JP2023043525A - Cell culture vessel, cell culture kit, and cell culture method - Google Patents

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

The present invention relates to cell incubators, cell culture kits, and cell culture methods.

When culturing cells or fungi, a special container made of glass or plastic called petri dish or microplate (hereinafter also referred to as a “cell culture vessel”) is generally used. In most of these cell culture vessels, the shape of the portion used for culture is a simple cylindrical shape with a flat bottom surface.

Here, as a cell culture method, solid culture is widely known in which an agar gel medium is placed in a cell culture vessel. In the solid culture, powdered agar and a liquid medium are added to distilled water, dissolved by heating, and the solution is poured into a cell culture vessel before cooling. Then, the medium is obtained by cooling and gelling the solution. In addition, gelling agents used for solid culture, not limited to agar, hardly change in size, and a gap between them and the cell culture vessel is less likely to occur.

Here, the present inventors found a phenomenon that when cancer cells are cultured using a specific synthetic polymer gel as a scaffold, some of them are converted into stem cells and proliferate, and have proposed a method for obtaining cancer stem cells at a high concentration. (For example, Patent Document 1, Non-Patent Documents 1 and 2, etc.). There have been methods to increase cancer stem cells, but they all required expensive equipment and reagents, and it took two to three weeks to obtain a small amount of cancer stem cells. On the other hand, according to the above method, it is possible to obtain the same amount of cancer stem cells as in the conventional method, just by culturing a small amount of cancer cells on a synthetic polymer gel for several days.

WO2018/151309

Jian Ping Gong, “Materials both Tough and Soft”, Science, Vol.344, pp.161-162. 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.

However, the synthetic polymer gel that causes the above phenomenon has a problem that the size is easily changed depending on the liquid properties of the liquid medium used for culture. In addition, there are many gels other than the above synthetic polymer gels whose size is likely to change due to changes in temperature, liquid properties of the liquid medium, and the like.

Here, when a hydrogel whose size is likely to change depending on the liquid properties of the liquid medium or the like (herein, these are collectively referred to as "hydrogel") is placed in a cell culture vessel, the hydrogel swells. In some cases, wrinkles or cracks may occur. In addition, when the hydrogel shrinks, the cells fall between the cell culture vessel and the hydrogel, or the hydrogel floats in the cell culture vessel, causing the cells to wrap around the back side of the hydrogel. This leads to problems in observing the state of cultured cells with a microscope and counting the number of cells.

Therefore, when a swelling-contracting hydrogel is used for cell culture, the hydrogel is immersed in a liquid medium until there is no change in size (herein also referred to as "equilibrium swelling"), and then the shape of the cell culture vessel is changed. and placed in the wells of the cell culture vessel one by one.

Here, if you want to screen many kinds of drugs at once using valuable cells and drugs, you should select a small-capacity 96-well microplate or the like. However, as described above, it cannot be said that the work of cutting the hydrogel according to the shape of the well and inserting it into each of the 96 holes (wells) one by one is very unrealistic.

The present invention provides a cell culture vessel, a cell culture kit, and the cell in which a hydrogel sheet can be cut according to the shape of a well of a cell culture vessel by a simple method, and the hydrogel after cutting can be easily accommodated in the well. An object of the present invention is to provide a cell culture method using an incubator.

The present invention is a cell culture vessel having wells for culturing cells on a hydrogel, comprising a body portion having through holes extending in the front and back directions, and an elastic portion arranged on the back side of the body portion. , wherein the main body includes 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 that constitutes a part of the side wall of the well; and an annular punching blade that is a part of the through hole and includes a second through hole that opens to the back side of the main body and that constitutes a part of the side wall of the well, wherein the well is A cell culture vessel is provided, which is 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.

The present invention also provides a cell culture kit including the above cell culture vessel and hydrogel sheet.

The present invention is a method for culturing cells using the above-described cell culture vessel, comprising a step of sandwiching a hydrogel sheet equilibrium-swollen with a liquid medium according to the purpose between the punching blade and the elastic portion; A cell culture comprising: a step of punching out the hydrogel sheet by pressing a blade toward the elastic portion, and housing the punched hydrogel in the well; and a step of culturing cells on the hydrogel. We also provide a method.

According to the present invention, a hydrogel can be accommodated in a well by a simple method. Provided are a cell culture vessel that can easily accommodate the hydrogel after cutting in the well of the cell, a cell culture kit containing the same, and a cell culture method using the cell culture vessel.

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. . FIG. 2A is a schematic cross-sectional view of the main body and the elastic part before punching out the hydrogel sheet in the cell culture vessel according to one embodiment of the present invention, and FIG. 2B is a schematic cross-sectional view of the main body after punching out the hydrogel sheet. and an elastic portion, and FIG. 2C is a schematic cross-sectional view of the body portion and the elastic portion after adding a liquid medium for cell culture into each well. 3A is the result of the water stop effect test of Example 2, FIG. 3B is the result of the water stop effect test of Comparative Example 1, and FIG. 3C is the result of the water stop effect test of Comparative Example 2. .

The cell culture vessel of the present invention is a container having wells for culturing cells, housing hydrogels in the wells, and culturing cells on the hydrogels. In this specification, a well refers to a concave structure for culturing cells, and is also referred to as a well-shaped structure. As described above, in the conventional cell culture vessel, it was necessary to cut the hydrogel sheet according to the shape of the well and put the cut hydrogel into the well one by one. On the other hand, in the cell culture vessel of the present invention, the hydrogel sheet can be punched according to the shape of the well, and the punched hydrogel can be accommodated in the well as it is. Therefore, even when the number of wells is large, the hydrogel can be accommodated in the wells with a simple operation.

(Configuration of cell incubator)
One embodiment of the cell incubator of the present invention will be described in detail below with reference to FIGS. 1 and 2. FIG. 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 hydrogel sheet 100, and FIG. 1B is a perspective view of the completed cell culture kit 110. FIG. 2A is a schematic cross-sectional view of the body portion 10 and the elastic portion 20 before the hydrogel sheet 100 is punched out, and FIG. 2B is a schematic view of the body portion 10 and the elastic portion 20 after the hydrogel sheet 100 is punched out. FIG. 2C is a schematic cross-sectional view showing a state after adding a liquid medium 60 to a well 50 composed of a punched hydrogel sheet 100 and first through holes 11a. 2A to 2C, the contour lines are partially omitted for the sake of convenience.

The cell culture vessel 1 has a body portion 10 having a through hole 11 extending in the front and back direction, and an elastic portion 20 arranged on the back side of the body portion 10 . The cell incubator 1 of this embodiment further has a housing 30 for housing the main body part 10 and the elastic part 20, and a cover (not shown) for preventing contamination of the inside. However, the cell incubator 1 does not necessarily have the housing 30 and the cover.

In the cell culture vessel 1 of the present embodiment, when the punching blade 13 of the main body 10 and the elastic portion 20 are brought into contact with each other, the area surrounded by the inner peripheral surface of the through-hole 11 and the elastic portion 20 is , well 50. In this embodiment, the main body 10 has a plurality of through holes 11 and punching blades 13 corresponding thereto, but the number of through holes 11 and punching blades 13 is not particularly limited, and may be one, for example. . However, when the number of through-holes 11 and punching blades 13 (the number of wells 50) is large, the effects of the present embodiment are particularly likely to be obtained. Therefore, the number of through-holes 11 and punching blades 13 is preferably two or more. Although the plurality of through-holes 11 may be randomly arranged in the main body 10, it is preferable that the through-holes 11 be arranged regularly from the viewpoint of operability during cell culture. Moreover, at this time, the distance between adjacent through holes 11 is appropriately selected according to the application of the cell culture vessel 1 .

As shown in FIG. 2A, the main body 10 of the cell culture vessel 1 has through holes 11 extending in the front and back directions. The through-holes 11 are a first through-hole 11 a opened on the front side of the body portion 10 and surrounded by the annular side wall portion 12 , and a second through-hole 11 a opened on the back side of the body portion 10 and surrounded by the annular punching blade 13 . 2 through holes 11b.

In the cell culture vessel of the present embodiment, the second through-holes 11b serve as areas for accommodating the punched hydrogels 100a, and the first through-holes 11a serve as areas for culturing cells on the hydrogels 100a.

Here, the shape of the first through hole 11a of the main body portion 10 is not particularly limited, but in the present embodiment, at the boundary between the first through hole 11a and the second through hole 11b, the horizontal direction of the first through hole 11a is The cross-sectional area is preferably set to be smaller than the horizontal cross-sectional area of the second through hole 11b. In the boundary region, 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, it becomes difficult for the hydrogel 100a after punching to enter the first through-hole 11a. expected to be effective. In this embodiment, the opening area of the entire first through-hole 11a is set smaller than the opening area of the second through-hole 11b so that the step surface 11c is formed between the first through-hole 11a and the second through-hole 11b. It is If the step surface 11c exists between the first through-hole 11a and the second through-hole 11b, it becomes easier to prevent the 100a from floating within the well 50. As shown in FIG.

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 step surface 11c. good. For example, at the boundary between the first through-hole 11a and the second through-hole 11b, the first through-hole 11a has a horizontal cross-sectional area smaller than the second through-hole 11b. A plurality of ridges, a plurality of protrusions, or the like projecting into the first through hole 11a may be arranged on the side wall portion 12 surrounding the through hole 11a. In this case, the ridges and projections may be arranged at least on the side wall portion 12 near the boundary between the first through-hole 11a and the second through-hole 11b. Further, the horizontal cross-sectional shape of the first through hole 11a is not particularly limited, and although it is circular in this embodiment, it may be polygonal or elliptical.

On the other hand, the shape of the outer side of the annular surface surrounding the first through hole 11a of the side wall portion 12 is not particularly limited, and in the present embodiment, as shown in FIG. has an integrated shape, but is not limited to that shape. For example, the structure may be such that a plurality of first through holes 11a are formed in a rectangular parallelepiped member.

Moreover, the horizontal cross-sectional area of the second through-hole 11b is appropriately selected according to the desired size of the hydrogel 100a. In addition, in order to suppress wrinkles on the surface of the hydrogel 100a after punching and to prevent unevenness from occurring on the surface, and to prevent a gap from forming between the hydrogel 100a and the punching blade 13, the second through-hole is formed. It is preferable that the horizontal cross-sectional area of the hole 11b is constant from the opening on the back side of the main body 10 to the stepped surface 11c. 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 be polygonal or elliptical.

On the other hand, the punching blade 13 surrounding the second through-hole 11b may be an annular blade capable of punching the hydrogel sheet 100 into a desired shape. is appropriately selected by The inner surface of the punching blade 13 preferably contacts the hydrogel sheet 100 substantially perpendicularly, and is preferably a single-edged blade. In other words, it is preferable that the outer side of the punching blade 13 is an inclined single-edged blade.

Moreover, it is preferable that the height of the punching blade 13 (second through hole 11b) is slightly higher than the thickness of the hydrogel sheet 100 . The punched hydrogel 100a is accommodated in the second through hole 11b. Therefore, if the height of the punching blade 13 is too low, the hydrogel sheet 100 and the stepped surface 11c abut against each other before the hydrogel sheet 100 is completely punched out in the main body 10 of the present embodiment. The punching of the gel sheet 100 tends to be incomplete. Also, a gap is generated between the cutting edge of the punching blade 13 and the elastic portion 20 . As a result, when culturing cells on the hydrogel 100a, the liquid medium 60 tends to move between the adjacent wells 50, easily causing contamination. On the other hand, if the height of the punching blade 13 is too high, the hydrogel 100a tends to float within the well 50 . On the other hand, when the height of the punching blade 13 is slightly higher than the thickness of the hydrogel sheet 100 , the hydrogel sheet 100 can be completely punched out, and the cutting edge of the punching blade 13 is located at the elastic portion 20 . can be sufficiently adhered to. It is preferable that the punching blade 13 has a height that allows the cutting edge of the punching blade 13 to hold the state of biting into the elastic portion 20 in order to ensure the water stoppage of the liquid medium 60 . According to the structure of the main body part 10 of the present embodiment, it is possible to suppress the floating of the hydrogel 100a.

Note that the main body portion 10 may have a structure other than the above as long as the object and effect of the present embodiment are not impaired. For example, the body portion 10 may have an engaging portion (not shown) for engaging with the housing 30 or the like. When the body portion 10 has an engaging portion for engaging with the housing 30 , after the body portion 10 (punching blade 13 ) and the elastic portion 20 are brought into contact with each other, the body portion 10 repels the elastic portion 20 . It is possible to suppress the occurrence of a gap between the punching blade 13 and the elastic portion 20 due to being pushed back by force. That is, it is possible to hold the main body portion 10 and the elastic portion 20 in a state of being in close contact with each other. The structure of such an engaging portion is not particularly limited, and can be a known structure.

Further, 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 the like.

Here, the material constituting the main body 10 may be any material that does not swell or erode with the liquid medium used for cell culture. For example, it may be resin, metal, ceramics, or a combination thereof. Examples of resins include olefin resins such as polyethylene and polypropylene; ester resins such as polyethylene terephthalate; polycarbonate resins; polystyrene; fluorine resins such as polytetrafluoroethylene; etc. are included. Examples of metals include stainless steel, fluororesin-treated stainless steel, and the like. Examples of ceramics include glass and the like. Among these, resins are preferred from the viewpoint of cost and moldability, and polystyrene and polypropylene, which are widely used in general cell culture vessels from the viewpoint of durability and hardness, are preferred. Note that only the punching blade 13 may be made of a different resin, metal, ceramics, or the like. In particular, it is preferable that the punching blade 13 is made of a material that is harder and less flexible than the hydrogel sheet 100 and the elastic portion 20 described later.

The manufacturing method of the body portion 10 is not particularly limited, and for example, the entire structure may be integrally formed by injection molding or the like, and the portions other than the punching blade 13 and the punching blade 13 are separately produced, and these are assembled. May be combined.

On the other hand, after the hydrogel sheet 100 is punched, the elastic part 20 can cover the opening of the second through hole 11b of the main body 10 (opening on the back side of the main body 10) without gaps. Any member may be used as long as it can be brought into close contact with. In the present embodiment, the elastic portion 20 is composed of one sheet, but the elastic portion 20 may be divided into a plurality of pieces so as to cover the openings of the plurality of punching blades 13 with different elastic portions 20. good.

Further, in this embodiment, the elastic portion 20 is detachable from the housing 30 , but the elastic portion 20 may be fixed to the housing 30 . Further, when the housing 30 is made of an elastic material, the housing 30 may be used as the elastic portion.

The hardness (Shore hardness A) of the elastic portion 20 is preferably 10° to 80°, 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 abuts thereon. In addition, the elastic deformation of the elastic portion 20 does not occur sufficiently, and as a result, it becomes difficult for them to adhere to each other. On the other hand, if the hardness of the elastic part 20 is less than 10°, the force required for the punching blade 13 to punch the hydrogel sheet 100 is absorbed by excessive deformation of the elastic part 20, or the punching blade 13 hits it. There is a high possibility that the elastic portion 20 will crack or collapse due to the contact. Shore hardness is a value measured by a method according to the new JIS standard (JIS K 6253) using a special device called a durometer.

The thickness of the elastic portion 20 is not particularly limited as long as the elastic portion 20 can be in close contact with the punching blade 13 without a gap and is not punched by the punching blade 13, and the material and physical properties of the elastic portion 20 are selected. determined accordingly.

Moreover, the material constituting the elastic portion 20 should not be swollen or eroded by the liquid used for cell culture, and should have the elasticity described above. Examples include silicone elastomers, ethylene-propylene-diene rubbers, acrylonitrile-butadiene rubbers, natural rubbers, styrene-butadiene rubbers, butyl rubbers, chloroprene rubbers, acrylic rubbers, epichlorohydrin rubbers, ethylene-vinyl acetate copolymers, polyurethanes, etc. is included. Among these, silicone elastomers are preferable from the viewpoint of heat resistance and stability to various liquids.

In addition, the housing 30 of the present embodiment may have a substantially rectangular parallelepiped concave portion as long as the elastic portion 20 and the main body portion 10 can be accommodated in the concave portion. In this embodiment, the housing 30 has a rectangular parallelepiped shape, but is not limited to this structure.

The housing 30 further has an engaging portion (not shown) for fixing the body portion 10 at a predetermined position and an engaging portion (not shown) for fixing the cover. good too. The shape of the engaging portion can be a known structure.

The material of the housing 30 is not particularly limited as long as it does not swell or erode with the liquid used for cell culture and can support the elastic portion 20 and the body portion 10 . The material of the housing 30 may be resin, metal, ceramics, or glass. These are similar to the materials available for the body portion 10 described above.

Moreover, the cover of the present embodiment is not limited to the structure as long as it can cover the first through hole 11a of the main body 10 and prevent impurities from entering the well 50 . Although the cover is detachable from the housing 30 in this embodiment, the cover may be attached to a part of the housing 30 so as to be rotatable. Also, the cover may be a film or the like that can be peeled off from the main body 10 as necessary.

The material of the cover may be any material that does not swell or erode with the liquid used for cell culture, and may be resin, metal, ceramics, or glass. good too. These are similar to the materials available for the body portion 10 described above.

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

(Other embodiments)
In the above description, the horizontal cross-sectional shapes of the first through-hole 11a and the second through-hole 11b are circular, respectively, and the boundary between the first through-hole 11a and the second through-hole 11b has an annular shape when viewed from the bottom. A stepped surface 11c was formed. However, the bottom view shape of the stepped surface 11c does not necessarily have to be annular, and may be any shape.

In the above description, the bottom surface of the well 50, that is, the surface of the elastic portion 20 on the main body portion 10 side is planar, but the surface of the elastic portion 20 does not necessarily have to be planar. For example, it may be recessed in a hemispherical shape, or may be recessed in a pyramidal or conical shape.

Further, when the horizontal cross-sectional area of the first through-holes 11a and the horizontal cross-sectional area of the second through-holes 11b are the same, after the hydrogel sheet 100 is punched out, the hydrogel 100a is suppressed from floating. , an annular member or the like may be inserted into the first through hole 11a.

(How to use the cell incubator)
A method of using the cell incubator 1 described above, that is, a cell culture method will be described with reference to FIGS. 2A, 2B, and 2C. In the cell culture method, the hydrogel sheet 100 is sandwiched between the punching blade 13 and the elastic portion 20 (hereinafter also referred to as "sandwiching step"). Then, the punching blade 13 is pushed toward the elastic portion 20 to punch out the hydrogel sheet 100, thereby accommodating the punched hydrogel 100a in the well 50 (second through hole 11b) (hereinafter referred to as "accommodating step"). also called). Thereafter, cells are cultured on the hydrogel 100a (inside the first through-holes 11a) (hereinafter also referred to as "cell culture step"). The cell culture method of the present embodiment may have steps other than these as long as the object and effect of the present embodiment are not impaired.

In the sandwiching step, the hydrogel sheet 100 is prepared, and the hydrogel sheet 100 is adjusted to the cells, tissues, bacteria, etc. to be cultured, and is balanced with a liquid medium according to the purpose of culture until the size does not change. It is preferably swollen. The type of the hydrogel sheet is not particularly limited, and may be the synthetic polymer gel described in the above-mentioned Patent Document 1 (International Publication No. 2018/151309). In addition, basement membrane components (product name: matrigel), gelatin, agar, agarose, fibrin, glycosaminoglycan, hyaluronic acid, proteoglycan, etc., and may be a gel sheet derived from a natural polymer. Hydrogel sheets derived from polyacrylamide, polydimethylacrylamide, polyvinyl alcohol, methylcellulose, polyethylene oxide, poly(II-hydroxyethyl methacrylate)/polycaprolactone may also be used. A hydrogel sheet may be obtained by selecting a plurality of them and combining them.

Moreover, the method for equilibrium swelling of the hydrogel sheet 100 can be the same as a known method, for example, a method of immersing the hydrogel sheet 100 in a liquid medium until the size does not change. Moreover, the hydrogel sheet 100 may be heated or cooled as necessary. The size of the hydrogel sheet 100 to be used is not particularly limited, but it may be cut according to the size of the housing 30, for example.

Then, as shown in FIG. 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 body portion 10 are arranged in this order inside the housing 30 . In addition, when the housing 30 and the elastic part 20 are integrated, or when the elastic part 20 is previously arranged in the housing 30, the hydrogel sheet 100 and the main body part 10 are placed on the elastic part 20. should be placed. Here, the body part 10 is arranged so that the punching blade 13 is in contact with the hydrogel sheet 100 .

Subsequently, as shown in FIG. 2B, in the accommodation step, the body portion 10 (punching blade 13) is pushed toward the elastic portion 20 until the punching blade 13 and the elastic portion 20 come into contact with each other. At this time, if necessary, a pressing auxiliary tool may be used. By this pressing, the punching blade 13 punches the hydrogel sheet 100 into a desired shape, and the punched hydrogel 100a is accommodated in the well 50 (the second through hole 11b). After that, as described above, when the body portion 10 and the housing 30 have a structure that allows engagement, the position of the body portion 10 may be fixed by the structure.

Further, when pushing the body portion 10 toward the elastic portion 20 in this step, the body portion 10 may be pushed after placing a cover on the body portion 10, or the body portion 10 may be pushed after removing the cover.

Then, as shown in FIG. 2C, cells and, if necessary, a liquid medium 60 or the like are put into the well 50, and the cells are cultured on the hydrogel 100a (cell culture step). The types of cells that can be cultured in this embodiment are not particularly limited, and various types of cells can be cultured.

(effect)
As described above, according to the cell culture vessel of the present invention, a hydrogel sheet of a desired shape can be formed inside the wells simply by preparing a hydrogel sheet, sandwiching it between the main body portion and the elastic portion, and punching the sheet. can accommodate. In addition, even if the cell culture vessel has a plurality of wells, the body (punching blade) and the elastic portion are in close contact with each other, thereby suppressing the movement of the liquid between the plurality of wells and preventing contamination. is difficult to occur.

Any hydrogel sheet can be used according to the cell culture vessel of the present invention. In particular, when the equilibrium-swollen hydrogel sheet is punched out with a punching blade, the size of the hydrogel is less likely to change in the well. Therefore, cracks in the well and wrinkles on the surface are less likely to occur. Furthermore, there is an advantage that there is little gap between the cell culture vessel and the like.

Furthermore, the cell culture vessel of the present invention can have a structure in which the hydrogel does not float when the liquid medium is added. Therefore, it is possible to suppress the breakage of the gel and the generation of voids due to the change in size of the hydrogel during the culture period, and the hydrogel, which could not be used as a medium until now, can be used for cell culture.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited by these examples.

[Example 1]
A resin-made cell culture vessel 1 having the structure shown in FIG. 1, the main body portion 10, the elastic portion 20, the housing 30, and a cover (not shown) was prepared. The elastic portion 20 of the cell culture vessel 1 was made of a silicone elastomer (KE-109E manufactured by Shin-Etsu Chemical Co., Ltd., Shore hardness A of about 25°) having a thickness of 0.8 mm. Then, a single network gel (sodium polystyrene sulfonate gel) having a thickness of 1.5 mm is placed between the punching blade 13 of the body portion 10 and the elastic portion 20, and the body portion 10 is moved to the elastic portion 20 while applying a load. side at 20 mm/min. At this time, the load required for cutting the high-strength hydrogel was measured in the compression test mode of a universal material testing machine (RTC-1310A manufactured by A&D).

[result]
The load required to cut the high-strength gel was about 5 kg. As described above, if the cell culture vessel of the present invention is used, it is possible to cut a single network hydrogel with a very small force, and it is demonstrated that even a very weak force can be used sufficiently in a clean bench. was done.

[Example 2]
A resin-made cell culture vessel 1 having the structure shown in FIG. 1, the main body portion 10, the elastic portion 20, the housing 30, and a cover (not shown) was prepared. The elastic portion 20 of the cell culture vessel 1 was made of a silicone elastomer (KE-109E manufactured by Shin-Etsu Chemical Co., Ltd., Shore hardness A of about 25°) having a thickness of 0.8 mm. Then, between the punching blade 13 of the main body 10 and the elastic part 20, a high-strength hydrogel (a double network composed of poly-2-acrylamide-2-methylpropanesulfonic acid and polydimethylacrylamide gel) was placed, and the body portion 10 was pushed toward the elastic portion 20 side at 20 mm/min while applying a load. At this time, the load required for cutting the high-strength hydrogel was measured in the compression test mode of a universal material testing machine (RTC-1310A manufactured by A&D).

[result]
The load required to cut the high-strength gel was about 25 kg weight. By using the cell culture vessel of the present invention, it was possible to cut the high-strength hydrogel with a relatively small force. As described above, depending on the type of hydrogel, a pressing aid may be used when punching with a punching blade.

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

[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 having a thickness of 0.5 mm. The water stoppage by the punching blade and the PET film was observed, and the result 3 minutes after adding the colored water is shown in FIG. 3B.

[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 and the bottom surface of the polystyrene housing 30 were brought into contact without using the elastic portion 20 . Then, the water stoppage by the punching blade and the housing was observed, and the result 3 minutes after adding the colored water is shown in FIG. 3C.

[result]
As shown in FIG. 3A, when a silicone elastomer was used as the elastic portion, no water leakage was observed even after two weeks (FIG. 3A). On the other hand, it was confirmed that water leaked out of all the wells in a short time when a PET film was used instead of the elastic part or when the polystyrene housing was directly contacted (Fig. 3B and Fig. 3B). 3C).

According to the cell culture vessel of the present invention, the hydrogel sheet can be cut by a simple method to match the shape of the well of the cell culture vessel, and the hydrogel after cutting can be easily accommodated in the well. Therefore, it is very useful for culturing various cells using hydrogel.

1 cell incubator 10 main body 11 through hole 11a first through hole 11b second through hole 11c stepped surface 12 side wall 13 punching blade 14 connecting part 20 elastic part 30 casing 50 well 60 liquid medium 100 hydrogel sheet 100a after punching Hydrogel 110 cell culture kit

Claims (8)

A cell culture vessel having wells for culturing cells on a hydrogel,
a body portion having through holes extending in the front and back directions;
an elastic portion arranged on the back side of the main body;
has
The main body is
an annular side wall portion that includes a first through hole that is a part of the through hole and opens to the front side of the body portion and that constitutes a part of the side wall of the well;
an annular punching blade that includes a second through hole that is a part of the through hole and opens to the back side of the main body and that constitutes a part of the side wall of the well;
has
The well is 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.
cell incubator. The hydrogel is accommodated in the well by punching out the equilibrium-swollen hydrogel sheet with the punching blade and bringing the punching blade into close contact with the elastic portion.
The cell incubator according to claim 1. At the boundary between the first through-hole and the second through-hole, the horizontal cross-sectional area of the first through-hole is smaller than the horizontal cross-sectional area of the second through-hole,
The cell incubator according to claim 1 or 2. The body portion has a plurality of the side wall portions and a plurality of the punching blades respectively corresponding to the plurality of side wall portions,
The cell culture vessel according to any one of claims 1-3. wherein the elastic portion comprises a silicone elastomer;
The cell culture vessel according to any one of claims 1-4. wherein the main body contains a resin,
The cell culture vessel according to any one of claims 1-5. A cell culture vessel according to any one of claims 1 to 6,
a hydrogel sheet;
A cell culture kit containing A method for culturing cells using the cell incubator according to any one of claims 1 to 6,
A step of sandwiching a hydrogel sheet equilibrium-swollen with a liquid medium according to the purpose between the punching blade and the elastic portion;
A step of pressing the punching blade toward the elastic portion to punch out the hydrogel sheet, thereby accommodating the punched hydrogel in the well;
culturing cells on the hydrogel;
A cell culture method, comprising:

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