Culture container, method for producing culture container, and method for culturing spheroid or adherent cell

Abstract

A culture container having: a frame body that has a through-hole; and a sheet part that has a gas permeation sheet layer and that closes a lower opening portion of the through-hole, wherein the gas permeation sheet layer has a plurality of micro recesses in the upper surface and has an oxygen permeability at 23°C of 3000-100000 cm3 / (m2×24 hr×atm).<sp / > <sp / >

Description

Culture vessel, method for manufacturing a culture vessel, and method for culturing spheroids or adherent cells

[0001] The present invention relates to a culture vessel, a method for manufacturing a culture vessel, and a method for culturing spheroids or adherent cells.

[0002] Conventionally, as disclosed in Patent Document 1, a culture vessel having a plurality of minute recesses formed on the surface is known. The space surrounded by the minute recesses constitutes a microwell. Cells are cultured while being accommodated in the microwell.

[0003] International Publication No. 2008 / 156041

[0004] In the culture vessel as described above, a culture vessel with high culture efficiency is desired.

[0005] An object of the present invention is to provide a culture vessel with high culture efficiency, a method for manufacturing a culture vessel, and a method for culturing spheroids or adherent cells.

[0006] One aspect of the present invention for solving the above problems relates to the culture vessel of the following [1] to

[15] . [1] A frame body having a through-hole, and a sheet portion having a gas permeable sheet layer and closing the lower opening of the through-hole. The gas permeable sheet layer has a plurality of minute recesses on the upper surface, and the oxygen permeability at 23 ° C is 3000 cm ３ / (m ２ ×24 hr×atm) or more and 100000 cm ３ / (m ２[1] The culture vessel is less than or equal to (24hr × atm). [2] The culture vessel according to [1], wherein the plurality of micro recesses are the same shape as each other. [3] The culture vessel according to [1] or [2], wherein the plurality of micro recesses have openings of the same shape as each other. [4] The culture vessel according to any one of [1] to [3], wherein the gas permeable sheet layer comprises at least one resin selected from the group consisting of polyethylene, polypropylene, polyethylene terephthalate, and 4-methyl-1-pentene (co)polymer. [5] The culture vessel according to any one of [1] to [4], wherein the sheet portion has a coating layer provided on the upper surface of the gas permeable sheet layer. [6] The culture vessel according to [5], wherein the coating layer comprises at least one superabsorbent resin selected from the group consisting of poly(vinyl alcohol) (PVA), poly(ethylene glycol) (PEG), PEG-acrylate, poly(vinylpyrrolidone) (PVP), polyethyleneimine (PEI), poly(L-lactide) (PLLA), poly(D-lactide) (PDLA), poly(L-lactide-co-D,L-lactide) (PLDLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PL-co-GA), poly(methyl methacrylate) (PMMA), poly(hydroxyethyl methacrylate) (PHEMA), polymethoxyethyl acrylate (PMEA), and poly(2-methacryloyloxyphosphorylcholine) (MPC). [7] The amount of coating layer attached is 0.1 μg / cm². ２ More than 1000μg / cm ２ The culture vessel described in [5] or [6] below. [8] The culture vessel described in any of [5] to [7], wherein the thickness of the gas permeable sheet layer is 50 μm or more and 1000 μm or less, and the thickness of the coating layer is 0.1 μm or more and 10 μm or less. [9] The culture vessel described in any of [1] to [8], wherein the gas permeable sheet layer has a bubble contact angle in water of 100° or more and 150° or less.

[10] The culture vessel described in any of [5] to [8], wherein the coating layer has a bubble contact angle in water of 25° or more and 100° or less.

[11] Maximum depth of micro recesses (H ｍａｘ ) and the maximum diameter (D) of the opening of the micro-recess. ｍａｘ)(1) satisfies the following formula (1), the culture vessel according to any one of [1] to

[10] .

[12] The maximum depth (H ｍａｘ ) is 50 μm or more and 500 μm or less, and the maximum diameter (D ｍａｘ ) of the opening of the micro recess is 100 μm or more and 1000 μm or less, the culture vessel according to any one of [1] to

[11] .

[13] The number per unit area of the micro recesses on the upper surface of the gas permeable sheet layer is 50 / cm ２ or more and 1000 / cm [[ID=z9]] ２ or less, the culture vessel according to any one of [1] to

[12] .

[14] The occupancy rate per unit area of the portion other than the micro recesses on the upper surface of the gas permeable sheet layer is from 2% to 50%, the culture vessel according to any one of [1] to

[13] .

[15] The minimum thickness (H ｍｉｎ ) is 10 μm or more and 500 μm or less, the culture vessel according to any one of [1] to

[14] .

[0007] Also, one aspect of the present invention for solving the above problems relates to a method for manufacturing a culture vessel according to the following

[16] to

[21] .

[16] A method for manufacturing the culture vessel according to [1], comprising: a step of obtaining a gas permeable sheet layer having micro recesses on the surface by extrusion film forming; a step of attaching the gas permeable sheet layer to the bottom surface of the frame body.

[17] In the step of obtaining the gas permeable sheet layer, a film-like molten resin is pressed against a cooling roll provided with concavo-convex portions on the surface, and while cooling the film-like molten resin, micro recesses are transferred to the surface of the film-like molten resin, the method for manufacturing the culture vessel according to

[16] .

[18] The maximum depth (H ｍａｘ ) and the maximum diameter (D ｍａｘ ) of the opening of the micro recess satisfy the following formula (1), the method for manufacturing the culture vessel according to

[16] or

[17] .

[19] The method for manufacturing a culture vessel according to

[16] to

[18] , wherein the gas permeable sheet layer has a bubble contact angle of 100° or more and 150° or less in water.

[20] The method for manufacturing a culture vessel according to

[16] to

[19] , further comprising the step of providing an adhesive layer on the lower surface of the frame before the bonding step.

[21] The method for manufacturing a culture vessel according to

[16] to

[20] , further comprising the step of applying a coating material to the surface of the gas permeable sheet layer after the bonding step to form a coating layer.

[0008] Furthermore, one aspect of the present invention for solving the above problems relates to the spheroid or adherent cell culture method described in

[22] below.

[22] A method for culturing spheroid or adherent cells using the culture vessel described above.

[0009] According to the present invention, it is possible to provide a culture vessel with high culture efficiency, a method for manufacturing a culture vessel, and a method for culturing spheroids or adherent cells.

[0010] Figure 1 is a perspective view of a culture vessel according to an embodiment. Figure 2 is a plan view of the culture vessel. Figure 3 is C of Figure 2. １ -C １ This is a cross-sectional view. Figure 4 shows X in Figure 2. １ This is an enlarged plan view showing a magnified section of Figure 3. Figure 5 is the X section of Figure 3. ２This is an enlarged cross-sectional view showing a magnified section. Figure 6A is a diagram similar to Figure 4, showing an example of a modified microwell. Figure 6B is a diagram similar to Figure 4, showing an example of a modified microwell. Figure 6C is a diagram similar to Figure 4, showing an example of a modified microwell. Figure 6D is a diagram similar to Figure 4, showing an example of a modified microwell. Figure 6E is a diagram similar to Figure 4, showing an example of a modified microwell. Figure 7 is a flowchart of the manufacturing method of the culture vessel. Figure 8 is a schematic diagram showing the configuration of the manufacturing apparatus for the gas permeable sheet. Figure 9A is a micrograph of the spheroid culture results when evaluated in the evaluation test of the cell culture apparatus. Figure 9B is a micrograph of the spheroid culture results when evaluated in the evaluation test of the cell culture apparatus. Figure 9C is a micrograph of the spheroid culture results when evaluated in the evaluation test of the cell culture apparatus. Figure 9D is a micrograph of the spheroid culture results when evaluated in the evaluation test of the cell culture apparatus. Figure 9E is a micrograph of the spheroid culture results when evaluated in the evaluation test of the cell culture apparatus. Figure 9F is a micrograph of the spheroid culture results evaluated in the cell culture device evaluation test. Figure 9G is a micrograph of the spheroid culture results evaluated in the cell culture device evaluation test. Figure 9H is a micrograph of the spheroid culture results evaluated in the cell culture device evaluation test. Figure 9I is a micrograph of the spheroid culture results evaluated in the cell culture device evaluation test. Figure 9J is a micrograph of the spheroid culture results evaluated in the cell culture device evaluation test. Figure 9K is a micrograph of the spheroid culture results evaluated in the cell culture device evaluation test. Figure 9L is a micrograph of the spheroid culture results evaluated in the cell culture device evaluation test. Figure 9M is a micrograph of the spheroid culture results evaluated in the cell culture device evaluation test. Figure 9N is a micrograph of the spheroid culture results evaluated in the cell culture device evaluation test. Figure 9O is a micrograph of the spheroid culture results evaluated in the cell culture device evaluation test.

[0011] The culture vessel according to the present invention will be described in detail below with reference to the drawings. The culture vessel, method for manufacturing the culture vessel, and method for culturing spheroids or adherent cells described below are examples of the culture vessel, method for manufacturing the culture vessel, and method for culturing spheroids or adherent cells according to the present invention, and the present invention is not limited to the embodiments described later.

[0012] [Embodiments] Referring to Figures 1 to 9, a culture vessel 1, a method for manufacturing the culture vessel 1, and a method for culturing spheroids or adherent cells according to an embodiment of the present invention will be described.

[0013] Figure 1 is a perspective view of the culture vessel 1. Figure 2 is a plan view of the culture vessel 1. The culture vessel 1 is used, for example, to culture cells derived from humans. Such a culture vessel 1 has wells 1a for culturing cells. The wells 1a have a plurality of microwells 7 (see Figure 5).

[0014] Culture vessel 1 is used for culturing cells to be cultured (hereinafter referred to as "target cells"). Cell culture means increasing the number of target cells, growing them, or maintaining them in a living cell state.

[0015] The culture vessel 1, with the cells to be cultured contained in the microwells 7, is placed in the culture space of a culture device (e.g., an incubator). Note that the culture vessel 1 does not need to be placed in a culture device (e.g., an incubator) during the culture process.

[0016] The culture vessel 1 according to this embodiment is used for culturing spheroids (cell aggregates) of target cells. The target cells may include, for example, cells used in cancer research, cells used in regenerative medicine research, cells used in organ models such as the liver or kidney, cells used in models such as blood vessels or the heart, and cells used in immunology research. Such target cells may be appropriately selected depending on the research subject and application field.

[0017] The specific configuration of the culture vessel 1 will be described below. In the following description, unless otherwise specified, when "inside" or "outside" is used, "inside" refers to the radially inner side of the culture vessel 1 and the components constituting the culture vessel 1. "Outside" refers to the radially outer side of the culture vessel 1 and the components constituting the culture vessel 1.

[0018] Furthermore, in the following explanation, the Cartesian coordinate system (X, Y, Z) shown in each figure may be used to describe the structure of the culture vessel 1. The X direction corresponds to the front-to-back direction of the culture vessel 1. The X-direction + side corresponds to the front side of the culture vessel 1. The X-direction - side corresponds to the rear side of the culture vessel 1. Note that the front and rear sides of the culture vessel 1 are not particularly limited.

[0019] Furthermore, the Y direction corresponds to the left-right and width directions of the culture vessel 1. The Y-direction + side corresponds to the left side when viewing the culture vessel 1 from the front. The Y-direction - side corresponds to the right side when viewing the culture vessel 1 from the rear.

[0020] The Z direction corresponds to the vertical and height directions of the culture vessel 1. The Z+ side corresponds to the upper side of the culture vessel 1. The Z- side corresponds to the lower side of the culture vessel 1. Note that the directions of the culture vessel 1 are not limited to the Cartesian coordinate system (X, Y, Z) shown in each figure.

[0021] (Culture vessel) The culture vessel 1 has a frame 2 and a sheet portion 3.

[0022] (Frame 2) Frame 2 is, for example, rectangular and made of synthetic resin. The synthetic resin constituting such frame 2 may be, for example, polystyrene or polyolefin. The polyolefin may be a cyclic olefin (co)polymer, a 4-methyl-1-pentene (co)polymer, polypropylene, or polyethylene.

[0023] From the viewpoint of gas permeability, the synthetic resin constituting the frame 2 is preferably a 4-methyl-1-pentene (co)polymer. Furthermore, from the viewpoint of heat resistance, the synthetic resin constituting the frame 2 is preferably polystyrene, polypropylene, or a cyclic olefin (co)polymer.

[0024] The instantaneous Shore D hardness of the frame 2 is between 60 and 90. The frame 2 has multiple (six in this embodiment) through holes 21. In this embodiment, three through holes 21 are arranged in the front-to-back direction, and two rows are provided in the left-to-right direction.

[0025] The through-hole 21 penetrates the frame 2 in the thickness direction (in other words, the vertical direction). The upper end of the through-hole 21 opens to the upper surface of the frame 2. The lower end of the through-hole 21 opens to the lower surface of the frame 2.

[0026] The through hole 21 has a frustoconical inner surface. The inner diameter of the through hole 21 is d ２１ (See Figure 3) In this case, the inner diameter d ２１ The diameter of the through-hole 21 decreases slightly downwards in the axial direction. That is, the inner circumferential surface of the through-hole 21 is a tapered surface in which the inner diameter changes in the vertical direction. However, the inner diameter of the through-hole 21 may be constant along its entire length in the vertical direction (in other words, the axial direction). In this case, the inner circumferential surface of the through-hole 21 becomes a cylindrical surface in which the inner diameter does not change. Thus, the shape of the through-hole is not limited to the shape of the through-hole 21 in this embodiment.

[0027] The inner surface of the through-hole 21, together with the upper surface of the sheet portion 3 described later, constitutes the well 1a. The well 1a is the space enclosed by the through-hole 21 and the sheet portion 3. The well 1a contains the culture medium, such as culture solution, during cultivation.

[0028] Furthermore, the number of through holes is not limited to six. The number of through holes (i.e., the number of wells) may be between one and five, or seven or more. The number of through holes may be, for example, 24, 96, or 384. Also, the shape of the frame is not limited to the shape of frame 2 described above. The shape of frame 2 may be various shapes having multiple through holes.

[0029] The sheet portion 3 is a rectangular sheet member in plan view. The sheet portion 3 is attached to the lower surface of the frame 2 by an adhesive layer 6 (see Figure 3). The sheet portion 3 closes the lower opening of the through hole 21.

[0030] Note that "plan view" means viewing the culture vessel 1 from above. The shape in the plan view refers to the shape of the culture vessel 1 when viewed from above.

[0031] The sheet portion 3 is preferably transparent from the viewpoint of improving the efficiency of cell observation. Specifically, the total light transmittance of the sheet portion 3, measured in accordance with JIS K 7361-1, is preferably 50% or more and 100% or less, and more preferably 70% or more and 100% or less.

[0032] The sheet portion 3 is composed of multiple layers (two layers in this embodiment) stacked vertically. Specifically, the sheet portion 3 has a gas permeable sheet layer 4 and a coating layer 5.

[0033] The gas permeable sheet layer 4 is a rectangular sheet member in plan view. The outer edge of the gas permeable sheet layer 4 coincides with the outer edge of the sheet portion 3. The gas permeable sheet layer 4 has a plurality of minute recesses 41 on its upper surface (see Figures 4 and 5). Note that the minute recesses 41 are omitted in Figure 3.

[0034] In the gas-permeable sheet layer 4, the space surrounded by the minute recesses 41 constitutes a microwell 7 (see Figure 5). The microwell 7 is a partially spherical space that opens upward.

[0035] On the upper surface of the gas permeable sheet layer 4, the portion other than the minute recess 41 is a flat portion 42 (see Figures 4 and 5). The flat portion 42 is provided so as to surround the minute recess 41. In this embodiment, the flat portion 42 is composed of a plurality of regular hexagonal planes.

[0036] In the gas permeable sheet layer 4, the region in which a plurality of minute recesses 41 (see Figures 4 and 5) are formed is referred to as the first region of the gas permeable sheet layer 4. On the other hand, in the gas permeable sheet layer 4, the region other than the first region (i.e., the region composed of the flat portion 42) constitutes the second region of the gas permeable sheet layer 4.

[0037] Furthermore, the portion of the upper surface of the gas permeable sheet layer 4 other than the minute recesses 41 (i.e., the second region of the gas permeable sheet layer 4) does not have to be planar. For example, the second region of the gas permeable sheet layer 4 may be composed of the leading edges of wall portions (not shown) that separate adjacent minute recesses 41.

[0038] The gas permeable sheet layer 4 has an oxygen permeability coefficient of 200 cm at 23°C. ３ × mm / ( m ２ ×24hr×atm) or more 4000cm ３ × mm / ( m ２ It is preferable that the oxygen permeability of the gas permeable sheet layer 4 is 4500 cm² or less (x 24hr x atm). Also, from a similar viewpoint, the oxygen permeability of the gas permeable sheet layer 4 is 4500 cm² or less. ３ / (m ２ ×24hr×atm) or more 100000cm ３ / (m ２ It is preferable that the value is less than or equal to (×24hr × atm).

[0039] Oxygen permeability was measured in accordance with the Japanese Industrial Standard JIS-K-7126-1 using a differential pressure gas permeability measuring device (Toyo Seiki Seisakusho, MT-C3) at a temperature of 23°C and 0% humidity [cm²]. ３ / (m ２ This is the value measured at [×24hr × atm].

[0040] The measurement sample is prepared by cutting a 90 x 90 mm test piece from the film material that makes up the gas permeable sheet layer 4. The diameter of the measurement area is 70 mm (permeable area is 38.46 cm²). ２ ) When high oxygen permeability is expected, an aluminum mask should be applied to the measurement sample beforehand, and the actual permeable area should be 5.0 cm². ２ It is preferable to do so.

[0041] The higher the oxygen permeability of the gas permeable sheet layer 4, the easier it is to promote cell proliferation and growth within the microwells 7. On the other hand, if the oxygen permeability of the gas permeable sheet layer 4 is moderately low, cell adhesion within the microwells 7 improves, allowing the culture vessel 1 to be used for culturing a wider variety of cells.

[0042] The gas permeable sheet layer 4 has a bubble contact angle of 100° or more and 150° or less in water. The bubble contact angle in water refers to the contact angle formed by bubbles (gas) in contact with the surface of the gas permeable sheet layer 4 when the gas permeable sheet layer 4 is placed in water.

[0043] The material constituting the gas permeable sheet layer 4 includes at least one resin selected from the group consisting of polyethylene, polypropylene, polyethylene terephthalate, and 4-methyl-1-pentene (co)polymers.

[0044] Furthermore, it is preferable that the resin used for the gas permeable sheet layer 4 is a different thermoplastic resin from the thermoplastic resin used for the frame 2. For example, the thermoplastic resin used for the frame 2 can be polystyrene or a cyclic olefin-based (co)polymer, and the resin used for the gas permeable sheet layer 4 can be a 4-methyl-1-pentene-based (co)polymer.

[0045] Furthermore, the thermoplastic resin used as the material for the frame 2 may be a 4-methyl-1-pentene (co)polymer, and the resin used as the material for the gas permeable sheet layer 4 may be a 4-methyl-1-pentene (co)polymer with different comonomer species or their ratios.

[0046] By using different thermoplastic resins for these materials, different properties can be imparted to the frame 2 and the gas permeable sheet layer 4. For example, by imparting rigidity to the frame 2 and oxygen permeability to the gas permeable sheet layer 4, a culture vessel 1 with both rigidity and oxygen permeability can be obtained.

[0047] Maximum thickness T of the gas permeable sheet layer 4 ４ｍａｘ (See Figure 5) The thickness is between 50 μm and 1000 μm. Maximum thickness T of the gas permeable sheet layer 4. ４ｍａｘ The thickness is preferably 100 μm or more and 600 μm or less. Maximum thickness T of the gas permeable sheet layer 4 ４ｍａｘ This is the thickness of the second region (i.e., the flat portion 42) of the gas permeable sheet layer 4.

[0048] Minimum thickness T of the gas permeable sheet layer 4 ４ｍｉｎ(See Figure 5) The thickness is between 10 μm and 500 μm. Minimum thickness T of the gas permeable sheet layer 4 ４ｍｉｎ The minimum thickness T of the gas permeable sheet layer 4 is preferably 30 μm or more and 200 μm or less. ４ｍｉｎ This is the thickness of the gas permeable sheet layer 4 in the central part of the minute recess 41.

[0049] Maximum thickness T of the gas permeable sheet layer 4 ４ｍａｘ and minimum thickness T ４ｍｉｎ By increasing this size, the mechanical strength, shape retention, and moisture resistance of the gas permeable sheet layer 4 (i.e., the sheet portion 3) can be improved.

[0050] On the other hand, the maximum thickness T of the gas permeable sheet layer 4 ４ｍａｘ and minimum thickness T ４ｍｉｎ By reducing this, the oxygen permeability, carbon dioxide permeability, water vapor permeability, shape conformability, lightness, handling, transparency, and moldability of the gas permeable sheet layer 4 (i.e., the sheet portion 3) can be improved.

[0051] Note that the maximum thickness T of the gas permeable sheet layer 4 is ４ｍａｘ and minimum thickness T ４ｍｉｎ This may be determined according to the required characteristics of the gas permeable sheet layer 4 (i.e., the sheet portion 3).

[0052] Each of the multiple micro recesses 41 is a recess with an open upper end. As shown in Figure 4, each of the multiple micro recesses 41 has the same shape as the others. In other words, as shown in Figure 4, each of the multiple micro recesses 41 has the same shape as the others.

[0053] Specifically, each of the multiple micro-recesses 41 has a regular hexagonal shape in plan view. The multiple micro-recesses 41 are arranged in a honeycomb pattern (in other words, a hexagonal lattice pattern). Such a configuration is preferable from the viewpoint of increasing the occupancy rate of the first region in the gas permeable sheet layer 4.

[0054] Here, with reference to Figures 4 and 5, the structure of one micro recess 41 will be described. The micro recess 41 has a partially spherical bottom surface. The depth of the micro recess 41 increases from the outer edge towards the center. The maximum depth of the micro recess 41 is H ４１ｍａｘ(See Figure 5). The maximum diameter of the opening of the minute recess 41 is D ４１ｍａｘ (See Figure 4.)

[0055] Maximum depth H of the micro-recess 41 ４１ｍａｘ and the maximum diameter D of the opening of the minute recess 41 ４１ｍａｘ This means that the following equation (1) is satisfied.

[0056]

[0057] Maximum depth H of the micro-recess 41 ４１ｍａｘ The diameter is between 50 μm and 500 μm. The maximum diameter D of the opening of the minute recess 41. ４１ｍａｘ The particle size is between 100 μm and 1000 μm.

[0058] From the viewpoint of improving the uniformity of the thickness of the coating layer 5 described later and improving the workability when recovering cells from the microwells 7, the maximum depth H of the micro recess 41 is set. ４１ｍａｘ It is preferable that it be small. On the other hand, from the viewpoint of increasing the volume of the microwell 7, the maximum depth H of the micro recess 41 ４１ｍａｘ Larger is preferable.

[0059] On the upper surface of the gas permeable sheet layer 4, the number of minute recesses 41 per unit area is 50 / cm². ２ More than 5000 pieces / cm ２ The following applies:

[0060] The occupancy rate per unit area of ​​the portion of the upper surface of the gas permeable sheet layer 4 other than the minute recess 41 (i.e., the second region of the gas permeable sheet layer 4) is between 2% and 50%.

[0061] The distance L between the minute recesses 41 on the upper surface of the gas permeable sheet layer 4 ４１ (See Figure 4) The distance L is between 1 μm and 50 μm. ４１ This is the width of the flat portion 42 surrounding the minute recess 41.

[0062] Note that the shape of the micro-recesses (in other words, microwells) is not limited to the shape shown in Figure 4. Figures 6A to 6E are similar to Figure 4 and show examples of modified shapes of the micro-recesses (in other words, microwells).

[0063] Figure 6A shows the shape and arrangement of the micro-recesses 41a (in other words, microwells 7a) according to the first example of the modified form. The micro-recesses 41a have a square shape in plan view. The micro-recesses 41a are arranged in a grid pattern aligned in the left-right and front-back directions.

[0064] The configuration of the other minute recesses 41a is substantially the same as that of the minute recess 41 described above. Therefore, the description of the minute recess 41 may be appropriately adapted and used to describe the configuration of the minute recess 41a.

[0065] Figure 6B shows the shape and arrangement of the micro-recesses 41b (in other words, microwells 7b) according to a second example of the modified form. The micro-recesses 41b have a circular shape in plan view. The micro-recesses 41b are arranged in a grid pattern aligned in the left-right and front-back directions.

[0066] The configuration of the other minute recesses 41b is substantially the same as that of the minute recesses 41 described above. Therefore, the description of the minute recesses 41 may be appropriately adapted and used to describe the configuration of the minute recesses 41b.

[0067] Figure 6C shows the shape and arrangement of the micro-recesses 41c (in other words, microwells 7c) according to a third modified example. The micro-recesses 41c have a circular shape in plan view. The micro-recesses 41c are arranged in a staggered pattern.

[0068] The configuration of the other minute recesses 41c is substantially the same as that of the minute recesses 41 described above. Therefore, the description of the minute recesses 41 may be appropriately adapted and used to describe the configuration of the minute recesses 41c.

[0069] Figure 6D shows the shape and arrangement of the micro-recesses 41d (in other words, microwells 7d) according to the fourth modified example. The micro-recesses 41d have an equilateral triangle shape in plan view. The micro-recesses 41d are arranged so that adjacent micro-recesses 41d in the left-right direction are oriented opposite to each other in the front-back direction.

[0070] The configuration of the other minute recesses 41d is substantially the same as that of the minute recesses 41 described above. Therefore, the description of the minute recesses 41 may be appropriately adapted and used to describe the configuration of the minute recesses 41d.

[0071] Figure 6E shows the shape and arrangement of the micro-recesses 41e (in other words, microwells 7e) according to the fifth modified example. The micro-recesses 41e have a regular octagonal shape in plan view. The micro-recesses 41e are arranged in a grid pattern aligned in the left-right and front-back directions.

[0072] The configuration of the other minute recesses 41e is substantially the same as that of the minute recess 41 described above. Therefore, the description of the minute recess 41 may be appropriately adapted and used to describe the configuration of the minute recess 41e.

[0073] Of the embodiments and their respective modifications described above, the shape and arrangement of the micro-recesses 41 in the embodiment, the micro-recesses 41a in the first example of the modifications, and the micro-recesses 41d in the fourth example of the modifications are preferable because they allow the distance between adjacent micro-recesses to be kept constant, thereby increasing the number of micro-recesses that can be placed per unit area.

[0074] On the other hand, in the embodiments and their respective modifications described above, the shape and arrangement of the minute recesses 41b in the second modification, the minute recesses 41c in the third modification, and the minute recesses 41e in the fifth modification result in a slightly smaller number of minute recesses that can be placed per unit area because there are areas where the distance between adjacent minute recesses is greater.

[0075] Furthermore, the shape and arrangement of the micro-recesses are not limited to the micro-recesses 41a to 41e shown in Figures 6A to 6E; other shapes and arrangements can also be adopted.

[0076] (Coating layer 5) As shown in Figure 5, the coating layer 5 is provided on the upper surface of the gas permeable sheet layer 4. The coating layer 5 covers the area on the upper surface of the gas permeable sheet layer 4 that faces the through hole 21 (in other words, the well 1a) of the frame 2 in the vertical direction (hereinafter referred to as the "coated area of ​​the gas permeable sheet layer 4").

[0077] In other words, the coating layer 5 is not provided on the upper surface of the gas permeable sheet layer 4 in areas other than the area of ​​the gas permeable sheet layer 4 to be coated (hereinafter referred to as the "adhesion area of ​​the gas permeable sheet layer 4"). The adhesion area of ​​the gas permeable sheet layer 4 is the part that is attached to the lower surface of the frame 2.

[0078] The coating layer 5 can be used to impart hydrophilicity to the upper surface of the gas permeable sheet layer 4 (in other words, the surface of the microwells 7) to enhance the adhesion of target cells, and as a coating for spheroid formation. From this viewpoint, it is preferable that the coating layer 5 contains a water-absorbing resin or gelatin.

[0079] The material constituting the coating layer 5 includes at least one superabsorbent resin selected from the group consisting of poly(vinyl alcohol) (PVA), poly(ethylene glycol) (PEG), PEG-acrylate, poly(vinylpyrrolidone) (PVP), polyethyleneimine (PEI), poly(L-lactide) (PLLA), poly(D-lactide) (PDLA), poly(L-lactide-co-D,L-lactide) (PLDLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PL-co-GA), poly(methyl methacrylate) (PMMA), poly(hydroxyethyl methacrylate) (PHEMA), polymethoxyethyl acrylate (PMEA), and poly(2-methacryloyloxyphosphorylcholine) (MPC).

[0080] Thickness T of coating layer 5 ５ (See Figure 5) It is preferable that the surface is uniform or nearly uniform throughout.

[0081] The amount of coating layer 5 that adheres is not particularly limited, but when culturing adherent cells, 0.1 μg / cm³ is recommended. ２ More than 800μg / cm ２ Preferably, it is 0.5 μg / cm³. ２ More than 70μg / cm ２ It is more preferable that the following is the case: 1.0 μg / cm³ ２ 60 μg / cm or more ２The following is even more preferable: 80 μg / cm³ when culturing spheroids. ２ More than 1000μg / cm ２ Preferably, it is 85 μg / cm³ ２ 900 μg / cm or more ２ More preferably, the following is 90 μg / cm³ ２ More than 800μg / cm ２ The following is even more preferable:

[0082] The greater the amount of coating layer 5 attached, the more the spheroid-forming ability of cells can be enhanced. The less coating layer 5 attached, the more cell adhesion and oxygen permeability can be enhanced.

[0083] The coating layer 5 has a bubble contact angle in water of 10° to 150°, preferably 20° to 130°, and more preferably 25° to 100°. The bubble contact angle in water refers to the contact angle formed by bubbles (gas) in contact with the surface of the coating layer 5 when the sheet portion 3 is placed in water.

[0084] In this embodiment, the bubble contact angle of the coating layer 5 is smaller than that of the gas permeable sheet layer 4. In other words, the coating layer 5 is more hydrophilic than the gas permeable sheet layer 4. Conversely, the gas permeable sheet layer 4 is less hydrophilic than the coating layer 5.

[0085] If the hydrophilicity of the gas permeable sheet layer 4 is low, and the coating layer 5 is absent, air bubbles may accumulate in the micro-recesses 41 of the gas permeable sheet layer 4. If air bubbles accumulate in the micro-recesses 41, the target cells may not be properly positioned within the micro-recesses 41.

[0086] In this embodiment, since a coating layer 5 with higher hydrophilicity than the gas permeable sheet layer 4 exists on the upper surface of the gas permeable sheet layer 4, air bubbles are less likely to accumulate in the micro-recesses 41 of the gas permeable sheet layer 4. As a result, the target cells are correctly positioned in the micro-recesses 41, improving culture efficiency.

[0087] The configuration of the sheet portion 3 has been described above. The sheet portion 3, having the configuration described above, is fixed to the lower surface of the frame 2 via the adhesive layer 6. As a result, the sheet portion 3 closes the lower surface of the frame 2 (in other words, the lower opening of the through hole 21) and forms the bottom surface of the culture container 1.

[0088] The adhesive layer 6 may be composed of known adhesives such as acrylic, silicone, urethane, and rubber-based adhesives. Of these, acrylic adhesives and silicone adhesives are preferred for the adhesive layer 6.

[0089] Next, the manufacturing method of the culture vessel 1 will be described with reference to Figure 7. Figure 7 is a flowchart showing the steps of the manufacturing method of the culture vessel 1. The manufacturing method shown in Figure 7 is carried out by a worker or a manufacturing device. The main performer may differ for each step. The following description will focus on the case where a worker carries out the manufacturing method.

[0090] First, in step S101 of Figure 7, the worker obtains the frame 2. The structure of the frame 2 is as previously described. The worker manufactures the frame 2, for example, by injection molding. The worker may manufacture the frame 2 using a manufacturing device. Alternatively, the worker may obtain a frame 2 that has been manufactured in advance by someone other than the worker.

[0091] Next, in step S102 of Figure 7, the worker obtains a gas permeable sheet layer. The size of the gas permeable sheet layer obtained in step S102 may differ from the size of the gas permeable sheet layer 4 described above. The configuration of the gas permeable sheet layer obtained in step S102 is the same as the configuration of the gas permeable sheet layer 4 described above, except for the size.

[0092] The gas permeable sheet layer is manufactured by extrusion film molding. The worker may manufacture the gas permeable sheet layer using manufacturing equipment. Alternatively, the worker may obtain a gas permeable sheet layer that has been pre-manufactured by someone other than themselves.

[0093] Here, we will briefly explain the manufacturing method of the gas permeable sheet layer. The gas permeable sheet layer is manufactured by extrusion film molding using an extruder.

[0094] A method for manufacturing a gas permeable sheet layer includes a first step of forming a molten resin into a film to obtain a first material, and a second step of forming minute recesses on the surface of the first material to obtain a gas permeable sheet layer. The first material is an example of a film-shaped molten resin.

[0095] The first material (not shown), which is in the form of a film obtained in the first step, is pressed against a cooling roll (not shown) in the second step. The cooling roll has an uneven surface and functions to cool the first material. As the first material is pressed against the cooling roll and cooled by the cooling roll, the uneven surface of the cooling roll is transferred to its surface. As a result, a gas-permeable sheet layer with minute recesses formed on its surface is obtained.

[0096] Next, in step S103 of Figure 7, the worker applies an adhesive layer 6 to the lower surface of the frame 2 obtained in step S101. The component produced in step S103 is sometimes referred to as the first intermediate (not shown).

[0097] Next, in step S104 of Figure 7, the worker attaches the gas permeable sheet layer to the lower surface of the frame 2 via the adhesive layer 6. In other words, the worker attaches the gas permeable sheet layer to the lower surface of the first intermediate.

[0098] The size of the gas permeable sheet layer attached to the lower surface of the frame 2 in step S104 is the same as the size of the sheet portion 3 described above. The component produced in step S104 is sometimes referred to as the second intermediate.

[0099] Next, in step S105 of Figure 7, the worker applies a coating layer 5 to the upper surface of the gas permeable sheet layer in the second intermediate.

[0100] In step S105, the operator applies a coating material containing a hydrophilic material to the upper surface of the gas permeable sheet layer in the second intermediate. As a result, a sheet portion is obtained in which a coating layer is formed on the upper surface of the gas permeable sheet layer. In this way, the culture container 1 is obtained.

[0101] In step S105, the coating layer is provided only on the upper surface of the gas permeable sheet layer in the second intermediate. In other words, the coating layer is not provided on the lower surface of the gas permeable sheet layer in the second intermediate.

[0102] Next, a method for culturing target cells using the culture vessel 1 prepared by the manufacturing method described above will be explained. The culture vessel 1 can be used to culture spheroids (cell aggregates) of target cells.

[0103] Cell culture can be performed by placing the drug (culture medium) and target cells in each well 1a and then placing them in the culture space of a culture device (incubator) set to a predetermined temperature and other conditions. The target cells are arranged in multiple microwells 7 within well 1a.

[0104] The following describes tests conducted to evaluate the performance of the culture vessel according to the present invention. In the tests, cell culture vessels 1 to 15 according to the examples were prepared, as shown in Table 1. The present invention will be described in detail below based on the examples, but the present invention is not limited to these examples.

[0105]

[0106] The glass transition temperature (Tg) of the materials used below was measured using a dynamic viscoelasticity analyzer (DVA-225, manufactured by IT Measurement Control Co., Ltd.) in tensile mode, at a temperature of -30 to 200°C (heating rate of 3°C / min), at a frequency of 1 Hz, and in an atmospheric environment. The value was determined from the peak of the loss modulus.

[0107] Furthermore, the number-average molecular weight (Mn) of the materials used below was measured by gel permulation chromatography (GPC).

[0108] Specifically, under the following conditions, the number-average molecular weight (Mn) of the polymer dissolved in orthodichlorobenzene was calibrated using a polystyrene standard to obtain the molecular weight: • Apparatus: Gel permeation chromatograph HLC-8321 GPC / HT type (manufactured by Tosoh Corporation) • Data analysis software: Empower3 (manufactured by Waters Inc.) • Detector: Differential refractometer • Series-connected columns: TSKgel GMH6-HT (2 tubes), and TSKgel GM H6-HTL (2 tubes) • Column temperature: 140°C • Flow rate: 1.0 ml / min • Sample concentration: 1.5 mg / ml

[0109] 1. Preparation of Gas Permeable Sheets First, 4-methyl-1-pentene polymer (manufactured by Mitsui Chemicals, Inc., TPX ("TPX" is a registered trademark of the company), molecular weight (Mw) = 428,000, molecular weight distribution (Mw / Mn) = 4.1, Tg = 40°C) was fed into an extruder 8 with a T-die equipped with a full-flight screw (see Figure 8). A molten web 82 was extruded from the T-die 81 of the extruder 8. This molten web was held between 82, a first roll 83, and a second roll 84 (a molded cooling roll) and formed into a sheet-like member (hereinafter referred to as the "first sheet-like member"). At this time, the surface shape of the second roll 84 was transferred to the surface of the first sheet-like member to become the second sheet-like member. Subsequently, the second sheet-like member was cooled by a third roll 85 (a metal cooling roll), and three types of gas permeable sheets with thicknesses of 135 μm, 185 μm, and 250 μm were obtained, with minute recesses formed on the surface.

[0110] In this example, a T-die 81 with an effective lip width of 300 mm was used. An elastic roll with a surface covered in silicone rubber was used as the first roll 83. Four types of metal rolls, each with a hexagonal or circular protrusion formed on its surface, were used as the second roll 84.

[0111] The first metal roll used as the second roll 84 has a hexagonal protrusion on its surface with a height of 85 μm and a side length of 115 μm. The second metal roll used as the second roll 84 has a hexagonal protrusion on its surface with a height of 85 μm and a side length of 202 μm. The third metal roll used as the second roll 84 has a hexagonal protrusion on its surface with a height of 85 μm and a side length of 289 μm. The fourth metal roll used as the second roll 84 has a circular protrusion on its surface with a height of 200 μm and a diameter of 350 μm. In this example, five types of gas permeable sheets were obtained by changing the second roll 84 in the extruder 8 to the four types of second roll 84 described above.

[0112] A gas permeable sheet with a thickness of 135 μm obtained by an extruder 8 equipped with a second roll 84 composed of a first metal roll is referred to as the first gas permeable sheet. The first gas permeable sheet is incorporated into the cell culture vessel 2 shown in Table 1.

[0113] A gas permeable sheet with a thickness of 135 μm obtained by an extruder 8 equipped with a second roll 84 composed of a second metal roll is referred to as the second gas permeable sheet. The second gas permeable sheet is incorporated into the cell culture vessels 3, 6, and 7 shown in Table 1.

[0114] A gas permeable sheet with a thickness of 135 μm obtained by an extruder 8 equipped with a second roll 84 composed of a third metal roll is referred to as the third gas permeable sheet. The third gas permeable sheet is incorporated into cell culture vessels 1, 4, 5, and 8 in Table 1.

[0115] A gas permeable sheet with a thickness of 185 μm obtained by an extruder 8 equipped with a second roll 84 composed of a first metal roll is referred to as the fourth gas permeable sheet. The fourth gas permeable sheet is incorporated into the cell culture vessels 9, 10, and 11 shown in Table 1.

[0116] A gas permeable sheet with a thickness of 250 μm obtained by an extruder 8 equipped with a second roll 84 composed of a fourth metal roll is referred to as the fifth gas permeable sheet. The fifth gas permeable sheet is incorporated into the cell culture vessels 12, 13, 14, and 15 in Table 1.

[0117] During molding, the temperature of the second roll 84 was set to 90°C. The temperature of the third roll 85 (sometimes referred to as the "cooling temperature") was set to 35°C. The extrusion temperature was set to 280°C. The extrusion temperature can be considered as the temperature of the molten web 82 extruded from the T-die 81. The take-up speed was set to 6 m / s. Microscopic observation of the surface of the finished film confirmed that the transfer rate was 99% or higher.

[0118] 2. Thickness Measurement of Gas Permeable Sheet The thickness of the gas permeable sheet was measured at five points on the sheet using a digital micrometer (Mitutoyo Corporation, MDC-25MXT) and the average thickness obtained was used.

[0119] 3. Observation of microscopic recesses in the gas permeable sheet (1) Shape of microscopic recesses Observation using a microscope confirmed that the shape of the microwell openings was hexagonal or circular, and that this matched the shape of the roll used.

[0120] (2) Number of microscopic recesses per unit area per well (number of recesses / cm²) ２ From the third gas-permeable sheet, the number of micro-indentations per unit area per well (in other words, microwells) was 444 / cm². ２ A substrate 1 was prepared. Furthermore, from the first gas permeable sheet, the number of micro-recesses (in other words, microwells) per unit area of ​​each well was 2618 / cm². ２ A substrate 2 was prepared. Furthermore, from the second gas permeable sheet, the number of micro-recesses (in other words, microwells) per unit area of ​​each well was 891 / cm². ２ A substrate 3 was prepared. Furthermore, from the fourth gas permeable sheet, the number of micro-recesses (in other words, microwells) per unit area of ​​each well was 444 / cm². ２A base material 4 was prepared. Furthermore, from the fifth gas permeable sheet, the number of micro-recesses (in other words, microwells) per unit area of ​​each well was 640 / cm². ２ A base material 5 was prepared.

[0121] The number of microscopic recesses (in other words, microwells) per unit area in each well can be calculated using the following formula (2).

[0122] The area of ​​one micro-recess is also the area of ​​one microwell. The area of ​​one microwell refers to the area of ​​one microwell in a plan view. For substrates 1 to 4, the area of ​​one microwell was calculated by measuring the length of one side of the regular hexagon at the opening of the microwell using a microscope and then using the formula for calculating the area of ​​a regular hexagon. For substrate 5, the diameter of the microwell was calculated using a microscope and then used the formula for calculating the area of ​​a circle.

[0123] Furthermore, a minute planar portion refers to the area surrounding a single minute recess (see minute recess 41 in Figures 4 and 5) on the planar portion (see planar portion 42 in Figures 4 and 5) on the upper surface of the gas permeable sheet layer (the area with the oblique grid in Figure 4). In this example, the minute planar portion is a regular hexagon or a circle in plan view.

[0124] Substrates 1 to 5 comprise the frame 2 and gas permeable sheet layer 4 (in other words, the components other than the coating layer 5) of the culture vessel 1 described above. Substrate 1 was used as the substrate for cell culture vessels 1, 4, 5, and 8 according to the example. Substrate 2 was used as the substrate for cell culture vessel 2 according to the example. Substrate 3 was used as the substrate for cell culture vessels 3, 6, and 7 according to the example. Substrate 4 was used as the substrate for cell culture vessels 9 to 11 according to the example. Substrate 5 was used as the substrate for cell culture vessels 12 to 15 according to the example.

[0125] (3) Occupancy rate of micro-planar areas per unit area of ​​the gas permeable sheet (%) For cell culture vessels 1 to 15, the occupancy rate of micro-planar areas per unit area (hereinafter referred to as the occupancy rate of planar areas) was determined using the following formula (3).

[0126]

[0127] The occupancy rates of the micro-flat surfaces for cell culture vessels 1-15 are as follows: • Cell culture vessels 1, 4, 5, 8: Micro-flat surface occupancy rate = 3.9% • Cell culture vessel 2: Micro-flat surface occupancy rate = 9.3% • Cell culture vessels 3, 6, 7, 9, 10, 11: Micro-flat surface occupancy rate = 5.5% • Cell culture vessels 12, 13, 14, 15: Micro-flat surface occupancy rate = 38.6%

[0128] Next, a 24-well culture plate (cell culture vessel) was prepared by attaching a gas-permeable sheet obtained by the above manufacturing method to the bottom surface of a 24-well container frame (frame body) made of polystyrene (hereinafter simply referred to as "PS"), which has a glass transition temperature of 94°C, via an acrylic adhesive (adhesive layer).

[0129] Next, a culture plate was placed inside the chamber of an atmospheric pressure plasma surface treatment device (manufactured by Sekisui Chemical Co., Ltd.), the chamber was filled with a nitrogen gas flow, and the culture plate was plasma-treated (irradiation time 2 seconds, output 10V, distance between the bottom of the container and the torch 2 mm).

[0130] Subsequently, the oxygen permeability and oxygen permeability coefficient of the gas permeable sheet were measured using a differential pressure type gas permeability measuring device (Toyo Seiki Seisakusho Co., Ltd., BT-3MT-C3) under conditions of 23°C and 0% RH. The measurement section diameter was 70 mm (permeability area was 38.47 cm²). ２ ) was used. Since a large oxygen permeability coefficient was expected, an aluminum mask was applied to the sample beforehand, and the actual permeability area was set to 5.0 cm². ２ That's what I decided.

[0131] 4. Formation of the coating layer 4-1. Types of coating materials The following materials were prepared as coating materials: ・pHEMA: Poly(2-hydroxyethyl methacrylate) (manufactured by Sigma-Aldrich Japan) ・MPC: Poly(2-methacryloyloxyphosphorylcholine) (manufactured by NOF Corporation, brand names: CM-5206, CM-1102) ・PMEA: Poly(2-methoxyethyl acrylate) (manufactured by Polymer Source Inc.)

[0132] 4-2. Preparation of coating material (1) Coating material 1 1 g of poly(2-hydroxyethyl methacrylate) (pHEMA) (manufactured by Sigma-Aldrich Japan) was dissolved in a mixed solution of 10 mL of ethanol and 10 mL of sterile water for injection and stirred with a stirrer for 1 hour. Then, it was diluted with ethanol and water (volume ratio 1:1) and sterilized by filtration to prepare the coating material. At this time, coating material 1 was obtained by changing the concentration of pHEMA after dilution.

[0133] (2) Coating material 2 In addition, 1 g of poly(2-methacryloyloxyphosphorylcholine) (manufactured by NOF Corporation) CM-5206 was weighed and dissolved in 10 mL of ethanol and stirred with a stirrer for 1 hour. Then, 10 mL of water was added and stirred for 1 hour. After that, it was diluted with ethanol and water (volume ratio 1:1) and filtered and sterilized to obtain coating material 2.

[0134] (3) Coating material 3 In addition, 1 g of poly(2-methacryloyloxyphosphorylcholine) (manufactured by NOF Corporation) CM-1102 was weighed and dissolved in 10 mL of ethanol and stirred with a stirrer for 1 hour. Then, 10 mL of water was added and stirred for 1 hour. After that, it was diluted with ethanol and water (volume ratio 1:1) and filtered and sterilized to obtain coating material 3.

[0135] (4) Coating material 4 Poly(2-hydroxyethyl methacrylate) (pHEMA) (manufactured by Sigma-Aldrich Japan) 1 g was dissolved in a mixed solution of 10 mL of ethanol and 10 mL of sterile water for injection and stirred with a stirrer for 1 hour. Then, the mixture was diluted with ethanol and water (volume ratio 1:1) and filtered to prepare the coating material. Coating material 4 was obtained by changing the concentration of pHEMA after dilution. Note that coating material 1 and coating material 4 differ only in the amount of adhesion, as shown in Table 1.

[0136] (5) Coating material 5 Poly(2-methoxyethyl acrylate) (PMEA) (manufactured by Polymer Source Inc.) 5 mg was dissolved in a mixed solution of 10 mL of ethanol and 10 mL of sterile water for injection and stirred with a stirrer for 1 hour. Then, the mixture was diluted with ethanol and water (volume ratio 1:1), filtered and sterilized to prepare the coating material. Coating material 5 was obtained by changing the concentration of PMEA after dilution.

[0137] 5. Placing the Coating Layers Coating materials 1 to 5 were applied to each pore of the sterilized cell culture vessel using a pipette, with a coating volume of 950 μl each. The vessels were then dried overnight at room temperature to obtain cell culture vessels 1 to 7 and 9 to 14 with the coating layers. Note that cell culture vessels 8 and 15 in the example do not have a coating layer.

[0138] Cell culture vessels 1 to 15 are cell culture vessels according to the example. Coating material 1 was used as the coating material for cell culture vessels 4, 10, and 13 according to the example. Coating material 2 was used as the coating material for cell culture vessel 5 according to the example. Coating material 3 was used as the coating material for cell culture vessels 1 to 3, 9, and 12 according to the example. Coating material 4 was used as the coating material for cell culture vessel 6 according to the example. Coating material 5 was used as the coating material for cell culture vessels 7, 11, and 14 according to the example.

[0139] 6. Terminal sterilization: Each of the cell culture vessels 1 to 15 obtained was packed into a culture plate gamma-ray resistant bag and sterilized by irradiation with gamma rays at 10 kGy to 40 kGy.

[0140] 7. Measurement and Evaluation 7-1. Bubble Contact Angle in Water The bubble contact angle in water of the coating layer surface before and after terminal sterilization, and the bubble contact angle in water of the gas permeable sheet surface were evaluated using a fully automated contact angle analyzer (KRUSS, Inc., device name: DSA30). The value 1 second after the bubble made contact was defined as the bubble contact angle. As shown in Table 1, cell culture vessels 8 and 15 do not have a coating layer, so evaluation of the bubble contact angle in water was not performed for them.

[0141] 7-2. Water Contact Angle The water contact angle of the obtained coating layer surface and the water contact angle of the gas permeable sheet were measured in accordance with JIS R 3257:1999. Under constant temperature and humidity conditions of 25±5℃ and 50±10%RH, 1.8 μL of distilled water droplets were dropped onto the above surface, and the angle of the contact interface between the surface and the water droplet within 1 minute from dropping was measured with a contact angle meter (Kyowa Interface Science Co., Ltd., A-XE type). The measurement position was the center of the well, and the average value obtained from measurements at three wells was taken as the water contact angle. The water contact angle was measured immediately after the coating layer was formed. As shown in Table 1, cell culture vessels 8 and 15 do not have a coating layer, so the water contact angle was not measured for them.

[0142] 7-3. Oxygen Permeability and Oxygen Permeability Coefficient The oxygen permeability and oxygen permeability coefficient of a gas permeable sheet after terminal sterilization were measured using a differential pressure type gas permeability measuring device (Toyo Seiki Seisakusho Co., Ltd., BT-3MT-C3) under conditions of 23°C and 0% RH. The diameter of the measurement section was 70 mm (permeable area was 38.47 cm²). ２ ) was used. Since a large oxygen permeability coefficient was expected, an aluminum mask was applied to the sample beforehand, and the actual permeability area was set to 5.0 cm². ２ That's what I decided.

[0143] 8. Formation of Spheroids or Adherent Cells 8-1. Cell Culture Process Cell culture was carried out using the cell culture vessels 1 to 15 described above under the following conditions. Then, it was evaluated whether spheroids and adherent cells were cultured in cell culture vessels 1 to 15. The cell culture conditions were the same for all cell culture vessels 1 to 15.

[0144] When adding culture medium to microwells, air bubbles can sometimes become trapped within the microwells. In this case, the cell culture vessel was pre-treated by pre-wetting with an ethanol aqueous solution or PBS (phosphate-buffered saline). After that, the cell culture vessel was washed with water or PBS, and the culture medium was added to the microwells.

[0145] (Culture conditions) Cells: TFK-1 cells Cell number: 1 x 10^5 cells / cm ２Culture medium volume: 2.0 mL / well; Culture environment temperature: 37°C; Culture environment CO2 concentration: 5%; Culture environment humidity: 100%; Culture period: 2 days

[0146] 8-2. Cell Observation and Image Acquisition Process The results of cell culture performed under the culture conditions described above were observed and images were acquired under the following conditions. For cell culture vessels 1 to 15, the area of ​​the acquired images was the same for all of them.

[0147] (Observation and imaging conditions) Microscope: BZ-X710 (manufactured by Keyence Corporation) Objective lens: S Plan Fluor ELWD 4X (manufactured by Nikon Corporation) Observation method: Phase contrast observation

[0148] 8-3. Evaluation of Spheroid and Adherent Cell Culture Cell Culture Vessels 1-15 were removed from the incubator, and the cells in the microwells were observed under a microscope. The results of the cell culture were evaluated according to the following criteria. Micrographs of cells cultured in the cell culture vessels for two days, observed under a phase-contrast microscope, are shown in Figures 9A-9O.

[0149] Figure 9A is a micrograph of cell culture vessel 1. Figure 9B is a micrograph of cell culture vessel 2. Figure 9C is a micrograph of cell culture vessel 3. Figure 9D is a micrograph of cell culture vessel 4. Figure 9E is a micrograph of cell culture vessel 5. Figure 9F is a micrograph of cell culture vessel 6. Figure 9G is a micrograph of cell culture vessel 7. Figure 9H is a micrograph of cell culture vessel 8. Figure 9I is a micrograph of cell culture vessel 9. Figure 9J is a micrograph of cell culture vessel 10. Figure 9K is a micrograph of cell culture vessel 11. Figure 9L is a micrograph of cell culture vessel 12. Figure 9M is a micrograph of cell culture vessel 13. Figure 9N is a micrograph of cell culture vessel 14. Figure 9O is a micrograph of cell culture vessel 15.

[0150] (Evaluation Criteria) A1: Circular spheroids were observed, and the spheroids moved when the cell culture vessel was gently shaken from side to side by hand. A2: Adherent cells were not attached to the rim of the microwells, but were attached within the microwells, and the adherent cells remained attached to the microwell surface and did not move when the cell culture vessel was gently shaken from side to side by hand. B1: Some non-circular spheroids (cell aggregates) were also observed. B2: There were areas where adherent cells were unevenly attached to the rims and flat areas of the microwells. C1: Most spheroids (cell aggregates) were not circular. C2: Adherent cells were unevenly attached overall.

[0151] The materials and evaluation results for the cell culture vessels are shown in Table 1 above. A rating of B2 or higher in the above evaluation criteria was considered acceptable, based on suitability for culturing spheroids or adherent cells. In Table 1, the unit for oxygen permeability coefficient is cm. ３ × mm / ( m ２ The formula is (×24hr×atm), and the unit of oxygen permeability is cm². ３ / (m ２ (×24hr×atm)

[0152] As is clear from Table 1 above, all of the cell culture vessels 1 to 15 in the examples were able to successfully culture spheroids or adherent cells.

[0153] All disclosures of the specifications, drawings, and abstracts contained in the Japanese applications 2024-227601, filed on 24 December 2024, and 2025-125633, filed on 28 July 2025, are incorporated herein by reference.

[0154] The culture vessel according to the present invention can be applied to the culture of various cells.

[0155] 1 Culture vessel 1a Well 2 Frame 21 Through hole 3 Sheet section 4 Gas permeable sheet layer 41, 41a, 41b, 41c, 41d, 41e Micro recess 42 Flat section 5 Coating layer 6 Adhesive layer 7, 7a, 7b, 7c, 7d, 7e Microwell 8 Extruder 81 T-die 82 Melt web 83 First roll 84 Second roll 85 Third roll

Claims

1. It comprises a frame having through holes, and a sheet portion having a gas permeable sheet layer that closes the lower opening of the through holes, wherein the gas permeable sheet layer has a plurality of minute recesses on its upper surface, and the oxygen permeability at 23°C is 3000 cm². ３ / (m ２ ×24hr×atm) or more 100000cm ３ / (m ２ A culture vessel with a temperature of 24 hours × atm or less.

2. The culture vessel according to claim 1, wherein the plurality of micro-recesses are of the same shape as each other.

3. The culture vessel according to claim 1, wherein the multiple micro-recesses have the same shape as the openings.

4. The culture vessel according to claim 1, wherein the gas permeable sheet layer comprises at least one resin selected from the group consisting of polyethylene, polypropylene, polyethylene terephthalate, and 4-methyl-1-pentene (co)polymer.

5. The culture container according to claim 1, wherein the sheet portion has a coating layer provided on the upper surface of the gas permeable sheet layer.

6. The culture vessel according to claim 5, wherein the coating layer comprises at least one superabsorbent resin selected from the group consisting of poly(vinyl alcohol) (PVA), poly(ethylene glycol) (PEG), PEG-acrylate, poly(vinylpyrrolidone) (PVP), polyethyleneimine (PEI), poly(L-lactide) (PLLA), poly(D-lactide) (PDLA), poly(L-lactide-co-D,L-lactide) (PLDLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PL-co-GA), poly(methyl methacrylate) (PMMA), poly(hydroxyethyl methacrylate) (PHEMA), polymethoxyethyl acrylate (PMEA), and poly(2-methacryloyloxyphosphorylcholine) (MPC).

7. The amount of the coating layer is 0.1 μg / cm³. ２ More than 1000μg / cm ２ The culture vessel according to claim 5, which is as follows:

8. The culture vessel according to claim 5, wherein the thickness of the gas permeable sheet layer is 50 μm or more and 1000 μm or less, and the thickness of the coating layer is 0.1 μm or more and 10 μm or less.

9. The culture vessel according to claim 1, wherein the gas permeable sheet layer has a bubble contact angle of 100° or more and 150° or less in water.

10. The culture vessel according to claim 5, wherein the coating layer has a bubble contact angle of 25° or more and 100° or less in water.

11. Maximum depth of the minute recess (H ｍａｘ ) and the maximum diameter (D) of the opening of the minute recess. ｍａｘ The culture vessel according to claim 1, wherein the following formula (1) is satisfied.

12. The maximum depth (H ｍａｘ ) of the micro recess is 50 μm or more and 500 μm or less, and the maximum diameter (D ｍａｘ ) of the opening of the micro recess is 100 μm or more and 1000 μm or less. The culture vessel according to claim 1.

13. The number of micro recesses per unit area on the upper surface of the gas permeable sheet layer is 50 / cm². ２ More than 1000 pieces / cm ２ The culture vessel according to claim 1, which is as follows:

14. The culture vessel according to claim 1, wherein the occupancy rate per unit area of ​​the portion of the upper surface of the gas permeable sheet layer other than the minute recess is 2% or more and 50% or less.

15. Minimum thickness of the gas permeable sheet layer (H ｍｉｎ The culture vessel according to claim 1, wherein the diameter of the element is 10 μm or more and 500 μm or less.

16. A method for manufacturing a culture vessel according to claim 1, comprising the steps of: obtaining the gas permeable sheet layer having the micro recesses on its surface by extrusion film molding; and attaching the gas permeable sheet layer to the bottom surface of the frame.

17. The method for manufacturing a culture vessel according to claim 16, wherein, in the step of obtaining the gas permeable sheet layer, a film-like molten resin is pressed against a cooling roll having an uneven surface, and while cooling the film-like molten resin, the minute recesses are transferred to the surface of the film-like molten resin.

18. Maximum depth of the minute recess (H ｍａｘ ) and the maximum diameter (D) of the opening of the minute recess. ｍａｘ A method for producing a culture vessel according to claim 16, wherein the following formula (1) is satisfied.

19. The method for manufacturing a culture vessel according to claim 16, wherein the gas permeable sheet layer has a bubble contact angle of 100° or more and 150° or less in water.

20. A method for manufacturing a culture container according to claim 16, comprising the step of providing an adhesive layer on the lower surface of the frame before the above-mentioned bonding step.

21. A method for manufacturing a culture vessel according to claim 16, comprising the step of applying a coating material to the surface of the gas permeable sheet layer after the bonding step to form a coating layer.

22. A method for culturing spheroids or adherent cells using the culture vessel described in claim 1.

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