Cell culture vessel, cell culture kit, and cell culture method

Abstract

To provide a cell culture vessel capable of accommodating hydrogels in wells of the cell culture vessel by a simple method.SOLUTION: Provided is a cell culture vessel having wells for culturing cells on a hydrogel, the cell culture vessel having a main body portion having a through hole extending in the front and back direction, and an elastic portion arranged on the back side of the body portion, the main body portion having: an annular side wall portion that comprises a first through hole that is a part of the through hole and opens to the front side of the main body portion and that constitutes a part of the side wall of the well; and an annular punching blade that comprises a second through hole that is a part of the through hole and opens to the back side of the main body portion and that constitutes a part of the side wall of the well, and the well being a region surrounded by the inner peripheral surface of the through hole and the elastic portion when the punching blade and the elastic portion are brought into contact with each other.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a cell culture vessel, a cell culture kit, and a cell culture method. [Background technology]

[0002] When culturing cells or bacteria, specialized glass or plastic containers called petri dishes or microplates (hereinafter also referred to as "cell culture vessels") are generally used. In most cases, the part used for culturing in these cell culture vessels is a simple cylinder with a flat bottom.

[0003] Here, solid culture, in which an agar gel medium is placed inside a cell culture vessel, is a widely known cell culture method. In this solid culture, powdered agar and liquid medium are added to distilled water, heated to dissolve, and the resulting solution is poured into the cell culture vessel before it cools. The medium is then obtained by cooling the solution and gelling it. In addition, gelling agents used in solid culture, including agar, rarely change in size, making it difficult for gaps to form between the cell culture vessel and the solid culture medium.

[0004] Here, the inventors have discovered a phenomenon in which, when cancer cells are cultured using a specific synthetic polymer gel as a scaffold, some of the cells transform into stem cells and proliferate, and have proposed this as a method for obtaining high concentrations of cancer stem cells (e.g., Patent Document 1, and Non-Patent Documents 1 and 2, etc.). Previous methods for increasing cancer stem cells have existed, but all required expensive equipment and reagents, and it took 2 to 3 weeks to obtain a small number of cancer stem cells. In contrast, with the above method, it is possible to obtain cancer stem cells in quantities comparable to those obtained by conventional methods by simply culturing a small number of cancer cells on a synthetic polymer gel for a few days. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2018 / 151309 [Non-patent literature]

[0006] [Non-Patent Document 1] Jian Ping Gong, “Materials both Tough and Soft”, Science, Vol.344, pp.161-162. [Non-patent document 2] Jun Suzuka, et al., “Rapid reprogramming of tumor cells into cancer stem cells on double-network hydrogels”, Nature Biomedical Engineering, Vol. 5, pp. 914-925. Summary of the Invention [Problem to be solved by the invention]

[0007] However, synthetic polymer gels that cause the above phenomenon have the problem that their size is easily changed depending on the properties of the liquid medium used for culture.In addition, there are many gels other than the above synthetic polymer gels that are easily changed in size due to temperature changes, the properties of the liquid medium, etc.

[0008] When a hydrogel (hereinafter, collectively referred to as "hydrogel"), whose size is easily changed depending on the properties of the liquid medium, is placed in a cell culture vessel, the hydrogel may swell and wrinkle or crack. Furthermore, when the hydrogel shrinks, cells may fall between the cell culture vessel and the hydrogel, or the hydrogel may float in the cell culture vessel, causing the cells to move around to the backside of the hydrogel. This may result in problems such as obstructing microscopic observation of the state of the cultured cells or cell counting.

[0009] Therefore, when using a swelling / shrinking hydrogel for cell culture, the hydrogel was immersed in a liquid medium until there was no change in size (also referred to as "equilibrium swelling" in this specification), and then cut to fit the shape of the cell culture vessel and placed one by one into the well of the cell culture vessel.

[0010] If one wishes to simultaneously screen multiple drugs using precious cells and drugs, a small-volume 96-well microplate should be selected. However, as mentioned above, cutting the hydrogel to fit the shape of the wells and then inserting it into each of the 96 wells is not practical.

[0011] The present invention aims to provide a cell culture vessel, a cell culture kit, and a cell culture method using the cell culture vessel, which allow a hydrogel sheet to be cut to fit the shape of a well in a cell culture vessel in a simple manner and which allows the cut hydrogel to be easily placed in the well. [Means for solving the problem]

[0012] The present invention provides a cell culture vessel having a well for culturing cells on a hydrogel, the cell culture vessel comprising: a main body portion having a through-hole extending in the front-to-back direction; and an elastic portion arranged on the back side of the main body portion; the main body portion including a first through-hole that is part of the through-hole and opens on the front side of the main body portion, and an annular side wall portion that constitutes part of the side wall of the well; and an annular punching blade that includes a second through-hole that is part of the through-hole and opens on the back side of the main body portion, and that constitutes part of the side wall of the well; the well is an area surrounded by the inner surface of the through-hole and the elastic portion when the punching blade and the elastic portion are abutted against each other.

[0013] The present invention also provides a cell culture kit comprising the above-mentioned cell culture vessel and a hydrogel sheet.

[0014] The present invention also provides a cell culture method using the above-mentioned cell culture vessel, comprising the steps of: sandwiching a hydrogel sheet that has been equilibrated and swollen with a liquid culture medium appropriate for the purpose between the punching blade and the elastic part; pressing the punching blade toward the elastic part to punch out the hydrogel sheet, thereby placing the punched hydrogel in the well; and culturing cells on the hydrogel. [Effects of the Invention]

[0015] According to the present invention, a cell culture vessel is provided which can contain hydrogel in a well in a simple manner, and even when there are multiple wells, a hydrogel sheet can be cut at once to fit the shape of the wells of the cell culture vessel, and the cut hydrogel can be easily contained in all of the wells; a cell culture kit including the same; and a cell culture method using the cell culture vessel. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1A is an exploded perspective view of a cell culture kit including a cell culture vessel and a hydrogel sheet according to one embodiment of the present invention, and FIG. 1B is a perspective view of the completed cell culture kit shown in FIG. 1A. [Figure 2] Figure 2A is a schematic cross-sectional view of the main body and elastic part in a cell culture vessel according to one embodiment of the present invention before punching out the hydrogel sheet, Figure 2B is a schematic cross-sectional view of the main body and elastic part after punching out the hydrogel sheet, and Figure 2C is a schematic cross-sectional view of the main body and elastic part after adding liquid medium for cell culture into each well. [Figure 3] 3A shows the results of the waterproofing effect test for Example 2, FIG. 3B shows the results of the waterproofing effect test for Comparative Example 1, and FIG. 3C shows the results of the waterproofing effect test for Comparative Example 2. As shown in FIG. DETAILED DESCRIPTION OF THE INVENTION

[0017] The cell culture vessel of the present invention has wells for culturing cells, contains a hydrogel in the well, and cultures cells on the hydrogel. In this specification, a "well" refers to a concave structure for culturing cells, also known as a well-shaped structure. As described above, conventional cell culture vessels require cutting a hydrogel sheet to fit the shape of the well and placing the cut hydrogels one by one into the wells. In contrast, the cell culture vessel of the present invention allows for punching of a hydrogel sheet to fit the shape of the wells, and the punched hydrogels can be placed directly into the wells. Therefore, even when there are a large number of wells, hydrogels can be placed in the wells with simple operations.

[0018] (Cell culture vessel configuration) One embodiment of the cell culture vessel of the present invention will be described in detail below with reference to FIGS. 1 and 2. However, the cell culture vessel of the present invention is not limited to this configuration. FIG. 1A is an exploded perspective view of a cell culture kit 110 including the cell culture vessel 1 and a hydrogel sheet 100, and FIG. 1B is a perspective view of the completed cell culture kit 110. Also, FIG. 2A is a schematic cross-sectional view of the main body 10 and elastic portion 20 before punching the hydrogel sheet 100. FIG. 2B is a schematic cross-sectional view of the main body 10 and elastic portion 20 after punching the hydrogel sheet 100. FIG. 2C is a schematic cross-sectional view showing the state after adding liquid medium 60 to a well 50 formed by the punched hydrogel sheet 100 and the first through-hole 11a. For convenience, outlines are partially omitted in FIGS. 2A to 2C.

[0019] The cell incubator 1 has a main body 10 with a through-hole 11 extending in the front-to-back direction, and an elastic part 20 arranged on the back side of the main body 10. The cell incubator 1 of this embodiment further has a housing 30 for housing the main body 10 and the elastic part 20, and a cover (not shown) for preventing internal contamination. However, the cell incubator 1 does not necessarily have to have the housing 30 and the cover.

[0020] In the cell culture vessel 1 of this embodiment, when the punching blade 13 of the main body 10 is brought into contact with the elastic portion 20, the inner circumferential surface of the through-hole 11 and the area surrounded by the elastic portion 20 form a well 50. In this embodiment, the main body 10 has multiple through-holes 11 and corresponding punching blades 13. However, the number of through-holes 11 and punching blades 13 is not particularly limited and may be, for example, one. However, the effect of this embodiment is particularly likely to be achieved when the number of through-holes 11 and punching blades 13 (the number of wells 50) is large. Therefore, the number of through-holes 11 and punching blades 13 is preferably two or more. Note that the multiple through-holes 11 may be randomly arranged in the main body 10, but a regular arrangement is preferable from the viewpoint of operability during cell culture. In this case, the distance between adjacent through-holes 11 is appropriately selected depending on the application of the cell culture vessel 1.

[0021] 2A, the main body 10 of the cell culture vessel 1 has through-holes 11 extending in the front-to-rear direction. The through-holes 11 have a first through-hole 11a that opens on the front side of the main body 10 and is surrounded by an annular side wall 12, and a second through-hole 11b that opens on the back side of the main body 10 and is surrounded by an annular punching blade 13.

[0022] In the cell culture vessel of this embodiment, the second through-holes 11b become areas for accommodating the punched hydrogel 100a, and the first through-holes 11a become areas for culturing cells on the hydrogel 100a.

[0023] Here, the shape of the first through hole 11a of the main body 10 is not particularly limited. However, in this embodiment, it is preferable that the horizontal cross-sectional area of ​​the first through hole 11a be smaller than the horizontal cross-sectional area of ​​the second through hole 11b at the boundary between the first through hole 11a and the second through hole 11b. If the horizontal cross-sectional area of ​​the first through hole 11a is smaller than the horizontal cross-sectional area of ​​the second through hole 11b at this boundary region, it is expected that the punched hydrogel 100a will be less likely to enter the first through hole 11a. Note that in this embodiment, the overall opening area of ​​the first through hole 11a is set smaller than the opening area of ​​the second through hole 11b so that a step surface 11c is formed between the first through hole 11a and the second through hole 11b. The presence of the step surface 11c between the first through hole 11a and the second through hole 11b makes it easier to prevent the hydrogel 100a from floating up in the well 50.

[0024] In this embodiment, the horizontal cross-sectional area of ​​the first through hole 11a is constant from the opening on the front side of the main body 10 to the stepped surface 11c, but may vary continuously or intermittently. For example, at the boundary between the first through hole 11a and the second through hole 11b, the side wall 12 surrounding the first through hole 11a may be provided with a plurality of ridges or protrusions extending into the first through hole 11a so that the horizontal cross-sectional area of ​​the first through hole 11a is smaller than the horizontal cross-sectional area of ​​the second through hole 11b. In this case, it is sufficient that the ridges or protrusions are at least disposed on the side wall 12 near the boundary between the first through hole 11a and the second through hole 11b. The horizontal cross-sectional shape of the first through hole 11a is not particularly limited. In this embodiment, the cross-sectional shape is circular, but it may also be polygonal or elliptical.

[0025] On the other hand, the outer shape of the annular surface surrounding the first through hole 11a of the side wall portion 12 is not particularly limited, and in this embodiment, as shown in Fig. 1A, the side wall portion 12 has a shape in which a plurality of cylindrical side wall portions 12 are integrated by connecting portions 14, but is not limited to this shape. For example, the side wall portion 12 may have a structure in which a plurality of first through holes 11a are formed in a rectangular parallelepiped member.

[0026] The horizontal cross-sectional area of ​​the second through hole 11b is selected appropriately depending on the size of the desired hydrogel 100a. Note that, in order to prevent the hydrogel 100a from wrinkling or becoming uneven on its surface after punching and to prevent a gap from being generated between the hydrogel 100a and the punching blade 13, it is preferable that the horizontal cross-sectional area of ​​the second through hole 11b is constant from the opening on the back side of the main body 10 to the step surface 11c. Note that the horizontal cross-sectional shape of the second through hole 11b is not particularly limited, and although it is circular in this embodiment, it may also be polygonal or elliptical.

[0027] On the other hand, the punching blade 13 surrounding the second through-hole 11b may be any annular blade capable of punching the hydrogel sheet 100 into a desired shape, and its horizontal thickness and other factors are appropriately selected depending on the type of hydrogel sheet 100. The inner surface of the punching blade 13 preferably abuts approximately perpendicularly against the hydrogel sheet 100, and is preferably a single-edged blade. In other words, the punching blade 13 is preferably a single-edged blade with an inclined outer surface.

[0028] Furthermore, the height of the punching blade 13 (second through-hole 11b) is preferably slightly higher than the thickness of the hydrogel sheet 100. The punched hydrogel 100a is contained within the second through-hole 11b. Therefore, if the height of the punching blade 13 is too low, the hydrogel sheet 100 and the step surface 11c may come into contact with each other before the hydrogel sheet 100 is completely punched out of the main body 10 of this embodiment, resulting in incomplete punching of the hydrogel sheet 100. Furthermore, a gap may be generated between the cutting edge of the punching blade 13 and the elastic portion 20. As a result, when cells are cultured on the hydrogel 100a, the liquid medium 60 may easily move between adjacent wells 50, resulting in contamination. On the other hand, if the height of the punching blade 13 is too high, the hydrogel 100a may easily float within the well 50. In contrast, if the height of the punching blade 13 is slightly higher than the thickness of the hydrogel sheet 100, it is possible to completely punch out the hydrogel sheet 100, and furthermore, the cutting edge of the punching blade 13 can be in sufficient contact with the elastic portion 20. Note that, in order to ensure watertightness of the liquid culture medium 60, it is preferable that the punching blade 13 has a height that allows the cutting edge of the punching blade 13 to remain embedded in the elastic portion 20. The structure of the main body portion 10 of this embodiment also makes it possible to prevent the hydrogel 100a from floating up.

[0029] Note that the main body 10 may have a structure other than that described above, as long as it does not impair the object and effect of this embodiment. For example, the main body 10 may have an engaging portion (not shown) for engaging with the housing 30. If the main body 10 has an engaging portion for engaging with the housing 30, after the main body 10 (punching blade 13) and the elastic portion 20 are brought into contact with each other, the main body 10 is pushed back by the repulsive force of the elastic portion 20, which can prevent a gap from being formed between the punching blade 13 and the elastic portion 20. In other words, the main body 10 and the elastic portion 20 can be held in a tightly contacted state. The structure of such an engaging portion is not particularly limited and can be a known structure.

[0030] The shape of the main body 10 in plan view is not particularly limited, and although it is rectangular in this embodiment, it may be circular, elliptical, polygonal, or any other shape.

[0031] The material constituting the main body 10 may be any material that is not swollen or corroded by the liquid medium used for cell culture. For example, it may be a resin, a metal, a ceramic, or a combination thereof. Examples of resins include olefin resins such as polyethylene and polypropylene; ester-based resins such as polyethylene terephthalate; polycarbonate resin; polystyrene; fluorine-based resins such as polytetrafluoroethylene; urethane resin; silicone resin; acrylic resin; and vinyl chloride. Examples of metals include stainless steel and fluorine-resin-treated stainless steel. Examples of ceramics include glass. Among these, resins are preferred from the standpoints of cost and moldability, and polystyrene and polypropylene, which are widely used in general cell culture vessels, are preferred from the standpoints of durability and hardness. The punching blade 13 alone may be made of a different resin, metal, ceramic, or the like. In particular, the punching blade 13 is preferably made of a material that is harder and less flexible than the hydrogel sheet 100 and the elastic portion 20 described below.

[0032] The manufacturing method of the main body 10 is not particularly limited, and the entire structure may be formed as a single unit, for example, by injection molding, or the parts other than the punching blade 13 may be manufactured separately from the punching blade 13 and then combined.

[0033] On the other hand, the elastic portion 20 may be any material that can cover the opening of the second through hole 11b of the main body portion 10 (the opening on the back side of the main body portion 10) without any gaps after punching the hydrogel sheet 100, and that can be in close contact with the cutting edge of the punching blade 13. In this embodiment, the elastic portion 20 is made of a single sheet, but the elastic portion 20 may be divided into multiple pieces so that different elastic portions 20 cover the openings of multiple punching blades 13.

[0034] In addition, in this embodiment, the elastic part 20 is configured to be detachable from the housing 30, but the elastic part 20 may be fixed to the housing 30. Furthermore, if the housing 30 is made of an elastic material, the housing 30 may be used as the elastic part.

[0035] The hardness (Shore hardness A) of the elastic portion 20 is preferably 10° to 80°, and more preferably 20° to 40°. If the hardness of the elastic portion 20 exceeds 80°, the punching blade 13 may be deformed or damaged when it comes into contact with the blade. Furthermore, the elastic portion 20 may not undergo sufficient elastic deformation, making it difficult to form a tight seal, increasing the possibility of leakage of the liquid medium 60 from the gap between the punching blade 13 and the elastic portion 20. On the other hand, if the hardness of the elastic portion 20 is less than 10°, the force required for the punching blade 13 to punch the hydrogel sheet 100 may be absorbed by excessive deformation of the elastic portion 20, or the elastic portion 20 may be cracked or crumbled when it comes into contact with the punching blade 13. The Shore hardness is a value measured using a dedicated device called a durometer in accordance with the new JIS standard (JIS K 6253).

[0036] The thickness of the elastic portion 20 is not particularly limited as long as it is thick enough to allow the elastic portion 20 to be in close contact with the punching blade 13 without any gaps and not be punched by the punching blade 13, and is determined appropriately depending on the material and physical properties of the elastic portion 20 selected.

[0037] The material constituting the elastic portion 20 need only be one that is not swollen or eroded by the liquid used for cell culture and that has the above-mentioned elasticity. Examples include silicone elastomer, ethylene-propylene-diene rubber, acrylonitrile-butadiene rubber, natural rubber, styrene-butadiene rubber, butyl rubber, chloroprene rubber, acrylic rubber, epichlorohydrin rubber, ethylene-vinyl acetate copolymer, polyurethane, etc. Among these, silicone elastomer is preferred from the viewpoints of heat resistance, stability against various liquids, etc.

[0038] Furthermore, the housing 30 of this embodiment has a recess in a substantially rectangular parallelepiped shape, and it is sufficient if the recess can accommodate the elastic portion 20 and the main body portion 10. In this embodiment, the housing 30 has a rectangular parallelepiped shape, but is not limited to this structure.

[0039] The housing 30 may further have an engaging portion (not shown) for fixing the main body 10 at a predetermined position, or an engaging portion (not shown) for fixing the cover. The shape of the engaging portion may be of a known structure.

[0040] The material of the housing 30 is not particularly limited as long as it is not swollen or eroded by the liquid used for cell culture and can support the elastic portion 20 and the main body portion 10. The material of the housing 30 may be resin, metal, ceramic, or glass. These are the same as the materials that can be used for the main body portion 10 described above.

[0041] Furthermore, the cover of this embodiment is not limited to a specific structure as long as it covers the first through-hole 11a of the main body 10 and prevents impurities from entering the well 50. In this embodiment, the cover is configured to be removable from the housing 30, but the cover may be rotatably attached to a part of the housing 30. Furthermore, the cover may be a film or the like that can be peeled off from the main body 10 as needed.

[0042] The cover may be made of any material that is not swollen or corroded by the liquid used for cell culture, such as resin, metal, ceramic, or glass, which are similar to the materials that can be used for the main body 10 described above.

[0043] Here, the cell culture vessel 1 may be distributed in a state including the main body 10, elastic portion 20, and housing 30, or in a state including the cover. Alternatively, for example, it may be distributed in a state including only the main body 10 and elastic portion 20. Furthermore, as shown in FIGS. 1A and 1B, it may be distributed as a cell culture kit including the cell culture vessel 1 and a hydrogel sheet 100.

[0044] (Other embodiments) In the above description, the horizontal cross-sectional shapes of first through hole 11a and second through hole 11b are each circular, and step surface 11c having a circular shape in bottom view is formed at the boundary between first through hole 11a and second through hole 11b. However, the shape of step surface 11c in bottom view does not necessarily have to be a circular shape and can be any shape.

[0045] In the above description, the bottom surface of the well 50, i.e., the surface of the elastic portion 20 facing the main body portion 10, is flat, but the surface of the elastic portion 20 does not necessarily have to be flat. For example, it may be recessed in a hemispherical shape, or in a pyramidal or conical shape.

[0046] Furthermore, when the horizontal cross-sectional area of ​​the first through hole 11a is the same as the horizontal cross-sectional area of ​​the second through hole 11b, after punching out the hydrogel sheet 100, a ring-shaped member or the like may be inserted into the first through hole 11a to prevent the hydrogel 100a from floating up.

[0047] (How to use the cell culture vessel) A method for using the cell culture vessel 1 described above, i.e., a cell culture method, will be described with reference to FIGS. 2A, 2B, and 2C. In this cell culture method, the hydrogel sheet 100 is sandwiched between the punching blade 13 and the elastic portion 20 (hereinafter also referred to as the "sandwiching step"). The punching blade 13 is then pressed toward the elastic portion 20 to punch the hydrogel sheet 100, thereby placing the punched hydrogel 100a in the well 50 (the second through-hole 11b) (hereinafter also referred to as the "storing step"). Thereafter, cells are cultured on the hydrogel 100a (inside the first through-hole 11a) (hereinafter also referred to as the "cell culture step"). The cell culture method of this embodiment may include other steps as long as they do not impair the purpose and effects of this embodiment.

[0048] In the sandwiching step, a hydrogel sheet 100 is prepared. Preferably, the hydrogel sheet 100 is swollen to equilibrium in a liquid medium appropriate for the cells, tissues, bacteria, etc. to be cultured and for the purpose of culture until no change in size occurs. The type of hydrogel sheet is not particularly limited, and it may be a synthetic polymer gel as described in the aforementioned Patent Document 1 (International Publication No. WO 2018 / 151309). Alternatively, it may be a gel sheet derived from natural polymers, such as collagen (types I, II, III, V, XI, etc.), basement membrane components (product name: Matrigel) reconstituted from mouse EHS tumor extract (including type IV collagen, laminin, heparan sulfate proteoglycan, etc.), gelatin, agar, agarose, fibrin, glycosaminoglycan, hyaluronic acid, proteoglycan, etc. The hydrogel sheet may also be derived from polyacrylamide, polydimethylacrylamide, polyvinyl alcohol, methylcellulose, polyethylene oxide, or poly(II-hydroxyethyl methacrylate) / polycaprolactone, or may be a hydrogel sheet made by combining multiple selected from these naturally occurring polymers and synthetic polymers.

[0049] The method for equilibrium swelling of the hydrogel sheet 100 can be the same as known methods, such as immersing the hydrogel sheet 100 in a liquid medium until no size change occurs. The hydrogel sheet 100 may be heated or cooled as needed. The size of the hydrogel sheet 100 used is not particularly limited, and it may be cut to fit the size of the housing 30, for example.

[0050] 2A, the equilibrium-swollen hydrogel sheet 100 is sandwiched between the punching blade 13 and the elastic portion 20. Specifically, the elastic portion 20, the hydrogel sheet 100, and the main body portion 10 are arranged in this order inside the housing 30. Note that when the housing 30 and the elastic portion 20 are integrated together or when the elastic portion 20 is already arranged inside the housing 30, the hydrogel sheet 100 and the main body portion 10 can be arranged on the elastic portion 20. Here, the main body portion 10 is arranged so that the punching blade 13 comes into contact with the hydrogel sheet 100.

[0051] Next, as shown in FIG. 2B , in the above-mentioned accommodation step, the main body 10 (punching blade 13) is pressed toward the elastic portion 20 until the punching blade 13 and the elastic portion 20 come into contact. At this time, an auxiliary pressing tool may be used if necessary. By this pressing, the punching blade 13 punches out the hydrogel sheet 100 into the desired shape, and the punched hydrogel 100a is accommodated in the well 50 (second through-hole 11b). Thereafter, as described above, if the main body 10 and the housing 30 have an engageable structure, the position of the main body 10 may be fixed by this structure.

[0052] Furthermore, when pushing the main body portion 10 toward the elastic portion 20 in this step, the cover may be placed on the main body portion 10 before pushing it in, or the main body portion 10 may be pushed in with the cover removed.

[0053] 2C, cells and, if necessary, a liquid medium 60 are placed in the well 50, and the cells are cultured on the hydrogel 100a (cell culture step). There are no particular limitations on the types of cells that can be cultured in this embodiment, and various types of cells can be cultured.

[0054] (effect) As described above, with the cell culture vessel of the present invention, a hydrogel sheet can be prepared, sandwiched between the main body and the elastic portion, and punched to accommodate a hydrogel of a desired shape inside the well. Furthermore, even if the cell culture vessel has multiple wells, the close contact between the main body (punching blade) and the elastic portion prevents liquid from moving between the multiple wells, making contamination less likely to occur.

[0055] Furthermore, the cell culture vessel of the present invention can be used with any hydrogel sheet. In particular, when a hydrogel sheet that has already reached equilibrium swelling is punched with a punching blade, the size of the hydrogel is less likely to change within the well. This makes it less likely for the sheet to crack or wrinkle on the surface. Another advantage is that there is less chance of gaps forming between the cell culture vessel and the sheet.

[0056] Furthermore, the cell culture vessel of the present invention can be designed so that the hydrogel does not float up when a liquid culture medium is poured in. This prevents damage to the gel and the generation of voids that accompany changes in the size of the hydrogel during the culture period, making it possible to use hydrogels for cell culture that could not previously be used as culture media. [Example]

[0057] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0058] [Example 1] A resin cell culture vessel 1 was prepared, having a main body 10, elastic portion 20, housing 30, and cover (not shown) structured as shown in FIG. 1 . The elastic portion 20 of the cell culture vessel 1 was a 0.8 mm-thick silicone elastomer (KE-109E, manufactured by Shin-Etsu Chemical Co., Ltd., Shore hardness A approximately 25°). A 1.5 mm-thick single-network gel (polystyrene sodium sulfonate gel) was placed between the punching blade 13 of the main body 10 and the elastic portion 20, and the main body 10 was pressed toward the elastic portion 20 at 20 mm / min while applying a load. The load required to cut the high-strength hydrogel was measured using a universal testing machine (RTC-1310A, manufactured by A&D) in compression test mode.

[0059] [result] The load required to cut the high-strength gel was approximately 5 kg. As described above, it was demonstrated that the cell culture vessel of the present invention can cut single-network hydrogels with a very small force, and that it can be used in a clean bench with a very small force.

[0060] [Example 2] A resin cell culture vessel 1 was prepared, having a main body 10, elastic portion 20, housing 30, and cover (not shown) as shown in FIG. 1 . The elastic portion 20 of the cell culture vessel 1 was a 0.8 mm-thick silicone elastomer (KE-109E, manufactured by Shin-Etsu Chemical Co., Ltd., Shore hardness A approximately 25°). A 1.5 mm-thick high-strength hydrogel (a double-network gel composed of poly-2-acrylamido-2-methylpropanesulfonic acid and polydimethylacrylamide) was placed between the punching blade 13 of the main body 10 and the elastic portion 20. The main body 10 was then pressed toward the elastic portion 20 at 20 mm / min while applying a load. The load required to cut the high-strength hydrogel was measured using a universal testing machine (RTC-1310A, manufactured by A&D) in compression test mode.

[0061] [result] The load required to cut the high-strength gel was approximately 25 kg. By using the cell culture vessel of the present invention, it was possible to cut high-strength hydrogels with a relatively small force. As mentioned above, depending on the type of hydrogel, a push-in auxiliary tool may be used when punching with the punching blade.

[0062] [Example 3] A resin cell culture vessel 1 was prepared, having a main body 10, elastic portion 20, housing 30, and cover (not shown) structured as shown in FIG. 1 . The elastic portion 20 of the cell culture vessel 1 was a 0.8 mm-thick silicone elastomer (KE-109E, manufactured by Shin-Etsu Chemical Co., Ltd., Shore hardness A approximately 25°). Then, the punching blade 13 of the main body 10 and the elastic portion 20 were fixed in close contact with each other, and water colored with an artificial coloring agent was poured into each well 50. The water-stopping ability of the punching blade 13 and elastic portion 20 was then confirmed over a long period of time (up to two weeks). The results are shown in FIG. 3A.

[0063] [Comparative Example 1] A cell culture vessel was prepared in the same manner as in Example 2, except that the elastic part 20 was changed to a polyethylene terephthalate (PET) film with a thickness of 0.5 mm. The water-stopping ability of the punching blade and the PET film was observed, and the results after 3 minutes of adding colored water are shown in Figure 3B.

[0064] Comparative Example 2 A cell culture vessel was prepared in the same manner as in Example 2, except that the punching blade 13 of the cell culture vessel 1 was brought into contact with the bottom surface of the polystyrene housing 30 without using the elastic part 20. The water-stopping ability of the punching blade and the housing was then observed, and the results after 3 minutes of adding colored water are shown in Figure 3C.

[0065] [result] As shown in Figure 3A, when silicone elastomer was used as the elastic part, no water leakage was observed even after two weeks (Figure 3A). On the other hand, when PET film was used instead of the elastic part or when the device was in direct contact with the polystyrene housing, water leaked out of all wells within a short period of time (Figures 3B and 3C). [Industrial Applicability]

[0066] The cell culture vessel of the present invention allows a hydrogel sheet to be cut to fit the shape of the well of the cell culture vessel in a simple manner, and the cut hydrogel can be easily placed in the well, making it extremely useful for culturing various cells using hydrogels. [Explanation of symbols]

[0067] 1 cell culture vessel 10 Main body 11 Through hole 11a 1st through hole 11b 2nd through hole 11c Step surface 12 Side wall 13 Punching blade 14 Connecting part 20 Elastic part 30 Case 50 wells 60 Liquid Medium 100 Hydrogel Sheets 100a Hydrogel after punching 110 Cell Culture Kit

Claims

1. A cell culture device having a well for culturing cells on a hydrogel, a main body portion having a through hole extending in a front-to-back direction; an elastic portion disposed on the rear side of the main body portion; and The main body portion is a first through-hole that is a part of the through-hole and opens to the front side of the main body, and an annular side wall portion that constitutes a part of the side wall of the well; a second through-hole that is a part of the through-hole and opens to the back side of the main body, and an annular punching blade that constitutes a part of the side wall of the well; and The well is a region surrounded by an inner circumferential surface of the through hole and the elastic portion when the punching blade and the elastic portion are brought into contact with each other. Cell culture vessel.

2. The hydrogel is contained in the well by punching the equilibrium swollen hydrogel sheet with the punching blade and bringing the punching blade into close contact with the elastic portion. The cell culture vessel according to claim 1 .

3. At a boundary between the first through hole and the second through hole, a horizontal cross-sectional area of ​​the first through hole is smaller than a horizontal cross-sectional area of ​​the second through hole. The cell culture vessel according to claim 1 or 2.

4. The main body portion has a plurality of side wall portions and a plurality of punching blades corresponding to the plurality of side wall portions, respectively. The cell culture vessel according to any one of claims 1 to 3.

5. The elastic portion includes a silicone elastomer. The cell culture vessel according to any one of claims 1 to 4.

6. The main body portion includes a resin. The cell culture vessel according to any one of claims 1 to 5.

7. The cell culture vessel according to any one of claims 1 to 6, A hydrogel sheet; Cell culture kit including:

8. A method for culturing cells using the cell culture vessel according to any one of claims 1 to 6, A step of sandwiching a hydrogel sheet swollen to equilibrium with a liquid medium according to the purpose between the punching blade and the elastic part; a step of inserting the punching blade into the elastic portion to punch the hydrogel sheet, thereby placing the punched hydrogel in the well; Culturing cells on the hydrogel; A cell culture method comprising:

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