Cell culture substrate, method for manufacturing the same, and cell culture kit

Abstract

The present invention relates to a cell culture medium comprising: a substrate and a layer containing a hydrophilic polymer covering at least a portion of the surface of the substrate, the cell culture medium having the following regions: (A) a region having cell adhesion and cell proliferation, and (B) a region adjacent to (A) but not having cell adhesion or cell proliferation, wherein region (A) is formed by a recess formed in the layer containing the hydrophilic polymer, the recess having an inclined surface and a bottom surface, the inclined surface having an inclination angle of 40° or more and 110° or less.

Description

Technical Field

[0001] This invention relates to cell culture materials, methods for manufacturing the same, and cell culture kits. Background Technology

[0002] Pluripotent stem cells, such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), are cells capable of differentiating into various tissues of an organism (differentiation pluripotency). They have attracted significant attention as cell sources for regenerative medicine and drug discovery screening. To apply pluripotent stem cells to regenerative medicine and drug discovery screening, they need to differentiate into target cells. This requires forming pluripotent stem cell aggregates. Furthermore, pluripotent stem cells can differentiate into various cell types, and the optimal size of these cell aggregates varies depending on the type of differentiated cells. Therefore, it is desirable to control the size to create uniformly sized cell aggregates.

[0003] As a cell culture medium for forming cell aggregates, for example, Patent Document 1 discloses a cell culture medium characterized by having a substrate and a stimulus-responsive polymer covering the substrate, wherein the stimulus-responsive polymer is a block copolymer having water-insoluble block segments and stimulus-responsive block segments, and the cell culture medium has: (A) cell proliferation and stimulus responsiveness, and an area of ​​0.001~5 mm². 2 (A) an island-like region; and (B) a region adjacent to the region (A) that does not have cell proliferation.

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: Japanese Patent Application Publication No. 2020-62009 Summary of the Invention

[0007] The problem the invention aims to solve

[0008] The cell culture medium disclosed in Patent Document 1 has a specific (A) region and a specific (B) region. In the (A) region, spherical bodies can be efficiently formed, and spherical bodies of uniform size and arbitrary shape can be formed. Furthermore, the cell culture medium disclosed in Patent Document 1 utilizes plasma treatment (isotropic etching: 20 Pa pressure, 20 mA conductive current, 10 seconds irradiation time) when forming the (A) region. However, the inventors have discovered that there is room for improvement in the uniformity of the shape of the (A) region and the clarity of the uneven shapes of the (A) and (B) regions in the cell culture medium manufactured using this method. By improving the uniformity of the shape of the (A) region and the clarity of the uneven shapes of the (A) and (B) regions, cell aggregates of uniform size and shape can be obtained more stably.

[0009] The object of this invention is to provide a cell culture medium capable of stably obtaining cell aggregates of uniform size and shape. A further object of this invention is to provide a method for manufacturing this cell culture medium.

[0010] Solution for solving the problem

[0011] This invention relates to a cell culture medium comprising:

[0012] A substrate, and a layer containing a hydrophilic polymer covering at least a portion of the surface of the substrate.

[0013] The cell culture medium has the following region (A) and the following region (B).

[0014] (A) Regions with cell adhesion and cell proliferation

[0015] (B) A region adjacent to region (A) that does not exhibit cell adhesion or cell proliferation.

[0016] (A) The region is formed by a recess in a layer containing a hydrophilic polymer, the recess having an inclined surface and a bottom surface, the inclined surface having an inclination angle of more than 40° and less than 110°.

[0017] The present invention also relates to a method for manufacturing the above-mentioned cell culture medium, comprising:

[0018] The coating process involves coating at least a portion of the surface of a substrate with a composition containing a hydrophilic polymer to form a layer containing a hydrophilic polymer.

[0019] In the patterning process, a portion of the surface of a layer containing hydrophilic polymers is subjected to plasma treatment to form region (A) in the plasma-treated portion.

[0020] The present invention also relates to a cell culture kit comprising: a cell culture medium of the present invention having a separator member provided therein, said separator member having a plurality of cylindrical separator walls capable of dividing the sides of cultured cells.

[0021] This invention includes, for example, the following inventions. [1]

[0023] A cell culture medium, comprising:

[0024] A substrate, and a layer containing a hydrophilic polymer covering at least a portion of the surface of the substrate.

[0025] The cell culture medium has the following region (A) and the following region (B).

[0026] (A) Regions with cell adhesion and cell proliferation

[0027] (B) A region adjacent to region (A) that does not exhibit cell adhesion or cell proliferation.

[0028] Region (A) is formed by a recess in the layer containing the hydrophilic polymer, the recess having an inclined surface and a bottom surface, the inclined surface having an inclination angle of 40° or more and 110° or less. [2]

[0030] According to the cell culture medium described in [1], the maximum depth of the (A) region is more than 1 nm and less than 500 nm. [3]

[0032] According to the cell culture medium of [1] or [2], at least a portion of the bottom surface of the (A) region is composed of the substrate. [4]

[0034] The cell culture medium according to any one of [1] to [3], wherein the thickness of the layer containing the hydrophilic polymer is 10 nm or more and 500 nm or less. [5]

[0036] According to any one of [1] to [4], the cell culture medium, wherein the planar region defined by the boundary between region (A) and region (B) has an area of ​​0.001 mm². 2 Above and 5mm 2 An ellipse that is less than or equal to 1 and less than 1.1 in length and width. [6]

[0038] The cell culture medium according to any one of [1] to [5], wherein the ratio (diameter / maximum depth) of the opening of the (A) region to the maximum depth (nm) of the (A) region is 500 or more and 3000 or less. [7]

[0040] The cell culture medium according to any one of [1] to [6], wherein the minimum distance between the (A) regions is more than 400µm and less than 10000µm. [8]

[0042] The cell culture medium according to any one of [1] to [7], wherein the water contact angle of the (A) region is 20° or more and 110° or less. [9]

[0044] The cell culture medium according to any one of [1] to [8], wherein the (A) region is formed by reactive ion etching.

[10]

[0046] The cell culture medium according to any one of [1] to [9], wherein the thickness of the medium is 0.01 mm or more and 0.5 mm or less.

[11]

[0048] A method for manufacturing a cell culture medium according to any one of [1] to

[10] , comprising:

[0049] In the coating process, at least a portion of the surface of a substrate is coated with a composition containing a hydrophilic polymer to form the layer containing the hydrophilic polymer.

[0050] In the patterning process, a portion of the surface of the layer containing the hydrophilic polymer is subjected to plasma treatment to form the region (A) in the plasma-treated portion.

[12]

[0052] According to the method for manufacturing cell culture materials described in

[11] , the hydrophilic polymer is reactive to active energy rays.

[0053] The coating process includes: irradiating the layer containing hydrophilic polymers with active energy rays and immobilizing the layer on the surface of the substrate.

[13]

[0055] The method for manufacturing cell culture medium according to

[11] or

[12] , wherein the plasma treatment is a reactive ion etching treatment.

[14]

[0057] According to any one of

[11] to

[13] , the manufacturing method further comprises, after the patterning process, forming a cross-sectional area having an in-plane direction of 0.05 cm². 2 Above and 100cm 2 The following is a bonding process in which the plate with the through hole is bonded to the substrate on the side of the substrate covered by the layer containing the hydrophilic polymer.

[15]

[0059] According to any one of

[11] to

[14] , the manufacturing method further comprises, after the patterning step, a temperature-responsive layer forming step of covering the surface of the plasma-treated layer containing the hydrophilic polymer with a composition containing the temperature-responsive polymer to form a layer containing the temperature-responsive polymer.

[16]

[0061] A cell culture kit comprising: cell culture medium,

[0062] The cell culture medium described in any one of [1] to

[10] is provided with a partition member, the partition member having a plurality of cylindrical partition walls capable of dividing the sides of the cultured cells.

[17]

[0064] The cell culture medium according to any one of [1] to

[10] , wherein the carbon ratio (R) of the carboxyl group in region (A) to the total carbon is... COOH The percentage is above 0.25%.

[0065] The effects of the invention

[0066] According to the present invention, a cell culture medium capable of stably obtaining cell aggregates of uniform size and shape can be provided. According to the present invention, a method for manufacturing the cell culture medium can also be provided. Attached Figure Description

[0067] Figure 1 This is a schematic diagram (cross-sectional view) of a cell culture medium according to one embodiment.

[0068] Figure 2 This is a schematic diagram illustrating the method for calculating the tilt angle.

[0069] Figure 3 This is a schematic diagram (perspective view) of a substrate having a layer containing a hydrophilic polymer formed on its surface after the covering process in one embodiment of the manufacturing method.

[0070] Figure 4 This is a schematic diagram (three-dimensional view) of the cell culture medium after the patterning process in one embodiment of the manufacturing method.

[0071] Figure 5 This is a schematic diagram (three-dimensional view) of the cell culture medium after the bonding process in one embodiment of the manufacturing method. Detailed Implementation

[0072] The following provides a detailed description of the methods for implementing the present invention. It should be noted that the present invention is not limited to the following embodiments, and modifications can be made to the following embodiments to achieve the aforementioned effects.

[0073] In this specification, "cell aggregate" refers to a three-dimensional aggregate of cells formed by the aggregation of multiple cells. The shape of the three-dimensional aggregate can be spherical or ellipsoidal, or hemispherical. These shapes can be formed by the folding of sheet-like cells with gaps, or they can be hollow. As an example of a cell aggregate, a spherical shape can be cited.

[0074] In this specification, "temperature responsiveness" refers to the degree of hydrophilicity / hydrophobicity changing with temperature. Furthermore, the boundary temperature at which the degree of hydrophilicity / hydrophobicity changes is denoted as the "response temperature".

[0075] In this specification, "substances derived from living organisms" refers to substances present in living organisms and substances equivalent to them, and which are chemically synthesized. Substances present in living organisms can be natural products or artificially synthesized through gene recombination technology. There are no particular limitations on substances derived from living organisms; examples include nucleic acids, proteins, and polysaccharides, which are the basic building blocks of living organisms; nucleotides, nucleosides, amino acids, and various sugars, which are their constituent elements; as well as lipids, vitamins, and hormones.

[0076] In this specification, "cell adhesion" refers to the ease with which cells adhere to a substrate or cell culture medium at the culture temperature, and "having cell adhesion" means that cells can adhere to the substrate or cell culture medium directly or with the aid of a biologically derived substance at the culture temperature. Conversely, "not having cell adhesion" means that cells cannot adhere to the substrate or cell culture medium directly or with the aid of a biologically derived substance at the culture temperature. Methods for evaluating the presence or absence of cell adhesion are not particularly limited, and include, for example, methods such as adding a fluorescently labeled or otherwise labeled substance to the area and confirming the adsorption of the substance; methods for measuring the water contact angle of the area; etc.

[0077] In this specification, "cell proliferation" refers to the ease with which cells proliferate at the culture temperature, and "having cell proliferation" means that cells can proliferate at the culture temperature. Conversely, "not having cell proliferation" means that cells cannot proliferate at the culture temperature. "High cell proliferation" means that more cells proliferate during the same culture period.

[0078] The cell culture medium of this embodiment has a substrate and a layer containing a hydrophilic polymer covering at least a portion of the surface of the substrate, the cell culture medium having region (A) and region (B) as described below.

[0079] (A) Island-like regions with cell adhesion and cell proliferation

[0080] (B) A region adjacent to region (A) that does not exhibit cell adhesion or cell proliferation.

[0081] Here, region (A) is formed by a recess in a layer containing a hydrophilic polymer, the recess having an inclined surface and a bottom surface, the inclined surface having an inclination angle of 40° or more and 110° or less.

[0082] Figure 1 This is a schematic diagram (cross-sectional view) of a cell culture medium according to one embodiment. Figure 1 The cell culture medium 10 shown has a substrate 1 and a layer 2 containing hydrophilic polymers covering the surface of the substrate 1. Figure 1 In the diagram, A and B represent regions (A) and (B), respectively. Additionally, Figure 1 In the diagram, A1 and A2 represent the inclined surface and bottom surface of the concave portion, respectively. A1 and A2 together form region (A). The thickness of layer 2 containing the hydrophilic polymer refers to the distance from the interface between the substrate 1 and layer 2 containing the hydrophilic polymer to region (B). Figure 1 The distance between the faces of the region (denoted by B in the text). Additionally, in... Figure 1 In the cell culture medium 10 shown, the bottom surface A2 of region (A) is a layer 2 containing hydrophilic polymers, but is not limited thereto. For example, at least part or all of the bottom surface A2 of region (A) may be substrate 1. Similarly, at least part of the inclined surface A1 of region (A) may also be substrate 1.

[0083] The maximum depth of region (A) refers to the out-of-plane distance between the bottom surface of region (A) and the surface of region (B). The maximum depth of region (A) can be, for example, 1 nm or more and 2000 nm or 1 nm or more and 500 nm or less. By making the maximum depth of region (A) 500 nm or less, the number of dead cells trapped by the uneven surface can be further suppressed, and the number of dead cells mixed into the cell aggregate can be further reduced. This further improves the cell viability of the cell aggregate. Furthermore, if the maximum depth of region (A) is 500 nm or less, the adhesion of air bubbles to the uneven surface is further suppressed. Suppressing air bubble adhesion eliminates the need for degassing or repeated dispensing and aspiration of culture medium using pipettes, further improving operability. By making the maximum depth of region (A) 1 nm or more, live cells that spontaneously move (migrate) on the cell culture medium are more likely to aggregate in region (A), thus further improving the cell viability of the cell aggregate. From the perspective of further improving the cell survival rate of the formed cell aggregate, the maximum depth of region (A) is more preferably 400 nm or less, and more preferably 350 nm or less.

[0084] In this specification, "inclination angle of the inclined surface" refers to the angle between the approximate curve of the inclined surface when approximating it as a straight line within a range of more than 20% and less than 70% of the maximum depth of the inclined surface in the plane that intersects the substrate perpendicularly along the axis passing through the center (centroid) of the bottom surface of the (A) region. Figure 2 This is a schematic diagram (cross-sectional view) used to illustrate the method for calculating the tilt angle. Figure 2 In this diagram, H represents the maximum depth of region (A) (i.e., the distance in the outward direction between the bottom surface A2 of region (A) and the surface of region (B)). The shape of region (A) in a plane perpendicular to the substrate along an axis passing through the center (centroid) of the bottom surface of region (A) can be measured, for example, using a stylus profilometer (manufactured by Bruker Co., Ltd., trade name: DEKTAKXT). Based on the maximum depth H of region (A), a straight approximation is made to the inclined surface A1 within a range of 20% (20% × H) to 70% (70% × H). The straight approximation can be performed, for example, using the least squares method. Next, the angle θ between the obtained approximate straight line L and the bottom surface A2 of region (A) is calculated. This angle θ is the inclination angle of the inclined surface. Figure 2 As shown, when the shape of region (A) narrows into a concave shape as it approaches the bottom surface A2, the angle θ is greater than 0° and less than 90°. Conversely, when the shape of region (A) widens into an inverted conical shape as it approaches the bottom surface A2, the angle θ is greater than 90° and less than 180°.

[0085] In this embodiment, the tilt angle of the tilted surface of the cell culture medium is 40° or more and 110° or less. When the tilt angle of the tilted surface is within this range, the uneven shape becomes clear, thus enabling the more stable production of cell aggregates with uniform size and shape. From the same viewpoint, the tilt angle of the tilted surface is preferably 50° or more, more preferably 60° or more, further preferably 65° or more, even more preferably 70° or more, even more preferably 75° or more, and particularly preferably 80° or more. From the same viewpoint, the tilt angle of the tilted surface is preferably 100° or less, more preferably 95° or less, even more preferably 90° or less, and even more preferably less than 90°. The inclination angle of the inclined surface can be 40° or higher and below 100°, 40° or higher and below 95°, 40° or higher and below 90°, 40° or higher and below 90°, 50° or higher and below 110°, 50° or higher and below 100°, 50° or higher and below 95°, 50° or higher and below 90°, 50° or higher and below 90°, 60° or higher and below 110°, 60° or higher and below 100°, 60° or higher and below 95°, 60° or higher and below 90°, 60° or higher and below 90°, 65° or higher and below 100°, 65° or higher and below 95°, 6 The angles are: 5° and 90° or less, 65° and 90° or more, 70° and 110° or less, 70° and 100° or more, 70° and 95° or more, 70° and 90° or more, 70° and 90° or more, 75° and 110° or less, 75° and 100° or more, 75° and 95° or more, 75° and 90° or more, 75° and 90° or more, 80° and 110° or more, 80° and 100° or more, 80° and 95° or more, 80° and 90° or more, or 80° and 90° or more. Here, when region (A) is formed by etching such as plasma treatment as described later, the more stringent the processing conditions, such as extending the processing time, the deeper region (A) becomes, and the easier it is for the tilt angle to increase. (A) When the maximum depth of the region is greater than 1 nm and less than 500 nm, and the tilt angle of the inclined surface is less than 90°, the cell culture medium exhibits excellent mass production properties and can be used to stably obtain cell aggregates of uniform size and shape.

[0086] The substrate used as the cell culture medium in this embodiment is not particularly limited, but is preferably formed from at least one selected from the group consisting of polystyrene, polyethylene, polyethylene terephthalate, polycarbonate, cyclic olefin polymers, cellulose acetate, nitrocellulose and polyvinylidene fluoride. More preferably, it is formed from at least one selected from the group consisting of polystyrene, polyethylene terephthalate, polycarbonate and cyclic olefin polymers. Even more preferably, it is formed from at least one selected from the group consisting of polystyrene, polyethylene terephthalate and polycarbonate. Most preferably, it is formed from polystyrene or polycarbonate.

[0087] From the perspective of being suitable for observing cells cultured on cell culture media using a high-magnification phase-contrast microscope, the refractive index of the substrate, measured using the D-line (wavelength 589 nm), is preferably 1.4 or higher and 1.6 or lower, more preferably 1.45 or higher and 1.6 or lower, and particularly preferably 1.5 or higher and 1.55 or lower. By keeping the refractive index of the substrate within these ranges, spherical aberration of the cells during phase-contrast microscopy can be reduced, resulting in clear phase-contrast images. Furthermore, to reduce spherical aberration, the thickness of the substrate is preferably 0.5 mm or lower, more preferably 0.4 mm or lower, particularly preferably 0.3 mm or lower, and most preferably 0.2 mm or lower. On the other hand, from the perspective of suppressing the inability to focus across the entire observation range due to substrate deflection during microscopic observation, the thickness of the substrate is preferably 0.01 mm or higher, more preferably 0.05 mm or higher, particularly preferably 0.1 mm or higher, and most preferably 0.15 mm or higher.

[0088] The thickness of the substrate is preferably 0.01 mm or more and 0.5 mm or less, more preferably 0.05 mm or more and 0.4 mm or less, even more preferably 0.1 mm or more and 0.3 mm or less, and particularly preferably 0.15 mm or more and 0.2 mm or less. When the substrate thickness is within this range, the phase difference image of the cells becomes clearer.

[0089] From the perspective of being suitable for observing cells cultured on cell culture media using high-magnification fluorescence microscopy, the fluorescence intensity (autofluorescence intensity) of the substrate at excitation wavelengths of 350 nm, 488 nm, and 647 nm (when irradiated with excitation light of these wavelengths) is preferably less than the fluorescence intensity (autofluorescence intensity) of a 1.2 mm thick polystyrene plate excited with light of the same excitation wavelength. More preferably, it is 80% or less of the fluorescence intensity of a 1.2 mm thick polystyrene plate; particularly preferably, it is 50% or less; and most preferably, it is 10% or less. Fluorescent dyes excited at excitation wavelengths of 350 nm, 488 nm, and 647 nm are frequently used in the fluorescence observation of cells. By keeping the autofluorescence intensity of the substrate at these wavelengths below a certain value, clear fluorescence images of the cells can be obtained.

[0090] The shape of the substrate is not particularly limited; it can be a planar shape like a plate or membrane, or it can be a fiber, porous particle, porous membrane, hollow fiber, etc. Alternatively, the substrate can also be the shape of containers commonly used for cell culture (such as Pictell's cell culture dishes, flasks, plates, bags, etc.). From the perspective of ease of culture operation, the preferred shape of the substrate is a planar shape like a plate or membrane, or the shape of a flat porous membrane.

[0091] The thickness of the layer containing the hydrophilic polymer can be, for example, 10 nm or more and 500 nm or less. By making the thickness of the layer containing the hydrophilic polymer 10 nm or more and 500 nm or less, cells can adhere only to region (A) and proliferate. In addition, cells can easily migrate and concentrate in region (A), thereby improving the cell survival rate of cell aggregates. Here, the "layer thickness" of the layer containing the hydrophilic polymer refers to the out-of-plane length from the interface between the substrate and the layer containing the hydrophilic polymer to the interface on the opposite side of the layer containing the hydrophilic polymer from the substrate (excluding region (A)). For the layer thickness greater than 10 nm, the thickness can be calculated by measuring the cross-sectional image of an ultrathin section of cell culture medium prepared by a microtome using a transmission electron microscope, measuring this distance at 10 randomly selected points, and averaging the results. For the layer thickness less than 10 nm, an ellipsometry can be used for measurement. To effectively inhibit cell adhesion to region (B), a layer thickness of 10 nm or more is preferred, and more preferably 30 nm or more, 40 nm or more, or 50 nm or more is further preferred. Furthermore, from the perspective of improving cell viability of cell aggregates by concentrating cells in region (A) through cell migration, a layer thickness of 200 nm or less is more preferred, and more preferably 180 nm or less, 160 nm or less, 150 nm or less, 140 nm or less, or 120 nm or less is further preferred. The layer thickness containing the hydrophilic polymer can be, for example, 10 nm or more and 200 nm or less, 10 nm or more and 180 nm or less, 10 nm or more and 160 nm or less, 10 nm or more and 150 nm or less, 10 nm or more and 140 nm or less, 10 nm or more and 120 nm or less, 30 nm or more and 200 nm or less, 30 nm or more and 180 nm or less, 30 nm or more and 160 nm or less, 30 nm or more and 150 nm or less, 30 nm or more and 140 nm or less, or 30 nm or more. And below 120nm, above 40nm and below 200nm, above 40nm and below 180nm, above 40nm and below 160nm, above 40nm and below 150nm, above 40nm and below 140nm, above 40nm and below 120nm, above 50nm and below 200nm, above 50nm and below 180nm, above 50nm and below 160nm, above 50nm and below 150nm, above 50nm and below 140nm, above 50nm and below 120nm.

[0092] Hydrophilic polymers contain phosphorylcholine or hydroxyl groups. By including phosphorylcholine or hydroxyl groups, the areas covered by these polymers become non-adhesive regions for cells. Furthermore, these hydrophilic polymers do not require complete degradation; through plasma treatment, the affected areas can be made into regions with cell adhesion and proliferation properties, and the degradation products of the hydrophilic polymer are less likely to enter the cells. Besides containing phosphorylcholine or hydroxyl groups, there are no particular limitations on the types of hydrophilic polymers. Commercially available examples include Lipidure(R)CM5206 (manufactured by Nippon Yu Co., Ltd.), Lipidure(R)CM2001 (manufactured by Nippon Yu Co., Ltd.), and BIOSURFINE(R)-AWP (manufactured by Toyosei Kogyo Co., Ltd.). In addition, as a substrate for commercially available products covered with hydrophilic polymers, PrimeSurface® (manufactured by Sumitomo Bakelite Co., Ltd.), EZ-BindShut® (manufactured by AGC Techno Glass Co., Ltd.), and EZ-BindShutII® (manufactured by AGC Techno Glass Co., Ltd.) are preferred.

[0093] The hydrophilic polymer preferably comprises a compound represented by the following general formula (1), a compound represented by the following general formula (2), or a compound represented by the following general formula (3).

[0094]

[0095] In general formula (1), R 1 and R 2 Each can independently represent a hydrogen atom or a methyl group, R 3 [This represents a hydrogen atom or any organic group, where m and n are independent positive integers.]

[0096]

[0097] In general formula (2), R 4 R 5 and R 6 Each can independently represent a hydrogen atom or a methyl group, R 7 [x, y, and z represent hydrogen atoms or any organic group, where x, y, and z each independently represent positive integers.]

[0098]

[0099] In general formula (3), R 8 and R 9 Each can independently represent a hydrogen atom or a methyl group, R 10 [A represents a hydrogen atom or any organic group, and 'a' and 'b' each independently represent a positive integer.]

[0100] By including the hydrophilic polymer in compounds of formula (1), formula (2), or formula (3), cell adhesion and proliferation are facilitated, making it suitable for forming uniformly shaped cell aggregates in region (A). From the viewpoint of being suitable for immobilizing hydrophilic polymers on a substrate, R in formula (1), formula (2), or formula (3)... 3 R 7 and R 10 Each group is preferably a hydrophobic group or a functional group reactive to active energy rays (e.g., UV, electron beams). As a hydrophobic group, straight-chain or cyclic alkyl groups such as methyl, ethyl, propyl, butyl, and cyclohexyl are preferred. As a functional group reactive to active energy rays (e.g., UV, electron beams), examples include azide, acrylate, methacrylate, vinyl, and epoxy groups, with azide being preferred.

[0101] Region (A) is formed by a recess in a layer containing a hydrophilic polymer. This recess has an inclined surface and a bottom surface. Furthermore, in the cell culture medium of this embodiment, the inclination angle of the inclined surface in region (A) is 40° or more and 110° or less. Region (A) may have both the inclined surface and the bottom surface within the layer containing the hydrophilic polymer, or a portion of the inclined surface and / or a portion or all of the bottom surface may be located within the substrate.

[0102] Region (A) can be understood as an island-like region when the cell culture medium is viewed from the side of the layer containing the hydrophilic polymer in a direction orthogonal to the substrate. In this case, the outer perimeter of the island-like region corresponds to the boundary between region (A) and region (B). In this specification, the planar region defined by the boundary between region (A) and region (B) (i.e., the outer perimeter of the aforementioned island-like region) is sometimes referred to as an opening. Figure 1 In this context, φ represents the diameter of the opening (also referred to as the dot diameter in this specification). It should be noted that an island-shaped region indicates that the region exists independently of the regions outside it. By making region (A) an island-shaped region as described above, compared to a region that is not island-shaped (e.g., a striped structure), living cells can be concentrated in region (A), thus enabling the more efficient production of cell aggregates.

[0103] The shape of the opening (the planar region defined by the boundary between region (A) and region (B)) is not particularly limited and can be appropriately set according to the target shape of the cell aggregate. For example, ellipses (including circles), polygons, or closed shapes formed by straight lines and curves can be used. In addition, from the perspective of being suitable for manufacturing cell aggregates with a near-spherical shape, the shape of the opening is preferably ellipse (including circles) or polygon, more preferably ellipse (including circles) or rectangle, further preferably ellipse (including circles) or square, and most preferably ellipse (including circles).

[0104] From the perspective of being suitable for manufacturing cell aggregates with a near-spherical shape, the aspect ratio of the shape of the opening (the planar region defined by the boundary between region (A) and region (B)) is preferably 1 or more and 2 or less, more preferably 1 or more and 1.5 or less, further preferably 1 or more and 1.1 or less, and most preferably 1 or more and 1.05 or less. Here, "aspect ratio" refers to the ratio of the maximum diameter (major axis) to the minimum diameter (minor axis) of the shape, i.e., major axis / minor axis.

[0105] From the perspective of being suitable for manufacturing cell aggregates of uniform size and shape, the standard deviation / average aspect ratio of the shape of the opening (the planar region defined by the boundary between region (A) and region (B)) is preferably 80% or less, more preferably 50% or less, further preferably 20% or less, and most preferably 5% or less.

[0106] The area of ​​the opening (the planar region defined by the boundary between region (A) and region (B)) can be, for example, 0.001 mm. 2 Above and 5mm 2 The area is preferably 0.005 mm². 2 Above and 1mm 2 Hereinafter, an area of ​​0.01 mm is preferred. 2 Above and 0.5mm 2 Hereinafter, an area of ​​0.015 mm is further preferred. 2 Above and 0.25mm 2 The preferred area is 0.02 mm². 2 Above and 0.2mm 2 the following.

[0107] From the perspective of being suitable for manufacturing cell aggregates of uniform size and shape, the standard deviation / average area of ​​the opening (the planar area defined by the boundary between region (A) and region (B)) is preferably 80% or less, more preferably 50% or less, further preferably 20% or less, and most preferably 5% or less.

[0108] (A) The shape of the bottom surface of the region can be the same as or different from the shape of the opening. (A) The shape of the bottom surface of the region is not particularly limited and can be appropriately set according to the target shape of the cell aggregate, such as an ellipse (including a circle), a polygon, or a closed shape formed by straight lines and curves. In addition, from the perspective of being suitable for manufacturing cell aggregates with a near-spherical shape, the shape of the opening is preferably an ellipse (including a circle) or a polygon, more preferably an ellipse (including a circle) or a rectangle, further preferably an ellipse (including a circle) or a square, and most preferably an ellipse (including a circle).

[0109] The aspect ratio of the bottom surface of region (A) can be the same as or different from that of the opening. From the perspective of being suitable for manufacturing cell aggregates with a near-spherical shape, the aspect ratio of the bottom surface of region (A) is preferably 1 or more and 2 or less, more preferably 1 or more and 1.5 or less, even more preferably 1 or more and 1.1 or less, and most preferably 1 or more and 1.05 or less.

[0110] From the perspective of being suitable for manufacturing cell aggregates of uniform size and shape, the standard deviation / average aspect ratio of the shape of the bottom surface of region (A) is preferably 80% or less, more preferably 50% or less, further preferably 20% or less, and most preferably 5% or less.

[0111] The area of ​​the bottom surface of region (A) can be the same as or different from the area of ​​the opening. For example, the area of ​​the bottom surface of region (A) can be 0.001 mm². 2 Above and 6mm 2 The area is preferably 0.001 mm². 2 Above and 5mm 2 Hereinafter, 0.005 mm is preferred. 2 Above and 1mm 2 Hereinafter, an area of ​​0.01 mm is further preferred. 2 Above and 0.5mm 2 Hereinafter, a more preferred area is 0.015 mm². 2 Above and 0.25mm 2 The preferred area is 0.02 mm². 2 Above and 0.2mm 2 the following.

[0112] From the perspective of being suitable for manufacturing cell aggregates of uniform size and shape, the standard deviation / average area of ​​the bottom surface of region (A) is preferably 80% or less, more preferably 50% or less, further preferably 20% or less, and most preferably 5% or less.

[0113] In region (A), the ratio of the diameter (nm) to the maximum depth (nm) of the opening in region (A) (diameter / maximum depth) can be set to, for example, 100 or more and 20,000 or less. The diameter / maximum depth ratio is preferably 500 or more and 3,000 or less. This results in a clearer uneven shape, enabling more stable acquisition of cell aggregates with uniform size and shape. A diameter / maximum depth ratio is more preferably 500 or more and 2,000 or less, and even more preferably 500 or more and 800 or less. It should be noted that "the diameter of the opening" refers to the average of the major axis diameter (maximum diameter) and the minor axis diameter (minimum diameter) of the opening. The diameter of the opening in… Figure 1 In this context, φ is used to represent it.

[0114] In addition, from the perspective of improving the oxygen concentration around the cells and improving the survival rate of cell aggregates, the minimum distance between regions (A) is preferably 400µm or more and 10000µm or less, more preferably 500µm or more and 8000µm or less, further preferably 1000µm or more and 5000µm or less, and most preferably 2000µm or more and 4000µm or less.

[0115] Region (A) can be formed, for example, by modifying a portion of the surface of a layer containing a hydrophilic polymer on the surface of a substrate using plasma treatment. Region (A) is the area where the surface of the layer containing the hydrophilic polymer is modified using plasma treatment or the like, thereby making region (A) a region with cell adhesion and cell proliferation properties. Since region (A) can be patterned by short-term plasma treatment or the like, the mass production of cell culture materials can be improved.

[0116] The aforementioned plasma treatment can also be carried out in the presence of introduced gases such as oxygen, hydrogen, nitrogen, ammonia, and argon. By using the aforementioned introduced gases, hydrophilic functional groups are generated in the material, enabling the formation of regions with cell adhesion and cell proliferation properties. Depending on the introduced gas used in the plasma treatment, the types of functional groups include hydroxyl, carbonyl, aldehyde, carboxyl, ether, ester, amino, and nitro groups. Using oxygen will induce the formation of a variety of functional groups, including hydroxyl, carbonyl, aldehyde, carboxyl, ether, ester, and nitro groups, which is therefore preferred. In particular, when carboxyl groups, which are highly polar functional groups, are generated, the hydrophilicity of the material is significantly modified, and cell adhesion and cell proliferation properties are also improved, which is also preferred.

[0117] When the surface material of the cell culture medium is organic and the functional group generated is carboxyl, the carbon ratio of the carboxyl group in region (A) to the total carbon (R) is... COOHPreferably, it is 0.25% or more, more preferably 0.3% or more, 0.5% or more, or 0.8% or more, and most preferably 1.2% or more. Additionally, the carbon ratio (RA) of the carboxyl group in region (A) relative to the total carbon is... COOH For example, it can be less than 10%, less than 8%, less than 6%, less than 5%, less than 4%, or less than 2%. (A) The carbon ratio of the carboxyl groups in the region to the total carbon (R) COOH For example, it can be 0.25% or higher and lower than 10%, 0.25% or higher and lower than 8%, 0.25% or higher and lower than 6%, 0.25% or higher and lower than 5%, 0.25% or higher and lower than 4%, 0.25% or higher and lower than 2%, 0.3% or higher and lower than 10%, 0.3% or higher and lower than 8%, 0.3% or higher and lower than 6%, 0.3% or higher and lower than 5%, 0.3% or higher and lower than 4%, 0.3% or higher and lower than 2%, 0.5% or higher and lower than 10%, 0.5% or higher and lower than 8%, 0.5% or lower. Above and below 6%, above 0.5% and below 5%, above 0.5% and below 4%, above 0.5% and below 2%, above 0.8% and below 10%, above 0.8% and below 8%, above 0.8% and below 6%, above 0.8% and below 5%, above 0.8% and below 4%, above 0.8% and below 2%, above 1.2% and below 10%, above 1.2% and below 8%, above 1.2% and below 6%, above 1.2% and below 5%, above 1.2% and below 4%, or above 1.2% and below 2%. By making the carbon ratio (R) of the carboxyl group in region (A) relative to the total carbon... COOH Within the scope described herein, cell culture media exhibiting cell adhesion and proliferation properties, as shown in this application, can be provided. Furthermore, from the viewpoint of reducing hypertrophy rate, R... COOH Preferably, it is 0.7% or higher and 1.0% or lower.

[0118] It should be noted that, in this specification, the carbon ratio (RA) of the carboxyl group relative to the total carbon in region (A) is... COOH The following formula was used to calculate the surface functional group content of region (A) after gas-phase chemical modification with trifluoroethanol, di-tert-butylcarbodiimide and pyridine was performed and the content of surface functional groups in region (A) was determined by X-ray photoelectron spectroscopy under the following conditions. The formula was derived from the reaction formula of gas-phase chemical modification.

[0119] R COOH (%) = (Furniture concentration (atom%)) / (3 × Carbon concentration (atom%) - 2 × F concentration (atom%)) / r COOH ×100

[0120] r COOH Reaction rate

[0121] <Measurement Conditions>

[0122] X-rays: Monochromatic Al-Kα rays (output: 25W)

[0123] Energy resolution: Wide-scan spectrum 117.40 eV

[0124] High-resolution spectrum 93.90 eV

[0125] Charge correction: C1s main peak (284.8 eV)

[0126] Furthermore, the ratio of the peak intensity of 287 eV to the peak intensity of 285 eV in the C1s spectrum measured by XPS (X-ray photoelectron spectroscopy) in region (A) is preferably 0.05 or greater than that in region (B). In this case, the difference in cell proliferation between region (A) and region (B) can be increased, thus enabling a large number of cells to proliferate in region (A), and making it easier to form uniform cell aggregates in region (A). From the viewpoint of being more suitable for forming uniform cell aggregates, the ratio of the peak intensity of 287 eV to the peak intensity of 285 eV in the C1s spectrum measured by XPS in region (A) is more preferably 0.07 or greater than that in region (B), further preferably 0.1 or greater, and most preferably 0.15 or greater.

[0127] As a method for adjusting the ratio of the peak intensity of 287 eV to the peak intensity of 285 eV in the C1s spectrum measured by XPS to the aforementioned range, there are no particular limitations, but a method of performing plasma treatment only on a portion of the layer containing hydrophilic polymers formed on the substrate surface (the portion predetermined as region (A)) is preferred. As a method for performing plasma treatment only on the portion predetermined as region (A), one example is to cover the layer containing hydrophilic polymers formed on the substrate surface with a mask fabricated by laser processing, and then perform plasma treatment from above the mask. Conditions such as plasma intensity and plasma irradiation time can be appropriately adjusted so that the difference between the ratio of the peak intensity of 287 eV to the peak intensity of 285 eV in the C1s spectrum measured by XPS in region (A) and the ratio of the peak intensity of 287 eV to the peak intensity of 285 eV in the C1s spectrum measured by XPS in region (B) converges to the aforementioned range. In the case of plasma treatment, using a gas containing oxygen and nitrogen as the introduction gas makes it easier to converge to the aforementioned range.

[0128] The plasma irradiation time is preferably 5 seconds or more and 10 minutes or less, more preferably 10 seconds or more and 5 minutes or less, and most preferably 30 seconds or more and 3 minutes or less. Air, nitrogen, or oxygen is preferably used as the introduction gas during plasma treatment, with oxygen being more preferred. The pressure of the introduction gas is preferably 1 Pa or more and less than 20 Pa, more preferably 1 Pa or more and 15 Pa or less, and even more preferably 1 Pa or more and 10 Pa or less. By not excessively increasing the gas pressure, it is easier to set the tilt angle of the tilted surface in region (A) within the aforementioned preferred range. Furthermore, from the perspective of making it easier to set the tilt angle of the tilted surface in region (A) within the aforementioned preferred range, plasma treatment is particularly preferably performed by reactive ion etching (RIE). RIE is also known as anisotropic etching.

[0129] The water contact angle of region (A) is preferably 20° or more and 110° or less. By modifying a portion of the surface of the layer containing the hydrophilic polymer using the plasma treatment described above, the wettability of region (A) can be improved. The water contact angle of region (A) is more preferably 40° or more and 80° or less, further preferably 50° or more and 70° or less, and most preferably 60° or more and 70° or less. When the water contact angle of region (A) is within the above range, the wettability of region (A) is further improved, and cell adhesion and cell proliferation are more excellent. In addition, from the viewpoint of reducing hypertrophy rate, the water contact angle of region (A) is preferably 20° or more and 40° or less, more preferably 25° or more and 45° or less. The water contact angle of region (A) can be achieved, for example, by adding a water droplet to the surface of region (A) and setting the angle between the liquid surface of the droplet when it is stationary and the surface of region (A) as θ. A And the following formula is used for determination.

[0130] θ A =2arctan(h / r)

[0131] (In the above formula, θ) A (where h represents the water contact angle, r represents the droplet height, and r represents the droplet radius.)

[0132] From the perspective of being suitable for peeling off cell aggregates from culture, region (A) can be temperature-responsive. When region (A) is temperature-responsive, from the perspective of enabling cell culture on cell culture media to be carried out at temperatures close to body temperature, the response temperature is preferably 50°C or lower, more preferably 35°C or lower. Furthermore, from the perspective of being suitable for suppressing cell peeling during operations such as changing the culture medium, the response temperature is particularly preferably 25°C or lower. Moreover, from the perspective of enabling the formation of cell aggregates through cooling operations at temperatures that do not damage the cells, the response temperature is preferably 4°C or higher, more preferably 10°C or higher, and even more preferably 15°C or higher.

[0133] In cases where region (A) exhibits temperature responsiveness, a layer containing temperature-responsive polymers with a thickness of 1 nm or more and 100 nm or less may be further provided on the surface of the layer containing the hydrophilic polymers (the surface including regions (A) and (B)). By having a layer containing temperature-responsive polymers with a thickness of 1 nm or more and 100 nm or less, temperature responsiveness in region (A) can be imparted without impairing the characteristics of regions (A) and (B) formed on the surface of the layer containing the hydrophilic polymers. To suit the imparting of temperature responsiveness to region (A) without impairing the characteristics of regions (A) and (B), the thickness of the layer containing the temperature-responsive polymers is more preferably 3 nm or more and 50 nm or less, more preferably 5 nm or more and 40 nm or less, and most preferably 10 nm or more and 35 nm or less. The preferred thickness of the temperature-responsive polymer layer, which enables the cells to be peeled off and recovered after culture based on temperature responsiveness without impairing cell proliferation, also varies depending on the cultured cells and can be appropriately adjusted within the range of thicknesses illustrated above.

[0134] The temperature-responsive polymer is preferably a block copolymer having water-insoluble block segments and temperature-responsive block segments. By making the temperature-responsive polymer such a block copolymer, the mass production of cell culture medium can be improved, and the incorporation of the temperature-responsive polymer into the manufactured cell aggregates can be suppressed. From the viewpoint of being suitable for rapidly separating cell aggregates from the cell culture medium, the ratio of structural units of temperature-responsive block segments contained in the temperature-responsive polymer is preferably 70 wt% or more, more preferably 80 wt% or more, particularly preferably 90 wt% or more, and most preferably 92 wt% or more.

[0135] Furthermore, by making the temperature-responsive polymer a block copolymer having water-insoluble block segments and temperature-responsive block segments, the surface of the cell culture medium can be given temperature responsiveness by a simple method such as dropping a solution containing the temperature-responsive polymer onto the surface of the cell culture medium and then drying it. Additionally, by making the layer formed at this time the thickness of the aforementioned preferred temperature-responsive polymer layer, even if the surface of the layer containing the hydrophilic polymer is entirely covered with the temperature-responsive polymer, the potential for damage to the properties of regions (A) and (B) can be further reduced.

[0136] Examples of monomeric units constituting temperature-responsive block segments include (meth)acrylamide compounds such as acrylamide and methacrylamide; N-alkyl-substituted (meth)acrylamide derivatives such as N,N-diethylacrylamide, N-ethylacrylamide, N-n-propylacrylamide, N-n-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-tert-butylacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, and N-tetrahydrofurfurylmethacrylamide; N,N-dialkyl-substituted (meth)acrylamide derivatives such as N,N-dimethyl(meth)acrylamide, N,N-ethylmethacrylamide, and N,N-diethylacrylamide; and 1-(1-oxo-2-propenyl)pyrrolidine, 1- (1-oxo-2-propenyl)-piperidine, 4-(1-oxo-2-propenyl)-morpholine, 1-(1-oxo-2-methyl-2-propenyl)-pyrrolidine, 1-(1-oxo-2-methyl-2-propenyl)-piperidine, 4-(1-oxo-2-methyl-2-propenyl)-morpholine and other (meth)acrylamide derivatives having cyclic groups; vinyl ethers such as methyl vinyl ethers; proline derivatives such as N-proline methyl ester acrylamide, preferably N,N-diethylacrylamide, N-n-propylacrylamide, N-isopropylacrylamide, N-n-propylmethylacrylamide, N-ethoxyethylacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethylacrylamide, more preferably N-n-propylacrylamide, N-isopropylacrylamide, and especially preferably N-isopropylacrylamide. Furthermore, when using room temperature culture medium during culture changes, N-n-propylacrylamide and N-proline methyl ester acrylamide are preferred from the perspective of setting the response temperature of the block copolymer to a temperature lower than room temperature.

[0137] Examples of monomeric units constituting the water-insoluble block segments include n-butyl acrylate, n-butyl methacrylate, isobutyl acrylate, isobutyl methacrylate, tert-butyl acrylate, tert-butyl methacrylate, n-hexyl acrylate, n-hexyl methacrylate, n-octyl acrylate, n-octyl methacrylate, n-decyl acrylate, n-decyl methacrylate, n-dodecyl acrylate, n-dodecyl methacrylate, n-tetradecyl acrylate, and n-tetradecyl methacrylate. Furthermore, from the perspective of being suitable for firmly immobilizing the block copolymer onto the substrate, those having reactive groups are preferred, for example, 4-azidophenyl acrylate, 4-azidophenyl methacrylate, 2-((4-azidobenzoyl)oxy)ethyl acrylate, and 2-((4-azidobenzoyl)oxy)ethyl methacrylate. Furthermore, from the perspective of improving cell proliferation, structures with aromatic rings are preferred, such as 2-hydroxyphenyl acrylate, 2-hydroxyphenyl methacrylate, 3-hydroxyphenyl acrylate, 3-hydroxyphenyl methacrylate, 4-hydroxyphenyl acrylate, 4-hydroxyphenyl methacrylate, N-(2-hydroxyphenyl)acrylamide, N-(2-hydroxyphenyl)methacrylamide, N-(3-hydroxyphenyl)acrylamide, N-(3-hydroxyphenyl)methacrylamide, N-(4-hydroxyphenyl)acrylamide, N-(4-hydroxyphenyl)methacrylamide, styrene, etc.

[0138] The water-insoluble block segments may also contain repeating units that control the response temperature of the block copolymer. Examples of repeating units controlling the response temperature of the block copolymer include hydrophilic or hydrophobic components, without particular limitation. Examples include 2-dimethylaminoethyl acrylate, 2-dimethylaminoethyl methacrylate, 2-diethylaminoethyl acrylate, 2-diethylaminoethyl methacrylate, N-[3-(dimethylamino)propyl]acrylamide, etc., which contain amino groups; and N-(3-sulfopropyl)-N-methacryloyloxyethyl-N,N-dimethylammonium betaine, N-methacryloyloxyethyl-N,N-dimethylammonium-α-N-methylcarboxylic betaine, etc., which contain betaine groups. Hydroxyethyl acrylate, hydroxyethyl methacrylate, N-(2-hydroxyethyl)acrylamide, polyethylene glycol monoacrylate, polyethylene glycol monomethacrylate, polypropylene glycol monoacrylate, polypropylene glycol monomethacrylate, methoxy polyethylene glycol monoacrylate, methoxy polyethylene glycol monomethacrylate, diethylene glycol monomethyl ether acrylate, diethylene glycol monomethyl ether methacrylate, diethylene glycol monoethyl ether methacrylate, diethylene glycol monoethyl ether methacrylate, 2-methoxyethyl acrylate, 2-methoxyethyl methacrylate, 2-ethoxyethyl acrylate, 2-methoxyethyl acrylate 3-Ethoxyethyl acrylate, 3-butoxyethyl acrylate, 3-butoxyethyl methacrylate, 3-butoxyethyl acrylamide, furfuryl acrylate, furfuryl methacrylate, tetrahydrofurfuryl acrylate, and other products containing polyethylene glycol groups or methoxyethyl groups; methoxymethyl acrylate, methoxymethyl methacrylate, 2-ethoxymethyl acrylate, 2-ethoxymethyl methacrylate, 3-butoxymethyl acrylate, 3-butoxymethyl methacrylate, 3-butoxymethyl acrylamide, and other products containing acrylate groups; 2-methacryloyloxyethyl phosphocholine, 2-Acryloyloxyethyl phosphoric acid choline, 3-(meth)acryloyloxypropyl phosphoric acid choline, 4-(meth)acryloyloxybutyl phosphoric acid choline, 6-(meth)acryloyloxyhexyl phosphoric acid choline, 10-(meth)acryloyloxydecyl phosphoric acid choline, ω-(meth)acryloyl(poly)oxyethylene phosphoric acid choline, 2-acrylamidoethyl phosphoric acid choline, 3-acrylamidopropyl phosphoric acid choline, 4-acrylamidobutyl phosphoric acid choline, 6-acrylamidohexyl phosphoric acid choline, 10-acrylamidodecyl phosphoric acid choline, ω-(meth)acrylamido(poly)oxyethylene phosphoric acid choline, etc., contain phosphoric acid choline groups.

[0139] Region (B) is adjacent to region (A) and does not exhibit cell adhesion or cell proliferation. If region (B) is adjacent to region (A) and does not exhibit cell proliferation, then during cell culture, cell aggregates can be formed only in region (A), resulting in a state where cells are absent from part or all of the surrounding area of ​​region (A). Furthermore, from the perspective of ensuring uniformity in the size and shape of the manufactured cell aggregates, it is preferable that region (B) exhibits neither cell proliferation nor cell adhesion.

[0140] The shape of region (B) is not limited except that it is adjacent to region (A). From the perspective of being suitable for manufacturing cell aggregates of uniform size and shape, it is preferable that region (B) is adjacent to the boundary line of region (A) for more than 20% of its length, more preferably more than 50%, further preferably more than 80%, and most preferably region (B) is completely surrounded by region (A).

[0141] The area ratio of region (A) to region (B) is not particularly limited. However, from the perspective of maximizing the number of cell aggregates that can be produced per unit area of ​​the cell culture medium, the area of ​​region (A) is preferably 10% or more, more preferably 30% or more, further preferably 50% or more, and most preferably 70% or more, relative to the total area of ​​regions (A) and (B). Furthermore, from the perspective of ensuring sufficient distance between multiple regions (A) and preventing the cell aggregates from merging and becoming uneven, the area of ​​region (B) is preferably 20% or more, more preferably 40% or more, further preferably 60% or more, and most preferably 80% or more, relative to the total area of ​​regions (A) and (B).

[0142] Cell culture media may have a layer containing organism-derived substances on its surface, as needed. This layer may exist entirely on the surface of the cell culture media or only on the surface of region (A). There are no particular limitations on the organism-derived substances; examples include matrix gum, laminin, fibronectin, hyalin, and collagen.

[0143] These substances derived from living organisms can be natural products, artificially synthesized substances using gene recombination technology, or synthetic proteins or peptides obtained by using restriction enzymes to cut fragments, or by chemically synthesizing substances equivalent to these substances derived from living organisms.

[0144] As a base adhesive, commercially available products such as Matrigel (manufactured by Corning Incorporated) and Geltrex (manufactured by Thermo Fisher Scientific) are preferred for ease of use.

[0145] There are no particular limitations on the type of laminin. For example, laminin 511, laminin 521, and laminin 511-E8 fragments, which have been reported to exhibit high activity against α6β1 integrin expressed on the surface of human iPS cells, can be used. Laminins can be natural products, artificially synthesized using gene recombination technology, or synthetic proteins or peptides obtained by chemical synthesis of substances equivalent to laminins. For ease of acquisition, commercially available products such as iMatrix-511 (manufactured by Nippi Co., Ltd.) are preferred.

[0146] Vitronectin can be a natural product, a synthetically produced substance using gene recombination technology, or a synthetic protein or peptide obtained by chemical synthesis of a substance equivalent to vitronectin. For ease of acquisition, commercially available vitronectin, human plasma-derived products (manufactured by Wako Pure Chemical Industries, Ltd.), synthesized protein (manufactured by Corning Incorporated), and vitronectin (VTN-N) (manufactured by Thermo Fisher Scientific) are preferred.

[0147] Fibronectin can be a natural product, a synthetically produced substance using gene recombination technology, or a synthetic protein or peptide obtained by chemical synthesis of a substance equivalent to fibronectin. For ease of acquisition, commercially available fibronectin solutions, human plasma-derived products (manufactured by Wako Pure Chemical Industries, Ltd.), and retrolin (manufactured by Takara Bio Co., Ltd.) are preferred.

[0148] There are no particular limitations on the type of collagen; for example, type I collagen, type IV collagen, etc., can be used. Collagen can be a natural substance, a substance artificially synthesized using gene recombination technology, or a synthetic peptide obtained by chemical synthesis of a substance equivalent to collagen. For ease of acquisition, commercially available collagen I, human (made by Corning Incorporated), collagen IV, human (made by Corning Incorporated), etc., are preferred.

[0149] From the perspective of inhibiting the denaturation of substances derived from organisms and enhancing cell proliferation, substances derived from organisms are preferably immobilized on cell culture media via non-covalent bonds. Here, "non-covalent bond" refers to bonding forces other than covalent bonds, which originate from intermolecular forces, such as electrostatic interactions, water-insoluble interactions, hydrogen bonds, π-π interactions, dipole-dipole interactions, dispersion forces, and other van der Waals interactions. The immobilization of substances derived from organisms into block copolymers can utilize a single bonding force or a combination of multiple bonding forces.

[0150] There are no particular limitations on the method of immobilizing substances derived from organisms. For example, preferred methods include coating a solution of a substance derived from an organism onto a cell culture medium for a specified time and immobilizing it, or adding the substance derived from an organism to the culture medium during cell culture to adsorb the substance onto the cell culture medium and immobilize it.

[0151] The cell culture medium of this embodiment can be modified as needed by setting a separator plate on the substrate (e.g., having an in-plane cross-sectional area of ​​0.05 cm²). 2 Above and 100cm 2 The following perforated plates and other structures are used to distinguish the individual cell aggregates.

[0152] The cell culture medium of this embodiment can be sterilized. The sterilization method is not particularly limited; autoclaving, UV sterilization, gamma-ray sterilization, and ethylene oxide gas sterilization can be used. From the viewpoint of suppressing the denaturation of block copolymers, autoclaving, UV sterilization, and ethylene oxide gas sterilization are preferred. From the viewpoint of suppressing substrate deformation, UV sterilization or ethylene oxide gas sterilization is further preferred. From the viewpoint of excellent mass production, ethylene oxide gas sterilization is preferred.

[0153] The cells cultured using the cell culture medium of this embodiment are not particularly limited as long as they are capable of adhering to a surface before stimulation is applied by lowering the temperature. Examples include various cell lines such as CHO cells derived from Chinese hamster ovaries, mouse connective tissue L929 cells, HEK293 cells derived from human fetal kidneys, and HeLa cells derived from human cervical cancer; as well as epithelial cells, endothelial cells, contractile skeletal muscle cells, smooth muscle cells, and cardiomyocytes that constitute various tissues and organs in an organism; neurons, glial cells, and fibroblasts that constitute the nervous system; liver parenchymal cells, non-parenchymal liver cells, and adipocytes that participate in the metabolism of an organism; stem cells existing in various tissues, such as mesenchymal stem cells, bone marrow cells, and Muse cells, which are cells with differentiation capacity; and pluripotent stem cells (multipotent stem cells) such as ES cells and iPS cells, and cells induced from their differentiation.

[0154] The cell culture medium of this embodiment can be manufactured, for example, by a manufacturing method comprising the following steps: a coating step, wherein at least a portion of the surface of a substrate is coated with a composition containing a hydrophilic polymer to form a layer containing a hydrophilic polymer; and a patterning step, wherein a portion of the surface of the layer containing the hydrophilic polymer is subjected to plasma treatment to form region (A) in the plasma-treated portion.

[0155] In the coating process, at least a portion of the surface of a substrate is coated with a composition containing a hydrophilic polymer to form a layer containing the hydrophilic polymer. By forming a layer containing the hydrophilic polymer, a state in which cell adhesion and cell proliferation are absent can be achieved. The method for forming the layer containing the hydrophilic polymer is not particularly limited; for example, a method of forming the layer by coating at least a portion of the surface of the substrate with a composition containing the hydrophilic polymer can be used. Various commonly known methods can be used for coating the composition containing the hydrophilic polymer, such as coating, brush coating, dip coating, spin coating, rod coating, flow coating, spray coating, roller coating, air knife coating, doctor blade coating, gravure coating, microgravure coating, slot coating, etc.

[0156] Figure 3 This is a schematic diagram (three-dimensional view) of a substrate with a layer containing hydrophilic polymers formed on its surface after the coating process. Figure 3 In this process, a layer 2 containing hydrophilic polymers is formed on one side of the surface of the substrate 1.

[0157] When the hydrophilic polymer is reactive to active energy rays, the coating process may include: irradiating the layer containing the hydrophilic polymer with active energy rays and immobilizing the layer containing the hydrophilic polymer on the surface of a substrate. Examples of hydrophilic polymers reactive to active energy rays include those having functional groups reactive to such active energy rays (e.g., UV rays, electron beams). Examples of active energy rays include UV rays and electron beams.

[0158] By irradiating a hydrophilic polymer that is reactive to reactive energy rays with reactive energy rays, a chemical reaction is induced between the hydrophilic polymers or between the hydrophilic polymer and the substrate, thereby immobilizing a layer containing the hydrophilic polymer onto the surface of the substrate. By immobilizing the layer containing the hydrophilic polymer onto the substrate surface, when coating a composition containing a temperature-responsive polymer in the temperature-responsive layer formation process described later, a layer containing the temperature-responsive polymer can be formed without deforming the layer containing the hydrophilic polymer. Furthermore, by immobilizing the layer containing the hydrophilic polymer onto the substrate surface, the shape of region (A) formed in the patterning process described later can also be maintained.

[0159] The patterning process involves plasma treating a portion of the surface of a layer containing a hydrophilic polymer to form region (A) in the plasma-treated portion. The plasma irradiation time is preferably 5 seconds to 10 minutes, more preferably 10 seconds to 5 minutes, and most preferably 30 seconds to 3 minutes. Air, nitrogen, oxygen, or water vapor are preferably used as the inlet gas during plasma treatment, with oxygen or water vapor being more preferred. The pressure of the inlet gas is preferably 1 Pa to less than 20 Pa, more preferably 1 Pa to 15 Pa, and even more preferably 1 Pa to 10 Pa. By avoiding excessively increasing the pressure, it is easier to set the tilt angle of the tilted surface in region (A) within the aforementioned preferred range. Furthermore, from the perspective of making it easier to set the tilt angle of the tilted surface in region (A) within the aforementioned preferred range, the plasma treatment is particularly preferably performed by reactive ion etching (RIE). RIE is also known as anisotropic etching. Furthermore, there are no particular limitations on the method for patterning region (A). For example, a method can be used to perform plasma processing on the desired part (the unmasked part) while it is covered by a metal mask, silicon mask, surface protective film, etc.

[0160] Figure 4 This is a schematic diagram (3D view) of the cell culture medium after the patterning process. Figure 4 In the cell culture medium 10 shown, there are equally spaced circular regions (region A shown, for example, the area of ​​this region is 0.001 mm²). 2 Above and 5mm 2 The area shown in A corresponds to the part that has undergone plasma treatment. The surface of layer 2 containing hydrophilic polymers in the region A is modified by plasma treatment, thereby exhibiting cell adhesion and cell proliferation properties. Additionally, the area outside the circular region (represented by region B) corresponds, for example, to the part protected by a metal mask or the like and not subjected to plasma treatment. The surface of region B is layer 2 containing hydrophilic polymers, and therefore does not exhibit cell adhesion or cell proliferation properties.

[0161] After the patterning process, a process can be performed to clean the aforementioned hydrophilic polymer with a solvent, dissolving and removing the unfixed hydrophilic polymer from the surface of the substrate. By removing the unfixed hydrophilic polymer, the surface of the substrate can be exposed, forming region (A). As the solvent used, it is preferable to contain water and an alcohol, considering its suitability for removing compounds byproducts of the self-reaction of hydrophilic polymers that are reactive to active energy rays, such as compounds containing azido-derived azene. By using a mixed solvent of water and alcohol for cleaning, the components derived from hydrophilic polymers remaining in region (A) can be reduced, thereby improving cell adhesion and cell proliferation in region (A). As the alcohol, lower alcohols are preferred, such as methanol, ethanol, 2-propanol, tert-butanol, isobutanol, pentanol, and hexanol, with methanol or ethanol being more preferred. Furthermore, the content of the alcohol is preferably 50-95%, more preferably 50-90%, particularly preferably 60-90%, and most preferably 70-90%.

[0162] It should be noted that the manufacturing method in other embodiments may also omit the patterning process and form the shape of region (A) solely through a coating process. In this case, the coating method in the coating process can utilize, for example, inkjet printing, injection printing such as on-demand printing, flexographic printing, letterpress printing such as letterpress printing, gravure printing, pad printing, offset printing, screen printing, etc. The process of modifying region (A) to make it a region with cell adhesion and cell proliferation properties can be performed before or after the coating process. Surface modification can be performed, for example, by corona discharge treatment, plasma irradiation, ultraviolet irradiation, etc.

[0163] In addition to the covering process and the patterning process, the manufacturing method of this embodiment may also include a bonding process and a temperature-responsive layer formation process as needed.

[0164] The bonding process occurs after the patterning process, where a cross-sectional area of ​​0.05 cm² in the in-plane direction is applied. 2 Above and 100cm 2 The following process involves bonding a plate with through holes to a substrate on the side covered by a layer containing a hydrophilic polymer. This is achieved by using a cross-sectional area of ​​0.05 cm² in the in-plane direction. 2 Above and 100cm 2 The following through-hole plate is bonded to the substrate, enabling the production of plates with spaces for inserting culture media in high-volume production. Figure 5 This is a schematic diagram (3D view) of the cell culture medium after the bonding process. Figure 5 The cell culture medium 11 shown is, for example, in Figure 4 The cell culture medium 10 shown is fitted with a separator 20 (with an in-plane cross-sectional area of ​​0.05 cm²).2 Above and 100cm 2 It is made of a plate with the following through holes. It has an in-plane cross-sectional area of ​​0.05 cm². 2 Above and 100cm 2 The following through-hole plate can be, for example, a partition member having multiple cylindrical partition walls with faces capable of dividing the sides of cultured cells. The cross-sectional area is 0.05 cm². 2 Above and 100cm 2 The sidewalls of the through holes below function as cylindrical partition walls.

[0165] The temperature-responsive layer formation process is a step following the patterning process, where the surface of a plasma-treated layer containing hydrophilic polymers is covered with a composition containing temperature-responsive polymers to form a layer containing temperature-responsive polymers. In this process, by forming a layer thickness of less than 100 nm, the molecular chains of the temperature-responsive polymers readily cover the surface of the formed region (A) in a sparse manner, which is suitable for imparting temperature responsiveness while maintaining the function of region (A) (cell adhesion and cell proliferation). Furthermore, by making the layer thickness of the temperature-responsive polymer layer 1 nm or more, it is preferable to manufacture cell culture media that can impart sufficient temperature responsiveness and rapidly form cell aggregates.

[0166] As a method for coating with a composition containing a temperature-responsive polymer, it is preferable to use the same method as the aforementioned coating method for a composition containing a hydrophilic polymer.

[0167] As a method of coating with a composition containing a temperature-responsive polymer, it is preferable to coat the entire surface of the cell culture medium with the temperature-responsive substance. When coating with the composition containing the temperature-responsive polymer, the mass production of the cell culture medium can be improved by using a conventional coating method without patterning. Furthermore, by coating the entire surface of the cell culture medium with the composition containing the temperature-responsive polymer, region (A) is given temperature responsiveness, while region (B) is also covered with the temperature-responsive polymer. By coating region (B) with the temperature-responsive polymer, cell adhesion in region (B) can be reduced.

[0168] The cell culture kit of this embodiment includes a cell culture medium. This cell culture medium may have a partition member having multiple cylindrical partition walls capable of dividing the sides of the cultured cells.

[0169] In addition to cell culture media, the cell culture kit of this embodiment may also contain temperature-responsive polymers or coating agents containing temperature-responsive polymers. This allows researchers performing the culture to easily adjust the thickness of the temperature-responsive polymer layer according to the type of cell.

[0170] The coating agent may contain a solvent. Examples of solvents that may be included in the coating agent include water, organic solvents, or mixtures thereof. Examples of organic solvents include alcohols such as methanol, ethanol, 1-propanol, 2-propanol, and 1-butanol; acetonitrile, formamide, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, 1,4-dioxane, and methyl ethyl ketone. From the perspective of achieving a uniform coating film thickness, a mixture of water and alcohols is preferred. Based on the total mass of the coating agent, the content of the temperature-responsive polymer can be set to 0.1–50% by weight, 0.2–10% by weight, or 0.5–5% by weight.

[0171] The coating agent may contain components other than temperature-responsive polymers and solvents. Examples of other components include those used to improve cell adhesion, such as polymers composed solely of water-insoluble block segments.

[0172] Example

[0173] The present invention will now be described in detail based on embodiments. However, this disclosure is not limited to the following embodiments. It should be noted that, unless otherwise stated, commercially available reagents are used.

[0174] [Example 1]

[0175] 0.9 mL of an 80 wt% aqueous ethanol solution containing 0.6 wt% polyvinyl alcohol (BIOSURFINE(R)-AWP, manufactured by Toyosei Kogyo Co., Ltd.) with a zizonated group as a hydrophilic polymer was added dropwise to a polycarbonate film (trade name: PANLITE (registered trademark), manufactured by Teijin Co., Ltd., thickness 0.18 mm) (substrate). Spin coating was performed using a spin coater (trade name: MS-B150, manufactured by MIKASA Co., Ltd.) at 2000 rpm for 60 seconds. The film was then left to stand under a high-pressure mercury lamp for 1 hour, thereby curing the hydrophilic polymer by UV irradiation, forming a hydrophilic polymer layer (layer thickness 75 nm). A metal mask with multiple circular holes (dots) of 0.2 mm in diameter (center-to-center distance 800 µm) was placed on a hydrophilic polymer layer. Plasma treatment was performed from above the metal mask using a plasma irradiation device (Samco Corporation, trade name: Aqua Plasma Cleaner AQ-500). This formed region (A) in the plasma-treated area. Region (B) was formed in the area masked by the metal mask. The plasma treatment conditions are shown in Table 1. In Table 1, "Anisotropic (RIE)" refers to plasma treatment that combines ion collision effects with a free radical-based chemical reaction by applying a high-frequency voltage to a device cavity containing the substrate. On the other hand, "Isotropic" refers to plasma treatment that utilizes a free radical-based chemical reaction without applying a high-frequency voltage to the device cavity.

[0176] [Examples 2-4]

[0177] Except for the changes in plasma treatment conditions as shown in Table 1, the cell culture medium was prepared in the same manner as in Example 1.

[0178] [Example 5]

[0179] The cell culture medium was prepared in the same manner as in Example 1, except that a plasma irradiation device (manufactured by Samco Corporation, trade name: 10-NR) was used instead of a plasma irradiation device (manufactured by Samco Corporation, trade name: Aqua Plasma (registered trademark) Cleaner AQ-500), and the plasma treatment conditions were as shown in Table 1.

[0180] [Example 6]

[0181] Except for using an ICP plasma irradiation device, a metal mask (with multiple circular holes (dots) of 0.1 mm in diameter and a center-to-center distance of 500 µm) was used instead of a metal mask (with multiple circular holes (dots) of 0.2 mm in diameter and a center-to-center distance of 800 µm) was used instead of a metal mask (with multiple circular holes (dots) of 0.2 mm in diameter and a center-to-center distance of 800 µm) and the plasma treatment conditions are shown in Table 1, cell culture materials were prepared in the same manner as in Example 1.

[0182] [Example 7]

[0183] The cell culture medium used in Example 6 was stored at 15-30°C and 40-90%RH for 6 months.

[0184] [Comparative Example 1]

[0185] Except that a plasma irradiation device (manufactured by SAKIGAKE Semiconductor Co., Ltd., trade name: Plasma Etcher-CPE-400) was used instead of a plasma irradiation device (manufactured by Samco Co., Ltd., trade name: Aqua Plasma (registered trademark) Cleaner AQ-500), and a metal mask (with multiple circular holes (dots) of 0.1 mm in diameter and a center-to-center distance of 500 µm) was used instead of a metal mask (with multiple circular holes (dots) of 0.2 mm in diameter and a center-to-center distance of 800 µm) was used instead of a metal mask, and the plasma treatment conditions are shown in Table 1, the cell culture medium was prepared in the same manner as in Example 1.

[0186] [Comparative Examples 2-4]

[0187] The cell culture medium was prepared in the same manner as in Example 1, except that a hydrophilic polymer layer was not formed and the plasma treatment conditions were changed as shown in Table 1.

[0188] [Comparative Example 5]

[0189] The polycarbonate membranes used in Examples 1-6 and Comparative Examples 1-4 (without hydrophilic polymer layer, without plasma treatment) were employed.

[0190] [Comparative Example 6]

[0191] The polycarbonate membrane with a hydrophilic polymer layer used in Examples 1-6 and Comparative Examples 1-4 (layer thickness: 75 nm, without plasma treatment) was used.

[0192] Evaluation of Cell Culture Materials

[0193] (Determination of the thickness of the hydrophilic polymer layer)

[0194] The thickness of the hydrophilic polymer layer formed on the substrate surface was calculated based on the substrate surface area, the concentration of the hydrophilic polymer in the solution, and the volume of solution added to the substrate. The specific gravity of the polymer was set to 1, and the coverage per unit area was calculated. The results are shown in Table 1.

[0195] (Evaluation of the shape of region (A))

[0196] The shape of region (A) formed by plasma-based etching was determined using a stylus profilometer (manufactured by Bruker Co., Ltd., trade name: DEKTAK XT). Cell culture medium was fixed to a glass substrate with adhesive tape, and the measurement was performed under the following conditions. It should be noted that the scanning direction was set along the axis passing through the center (centroid) of the bottom surface of region (A).

[0197] Z-axis measurement range: 6.5µm

[0198] X-axis measurement range: 500µm

[0199] Measurement time: 30 seconds

[0200] Load capacity: 1mg

[0201] The maximum depth (nm), etching tilt (µm-Z axis / mm-X axis), and tilt angle (°) are determined from the obtained profile.

[0202] The maximum depth is defined as the vertical distance from the point on the opposite side of the interface between the hydrophilic polymer layer and the substrate. That is, it is synonymous with the out-of-plane distance between the bottom surface of region (A) and the surface of region (B).

[0203] The etching tilt is the slope of an approximate straight line obtained by approximating the tilt surface of region (A) within the range of 20% to 70% of the depth when the maximum depth is set to 100% (least square method).

[0204] The tilt angle is the angle between the bottom surface of region (A) and the approximate straight line.

[0205] The results are shown in Table 2.

[0206] (Evaluation of the surface functional group content in region (A))

[0207] The surface functional group content of the (A) region formed by plasma treatment was determined using X-ray photoelectron spectroscopy. The polycarbonate film was subjected to full-surface plasma or RIE treatment under the conditions shown in Table 1, and the sample was prepared by gas-phase chemical modification with trifluoroethanol, di-tert-butylcarbodiimide, and pyridine. Measurements were performed using a PH15000 VersaProbe II (manufactured by ULVAC·PHI) under the following conditions.

[0208] X-ray source: Monochromatic Al-Kα rays (output: 25W)

[0209] Analysis area: 1000×300µm

[0210] Energy resolution: Wide-scan spectrum 117.40 eV

[0211] High-resolution spectrum 93.90 eV

[0212] Charge correction: C1s main peak (284.8 eV)

[0213] Based on the following formula derived from the reaction equation of gas-phase chemical modification, calculate the carbon ratio (R) of the carboxyl group relative to the total carbon in region (A). COOH ).

[0214] R COOH (%) = (F concentration (atom%)) / (3 × C concentration (atom%) - 2 × F concentration (atom%)) / r COOH ×100

[0215] r COOH Reaction rate (calculated from polyacrylic acid (standard))

[0216] The results are shown in Table 3. It should be noted that in Comparative Examples 5 and 6, the region near the center of the membrane was measured.

[0217] (Evaluation of water contact angle)

[0218] The water contact angle of the plasma-modified membrane surface was evaluated using a contact angle measuring machine (model: DM300, manufactured by Kyowa Interface Science Co., Ltd.). Polycarbonate membranes were treated under the conditions shown in Table 1, and test samples were prepared. 1 µL of water was dropped onto the surface-modified area, and the water contact angle (°) was measured after 20 seconds. The results are shown in Table 3. It should be noted that in Comparative Examples 5 and 6, the measurement was performed in the area near the center of the membrane.

[0219] (Evaluation of the shape of the opening)

[0220] A solution of fluorescently labeled cell culture protein matrix (manufactured by Matrixome Co., Ltd., trade name: iMatrix-511) was added to the opening (region (A)) to allow the protein matrix to be adsorbed. After removing the protein matrix solution and washing with PBS (-) (Fujifilm and Koujun Pharmaceutical Co., Ltd.), the cell culture medium was observed and fluorescent images were captured using a fluorescence microscope from the hydrophilic polymer layer side in a direction orthogonal to the substrate. The major axis (µm) and minor axis (µm) of the fluorescence images corresponding to each opening were measured, and the aspect ratio (major axis (µm) / minor axis (µm)) was calculated. Measurements were performed on each cell culture medium at n numbers of approximately 1200-1300, and the average value was calculated. Additionally, the CV (standard deviation divided by the average value and expressed as a percentage) was also calculated. The results are shown in Table 2. It should be noted that, since the protein matrix is ​​adsorbed on the inclined and bottom surfaces of region (A), the fluorescence image of the cell culture medium observed from the hydrophilic polymer layer side in a direction orthogonal to the substrate becomes the shape of the opening (the planar region defined by the boundary between region (A) and region (B)).

[0221] (Cell Culture Evaluation)

[0222] Cell culture medium was attached to the bottom of a bottomless 6-well plate and sterilized, serving as the culture container. Human iPS cell line 201B7 was used to achieve a cell density of 15,000 cells / cm². 2 Cells were inoculated using AK02N medium (manufactured by Ajinomoto Co., Ltd.) (2 mL / well) and cultured at 37°C with 5% CO2. Twenty-four hours after inoculation, Y-27632 (manufactured by Wako Pure Chemical Industries, Ltd.) (10 µM) and a protein matrix for cell culture (manufactured by Matrixome Co., Ltd., trade name: iMatrix-511) (1.25 µg / mL) were added to the medium. The medium was changed at 1, 3, and 5 days after culture began. Six days after culture began, the shape of cell aggregates was evaluated using a phase-contrast microscope. The size of the cell aggregates (average of the major axis (µm) and minor axis (µm)) was measured, and the hypertrophy rate (%) was calculated. The hypertrophy rate was defined as the percentage obtained by dividing the size of the cell aggregates by the diameter of the points on the metal mask. The size of the cell aggregates and the hypertrophy rate were measured for each cell culture medium at n=15, and the average values ​​were calculated. The results are shown in Table 2.

[0223] [Table 1]

[0224]

[0225] [Table 2]

[0226]

[0227] [Table 3]

[0228]

[0229] The cell culture media of Examples 1-3 and 5-7, which formed region (A) through anisotropic etching, have large tilt angles (85.9° to 88.7°) and form clear uneven shapes. Furthermore, although it is isotropic etching, the cell culture media of Example 4, which formed region (A) by etching with reduced gas pressure, has a significantly larger tilt angle compared to the cell culture media of Comparative Example 1, which had high gas pressure, and forms clear uneven shapes. Additionally, the aspect ratio of the opening shape of the cell culture media of Examples 1-7 is 1.1 or less. Furthermore, the hypertrophy rate of cell aggregates in the cell culture media of Examples 1-5 is 140% or less, and the hypertrophy rate of cell aggregates in the cell culture media of Examples 6-7 is 165% or less. This can be understood as forming region (A) with a size and shape close to the target size and shape (circular holes with diameters of 0.2 mm and 0.1 mm in the metal mask used). In particular, the cell culture substrates of Examples 1-3 and 5-7 have smaller aspect ratios (CV) at the openings, resulting in more uniform size and shape. Furthermore, regarding the diameter-to-maximum depth ratio of the opening in region (A), the substrates treated with anisotropic etching and isotropic etching under low-pressure conditions exhibit values ​​in the range of 500 to 3000, displaying a clear uneven shape. On the other hand, in Comparative Example 1, it was confirmed that the etching tilt angle was small and the shape was unclear. Additionally, in Comparative Examples 2-4, since region (B) was absent, cells non-specifically adhered and proliferated outside region (A), making size control difficult. Furthermore, in Comparative Examples 5-6, since region (A) was absent, cells did not adhere and could not be cultured. The carbon ratio (R) of carboxyl groups to total carbon on the surface of the substrates etched under the conditions of Examples 1-7 is... COOH The values ​​showed a value above 0.25%, indicating good culture performance. Furthermore, the cell culture results showed that using these cell culture media resulted in more stable cell aggregates of uniform size and shape.

[0230] Explanation of reference numerals in the attached figures

[0231] A…(A) region, A1…sloping surface, A2…bottom surface, B…(B) region, H…(A) region maximum depth, 1…substrate, 2…layer containing hydrophilic polymer, 10, 11…cell culture medium, 20…partition plate.

Claims

1. A cell culture medium, comprising: A substrate, and a layer containing a hydrophilic polymer covering at least a portion of the surface of the substrate. The cell culture medium has the following region (A) and the following region (B). (A) Regions with cell adhesion and cell proliferation (B) A region adjacent to region (A) that does not exhibit cell adhesion or cell proliferation. Region (A) is formed by a recess in the layer containing the hydrophilic polymer, the recess having an inclined surface and a bottom surface, the inclined surface having an inclination angle of 40° or more and 110° or less.

2. The cell culture medium according to claim 1, wherein, The maximum depth of region (A) is greater than 1 nm and less than 500 nm.

3. The cell culture medium according to claim 1 or 2, wherein, At least a portion of the bottom surface of region (A) is formed by the substrate.

4. The cell culture medium according to claim 1 or 2, wherein, The thickness of the layer containing the hydrophilic polymer is 10 nm or more and 500 nm or less.

5. The cell culture medium according to claim 1 or 2, wherein, The planar region defined by the boundary between region (A) and region (B) has an area of ​​0.001 mm². 2 Above and 5mm 2 An ellipse that is less than or equal to 1 and less than 1.1 in length and width.

6. The cell culture medium according to claim 5, wherein, The ratio (diameter / maximum depth) of the opening of region (A) to the maximum depth of region (A) is 500 or more and 3000 or less.

7. The cell culture medium according to claim 1 or 2, wherein, The minimum distance between the regions (A) is more than 400µm and less than 10000µm.

8. The cell culture medium according to claim 1 or 2, wherein, The water contact angle in region (A) is greater than 20° and less than 110°.

9. The cell culture medium according to claim 1 or 2, wherein, Region (A) was formed by reactive ion etching.

10. The cell culture medium according to claim 1 or 2, wherein, The carbon ratio (R) of the carboxyl groups in region (A) to the total carbon. COOH The percentage is above 0.25%.

11. The cell culture medium according to claim 1 or 2, wherein, The thickness of the substrate is 0.01 mm or more and 0.5 mm or less.

12. A method for manufacturing the cell culture medium according to claim 1, comprising: In the coating process, at least a portion of the surface of a substrate is coated with a composition containing a hydrophilic polymer to form the layer containing the hydrophilic polymer. In the patterning process, a portion of the surface of the layer containing the hydrophilic polymer is subjected to plasma treatment to form the region (A) in the plasma-treated portion.

13. The method for manufacturing cell culture medium according to claim 12, wherein, The hydrophilic polymer is reactive to active energy rays. The coating process includes: irradiating the layer containing hydrophilic polymers with active energy rays to immobilize the layer on the surface of the substrate.

14. The method for manufacturing cell culture medium according to claim 12 or 13, wherein, The plasma treatment is a reactive ion etching process.

15. The manufacturing method according to claim 12 or 13, wherein, After the patterning process, a cross-sectional area of ​​0.05 cm² in the in-plane direction is further formed. 2 Above and 100cm 2 The following is a bonding process in which the plate with the through hole is bonded to the substrate on the side of the substrate covered by the layer containing the hydrophilic polymer.

16. The manufacturing method according to claim 12 or 13, wherein, After the patterning process, there is a temperature-responsive layer forming process in which the surface of the plasma-treated layer containing the hydrophilic polymer is covered with a composition containing a temperature-responsive polymer to form a layer containing the temperature-responsive polymer.

17. A cell culture kit comprising: cell culture medium, The cell culture medium according to claim 1 or 2 is provided with a partition member, the partition member having a plurality of cylindrical partition walls capable of dividing the sides of the cultured cells.

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