 and modifying it into a boundary flattening portion 34, a peripheral projection 22 for pressing the thin-walled portion 33 and modifying it into a peripheral flattening portion 35, and a support portion 23 for supporting these. The boundary projection 21 and the peripheral projection 22 are at the same height from the bottom surface of the support portion 23.

[0043] Furthermore, as shown in Figure 4(a), a ridged pattern structure 31 can be formed on the container material 30 by repeatedly pressing using a single primary mold 10, without using two primary molds 10. In this case, a thin-walled portion 33 is formed in the container material 30 in the area corresponding to the boundary line of the primary mold 10 that has been used repeatedly, and a thin-walled portion 33 is also formed in the area corresponding to the peripheral edge of the container material 30 excluding the boundary line of the primary mold 10.

[0044] Even when the container material 30 is pressed using the primary mold 10 in this manner, the thin-walled portion 33 can be transformed into a flattened portion by using the secondary mold 20 equipped with the boundary protrusion 21 and peripheral protrusion 22 shown in Figure 4(b). In other words, the boundary protrusion 21 can modify the thin-walled portion 33 formed in the area corresponding to the boundary line of the primary mold 10 into a boundary flattening portion 34, and the peripheral protrusion 22 can modify the thin-walled portion 33 formed on the peripheral edge of the primary mold 10 into a peripheral flattening portion 35.

[0045] Furthermore, in the method for manufacturing a cell culture vessel according to this embodiment, it is preferable to form the peripheral protrusion 22 to be wide enough to be pressed up to a predetermined area inside the thin-walled portion 33, thereby allowing the secondary mold 20 to press down on the thin-walled portion 33 and the predetermined area inside the thin-walled portion 33. The reason is that when forming a bag-shaped container with a cell culture surface inside as a cell culture vessel, the area around the culture surface can be heat-sealed (heat-welded) to prevent leakage.

[0046] In other words, when heat-sealing the container material 30 into a bag shape, if a V-pattern is formed as, for example, an uneven pattern structure 31, the surface on which the V-pattern is formed must be the inner surface of the bag. Therefore, since the surface on which the V-pattern is formed is located on the opposite side of the container material 30 from the heat sealer, heat is not efficiently transferred, making it difficult to completely seal the bag. Furthermore, if the heat sealer temperature is raised until the V-pattern forming surface is completely softened (welded), it is possible to completely weld the V-pattern (the V-pattern will disappear), but this is undesirable because the parts in direct contact with the heat sealer will become too hot and burn (carbonize). Therefore, in the method for manufacturing a cell culture vessel of this embodiment, the predetermined area in which heat sealing is performed to form the container material 30 into a bag shape is also modified in advance by the secondary mold 20 to form a flattened portion, thereby enabling suitable heat sealing.

[0047] Next, with reference to Figure 5, the arrangement of the primary mold and modified secondary molds in the method for manufacturing cell culture vessels of this embodiment will be described. Figure 5(a) shows a state in which four primary molds 10 are arranged in the container equipment 30, and Figure 5(b) is a schematic diagram showing the structure of the secondary mold 20b used in this case, with the upper side showing a plan view and the lower side showing a front view.

[0048] As shown in Figure 5(a), when four primary molds 10 are placed on the container material 30 and pressed against the container material 30, a recessed pattern structure 31 is formed on the container material 30. As a result, protrusions 32 are formed in the parts of the container material 30 corresponding to the boundaries of the primary molds 10. In addition, thin-walled portions 33 are formed in the parts of the container material 30 corresponding to the peripheral edges excluding the boundaries of the primary molds 10.

[0049] Therefore, in this embodiment, a container material 30 without the protruding portion 32 and the thin-walled portion 33 is obtained by pressing a secondary mold 20b equipped with a boundary protrusion 21b and a peripheral protrusion 22b, as shown in Figure 5(b). In other words, the secondary mold 20b includes a boundary projection 21b for pressing the protruding portion 32 and modifying it into a boundary flattening portion 34, a peripheral projection 22b for pressing the thin-walled portion 33 and modifying it into a peripheral flattening portion 35, and a support portion 23b for supporting these. The boundary projection 21b and the peripheral projection 22b are at the same height from the bottom surface of the support portion 23b.

[0050] Furthermore, as shown in Figure 5(a), instead of using four primary molds 10, the uneven pattern structure 31 can also be formed on the container material 30 by repeatedly pressing using one or two primary molds 10. In this case, a thin-walled portion 33 is formed in the container material 30 in the area corresponding to the boundary line of the primary mold 10 that has been used repeatedly, and a thin-walled portion 33 is also formed in the area corresponding to the peripheral edge of the container material 30 excluding the boundary line of the primary mold 10.

[0051] Even when the container material 30 is pressed using the primary mold 10 in this manner, the thin-walled portion 33 can be transformed into a flattened portion by using the secondary mold 20b which has boundary protrusions 21b and peripheral protrusions 22b as shown in Figure 5(b). In other words, the boundary protrusion 21b can modify the thin-walled portion 33 formed in the area corresponding to the boundary line of the primary mold 10 into a boundary flattening portion 34, and the peripheral protrusion 22b can modify the thin-walled portion 33 formed on the peripheral edge of the primary mold 10 into a peripheral flattening portion 35.

[0052] In the method for manufacturing a cell culture vessel according to this embodiment, as a method for forming a cell culture vessel using the surface of the container equipment 30, which includes such flattened portions 34, 35 and uneven pattern structure 31, as the culture surface, for example, two container equipment 30 can be prepared, the container equipment 30 can be stacked with the culture surface facing inward, and the edges can be heat-sealed. Alternatively, one container 30 and one resin film without a culture surface can be prepared, and these can be stacked with the culture surface facing inward, and the edges can be heat-sealed. Furthermore, regarding the method for forming the cell culture vessel, it is sufficient that the surface of the container material 30, which includes the flattened portion and the uneven pattern structure 31 described above, can be used as the culture surface, and the specific means are not particularly limited.

[0053] Here, with reference to Figure 6, a primary mold and a reference example of the method for manufacturing a cell culture vessel according to this embodiment will be described. Figure 6(a) shows the primary mold 10 used in the method for manufacturing a cell culture vessel of this embodiment, and how a thin-walled portion 33 is formed in the portion of the container equipment 30 corresponding to the peripheral edge of the primary mold 10 by pressing the primary mold 10 against the container equipment 30.

[0054] Prior to completing the present invention, the inventors conducted an experiment to see if, as shown in Figure 6(b), cutting off the protrusion 110 on the periphery of the primary mold 100 and pressing the primary mold 100 against the container equipment 30 would increase the thickness of the portion of the container equipment 30 corresponding to the periphery of the primary mold 10, thereby preventing the formation of a thin-walled portion 33.

[0055] However, it was found that even when using a primary mold 100 with the protrusions 110 on the periphery cut off in this way, resin still flows out from the periphery of the primary mold 10 when pressed against the container material 30, resulting in the formation of a thin-walled portion 33. Therefore, after much research, the inventors have found that by using a primary mold 10 and a secondary mold 20 in combination, and by modifying the thin-walled portion 33 into a flattened portion using the secondary mold 20, the problem of the thin-walled portion 33 being formed in the portion corresponding to the peripheral edge of the primary mold 10 is resolved. Furthermore, even when using such a primary mold 100, the thin-walled portion can be eliminated by using the secondary mold 20, so it is possible to use the primary mold 100 in this embodiment.

[0056] Next, with reference to Figure 7, a reference example of a secondary mold in the method for manufacturing cell culture vessels according to this embodiment will be described. Figure 7 is a schematic diagram showing the configuration of a secondary mold 200 that has boundary protrusions 210 but no peripheral protrusions, with the top showing a plan view and the bottom showing a front view.

[0057] When using such a secondary mold 200, although it was possible to flatten the protruding portion 32 of the container material 30, it was somewhat difficult to properly transform the protruding portion 32 into a flattened portion. In other words, when using the secondary mold 200, deformation often occurred throughout the container material 30.

[0058] Furthermore, when using such a secondary mold 200, a flattened area is not formed around the culture surface. As a result, when the container material 30 is heat-sealed into a bag shape, the gaps in the uneven pattern structure 31 may not be sufficiently welded, sometimes leading to liquid leakage. Therefore, in the method for manufacturing a cell culture vessel of this embodiment, by using a secondary mold 20 equipped with peripheral protrusions 22 along with boundary protrusions 21, it is possible to stably modify the protruding portion 32 into a flattened portion.

[0059] The cell culture vessel of this embodiment is obtained by the method for manufacturing a cell culture vessel described above, and is a bag-shaped cell culture vessel having a cell culture surface inside, characterized in that it is provided with a plurality of regions on the culture surface in which an uneven pattern structure 31 is formed, flattened portions 34 are provided between the plurality of regions in which the uneven pattern structure 31 is formed, and flattened portions 35 are provided around the entire periphery of the culture surface.

[0060] Furthermore, in the cell culture vessel of this embodiment, the shape of the uneven pattern structure 31 is not particularly limited as long as it is an uneven pattern, but for example, it may be a plurality of triangular prisms arranged in a mountain range-like manner as shown in the schematic diagram of Figure 1, or a plurality of triangular prisms arranged in a mountain range-like manner with gaps in between. It may also be a structure in which a plurality of recesses (wells) are formed, and the bottom of the recesses may or may not have fine holes.

[0061] The cells cultured using the cell culture vessel of this embodiment are not particularly limited and may be suspension cells, adherent cells, spheres (cell aggregates), or organoids created by aggregating several types of cells.

[0062] As described above, according to the method for manufacturing a cell culture vessel and the cell culture vessel of this embodiment, even when multiple primary molds are used or when a primary mold is used repeatedly to form an uneven pattern structure on the container material, by using a secondary mold, it is possible to obtain a container material without protrusions or thin walls. Using this, it is possible to obtain a cell culture vessel that is less prone to cracking, facilitates the delivery of culture medium, and reduces the amount of culture medium used. [Examples]

[0063] The following describes tests conducted to confirm the effectiveness of the method for producing a cell culture vessel according to the present invention. [Test 1] In this experiment, two primary molds were used simultaneously to form an uneven pattern structure on the container material, and then a secondary mold was used to modify the protruding and thin-walled portions formed on the container material into flattened portions.

[0064] First, the following were fabricated as the primary and secondary molds to be used in this test. As the primary mold, a 120 x 90 mm silicon (Si) material was used, and dicing was performed with a blade at a 75-degree angle to the horizontal to create a pattern of multiple triangular prisms arranged in a mountain range-like fashion along the longitudinal direction of the silicon material, as shown in the schematic diagram of Figure 1, for the primary mold 10 (hereinafter sometimes referred to as a V-pattern). In the same figure, the primary mold 10 is shown as a cross-sectional view of the center in the depth direction as seen from the front, and the multiple protrusions 11 of the primary mold 10 are formed in a triangular prism shape in the depth direction. The multiple protrusions in the primary mold were formed continuously without any gaps between them, and their pitch (the width of the base of one protrusion) was 0.11 mm. The height of each protrusion (the height from the base to the apex of the protrusion) was 0.2 mm.

[0065] As a secondary mold, a mold was fabricated that could modify the protruding and thin-walled portions formed when two primary molds are placed adjacent to each other in the longitudinal direction into flattened portions. Specifically, using 180 x 120 aluminum material, a mold was fabricated having a rectangular parallelepiped-shaped boundary protrusion 21 in the short direction of the aluminum material, as shown in the schematic diagram of Figure 1, for the secondary mold 20. In this case, the boundary protrusion 21 was fabricated by machining. Note that in the same figure, the secondary mold 20 is shown as a cross-sectional view of the center in the depth direction as seen from the front, and the boundary protrusion 21 of the secondary mold 20 is formed in a rectangular parallelepiped shape in the depth direction.

[0066] Furthermore, peripheral protrusions 22 were formed on the periphery of the secondary mold 20. As shown in Figure 4, the peripheral protrusions 22 were formed around the entire circumference of the secondary mold 20, and were wider than the boundary protrusions 21. The width of the boundary protrusion in the secondary mold was set to 2 mm, and the width of the peripheral protrusion 22 was set to 6 mm. Furthermore, the height of both the boundary protrusion and the peripheral protrusion was set to 5 mm.

[0067] In this test, the width of the boundary protrusion was set to 2 mm, but its size is not particularly limited, and a narrower width is desirable from the viewpoint of reducing the area that crushes the uneven pattern structure 31. On the other hand, if the width of the boundary protrusion is made too narrow, it becomes difficult to align the protruding portion 32 to modify it into a flattened portion, so in this test, it was set to 2 mm for safety reasons.

[0068] The container material used was a linear low-density polyethylene film (manufactured by Toyo Seikan Group Holdings Co., Ltd.) with a thickness of 0.11 mm and dimensions of 200 x 140 mm. This container material was then bonded to a reinforcing material made of polyethylene terephthalate using an acrylic adhesive. The thickness of the reinforcing material was 0.06 mm, and the thickness of the adhesive layer was 0.02 mm.

[0069] Next, as shown in Figure 4(a), two primary molds were placed adjacent to each other in the longitudinal direction on the opposite side of the adhesive surface of the reinforcing material in the container material, and a textured pattern structure was processed onto the surface of the container material using a heat transfer device (manufactured by Toyo Seikan Group Holdings Co., Ltd.). At this time, the temperature of the primary molds was set to 130°C, and pressure of 4000N was applied to the two primary molds for 20 seconds. Then, the surface of this container material was used as the culture surface in the cell culture vessel, and a method for manufacturing a cell culture vessel using only this primary mold was designated as Comparative Example 1.

[0070] In Comparative Example 1, the culture surface had protrusions formed in areas corresponding to the boundaries of adjacent primary molds. In addition, thin-walled areas were formed around the periphery, excluding the boundaries of the primary molds. These protrusions and thin-walled areas were photographed from the side using a microscope, and the height of the protrusions from the bottom surface of the container and the thickness of the container in the thin-walled areas were measured.

[0071] Next, a secondary mold was attached to the heat transfer device, and the secondary mold was pressed against the surface of the container material that had been processed with a textured pattern. At this time, the temperature of the secondary mold was set to 130°C, and pressure of 4000N was applied to the secondary mold for 20 seconds. This process involves pressing the secondary mold against the protruding and thin-walled portions formed at the boundary lines of adjacent primary molds, thereby transforming the protruding and thin-walled portions into flattened portions.

[0072] The surface of this container material was used as the culture surface in a cell culture vessel, and a method for manufacturing a cell culture vessel using a primary mold and a secondary mold was designated as Example 1. Then, a microscope was used to photograph the side of the flattened section to measure the height of the flattened section with the modified protrusion from the bottom surface of the container material, and the thickness of the container material in the flattened section with the modified thin-walled section.

[0073] Figure 8 shows the results and effects of Comparative Example 1 and Example 1. As shown in the figure, the height of the protrusion in Comparative Example 1 was 0.3 to 0.8 mm, and the height of the highest point of the protrusion was 0.8 mm. In contrast, the height of the flattened portion in Example 1, where the protrusion was modified, was 0.02 to 0.05 mm, and the height of the highest point of the flattened portion was 0.05 mm.

[0074] Thus, in Comparative Example 1, the presence of protrusions on the culture surface may cause problems when supplying the culture medium, whereas in Example 1, the protrusions are modified into flattened sections, thus preventing the risk of problems occurring when supplying the culture medium. Furthermore, the minimum liquid thickness when draining the culture medium from the cell culture vessel was 0.8 mm in Comparative Example 1, but it could be reduced to 0.05 mm in Example 1. This demonstrated that the liquid thickness inside the cell culture vessel can be reduced during medium exchange, thereby reducing the amount of culture medium consumed.

[0075] Furthermore, the thickness of the thin-walled portion of Comparative Example 1 was 0.01 to 0.015 mm, and the thickness of the thinnest part of the thin-walled portion was 0.01 mm. In contrast, the thickness of the flattened portion, in which the thin-walled portion was modified in Example 1, was 0.025 to 0.03 mm, and the thickness of the thinnest part of the flattened portion was 0.025 mm. In other words, the container material obtained in Example 1 did not have thin-walled sections and was less prone to cracking than the one obtained in Comparative Example 1.

[0076] [Exam 2] In this experiment, a single primary mold was used twice to form an uneven pattern structure on the container material, and then a secondary mold was used to modify the thin-walled sections formed on the container material into flattened sections.

[0077] The same primary mold used in Test 1 was employed. As a secondary mold, we fabricated one that could convert the protruding and thin-walled sections that occur when two primary molds are placed adjacent to each other in the short direction into flattened sections. Specifically, it was fabricated using 240 x 90 mm aluminum material in the same manner as in Test 1. The width of the boundary protrusion in the secondary mold was set to 2 mm, and the width of the peripheral protrusion 22 was set to 6 mm. Furthermore, the height of both the boundary protrusion and the peripheral protrusion was set to 5 mm.

[0078] The container material used was a linear low-density polyethylene film (manufactured by Toyo Seikan Group Holdings Co., Ltd.) with a thickness of 0.11 mm and dimensions of 300 x 140 mm. This container material was then bonded to a reinforcing material made of polyethylene terephthalate using an acrylic adhesive. The thickness of the reinforcing material was 0.06 mm, and the thickness of the adhesive layer was 0.02 mm.

[0079] Next, on the opposite side of the adhesive surface of the reinforcing material in the container, one primary mold was placed at one of the positions where two primary molds could be placed adjacent to each other in the short direction, and a heat transfer device (manufactured by Toyo Seikan Group Holdings Co., Ltd.) was used to process an uneven pattern structure onto the surface of the container. At this time, the temperature of the primary mold was set to 130°C, and a pressure of 2000N was applied to one primary mold for 20 seconds.

[0080] Next, the primary mold was removed, and one primary mold was placed in the other position where the two primary molds could be placed adjacent to each other in the short direction. The same process was then carried out to create a second uneven pattern structure on the surface of the container material. Then, the surface of this container material was used as the culture surface in the cell culture vessel, and a method for manufacturing a cell culture vessel using only this primary mold was designated as Comparative Example 2.

[0081] In Comparative Example 2, the culture surface showed thin-walled areas in the regions corresponding to the boundaries of the repeatedly used primary mold. Thin-walled areas were also formed around the periphery of the primary mold, excluding the boundaries. Microscopic photographs were taken from the sides of these thin-walled areas to measure the thickness of the container material in those areas.

[0082] Next, a secondary mold was attached to the heat transfer device, and the secondary mold was pressed against the surface of the container material that had been processed with a textured pattern. At this time, the temperature of the secondary mold was set to 130°C, and pressure of 4000N was applied to the secondary mold for 20 seconds. This process involves pressing a secondary mold against the thin-walled section, thereby transforming it into a flattened section.

[0083] The surface of this container material was used as the culture surface in a cell culture vessel, and a method for manufacturing a cell culture vessel using a primary mold and a secondary mold was presented as Example 2. Then, a microscope was used to photograph the side of the flattened section, and the thickness of the container material in the flattened section where the thin-walled section had been modified was measured. Furthermore, the tensile strength of the container materials with the uneven pattern structure obtained in Comparative Example 2 and Example 2 was measured using a precision universal testing machine (Shimadzu Corporation, model number AG-IS). At this time, tensile strength was applied in a direction parallel to the V-pattern formed on the container material (the short side direction of the primary mold).

[0084] The results and effects of Comparative Example 2 and Example 2 are shown in Figure 9. As shown in the figure, the thickness of the thin-walled section of Comparative Example 2 was 0.008 to 0.012 mm, and the thickness of the thinnest part of the thin-walled section was 0.008 mm. In contrast, the height of the flattened section, in which the thin-walled section was modified, was 0.022 to 0.028 mm, and the thickness of the thinnest part of the flattened section was 0.022 mm. Furthermore, the tensile strength of the container material obtained in Comparative Example 2 was 2N, while the tensile strength of the container material obtained in Example 2 was 8N.

[0085] Therefore, it became clear that the container material obtained in Example 2, as a result of modifying the thin-walled portion into a flattened portion, has a significantly reduced risk of cracking compared to the container material obtained in Comparative Example 2.

[0086] [Exam 3] In this experiment, four primary molds were used simultaneously to form an uneven pattern structure on the container material, and then secondary molds were used to modify the protruding and thin-walled portions formed on the container material into flattened portions.

[0087] The same primary mold used in Test 1 was employed. As a secondary mold, as shown in Figure 5, a mold was fabricated that could modify the protruding and thin-walled portions that occur when four primary molds are placed adjacent to each other into flattened portions. Specifically, it was fabricated using 240 x 180 aluminum material in the same manner as in Test 1. The width of the boundary protrusion in the secondary mold was set to 2 mm, and the width of the peripheral protrusion 22 was set to 6 mm. Furthermore, the height of both the boundary protrusion and the peripheral protrusion was set to 5 mm.

[0088] As the container material, a linear low-density polyethylene film (manufactured by Toyo Seikan Group Holdings Co., Ltd.) with a thickness of 0.11 mm and measuring 300 x 240 mm was used. This container material was then bonded to a reinforcing material made of polyethylene terephthalate using an acrylic adhesive. At this time, the thickness of the reinforcing material was 0.06 mm, and the thickness of the adhesive layer was 0.02 mm.

[0089] Next, as shown in Figure 5(a), four primary molds were placed adjacent to each other in the longitudinal and transverse directions on the opposite side of the adhesive surface of the reinforcing material in the container material, and a textured pattern structure was processed onto the surface of the container material using a heat transfer device (manufactured by Toyo Seikan Group Holdings Co., Ltd.). At this time, the temperature of the primary molds was set to 130°C, and the four primary molds were pressurized twice at 4000N for 20 seconds each. Then, the surface of this container material was used as the culture surface in the cell culture vessel, and a method for manufacturing a cell culture vessel using only this primary mold was designated as Comparative Example 3.

[0090] In Comparative Example 3, the culture surface had protrusions formed in areas corresponding to the boundaries of adjacent primary molds. In addition, thin-walled areas were formed around the periphery, excluding the boundaries of the primary molds. These protrusions and thin-walled areas were photographed from the side using a microscope, and the height of the protrusions from the bottom surface of the container and the thickness of the container in the thin-walled areas were measured. Figure 10 shows the photographs of the protrusions and thin-walled areas taken at that time.

[0091] Next, a secondary mold was attached to the heat transfer device, and the secondary mold was pressed against the surface of the container material that had been processed with a textured pattern. At this time, the temperature of the secondary mold was set to 130°C, and pressure of 4000N for 20 seconds was applied twice to the four primary molds. This process involves pressing the secondary mold against the protruding and thin-walled portions formed at the boundary lines of adjacent primary molds, thereby transforming the protruding and thin-walled portions into flattened portions.

[0092] The surface of this container material was used as the culture surface in a cell culture vessel, and a method for manufacturing a cell culture vessel using a primary mold and a secondary mold was designated as Example 3. Then, using a microscope, photographs were taken from the side of the flattened section to measure the height of the flattened section with the modified protrusion from the bottom surface of the container material and the thickness of the container material in the flattened section with the modified thin-walled section. Figure 11 shows the photographs of the flattened section with the modified protrusion and the flattened section with the modified thin-walled section taken at this time.

[0093] The results and effects of Comparative Example 3 and Example 3 are shown in Figure 12. As shown in the figure, the height of the protrusion in Comparative Example 3 was 0.3 to 0.7 mm, and the height of the highest point of the protrusion was 0.7 mm. In contrast, the height of the flattened portion, in which the protrusion was modified, was 0.02 to 0.06 mm, and the height of the highest point of the flattened portion was 0.06 mm.

[0094] Thus, in Comparative Example 3, the presence of protrusions on the culture surface may cause problems when supplying the culture medium, whereas in Example 3, the protrusions are modified into flattened sections, thus preventing the risk of problems occurring when supplying the culture medium. Furthermore, while the minimum liquid thickness when draining the culture medium from the cell culture vessel was 0.7 mm in Comparative Example 3, it could be reduced to 0.06 mm in Example 3. This demonstrated that the liquid thickness inside the cell culture vessel can be reduced during medium exchange, thereby reducing the amount of culture medium consumed.

[0095] Furthermore, the thickness of the thin-walled portion of Comparative Example 3 was 0.009 to 0.013 mm, and the thickness of the thinnest part of the thin-walled portion was 0.009 mm. In contrast, the height of the flattened portion, in which the thin-walled portion was modified, was 0.024 to 0.03 mm, and the thickness of the thinnest part of the flattened portion of Example 3 was 0.024 mm. In other words, the container material obtained in Example 3 did not have thin-walled sections and was less prone to cracking than the one obtained in Comparative Example 3.

[0096] The present invention is not limited to the embodiments and examples described above, and it goes without saying that various modifications can be made within the scope of the present invention. For example, the number of primary molds used to form one culture surface can be increased to, for example, six or eight, and the secondary mold can be pressed multiple times to modify protruding or thin-walled portions of the container material formed by the primary mold into flattened portions. [Industrial applicability]

[0097] The present invention can be suitably used when manufacturing cell culture vessels having a large-sized culture surface. [Explanation of symbols]

[0098] 10 Primary mold 11 Convex part 12 Support part 20,20b Secondary mold 21,21b Boundary protrusion 22,22b Peripheral protrusions 23,23b Support part 30 Containers and equipment 31. Uneven pattern structure 32 Protrusion 33 Thin-walled section 34 Boundary flattening part 35 Peripheral flattening portion 60 Reinforcement material

Claims

1. A method for manufacturing a cell culture vessel having a cell culture surface, A primary mold is used which has protrusions that form an uneven pattern structure on the surface of the container material, and a secondary mold is used which has protrusions that flatten a part of the surface of the container material. The primary mold is pressed against the container material to form the uneven pattern structure on the container material. The secondary mold is pressed against the thin-walled portion formed in the container material to transform the thin-walled portion into a flattened portion. The surface of the container material, which includes at least a portion of the modified flattened portion and the uneven pattern structure formed thereon, is used as the culture surface to form the cell culture vessel. A method for producing a cell culture vessel, characterized by the following:

2. Multiple primary molds are arranged adjacent to each other along a boundary line and pressed against the container material to form the uneven pattern structure on the container material. The secondary mold is pressed against the protrusions and thin-walled portions formed in the container material at the boundaries of the multiple primary molds, thereby modifying the protrusions and thin-walled portions into flattened portions. A method for producing a cell culture vessel according to claim 1, characterized by its features.

3. Using one primary mold, the container material is pressed multiple times to form the uneven pattern structure on the container material. The secondary mold is pressed against the thin-walled portion formed in the portion of the container material corresponding to the peripheral edge of the primary mold, thereby transforming the thin-walled portion into a flattened portion. A method for producing a cell culture vessel according to claim 1, characterized by its features.

4. A method for manufacturing a cell culture vessel according to any one of claims 1 to 3, characterized in that the thin-walled portion and a predetermined area inside the thin-walled portion are pressed by the secondary mold.

5. A method for manufacturing a cell culture vessel according to any one of 1 to 4, characterized in that the container material is bonded to a reinforcing material, and the primary mold and the secondary mold are pressed against the surface of the container material opposite to the bonded surface of the reinforcing material.

6. A method for manufacturing a cell culture vessel according to any one of claims 1 to 5, characterized in that the container material is made of a thermoplastic resin, and the uneven pattern structure and flattened portion are formed on the container material by pressing the primary mold and the secondary mold and performing thermal transfer or melt extrusion molding.

7. A method for manufacturing a cell culture vessel according to any one of claims 1 to 6, characterized in that the aforementioned uneven pattern structure is formed by arranging a plurality of substantially triangular prisms in parallel in a mountain range-like manner, or by forming a plurality of recesses having or not having micropores.

8. A method for manufacturing a cell culture vessel according to any one of claims 1 to 7, characterized in that the cell culture vessel is a bag-shaped container having a cell culture surface inside.

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