Method for manufacturing culture vessel, culture vessel, method for culturing cells, and cell observation method

Abstract

To provide a culture vessel by which cells can be easily observed.SOLUTION: In a method for manufacturing a culture vessel, a culture vessel having a body part which has a through hole, and a sheet stuck on a lower face of the body part so as to close a lower end part of the through hole is manufactured. The method includes the steps of: adding tensile force to a sheet material with a thickness dimension of less than 50 μm; obtaining a first intermediate body in which the sheet material is stuck on a substrate; and sticking a surface of the sheet material side of the first intermediate body to the lower face of the body part.SELECTED DRAWING: Figure 2

Description

[Technical Field]

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

[0002] Conventionally, as disclosed in Patent Document 1, cells have been cultured using a culture vessel having a container for culturing cells. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2022-149254 Summary of the Invention [Problem to be solved by the invention]

[0004] In cell culture using the above-described culture vessel, a user of the culture vessel may observe the cells in the culture vessel using a microscope. In such observation work, it is desirable that the cells be easily observed.

[0005] An object of the present invention is to provide a method for manufacturing a culture vessel that makes it easy to observe cells, a culture vessel, a cell culture method, and a cell observation method. [Means for solving the problem]

[0006] One aspect of the method for producing a culture vessel according to the present invention is to A method for manufacturing a culture vessel having a main body portion having a through-hole and a sheet attached to a lower surface of the main body portion so as to close a lower end of the through-hole, applying tension to a sheet material having a thickness dimension of less than 50 μm; a step of attaching the sheet material to a substrate to obtain an intermediate; and adhering the surface of the intermediate body on the sheet material side to the lower surface of the main body.

[0007] One aspect of the culture vessel according to the present invention is a main body portion having a through hole; a sheet attached to the lower surface of the main body portion so as to close the lower end of the through hole, The thickness dimension of the sheet is less than 50 μm.

[0008] The cell culture method according to the present invention comprises: This is carried out using the culture vessel described above.

[0009] The cell observation method according to the present invention comprises: This is carried out using the culture vessel described above. [Effects of the Invention]

[0010] According to the present invention, a method for manufacturing a culture vessel that allows cells to be easily observed, a culture vessel, a cell culture method, and a cell observation method can be provided. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a perspective view of a culture vessel according to an embodiment. [Figure 2] FIG. 2 is a cross-sectional view taken along the line C1-C1 in FIG. [Figure 3] FIG. 3 is a plan view showing a part of the gas-permeable sheet. [Figure 4] FIG. 4 is a schematic diagram showing a state in which a sheet piece is attached to a flat surface. [Figure 5] FIG. 5 is a flowchart showing the steps of manufacturing the culture vessel. [Figure 6A] FIG. 6A is a schematic diagram showing steps in a method for producing a culture vessel. [Figure 6B] FIG. 6B is a schematic diagram showing steps in a method for producing a culture vessel. [Figure 6C] FIG. 6C is a schematic diagram showing steps in a method for producing a culture vessel. [Figure 6D] FIG. 6D is a schematic diagram showing steps in a method for producing a culture vessel. [Figure 6E]FIG. 6E is a schematic diagram showing steps in a method for producing a culture vessel. [Figure 6F] FIG. 6F is a schematic diagram showing steps in a method for producing a culture vessel. [Figure 6G] FIG. 6G is a schematic diagram showing steps in a method for producing a culture vessel. [Figure 6H] FIG. 6H is a schematic diagram showing steps in the method for producing a culture vessel. [Figure 6I] FIG. 6I is a schematic diagram showing steps in a method for producing a culture vessel. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, a method for manufacturing a culture vessel, a culture vessel, a cell culture method, and a cell observation method according to the present invention will be described in detail with reference to the drawings. Note that the culture vessel described below is an example of the culture vessel according to the present invention, and the present invention is not limited to the embodiments described below.

[0013] [Embodiment] A method for manufacturing a culture vessel, a culture vessel, a cell culture method, and a cell observation method according to embodiments of the present invention will be described with reference to FIGS. 1 to 6I.

[0014] Fig. 1 is a perspective view of a culture vessel 1 according to an embodiment of the present invention. Fig. 2 is a cross-sectional view taken along C1-C1 in Fig. 1, showing a part of the culture vessel 1.

[0015] The culture vessel 1 is used, for example, for culturing cells derived from a human. Specifically, the culture vessel 1 of this embodiment can be suitably used for culturing spheroids (cell aggregates) of target cells. Such a culture vessel 1 has a plurality of storage sections 10 for culturing cells. The culture vessel 1 is housed in the culture space of a culture device (for example, an incubator) with cells to be cultured (hereinafter referred to as "target cells") housed in the storage sections 10.

[0016] The culture vessel 1 does not need to be used while housed in a culture device. The culture vessel 1 may be used in various situations depending on the target cells. The culture vessel 1 of this embodiment is also used when observing the cultured cells.

[0017] The specific configuration of the culture vessel 1 will be described below, followed by a description of a method for manufacturing the culture vessel 1.

[0018] The culture vessel 1 has a main body 2 , a gas permeable sheet 3 , and an adhesive layer 4 .

[0019] 1 and 2 may be used to explain the structure of the culture vessel 1. The Cartesian coordinate system (X, Y, Z) represents the directions of the culture vessel 1 when in use.

[0020] The X direction corresponds to the front-to-rear direction of the culture vessel 1. The positive side of the X direction corresponds to the front side of the culture vessel 1. The negative side of the X direction corresponds to the rear side of the culture vessel 1. In this embodiment, the X direction is also the longitudinal direction of the culture vessel 1. However, the X direction may also be the lateral direction of the culture vessel 1.

[0021] The Y direction corresponds to the left-right direction of the culture vessel 1. The + side of the Y direction corresponds to the left side of the culture vessel 1. The - side of the Y direction corresponds to the right side of the culture vessel 1. In this embodiment, the Y direction is also the short-side direction of the culture vessel 1. However, the Y direction may also be the long-side direction of the culture vessel 1.

[0022] The Z direction corresponds to the up-down direction of the culture vessel 1. The +Z direction side corresponds to the upper side of the culture vessel 1. The -Z direction side corresponds to the lower side of the culture vessel 1.

[0023] Furthermore, the left side, right side, front side, and rear side of the culture vessel 1 differ depending on the direction from which the culture vessel 1 is viewed, and are not limited to the left side, right side, front side, and rear side of the culture vessel 1 shown in Figure 1.

[0024] The main body 2 is a substantially rectangular box. The main body 2 is made of, for example, synthetic resin and is an integrally molded product produced by injection molding. Examples of synthetic resins that make up the main body 2 include polystyrene and polyolefin.

[0025] The polyolefin may be a cyclic olefin (co)polymer, a 4-methyl-1-pentene (co)polymer, polypropylene, or polyethylene. From the viewpoint of gas permeability, a 4-methyl-1-pentene (co)polymer is preferred. From the viewpoint of heat resistance, polystyrene, polypropylene, or a cyclic olefin (co)polymer is preferred.

[0026] The instantaneous value of Shore D hardness of the main body portion 2 is, for example, 60 to 90. The instantaneous value of Shore D hardness is a value obtained by using a durometer (D type) to bring the indenter into contact with a part of the main body portion 2 and then immediately reading the scale (in accordance with ASTM D2240). If the instantaneous value of Shore D hardness is in the range of 60 to 90, the gas permeable sheet can be attached to the main body portion 2 with a good appearance in the step of attaching the gas permeable sheet (the step carried out in step S105 in the flowchart shown in FIG. 5, which will be described later).

[0027] The main body 2 has a plurality of cylindrical portions 21. Each cylindrical portion 21 is cylindrical and opens in the vertical direction. In this embodiment, the shape of the opening of each cylindrical portion 21 (in other words, the outer shape in a plan view) is circular.

[0028] The cylindrical portions 21 are arranged side by side in the left-right and front-rear directions. The number of cylindrical portions 21 corresponds to the number of storage portions 10 for culturing cells. The number of cylindrical portions 21 may be, for example, 6, 24, 96, or 384. Of course, the number of cylindrical portions 21 may be other than 6, 24, 96, and 384. The number of cylindrical portions 21 corresponds to the number of wells in the culture vessel 1.

[0029] Adjacent cylindrical portions 21 are connected to each other by the upper surface 22 and the lower surface 23 of the main body 2. Specifically, the upper ends of adjacent cylindrical portions 21 are connected to each other by the upper surface 22 of the main body 2. Furthermore, the lower ends of adjacent cylindrical portions 21 are connected to each other by the lower surface 23 of the main body 2. The upper surface 22 and the lower surface 23 of the main body 2 are flat surfaces.

[0030] The space defined by the inner peripheral surface of the cylindrical portion 21 (hereinafter referred to as the "internal space of the cylindrical portion 21") constitutes the through-hole 210 of the main body portion 2. The internal space of the cylindrical portion 21 (i.e., the through-hole 210), together with the gas-permeable sheet 3 described below, constitutes the storage portion 10 for culturing cells. The internal space of the cylindrical portion 21 may be cylindrical in shape with an internal diameter that does not change over its entire length, or may be cylindrical in shape with an internal diameter that changes over its entire length (i.e., the internal peripheral surface of the cylindrical portion 21 is inclined). Specifically, the internal space of the cylindrical portion 21 may be truncated cone in shape with an internal diameter that decreases toward the gas-permeable sheet 3.

[0031] The outer shape of the through-hole 210 of the main body 2 (in other words, the outer shape of the opening of the tubular portion 21) may be a circle, a triangle, a rectangle, a pentagon, a hexagon, or any other polygon.

[0032] The outer shape of the through-hole 210 is preferably circular or rectangular. From the viewpoint of improving the adhesive strength between the gas permeable sheet 3 and the periphery of the through-hole 210, the outer shape of the through-hole 210 is more preferably circular. The reason for this is that when the outer shape of the through-hole 210 is circular, the adhesive can be more evenly applied to the adhesive portion between the periphery of the through-hole 210 and the gas permeable sheet 3.

[0033] Furthermore, from the viewpoint of increasing the volume occupied by the through holes 210 (in other words, the capacity of the storage section 10), the outer shape of the through holes 210 is preferably rectangular. For example, when there are a large number of through holes 210 (e.g., 384), if the outer shape of the through holes 210 is circular, the volume of the storage section 10 may become small. In this case, if the outer shape of the through holes 210 is polygonal, such as rectangular, it is easier to increase the volume of the storage section 10 compared to when the outer shape of the through holes 210 is circular.

[0034] Next, the gas permeable sheet 3 will be described with reference to Fig. 2. Fig. 2 shows a cross-sectional view of a portion of the culture vessel 1 that corresponds to one of the cylindrical portions 21.

[0035] The gas permeable sheet 3 is an example of a sheet, and is attached to the lower surface 23 of the main body 2 via an adhesive layer 4. The area of ​​the gas permeable sheet 3 is slightly larger than the area of ​​the lower surface 23 of the main body 2. Such a gas permeable sheet 3 closes the lower end (in other words, the lower opening) of the tubular portion 21 in the main body 2 (in other words, the through hole 210).

[0036] The gas permeable sheet 3 has an oxygen permeability of 1000 mL / m 2 The gas-permeable sheet 3 is a sheet-like member containing, for example, at least one of a polyolefin polymer and a silicone polymer.

[0037] When the gas-permeable sheet 3 contains a polyolefin-based polymer, the polyolefin-based polymer may contain at least one selected from the group consisting of polyethylene, polypropylene, and 4-methyl-1-pentene-based polymer.

[0038] When the gas-permeable sheet 3 is made of a 4-methyl-1-pentene (co)polymer, it is preferable that the main body 2 is also made of a 4-methyl-1-pentene (co)polymer. When the gas-permeable sheet 3 is made of a 4-methyl-1-pentene (co)polymer, it is also preferable that the main body 2 is made of polystyrene.

[0039] The thickness of the gas permeable sheet 3 is uniform or approximately uniform throughout. The thickness of the gas permeable sheet 3 is, for example, less than 50 μm. In this embodiment, the thickness of the gas permeable sheet 3 is 12.5 μm.

[0040] Here, the reason why the thickness of the gas-permeable sheet is set to less than 50 μm will be explained. When the thickness of the sheet material that forms the base of the gas-permeable sheet of the culture vessel is 50 μm or more, wrinkles are less likely to occur in the sheet material. Therefore, if the thickness of the sheet material is 50 μm or more, wrinkles can be prevented from occurring in the sheet material even when the culture vessel is manufactured by a manufacturing method different from the manufacturing method of the culture vessel according to the present embodiment described below (hereinafter referred to as the "other manufacturing method"). In other words, wrinkles are less likely to occur in the gas-permeable sheet of the culture vessel. However, when the thickness of the gas-permeable sheet is less than 50 μm, wrinkles will occur in the gas-permeable sheet when the culture vessel is manufactured by the other manufacturing method. On the other hand, when the thickness of the gas-permeable sheet is less than 50 μm, wrinkles can be prevented from occurring in the gas-permeable sheet when the culture vessel is manufactured by the manufacturing method of the culture vessel according to the present embodiment. For these reasons, the thickness of the gas-permeable sheet 3 of the culture vessel 1 according to the present embodiment is set to less than 50 μm.

[0041] The portion of the gas permeable sheet 3 that closes the lower end of the tubular portion 21 (hereinafter referred to as the closing portion of the gas permeable sheet 3) constitutes the storage portion 10 together with the inner space of the tubular portion 21.

[0042] 3 is a plan view of the closing portion of the gas permeable sheet 3. The plan view is a view showing the shape of the culture vessel 1 viewed from above (+Z direction side) of the culture vessel 1. The plan view also means that the culture vessel 1 is viewed from above (+Z direction side) of the culture vessel 1.

[0043] In this embodiment, the area S of a sheet piece 31 (see Figure 3) cut out from a closed portion of the gas-permeable sheet 3 and the area S' of the sheet piece 31 when the cut-out sheet piece 31 is attached to a flat surface 51 (see Figure 4) without wrinkles satisfy the following relational expression (1).

[0044]

number

[0045] The area S of the sheet piece 31 is the area of ​​the sheet piece 31 in a state where it constitutes a part of the gas permeable sheet 3 (i.e., the state shown in FIG. 3). The area S' of the sheet piece 31 is the area of ​​the sheet piece 31 in a state where the sheet piece 31 is cut out from the gas permeable sheet 3 and attached to a flat surface 51 (i.e., the state shown in FIG. 4). In the state shown in FIG. 4, no wrinkles are generated in the sheet piece 31.

[0046] The area S and the area S′ satisfying the above relational expression (1) means that there are no wrinkles in the closed portion of the gas-permeable sheet 3.

[0047] Also, the mass per unit area of ​​the closed portion of the gas permeable sheet 3 is X mg / cm 2 and the thickness Y μm of the closed portion of the gas-permeable sheet 3 satisfy the following relational expression (2).

[0048]

number

[0049] The mass X and thickness Y satisfying the above relational expression (2) means that no wrinkles exist in the closed portions of the gas-permeable sheet 3.

[0050] 6.84×10 in the above relational expression (2) -2 The constants are values ​​derived by the inventor of the present invention (hereinafter referred to as the inventor). Here, a brief description will be given of how the above constants are derived.

[0051] First, the inventor of the present invention (hereinafter referred to as the present inventor) obtained a culture vessel 1 manufactured by the culture vessel manufacturing method according to the present invention described below. Then, the inventor cut out a sheet piece (not shown) from the closed portion of the gas permeable sheet 3 in the obtained culture vessel 1. Hereinafter, this sheet piece will be referred to as the first sheet piece.

[0052] The weight of the first sheet piece is 47 mg. The area of ​​the first sheet piece in plan view is 56.15 cm. 2The thickness of the first sheet piece is 12.5 μm.

[0053] The inventor then calculated the mass per unit area of ​​the closed portion of the gas-permeable sheet 3 in the culture vessel 1 from the weight and area of ​​the first sheet piece: X mg / cm 2 The calculated mass was X mg / cm 2 is 0.837 mg / cm 3 is.

[0054] The present inventors also produced a culture vessel (not shown) using a manufacturing method different from the manufacturing method of the culture vessel according to the present invention described below. Hereinafter, this culture vessel will be referred to as a reference culture vessel. The present inventors then cut out a sheet piece (not shown) from the closed portion of the gas-permeable sheet of the obtained reference culture vessel. Hereinafter, this sheet piece will be referred to as a reference sheet piece.

[0055] The reference culture vessel manufacturing method differs from the culture vessel manufacturing method according to the present invention in that step S103 (see FIG. 5) in the culture vessel manufacturing method according to the present invention, which will be described later, is not performed.

[0056] The weight of the reference sheet piece is 48 mg. The area of ​​the reference sheet piece in plan view is 56.15 cm. 2 The thickness of the reference sheet piece is 12.5 μm.

[0057] The inventor then calculated the mass per unit area of ​​the closed portion of the gas-permeable sheet for the reference culture vessel from the weight and area of ​​the reference sheet piece: X mg / cm 2 The calculated mass was X mg / cm 2 is 0.855 mg / cm 3 is.

[0058] As a result of the above test, the mass per unit area of ​​the first sheet piece was X mg / cm 2 is the mass per unit area of ​​the reference sheet piece, X mg / cm 2The inventors then calculated the mass per unit area of ​​the reference sheet piece as X mg / cm in order to eliminate the influence of the thickness dimension of the sheet piece. 2 (i.e., 0.855 mg / cm 3 ) divided by the thickness of the reference sheet piece, 12.5 μm (i.e., 6.84 × 10 -2 ) was used as a constant in the above relational expression (2).

[0059] Next, a method for manufacturing the culture vessel 1 of this embodiment will be described with reference to Figures 5 to 6I. Figure 5 is a flowchart showing the steps for manufacturing the culture vessel 1. Figures 6A to 6I are schematic diagrams showing the steps for manufacturing the culture vessel 1.

[0060] The subject of the method for manufacturing the culture vessel 1 described below is an operator. However, the subject of the method for manufacturing the culture vessel 1 may also be, for example, a robot.

[0061] First, in step S101 of Fig. 5, an operator obtains a sheet material 61. As shown in Fig. 6A, the sheet material 61 has a rectangular shape in a plan view. The sheet material 61 is a member that will become the gas permeable sheet 3 in the culture vessel 1 after the manufacturing method of the culture vessel 1 is carried out.

[0062] Such sheet material 61 may be cut out from a rolled sheet. Alternatively, sheet material 61 may be cut out from a sheet larger than sheet material 61. The area of ​​sheet material 61 in a plan view is larger than the area of ​​main body portion 2 in a plan view.

[0063] The sheet material 61 has an oxygen permeability of 1000 mL / m 2 The sheet material 61 is a sheet-like member having a thermal resistance of 1000 psi or more per day atm. The sheet material 61 contains, for example, at least one of a polyolefin polymer and a silicone polymer.

[0064] When the sheet material 61 contains a polyolefin polymer, the polyolefin polymer may contain at least one selected from the group consisting of polyethylene, polypropylene, and 4-methyl-1-pentene polymer.

[0065] Next, in step S102 of Fig. 5, the worker temporarily fixes two of the four corners of the sheet material 61 (the two upper corners in Fig. 6B) to the upper surface 621 of the workbench 62 using fixing means such as tape 63. Note that the fixing means is not limited to tape. The fixing means may be, for example, an adhesive or pins.

[0066] Next, in step S103 of FIG. 5, the worker applies tension to the sheet material 61. The direction of the tension applied to the sheet material 61 is indicated by arrows A1 and A2 in FIG. 6C. The directions indicated by arrows A1 and A2 are parallel to the diagonal directions of the sheet material 61 and are parallel to the center O of the sheet material 61. 61 The direction is away from.

[0067] Then, the worker temporarily fixes two of the four corners of sheet material 61 (the two lower corners in FIG. 6C ) to upper surface 621 of workbench 62 with fixing means such as tape 63. In the state shown in FIG. 6C , tension is applied to sheet material 61 to an extent that does not cause wrinkles in sheet material 61. In other words, in the state shown in FIG. 6C , no wrinkles or almost no wrinkles are generated in sheet material 61.

[0068] The tension applied to the sheet material 61 may be large enough to prevent wrinkles from forming in the sheet material 61. The tension applied to the sheet material 61 may also be large enough to slightly stretch the sheet material 61.

[0069] Furthermore, the direction of the tension applied to the sheet material 61 is not limited to the direction of the tension in this embodiment. The direction of the tension applied to the sheet material 61 may be any direction within the plane of the sheet material 61.

[0070] 5, the worker obtains the release film 64 shown in FIG. 6D. The release film 64 is cut out from a release film (not shown) that is larger than the release film 64.

[0071] The release film 64 includes a film layer 641 and a release layer 642 .

[0072] The film layer 641 is a member that supports the sheet material 61 when the sheet material 61 is attached to the main body part 2 in step S108 described below. The film layer 641 is an example of a substrate, and is in the form of a transparent film.

[0073] Film layer 641 is peelable layer 642 and is made of polyethylene terephthalate (PET). Film layer 641 has an adhesive surface on a first surface. The thickness of film layer 641 is, for example, 30 μm or more and 200 μm or less.

[0074] Release layer 642 is in the form of a transparent film. Release layer 642 is removably attached to the first surface (i.e., the adhesive surface) of film layer 641. The thickness of release layer 642 is, for example, not less than 10 μm and not more than 200 μm.

[0075] Next, in step S105 of FIG. 5, the worker attaches the sheet material 61 to the film layer 641 of the release film 64 as shown in FIG. 6E.

[0076] Specifically, the worker peels off the release layer 642 from the release film 64. Then, the worker attaches the first surface (in other words, the adhesive surface) of the film layer 641 to the upper surface of the sheet material 61 that is temporarily fixed to the upper surface 621 of the workbench 62.

[0077] 6E is a diagram showing a state in which the first surface (in other words, the adhesive surface) of the film layer 641 is attached to the upper surface of the sheet material 61 that is temporarily fixed to the upper surface 621 of the workbench 62. The film layer 641 and the sheet material 61 in the state shown in FIG. 6E constitute a first intermediate 66.

[0078] 5, the worker removes air bubbles present between the film layer 641 and the sheet material 61. Specifically, the worker removes air bubbles present between the film layer 641 and the sheet material 61 using roller 65 shown in FIG.

[0079] 5, the worker cuts the first intermediate 66 to a predetermined size. Specifically, the worker cuts the first intermediate 66 along the portion indicated by the two-dot chain line α in FIG. 6F. The distance between the two-dot chain line α in FIG. 6F and the outer periphery of the film layer 641 in the first intermediate 66 is, for example, 0.1 mm or more and 3 mm or less.

[0080] Then, the worker obtains a second intermediate body 67 (see FIG. 6G). The second intermediate body 67 corresponds to an example of an intermediate body. The second intermediate body 67 is composed of a film layer 641 and a sheet material 61.

[0081] 5, the operator attaches the sheet material 61 of the second intermediate 67 to the first surface of the main body 2. The first surface of the main body 2 corresponds to the lower surface 23 of the main body 2 in the culture vessel 1 shown in FIG.

[0082] 6G, with the sheet material 61 facing downward, the worker aligns the four corners of the second intermediate 67 with the first surface of the main body 2. In this state, the adhesive layer 4 is provided on the first surface of the main body 2.

[0083] 6G, the worker moves second intermediate 67 closer to main body 2, and adheres sheet material 61 to adhesive layer 4. As a result, sheet material 61 is adhered to the first surface of main body 2 via adhesive layer 4.

[0084] Thereafter, the worker presses the sheet material 61 toward the adhesive layer 4 using a roller 65 (see FIG. 6H) to reliably attach the sheet material 61 to the adhesive layer 4 (that is, the main body portion 2).

[0085] During such a bonding operation, the sheet material 61 is supported without any wrinkles by the film layer 641. Therefore, the sheet material 61 is bonded to the main body 2 without any wrinkles.

[0086] 5 is the third intermediate body 68 (see FIG. 6H). The third intermediate body 68 is composed of the main body 2, the sheet material 61, and the film layer 641.

[0087] The method for manufacturing a culture vessel according to this embodiment can also be ended when step S108 in Fig. 5 is completed. In other words, the third intermediate 68 can be considered as a culture vessel manufactured by the method for manufacturing a culture vessel according to this embodiment.

[0088] The third intermediate 68 is distributed in a state of being housed in a container such as a box. In this case, the film layer 641 of the third intermediate 68 functions as a protective layer that protects the sheet material 61.

[0089] Next, in step S109 of Fig. 5, the operator peels off the film layer 641 of the third intermediate 68 from the sheet material 61, as shown in Fig. 6I. As a result, the culture vessel 1 shown in Fig. 1 is obtained. In the culture vessel 1 shown in Fig. 6I, the sheet material 61 constitutes the gas permeable sheet 3.

[0090] (Actions and Effects of This Embodiment) According to the method for manufacturing a culture vessel according to this embodiment as described above, it is possible to obtain a culture vessel 1 in which cells can be easily observed. The reason for this will be explained below.

[0091] First, the method for manufacturing a culture vessel according to this embodiment includes a step of applying tension to the sheet material 61, as shown in step S103 of Fig. 5. Therefore, in the case of a culture vessel 1 manufactured by the method for manufacturing a culture vessel according to this embodiment, wrinkles are less likely to occur in the gas-permeable sheet 3.

[0092] If wrinkles exist in the gas-permeable sheet 3, it would be difficult for a user of the culture vessel 1 to observe the cells in the culture vessel 1 under a microscope. In the case of the culture vessel 1 according to this embodiment, there are no wrinkles in the gas-permeable sheet 3, so that a user of the culture vessel 1 can easily observe the cells in the culture vessel 1 under a microscope.

[0093] In particular, in the case of the culture vessel 1 according to this embodiment, the thickness of the gas permeable sheet 3 is less than 50 μm. If the thickness of the gas permeable sheet 3 is less than 50 μm, wrinkles are likely to occur in the sheet material 61 (see FIG. 6A) that forms the base of the gas permeable sheet 3. For this reason, the method for manufacturing a culture vessel according to this embodiment is more effective when applied to a thin gas permeable sheet 3 with a thickness of less than 50 μm.

[0094] Furthermore, in the case of a thin gas-permeable sheet 3 with a thickness of less than 50 μm, areas with low transparency (in other words, hazy areas) are unlikely to occur.

[0095] If there are areas of low transparency in the gas-permeable sheet 3 (in other words, areas that appear hazy when observed with a phase-contrast microscope), it will be difficult for a user of the culture vessel 1 to distinguish between the cells and the areas of low transparency in the gas-permeable sheet 3 when observing the cells in the culture vessel 1 with a microscope.

[0096] On the other hand, in the case of the culture vessel 1 according to this embodiment, portions with low transparency (in other words, hazy portions when observed with a phase-contrast microscope) are unlikely to occur in the gas-permeable sheet 3, making it easy for the user of the culture vessel 1 to observe the cells in the culture vessel 1. From this perspective as well, the method for manufacturing a culture vessel according to this embodiment can realize a culture vessel 1 in which cells can be easily observed.

[0097] Tests carried out to confirm that the culture vessel 1 according to this embodiment is suitable for cell observation will be described below. Note that the present invention is not limited to the configurations related to the tests below.

[0098] [test] The test was carried out in the following order: culture vessel manufacturing process, cell culture process, cell observation and image acquisition process, and image analysis process. Each process is explained below.

[0099] 1. Culture vessel manufacturing process For this test, the following culture vessels A to C were manufactured. The basic configuration of the culture vessels A to C is the same as the basic configuration of the culture vessel 1 according to the above-described embodiment.

[0100] 1-1.Culture container A A culture vessel A having the following configuration was manufactured by the above-described method for manufacturing a culture vessel according to this embodiment. Number of wells (cylindrical parts 21) in the culture vessel: 24 Inner diameter of the bottom end of the well (cylindrical part 21) of the culture vessel: 15.55 mm Inner diameter of the upper end of the well (cylindrical part 21) of the culture vessel: 16.20 mm Gas permeable sheet material: 4-methyl-1-pentene copolymer (TPX, manufactured by Mitsui Chemicals, Inc.) Gas permeable sheet thickness: 12.5 μm

[0101] 1-2.Culture container B A culture vessel B having the following configuration was manufactured by a manufacturing method different from the manufacturing method of the culture vessel according to the present embodiment described above. The manufacturing method of the culture vessel B is a manufacturing method in which steps S101 and S108 of the manufacturing process of the culture vessel shown in FIG. 5 are performed. Number of wells (cylindrical parts 21) in the culture vessel: 24 Inner diameter of the bottom end of the well (cylindrical part 21) of the culture vessel: 15.55 mm Inner diameter of the upper end of the well (cylindrical part 21) of the culture vessel: 16.20 mm Gas permeable sheet material: 4-methyl-1-pentene copolymer (TPX, manufactured by Mitsui Chemicals, Inc.) Gas permeable sheet thickness: 50 μm

[0102] 1-3.Culture container C A culture vessel C having the following configuration was produced by the same production method as the culture vessel B described above. Number of wells (cylindrical parts 21) in the culture vessel: 24 Inner diameter of the bottom end of the well (cylindrical part 21) of the culture vessel: 15.55 mm Inner diameter of the upper end of the well (cylindrical part 21) of the culture vessel: 16.20 mm Gas permeable sheet material: 4-methyl-1-pentene copolymer (TPX, manufactured by Mitsui Chemicals, Inc.) Gas permeable sheet thickness: 100 μm

[0103] 2. Cell culture process Cell culture was carried out under the following conditions using the above-mentioned culture vessels A to C. The conditions for cell culture carried out in culture vessels A to C were all the same.

[0104] 2.1.Culture conditions Cell:TFK-1 cell Number of cells: 1×10 4 cell / cm 2 Culture medium volume: 0.5mL / well Temperature of the culture environment: 37℃ CO2 concentration in the culture environment: 5% Humidity of the culture environment: 100% Culture period: 4 days

[0105] 3. Cell observation and image acquisition process The results of cell culture carried out under the above conditions were observed and images were acquired under the following conditions. The area of ​​the images acquired was the same for all culture vessels A to C. The microscope and objective lens used were the same for all culture vessels A to C.

[0106] 3.1. Observation and photography conditions Microscope: BZ-X710 (Keyence Corporation) Objective lens: S Plan Fluor ELWD 20X (Nikon Corporation) Exposure time for culture vessels A to C: 1 / 100 s

[0107] 4.Image analysis process Of the images acquired in the above-mentioned cell observation and image acquisition process, the areas where no cells existed were analyzed under the following conditions. The analysis conditions were all the same for culture vessels A to C.

[0108] 3.1.Analysis conditions Image analysis software: ImageJ

[0109] 3.2.Analysis Procedure First, the image to be analyzed (hereinafter referred to as the analysis target image) was opened in ImageJ. Next, ImageJ was used to select the part to be analyzed (hereinafter referred to as the analysis target part) from the analysis target image. Next, ImageJ was used to calculate the average value and standard deviation (StdDev) of the gray value (brightness) of the analysis target part. The standard deviation is the distribution of gray values ​​(brightness) in the analysis target part.

[0110] Table 1 below shows the standard deviations for culture vessels A to C determined using ImageJ.

[0111] [Table 1]

[0112] The lower the standard deviation, the smaller the distribution of gray values ​​(brightness), which is preferable. As shown in Table 1 above, when the material of the gas-permeable sheet is 4-methyl-1-pentene copolymer, the standard deviation of culture vessel A manufactured by the method for manufacturing a culture vessel according to this embodiment is smaller than the standard deviations of culture vessel B and culture vessel C.

[0113] Table 2 below shows the difference in height between the center and outer edge of the gas permeable sheet for culture vessels A to C. As is clear from Table 2 below, culture vessel A manufactured by the culture vessel manufacturing method according to this embodiment has a clearly smaller difference in height between the center and outer edge of the gas permeable sheet than culture vessels B and C.

[0114] [Table 2] [Industrial Applicability]

[0115] The culture vessel manufactured by the method for manufacturing a culture vessel according to the present invention can be applied to the culture of various cells. [Explanation of symbols]

[0116] 1 Culture vessel 10 Storage section 2 Main body 21 Cylindrical part 210 Through hole 22 Top side 23 Bottom side 3 Gas permeable sheet 31 Sheet pieces 4 Adhesive layer 51 Flat surface 61 Sheet Material 62 Workbench 621 Top surface 63 Tape 64 Release film 641 Film Layer 642 Peeling layer 65 Laura 66 First intermediate 67 Second intermediate 68 Third intermediate

Claims

1. A method for manufacturing a culture vessel having a main body portion having a through-hole and a sheet attached to a lower surface of the main body portion so as to close a lower end of the through-hole, applying tension to a sheet material having a thickness dimension of less than 50 μm; a step of attaching the sheet material to a substrate to obtain an intermediate; and adhering the surface of the intermediate body on the sheet material side to the lower surface of the main body. A method for manufacturing a culture vessel.

2. The oxygen permeability of the sheet material is 1000 mL / m ２ ・Day・ATM or more, The method for producing the culture vessel according to claim 1 .

3. the sheet material contains at least one of a polyolefin-based polymer and a silicone-based polymer; The method for producing the culture vessel according to claim 1 .

4. the sheet material contains the polyolefin polymer, The polyolefin polymer contains at least one selected from the group consisting of polyethylene, polypropylene, and a 4-methyl-1-pentene polymer. The method for producing the culture vessel according to claim 3 .

5. a main body portion having a through hole; a sheet attached to the lower surface of the main body portion so as to close the lower end of the through hole, The thickness of the sheet is less than 50 μm. Culture container.

6. The sheet is tensioned in a diagonal direction of the sheet and in a direction away from the center of the sheet. The culture vessel according to claim 5 .

7. The area S of a sheet piece cut out from the sheet; The area S' of the sheet piece when the sheet piece is attached to a flat surface without wrinkles, The relationship: S≧S′ is satisfied. The culture vessel according to claim 5 .

8. Mass per unit area of ​​the sheet X mg / cm ２ and, The thickness of the sheet is Y μm, Relational formula: X / Y<6.84×10 －２ fulfill, The culture vessel according to claim 5 .

9. The oxygen permeability of the sheet is 1000 mL / m ２ ・Day・ATM or more, The culture vessel according to claim 5 .

10. The sheet contains at least one of a polyolefin polymer and a silicone polymer. The culture vessel according to claim 5 .

11. the sheet contains a polyolefin polymer, The polyolefin polymer contains at least one selected from the group consisting of polyethylene, polypropylene, and a 4-methyl-1-pentene polymer. The culture vessel according to claim 9 .

12. A cell culture method using the culture vessel according to any one of claims 6 to 11.

13. A cell observation method using the culture vessel according to any one of claims 6 to 11.

Images