Cell culture substrate, method for producing same, and cell culture kit

Abstract

The present invention relates to a cell culture substrate comprising a substrate and a layer containing a hydrophilic polymer that covers at least part of the surface of the substrate, where the cell culture substrate has an (A) region and a (B) region, the (A) region has cell adhesion and cell proliferation properties, and the (B) region is adjacent to the (A) region and does not have cell adhesion or cell proliferation properties, wherein: the (A) region comprises a recess formed in the layer containing the hydrophilic polymer; the recess has an inclined surface and a bottom surface; and the angle of inclination of the inclined surface is 40° to 110°.

Description

Cell culture substrate, its manufacturing method, and cell culture kit

[0001] The present invention relates to a cell culture substrate, a method for producing the same, and a cell culture kit.

[0002] Pluripotent stem cells, such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), have the ability to differentiate into various tissues in the body (pluripotency), and have attracted considerable attention as a cell source for regenerative medicine and drug discovery screening. To apply pluripotent stem cells to regenerative medicine and drug discovery screening, it is necessary to differentiate the pluripotent stem cells into the target cells, which requires the formation of cell aggregates of pluripotent stem cells. Furthermore, while pluripotent stem cells can differentiate into various cells, it is known that the optimal size of the cell aggregate varies depending on the type of cell after differentiation. Therefore, it is desirable to control the size and produce cell aggregates of uniform size.

[0003] As a cell culture substrate for forming cell aggregates, for example, Patent Document 1 discloses a cell culture substrate having a substrate and a stimuli-responsive polymer coated on the substrate, wherein the stimuli-responsive polymer is a block copolymer having a water-insoluble block segment and a stimuli-responsive block segment, and the cell culture substrate has (A) cell proliferation properties and stimuli-responsive properties, and has an area of ​​0.001 to 5 mm ２ and (B) an area adjacent to the area (A) where no cell proliferation is possible.

[0004] Japanese Patent Application Laid-Open No. 2020-62009

[0005] The cell culture substrate disclosed in Patent Document 1 has a specific (A) region and a specific (B) region, and in the (A) region, spheroids can be efficiently formed, and spheroids of uniform size and arbitrary shape can be formed. The cell culture substrate disclosed in Patent Document 1 utilizes plasma treatment (isotropic etching: 20 Pa gas pressure, 20 mA current, 10 seconds of irradiation) to form the (A) region. However, the inventors have found that the cell culture substrate produced by this method has room for improvement in terms of the uniformity of the shape of the (A) region and the clarity of the uneven shapes of the (A) and (B) regions. 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 more reliably obtained.

[0006] An object of the present invention is to provide a cell culture substrate that enables stable production of cell aggregates that are uniform in size and shape, and a method for producing the cell culture substrate.

[0007] The present invention relates to a cell culture substrate comprising a substrate and a layer containing a hydrophilic polymer that covers at least a portion of the surface of the substrate, and having the following region (A) and region (B): (A) a region that has cell adhesive properties and cell proliferation properties; and (B) a region that is adjacent to region (A) and does not have cell adhesive properties or cell proliferation properties. Region (A) comprises a recess formed in the layer containing the hydrophilic polymer, and the recess has an inclined surface and a bottom surface, and the inclination angle of the inclined surface is 40° or more and 110° or less.

[0008] The present invention also relates to a method for producing the above-mentioned cell culture substrate, which comprises a coating step of coating at least a portion of the surface of the substrate with a composition containing a hydrophilic polymer to form a layer containing the hydrophilic polymer, and a patterning step of performing a plasma treatment on a portion of the surface of the layer containing the hydrophilic polymer to form an (A) region in the plasma-treated portion.

[0009] The present invention further relates to a cell culture kit comprising the cell culture substrate of the present invention provided with a partition member having a plurality of cylindrical partition walls capable of dividing the surface on which cells are cultured.

[0010] The present invention includes, for example, the following inventions. [1] A cell culture substrate comprising a substrate and a layer containing a hydrophilic polymer covering at least a portion of the surface of the substrate, the cell culture substrate having the following regions (A) and (B): (A) a region having cell adhesiveness and cell proliferation properties, and (B) a region adjacent to the region (A) that does not have cell adhesiveness or cell proliferation properties. The region (A) comprises a recess formed in the layer containing the hydrophilic polymer, the recess having an inclined surface and a bottom surface, the inclination angle of the inclined surface being 40° to 110°. [2] The cell culture substrate according to [1], wherein the maximum depth of the region (A) is 1 nm to 500 nm. [3] The cell culture substrate according to [1] or [2], wherein at least a portion of the bottom surface of the region (A) is constituted by the substrate. [4] The cell culture substrate according to any of [1] to [3], wherein the layer containing the hydrophilic polymer has a thickness of 10 nm to 500 nm. [5] The planar area defined by the boundary between the area (A) and the area (B) has an area of ​​0.001 mm ２ More than 5 mm ２The cell culture substrate according to any one of [1] to [4], wherein the (A) region is an ellipse having a diameter (nm) of 100 to 1200 mm and an aspect ratio of 1 to 1.1. [6] The cell culture substrate according to any one of [1] to [5], wherein the ratio of the diameter (nm) of the opening of the (A) region to the maximum depth (nm) of the (A) region (diameter / maximum depth) is 500 to 3000. [7] The cell culture substrate according to any one of [1] to [6], wherein the minimum distance between the (A) regions is 400 μm to 10,000 μm. [8] The cell culture substrate according to any one of [1] to [7], wherein the (A) region has a water contact angle of 20° to 110°. [9] The cell culture substrate according to any one of [1] to [8], wherein the (A) region is formed by reactive ion etching.

[10] The cell culture substrate according to any one of [1] to [9], wherein the thickness of the substrate is 0.01 mm to 0.5 mm.

[11] The method for producing a cell culture substrate according to any one of [1] to

[10] , comprising: a coating step of coating at least a portion of the surface of a substrate with a composition containing a hydrophilic polymer to form a layer containing the hydrophilic polymer; and a patterning step of performing plasma treatment on a portion of the surface of the layer containing the hydrophilic polymer to form the region (A) in the plasma-treated portion.

[12] The method for producing a cell culture substrate according to

[11] , wherein the hydrophilic polymer is reactive to active energy rays, and the coating step comprises irradiating the layer containing the hydrophilic polymer with active energy rays to fix the layer to the surface of the substrate.

[13] The method for producing a cell culture substrate according to

[11] or

[12] , wherein the plasma treatment is reactive ion etching. [14 ... any one of [1] to

[10] , wherein the cross-sectional area in the in-plane direction after the patterning step is 0.05 cm. ２ More than 100cm ２

[15] The manufacturing method according to any one of

[11] to

[14] , further comprising a laminating step of laminating a plate having the following through-holes to the substrate on the side of the substrate coated with the layer containing the hydrophilic polymer.

[15] The manufacturing method according to any one of

[11] to

[14] , further comprising a temperature-responsive layer forming step of coating the surface of the layer containing the hydrophilic polymer, which has been subjected to plasma treatment, with a composition containing a temperature-responsive polymer, to form a layer containing the temperature-responsive polymer.

[16] A cell culture kit including a cell culture substrate, wherein the cell culture substrate is the cell culture substrate according to any one of [1] to

[10] , and is provided with a partition member having a plurality of cylindrical partitions that can partition the surface on which cells are cultured.

[17] The cell culture kit includes a cell culture substrate according to any one of [1] to

[10] , further comprising a ratio of carbon of carboxyl groups to total carbon (R ＣＯＯＨ The cell culture substrate according to any one of [1] to

[10] , wherein the content of the cellulose acetate layer is 0.25% or more.

[0011] According to the present invention, a cell culture substrate that enables stable production of cell aggregates with uniform size and shape can be provided. The present invention also provides a method for producing the cell culture substrate.

[0012] FIG. 1 is a schematic diagram (cross-sectional view) of a cell culture substrate according to one embodiment. FIG. 2 is a schematic diagram for explaining a method for calculating an inclination angle. FIG. 3 is a schematic diagram (perspective view) of a substrate having a layer containing a hydrophilic polymer formed on its surface after a coating step in a manufacturing method according to one embodiment. FIG. 4 is a schematic diagram (perspective view) of a cell culture substrate after a patterning step in a manufacturing method according to one embodiment. FIG. 5 is a schematic diagram (perspective view) of a cell culture substrate after a bonding step in a manufacturing method according to one embodiment.

[0013] Hereinafter, embodiments of the present invention will be described in detail. Note that the present invention is not limited to the following embodiments, and the following embodiments can be modified and implemented within the scope in which the above-described effects can be obtained.

[0014] As used herein, the term "cell aggregate" refers to a three-dimensional aggregate of cells formed by the aggregation of multiple cells. The shape of the three-dimensional aggregate may be an ellipsoid such as a sphere, or may be a hemisphere or other shape. These shapes may have gaps formed by folding a sheet of cells, or may be hollow. An example of a cell aggregate is a spheroid.

[0015] In this specification, "temperature responsive" means that the degree of hydrophilicity / hydrophobicity changes with temperature. Furthermore, the boundary temperature at which the degree of hydrophilicity / hydrophobicity changes is referred to as the "responsive temperature."

[0016] As used herein, the term "biologically derived substance" refers to a substance present in the body of a living organism, or a chemically synthesized substance equivalent to such a substance. Substances present in the body of a living organism may be natural products, or may be artificially synthesized using recombinant DNA technology or the like. There are no particular limitations on biologically derived substances, and examples include nucleic acids, proteins, and polysaccharides, which are the basic materials that make up living organisms, and their constituent elements, such as nucleotides, nucleosides, amino acids, and various sugars, as well as lipids, vitamins, and hormones.

[0017] As used herein, "cell adhesiveness" refers to the ease with which cells adhere to a substrate or cell culture substrate at culture temperature, and "having cell adhesiveness" refers to the ability of cells to adhere to a substrate or cell culture substrate at culture temperature, either directly or via a biologically derived substance. Furthermore, "not having cell adhesiveness" refers to the inability of cells to adhere to a substrate or cell culture substrate at culture temperature, either directly or via a biologically derived substance. Methods for assessing the presence or absence of cell adhesiveness are not particularly limited, and include, for example, adding the aforementioned biologically derived substance, which has been labeled with a fluorescent label or the like, to the region and confirming whether or not the substance is adsorbed, or measuring the water contact angle of the region.

[0018] As used herein, "cell proliferation" refers to the ease with which cells proliferate at the culture temperature, "having cell proliferation" refers to the ability of cells to proliferate at the culture temperature, and "not having cell proliferation" refers to the inability of cells to proliferate at the culture temperature. "High cell proliferation" refers to the proliferation of more cells when compared over the same culture period.

[0019] The cell culture substrate according to this embodiment comprises a substrate and a layer containing a hydrophilic polymer that covers at least a portion of the surface of the substrate, and has the following regions (A) and (B): (A) island-like regions that have cell adhesiveness and cell proliferation properties, and (B) a region that is adjacent to region (A) and does not have cell adhesiveness or cell proliferation properties. Region (A) consists of a recess formed in the layer containing the hydrophilic polymer, and the recess has an inclined surface and a bottom surface, and the inclination angle of the inclined surface is 40° or more and 110° or less.

[0020] FIG. 1 is a schematic diagram (cross-sectional view) of a cell culture substrate according to one embodiment. The cell culture substrate 10 shown in FIG. 1 includes a substrate 1 and a layer 2 containing a hydrophilic polymer that coats the surface of the substrate 1. In FIG. 1, A and B represent region (A) and region (B), respectively. Also, in FIG. 1, A1 and A2 represent the inclined surface and bottom surface of the recess, respectively, and A1 and A2 together constitute region (A). The thickness of the layer 2 containing a hydrophilic polymer refers to the distance from the surface where the substrate 1 and the layer 2 containing a hydrophilic polymer contact each other to the surface of region (B) (region B in FIG. 1). In addition, in the cell culture substrate 10 shown in FIG. 1, the bottom surface A2 of region (A) is the layer 2 containing a hydrophilic polymer, but this is not limited thereto. For example, at least a portion or all of the bottom surface A2 of region (A) may be substrate 1. Similarly, at least a portion of the inclined surface A1 of region (A) may also be substrate 1.

[0021] The maximum depth of the (A) region refers to the out-of-plane distance between the bottom surface of the (A) region and the surface of the (B) region. The maximum depth of the (A) region may be, for example, 1 nm or more and 2000 nm or less, or 1 nm or more and 500 nm or less. When the maximum depth of the (A) region is 500 nm or less, the number of dead cells captured by the unevenness can be further suppressed, and the number of dead cells mixed into the cell aggregate can be further reduced. This makes it possible to further increase the cell viability of the cell aggregate. Furthermore, when the maximum depth of the (A) region is 500 nm or less, adhesion of air bubbles to the unevenness portion can be further suppressed. Suppression of air bubble adhesion eliminates the need for degassing or repeated dispensing and aspirating of the medium using a pipettor to remove air bubbles, thereby further improving operability. When the maximum depth of the (A) region is 1 nm or more, live cells that spontaneously migrate (migrate) on the cell culture substrate are more likely to gather in the (A) region, thereby further increasing the cell viability of the cell aggregate. The maximum depth of region (A) is more preferably 400 nm or less, and even more preferably 350 nm or less, since this is suitable for further increasing the cell viability of the cell aggregates that are formed.

[0022] In this specification, the "inclination angle of the inclined surface" refers to the angle formed by the approximate curve obtained by linearly approximating the inclined surface in a range of 20% to 70% of the maximum depth of the (A) region on a plane perpendicular to the substrate along an axis passing through the center (center of gravity) of the bottom surface of the (A) region. FIG. 2 is a schematic diagram (cross-sectional view) for explaining a method for calculating the inclination angle. In FIG. 2, H indicates the maximum depth of the (A) region (i.e., the out-of-plane distance between the bottom surface A2 of the (A) region and the surface of the (B) region). The shape of the (A) region on a plane perpendicular to the substrate along an axis passing through the center (center of gravity) of the bottom surface of the (A) region can be measured, for example, using a stylus profiling system (manufactured by Bruker Corporation, product name: DEKTAK XT). The maximum depth H of the (A) region is used as a reference, and the inclined surface A1 is linearly approximated in a range of 20% (20% × H) to 70% (70% × H) of that. The linear approximation can be performed, for example, by the least squares method. Next, the angle θ between the obtained approximation line L and the bottom surface A2 of the (A) region is calculated. This angle θ is the inclination angle of the inclined surface. As shown in FIG. 2, if the shape of the (A) region narrows in a tapered manner as it approaches the bottom surface A2, the angle θ is greater than 0° and less than 90°. Also, if the shape of the (A) region widens in an inverse tapered manner as it approaches the bottom surface A2, the angle θ is greater than 90° and less than 180°.

[0023] In the cell culture substrate according to this embodiment, the inclination angle of the inclined surface is 40° or more and 110° or less. When the inclination angle of the inclined surface is within this range, the uneven shape becomes clear, and cell aggregates of uniform size and shape can be more reliably obtained. From the same viewpoint, the inclination angle of the inclined surface is preferably 50° or more, more preferably 60° or more, even more 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 inclination angle of the inclined 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 is 40° or more and 100° or less, 40° or more and 95° or less, 40° or more and 90° or less, 40° or more and less than 90°, 50° or more and 110° or less, 50° or more and 100° or less, 50° or more and 95° or less, 50° or more and 90° or less, 50° or more and less than 90°, 60° or more and 110° or less, 60° or more and 100° or less, 60° or more and 95° or less, 60° or more and 90° or less, 60° or more and less than 90°, 65° or more and 100° or less, 65° or more and 95° or less, 65° or more The inclination angle may be 90° or less, 65° or more but less than 90°, 70° or more but less than 110°, 70° or more but less than 100°, 70° or more but less than 95°, 70° or more but less than 90°, 75° or more but less than 110°, 75° or more but less than 100°, 75° or more but less than 95°, 75° or more but less than 90°, 80° or more but less than 110°, 80° or more but less than 100°, 80° or more but less than 95°, 80° or more but less than 90°, or 80° or more but less than 90°. Here, when region (A) is formed by etching such as plasma treatment described below, the stronger the treatment conditions, such as increasing the treatment time, the deeper region (A) and the larger the inclination angle tend to be. When the maximum depth of the (A) region is 1 nm or more and 500 nm or less and the inclination angle of the inclined surface is less than 90°, the cell culture substrate is excellent in mass productivity and can be used to stably obtain cell aggregates of uniform size and shape.

[0024] The substrate used for the cell culture substrate according to 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, cycloolefin polymer, cellulose acetate, nitrocellulose, and polyvinylidene fluoride, more preferably formed from at least one selected from the group consisting of polystyrene, polyethylene terephthalate, polycarbonate, and cycloolefin polymer, even more preferably formed from at least one selected from polystyrene, polyethylene terephthalate, and polycarbonate, and most preferably formed from polystyrene or polycarbonate.

[0025] Since the cell culture substrate is suitable for observing cells cultured on it using a high-magnification phase-contrast microscope, the refractive index of the substrate measured with the D-line (wavelength 589 nm) is preferably 1.4 to 1.6, more preferably 1.45 to 1.6, and particularly preferably 1.5 to 1.55. Having the refractive index of the substrate within these ranges reduces spherical aberration during phase-contrast microscopic observation of cells, allowing for clear phase-contrast images to be obtained. Furthermore, to reduce spherical aberration, the thickness of the substrate is preferably 0.5 mm or less, more preferably 0.4 mm or less, particularly preferably 0.3 mm or less, and most preferably 0.2 mm or less. On the other hand, since the thickness of the substrate is suitable for preventing the entire observation range from being out of focus due to bending of the substrate during microscopic observation, the thickness of the substrate is preferably 0.01 mm or more, more preferably 0.05 mm or more, particularly preferably 0.1 mm or more, and most preferably 0.15 mm or more.

[0026] The thickness of the substrate is preferably 0.01 mm to 0.5 mm, more preferably 0.05 mm to 0.4 mm, even more preferably 0.1 mm to 0.3 mm, and particularly preferably 0.15 mm to 0.2 mm. When the thickness of the substrate is within this range, the phase contrast image of the cells becomes clearer.

[0027] Because the cell culture substrate is suitable for observing cells cultured on it using a high-magnification fluorescence microscope, the fluorescence intensity (autofluorescence intensity) of the substrate at excitation wavelengths of 350 nm, 488 nm, and 647 nm (when irradiated with excitation light having these wavelengths) is preferably smaller than the fluorescence intensity (autofluorescence intensity) of a 1.2 mm thick polystyrene plate excited with light having the same excitation wavelength, more preferably 80% or less of the fluorescence intensity of a 1.2 mm thick polystyrene plate, particularly preferably 50% or less of the fluorescence intensity of a 1.2 mm thick polystyrene plate, and most preferably 10% or less of the fluorescence intensity of a 1.2 mm thick polystyrene plate. Fluorescent dyes excited at excitation wavelengths of 350 nm, 488 nm, and 647 nm are frequently used in fluorescent observation of cells. By keeping the autofluorescence intensity of the substrate at these wavelengths below a certain value, clear fluorescent images of cells can be obtained.

[0028] The shape of the substrate is not particularly limited, and may be a planar shape such as a plate or a film, or may be a shape such as a fiber, porous particle, porous membrane, hollow fiber, etc. The shape of the substrate may also be the shape of a container generally used for cell culture, etc. (cell culture dish such as a Petri dish, a flask, a plate, a bag, etc.) From the viewpoint of ease of culture operation, the shape of the substrate is preferably a planar shape such as a plate or a film, or a flat porous membrane shape.

[0029] The thickness of the hydrophilic polymer-containing layer may be, for example, 10 nm to 500 nm. Having a thickness of 10 nm to 500 nm allows cells to adhere and proliferate only in region (A), and facilitates cell migration to region (A), thereby increasing the cell viability of cell aggregates. Here, the "thickness" of the hydrophilic polymer-containing layer refers to the out-of-plane length from the interface between the substrate and the hydrophilic polymer-containing layer to the interface of the hydrophilic polymer-containing layer on the opposite side of the substrate (excluding region (A)). For layer thicknesses exceeding 10 nm, cross-sectional images of ultrathin sections of the cell culture substrate prepared with a microtome are measured using a transmission electron microscope, and the distances at 10 randomly selected points are measured and averaged to calculate the distance. Furthermore, for layer thicknesses of 10 nm or less, measurements can be made using an ellipsometer. Since it is suitable for suppressing adhesion of cells to the (B) region, the layer thickness is more preferably 10 nm or more, and even more preferably 30 nm or more, 40 nm or more, or 50 nm or more. Furthermore, since it is suitable for increasing the cell viability of the cell aggregate by collecting cells in the (A) region through cell migration, the layer thickness is more preferably 200 nm or less, and even more preferably 180 nm or less, 160 nm or less, 150 nm or less, 140 nm or less, or 120 nm or less. The layer thickness of the layer containing a hydrophilic polymer may be, for example, 10 nm or more to 200 nm or less, 10 nm or more to 180 nm or less, 10 nm or more to 160 nm or less, 10 nm or more to 150 nm or less, 10 nm or more to 140 nm or less, 10 nm or more to 120 nm or less, 30 nm or more to 200 nm or less, 30 nm or more to 180 nm or less, 30 nm or more to 160 nm or less, 30 nm or more to 150 nm or less, 30 nm or more to 140 nm or less, 30 nm or more to 1 The thickness may be 20 nm or less, 40 nm or more to 200 nm or less, 40 nm or more to 180 nm or less, 40 nm or more to 160 nm or less, 40 nm or more to 150 nm or less, 40 nm or more to 140 nm or less, 40 nm or more to 120 nm or less, 50 nm or more to 200 nm or less, 50 nm or more to 180 nm or less, 50 nm or more to 160 nm or less, 50 nm or more to 150 nm or less, 50 nm or more to 140 nm or less, or 50 nm or more to 120 nm or less.

[0030] The hydrophilic polymer contains a phosphorylcholine group or a hydroxyl group. By including a phosphorylcholine group or a hydroxyl group in the hydrophilic polymer, it is possible to make the area coated with the hydrophilic polymer an area to which cells do not adhere. In addition, since such hydrophilic polymers do not need to be completely decomposed and removed, by performing plasma treatment, the area can be made into an area with cell adhesiveness and cell proliferation properties, and cells are less likely to be contaminated with decomposition products of the hydrophilic polymer. Other than containing a phosphorylcholine group or a hydroxyl group, the type of hydrophilic polymer is not particularly limited. Commercially available products include, for example, Lipidure® CM5206 (manufactured by NOF Corporation), Lipidure® CM2001 (manufactured by NOF Corporation), and BIOSURFINE®-AWP (manufactured by Toyo Gosei Co., Ltd.). In addition, examples of commercially available substrates coated with a hydrophilic polymer that can be suitably used include PrimeSurface® (manufactured by Sumitomo Bakelite Co., Ltd.), EZ-BindShut® (manufactured by AGC Technoglass Co., Ltd.), and EZ-BindShutII® (manufactured by AGC Technoglass Co., Ltd.).

[0031] The hydrophilic polymer preferably contains 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).

[0032] [In general formula (1), R １ and R ２ each independently represents a hydrogen atom or a methyl group, R ３ represents a hydrogen atom or an arbitrary organic group, and m and n each independently represent a positive integer.

[0033] [In general formula (2), R ４ , R ５ and R ６ each independently represents a hydrogen atom or a methyl group, R ７ represents a hydrogen atom or an arbitrary organic group, and x, y, and z each independently represent a positive integer. [In general formula (3), R ８ and R ９ each independently represents a hydrogen atom or a methyl group, R １０represents a hydrogen atom or an arbitrary organic group, and a and b each independently represent a positive integer.

[0034] When the hydrophilic polymer contains a compound represented by the general formula (1), a compound represented by the general formula (2), or a compound represented by the general formula (3), cells can be easily attached and proliferated, and it is suitable for forming cell aggregates of uniform shape in the (A) region. ３ , R ７ , and R １０ are suitable for immobilizing a hydrophilic polymer on a substrate, and therefore are preferably hydrophobic groups or functional groups reactive to active energy rays (e.g., UV, electron beams, etc.). As the hydrophobic group, linear or cyclic alkyl groups such as methyl, ethyl, propyl, butyl, and cyclohexyl groups can be preferably used. Furthermore, as the functional group reactive to active energy rays (e.g., UV, electron beams, etc.), azide groups, acrylate groups, methacrylate groups, vinyl groups, and epoxy groups can be used, and an azide group can be preferably used.

[0035] Region (A) consists of a recess formed in a layer containing a hydrophilic polymer. Here, the recess has an inclined surface and a bottom surface. In the cell culture substrate according to this embodiment, the inclination angle of the inclined surface in region (A) is 40° or more and 110° or less. In region (A), both the inclined surface and the bottom surface may be in the layer containing a hydrophilic polymer, or part of the inclined surface and / or part or all of the bottom surface may be in the substrate.

[0036] Region (A) is recognized as an island-like region when the cell culture substrate is observed from the hydrophilic polymer-containing layer side in a direction perpendicular to the substrate. In this case, the periphery 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 periphery of the island-like region) is sometimes referred to as an opening. In FIG. 1, φ indicates the diameter of the opening (also referred to as the spot diameter in this specification). Note that an island-like region indicates that the region exists independently from other regions. When region (A) is an island-like region as described above, living cells are concentrated in region (A) compared to regions that are not island-like (e.g., striped structures, etc.), allowing for more efficient production of cell aggregates.

[0037] 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 set appropriately depending on the shape of the desired cell aggregate, and examples include an ellipse (including a circle), a polygon, or a closed shape formed by straight lines and curves. Furthermore, since this is suitable for producing cell aggregates with a shape close to a sphere, 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, even more preferably an ellipse (including a circle) or a square, and most preferably an ellipse (including a circle).

[0038] Since this is suitable for producing cell aggregates with a shape close to a sphere, 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 to 2, more preferably 1 to 1.5, even more preferably 1 to 1.1, and most preferably 1 to 1.05. Here, "aspect ratio" refers to the ratio of the maximum diameter (major diameter) to the minimum diameter (minor diameter) of the shape, i.e., major diameter / minor diameter.

[0039] Since this is suitable for producing cell aggregates of uniform size and shape, it is preferable that the standard deviation / average aspect ratio of the aspect ratio of the shape of the opening (the planar area defined by the boundary between area (A) and area (B)) is 80% or less, more preferably 50% or less, even more preferably 20% or less, and most preferably 5% or less.

[0040] The area of ​​the opening (the planar area defined by the boundary between the (A) region and the (B) region) is, for example, 0.001 mm ２ More than 5 mm ２ The area may be 0.005 mm or less. ２ More than 1 mm ２ Preferably, the area is 0.01 mm or less. ２ More than 0.5 mm ２ More preferably, the area is 0.015 mm or less. ２ 0.25mm or more ２ More preferably, the area is 0.02 mm or less. ２ More than 0.2 mm ２ The following are most preferred:

[0041] Since this is suitable for producing cell aggregates of uniform size and shape, it is preferable that the standard deviation / average area of ​​the opening (the planar area defined by the boundary between area (A) and area (B)) is 80% or less, more preferably 50% or less, even more preferably 20% or less, and most preferably 5% or less.

[0042] The shape of the bottom surface of region (A) may be the same as or different from the shape of the opening. The shape of the bottom surface of region (A) is not particularly limited and can be set appropriately depending on the shape of the desired cell aggregate, but examples include an ellipse (including a circle), a polygon, or a closed shape formed by straight lines and curves. Furthermore, since this is suitable for producing cell aggregates with a shape close to a sphere, 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, even more preferably an ellipse (including a circle) or a square, and most preferably an ellipse (including a circle).

[0043] The aspect ratio of the shape of the bottom surface of region (A) may be the same as or different from the aspect ratio of the shape of the opening. Because this is suitable for producing cell aggregates with a shape close to a sphere, the aspect ratio of the shape of the bottom surface of region (A) is preferably 1 to 2, more preferably 1 to 1.5, even more preferably 1 to 1.1, and most preferably 1 to 1.05.

[0044] Since this is suitable for producing cell aggregates of uniform size and shape, it is preferable that the standard deviation / average aspect ratio of the aspect ratio of the shape of the bottom surface of region (A) is 80% or less, more preferably 50% or less, even more preferably 20% or less, and most preferably 5% or less.

[0045] The area of ​​the bottom surface of the (A) region may be the same as or different from the area of ​​the opening. The area of ​​the bottom surface of the (A) region is, for example, 0.001 mm ２ More than 6mm ２ The area may be 0.001 mm or less. ２ More than 5 mm ２ Preferably, 0.005 mm or less ２ More than 1 mm ２ More preferably, the area is 0.01 mm or less. ２ More than 0.5 mm ２ More preferably, the area is 0.015 mm or less. ２ 0.25mm or more ２ Even more preferably, the area is 0.02 mm or less. ２ More than 0.2 mm ２ The following are most preferred:

[0046] Since this is suitable for producing cell aggregates of uniform size and shape, it is preferable that the standard deviation / average area of ​​the bottom surface of region (A) is 80% or less, more preferably 50% or less, even more preferably 20% or less, and most preferably 5% or less.

[0047] The ratio (diameter / maximum depth) of the diameter (nm) of the opening of region (A) can be, for example, 100 or more and 20,000 or less. The diameter / maximum depth is preferably 500 or more and 3,000 or less. This makes the uneven shape clearer, and cell aggregates of uniform size and shape can be more consistently obtained. The diameter / maximum depth is more preferably 500 or more and 2,000 or less, and even more preferably 500 or more and 800 or less. Note that the "diameter of the opening" refers to the average of the major axis diameter (maximum diameter) and minor axis diameter (minimum diameter) of the opening. The diameter of the opening is indicated by φ in Figure 1.

[0048] Furthermore, since this is suitable for increasing the oxygen concentration around the cells and increasing the survival rate of the cell aggregates, the minimum distance between the (A) regions is preferably 400 μm or more and 10,000 μm or less, more preferably 500 μm or more and 8,000 μm or less, even more preferably 1,000 μm or more and 5,000 μm or less, and most preferably 2,000 μm or more and 4,000 μm or less.

[0049] Region (A) can be formed, for example, by forming a layer containing a hydrophilic polymer on the surface of a substrate and then modifying a portion of the surface of the layer containing the hydrophilic polymer by plasma treatment. Region (A) is a region in which the surface of the layer containing the hydrophilic polymer has been modified by plasma treatment or the like, so that region (A) has cell adhesive properties and cell proliferation properties. Region (A) can be patterned by plasma treatment or the like in a short time, thereby improving the mass productivity of cell culture substrates.

[0050] The plasma treatment described above can also be performed in the presence of an introduced gas such as oxygen, hydrogen, nitrogen, ammonia, or argon. Using the introduced gas described above generates hydrophilic functional groups in the material, forming regions with cell adhesiveness and cell proliferation properties. The types of functional groups include hydroxyl groups, carbonyl groups, aldehyde groups, carboxyl groups, ether bonds, ester bonds, amino groups, and nitro groups, depending on the introduced gas used in the plasma treatment. The use of oxygen is preferred because it generates a variety of functional groups, such as hydroxyl groups, carbonyl groups, aldehyde groups, carboxyl groups, ether bonds, ester bonds, and nitro groups from the aforementioned groups. The generation of carboxyl groups, which are highly polar functional groups, is particularly preferred because it significantly improves the hydrophilicity of the material and also improves cell adhesiveness and cell proliferation properties.

[0051] When the surface material of the cell culture substrate is an organic substance and the functional group generated is a carboxyl group, the carbon ratio of the carboxyl group to the total carbon in the (A) region (R ＣＯＯＨ ) is preferably 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. In addition, the carbon ratio of carboxyl groups to the total carbon in the (A) region (R ＣＯＯＨ ) may be, for example, 10% or less, 8% or less, 6% or less, 5% or less, 4% or less, or 2% or less. ＣＯＯＨ) is, for example, 0.25% or more and 10% or less, 0.25% or more and 8% or less, 0.25% or more and 6% or less, 0.25% or more and 5% or less, 0.25% or more and 4% or less, 0.25% or more and 2% or less, 0.3% or more and 10% or less, 0.3% or more and 8% or less, 0.3% or more and 6% or less, 0.3% or more and 5% or less, 0.3% or more and 4% or less, 0.3% or more and 2% or less, 0.5% or more and 10% or less, 0.5% or more and 8% or less, 0.5% or more and 6% or less, 0.5% to 5%, 0.5% to 4%, 0.5% to 2%, 0.8% to 10%, 0.8% to 8%, 0.8% to 6%, 0.8% to 5%, 0.8% to 4%, 0.8% to 2%, 1.2% to 10%, 1.2% to 8%, 1.2% to 6%, 1.2% to 5%, 1.2% to 4%, or 1.2% to 2%. ＣＯＯＨ ) is within the above range, it is possible to provide a cell culture substrate having the cell adhesiveness and proliferation properties described in the present application. ＣＯＯＨ From the viewpoint of reducing the rate of hypertrophy, it is preferable that the content of Cr is 0.7% or more and 1.0% or less.

[0052] In this specification, the carbon ratio of carboxyl groups to the total carbon atoms in the (A) region (R ＣＯＯＨ ) was calculated from the following formula derived from the reaction formula of the gas-phase chemical modification after performing gas-phase chemical modification using trifluoroethanol, di-t-butylcarbodiimide, and pyridine, and measuring the amount of surface functional groups in the (A) region by X-ray photoelectron spectroscopy under the following conditions: ＣＯＯＨ (%) = (fluorine (F) concentration (atom %)) / (3 × carbon (C) concentration (atom %) - 2 × F concentration (atom %)) / r ＣＯＯＨ ×100 r ＣＯＯＨ : Reaction rate <Measurement conditions> X-ray: Monochrome Al-Kα ray (output: 25 W) Energy resolution: Wide scan spectrum 117.40 eV High resolution spectrum 93.90 eV Charge correction: C1s main peak (284.8 eV)

[0053] Furthermore, when XPS (X-ray photoelectron spectroscopy) is measured for region (A), the ratio of the peak intensity at 287 eV to the peak intensity at 285 eV in the C1s spectrum is preferably 0.05 or more greater than the ratio of the peak intensity at 287 eV to the peak intensity at 285 eV in the C1s spectrum of XPS measurement for region (B). In this case, the difference in cell proliferation between region (A) and region (B) can be increased, making it possible to proliferate many cells in region (A), and facilitating the formation of uniform cell aggregates in region (A). Since this is more suitable for forming uniform cell aggregates, it is more preferable that the ratio of the peak intensity at 287 eV to the peak intensity at 285 eV in the C1s spectrum of XPS measurement for region (A) is 0.07 or more greater, even more preferable that it is 0.1 or more greater, and most preferable that it is 0.15 or more greater, than the ratio of the peak intensity at 287 eV to the peak intensity at 285 eV in the C1s spectrum of XPS measurement for region (B).

[0054] There are no particular limitations on the method for adjusting the ratio of the 287 eV peak intensity / 285 eV peak intensity in the C1s spectrum of XPS measurement to fall within the above-mentioned range, but a preferred method is to perform plasma treatment only on a portion of the layer containing a hydrophilic polymer formed on the surface of the substrate (the portion intended to become region (A)). An example of a method for performing plasma treatment only on the portion intended to become region (A) is to cover the layer containing a hydrophilic polymer formed on the surface of the substrate with a mask prepared by laser processing and then perform plasma treatment through the mask. Conditions such as plasma intensity and plasma exposure time can be appropriately adjusted so that the difference between the ratio of the 287 eV peak intensity / 285 eV peak intensity in the C1s spectrum of XPS measurement for region (A) and the ratio of the 287 eV peak intensity / 285 eV peak intensity in the C1s spectrum of XPS measurement for region (B) falls within the above-mentioned range. In the case of plasma treatment, using a gas containing oxygen and nitrogen as the introduced gas makes it easier to fall within the above-mentioned range.

[0055] 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. The gas introduced during plasma treatment is preferably air, nitrogen, or oxygen, and more preferably oxygen. The gas pressure of the introduced gas is preferably 1 Pascal (Pa) or more but less than 20 Pascals, more preferably 1 Pascal to 15 Pascals, and even more preferably 1 Pascal to 10 Pascals. By not increasing the gas pressure too much, it becomes easier to keep the inclination angle of the inclined surface in region (A) within the above-mentioned preferred range. Furthermore, since it becomes even easier to keep the inclination angle of the inclined surface in region (A) within the above-mentioned preferred range, it is particularly preferable to perform the plasma treatment by reactive ion etching (RIE). RIE is also called anisotropic etching.

[0056] 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 a hydrophilic polymer by the above-mentioned plasma treatment, the wettability of region (A) can be increased. The water contact angle of region (A) is more preferably 40° or more and 80° or less, even more 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 increased, resulting in better cell adhesiveness and cell proliferation. Furthermore, from the viewpoint of reducing the swelling 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 determined, for example, by dropping a water droplet on the surface of region (A) and defining the angle θ between the liquid surface and the surface of region (A) when the droplet is stationary. Ａ It can be measured using the following formula: θ Ａ = 2 arctan(h / r) (where θ Ａ is the water contact angle, h is the droplet height, and r is the droplet radius.)

[0057] The (A) region may be temperature-responsive, since it is suitable for detaching cultured cell aggregates. When the (A) region is temperature-responsive, since cells can be cultured at a temperature close to body temperature when culturing cells on the cell culture substrate, the response temperature is preferably 50°C or lower, and more preferably 35°C or lower. Furthermore, since it is suitable for preventing cells from detaching when performing operations such as changing the culture medium during culture, the response temperature is particularly preferably 25°C or lower. Furthermore, since it is possible to form cell aggregates by cooling at a temperature that does 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.

[0058] When the (A) region is temperature-responsive, for example, a layer containing a temperature-responsive polymer having a layer thickness of 1 nm to 100 nm may be further provided on the surface of the layer containing a hydrophilic polymer (the surface including the (A) region and the (B) region). By providing a layer containing a temperature-responsive polymer with a layer thickness of 1 nm to 100 nm, temperature responsiveness can be imparted to the (A) region without impairing the respective properties of the (A) region and the (B) region formed on the surface of the layer containing a hydrophilic polymer. To be suitable for imparting temperature responsiveness to the (A) region without impairing the respective properties of the (A) region and the (B) region, the layer thickness of the layer containing a temperature-responsive polymer is preferably 3 nm to 50 nm, more preferably 5 nm to 40 nm, and most preferably 10 nm to 35 nm. The layer thickness of the temperature-responsive polymer suitable for imparting temperature responsiveness to the (A) region without impairing cell proliferation and for enabling cells to be detached and recovered by temperature responsiveness after culture varies depending on the cells to be cultured and can be appropriately adjusted within the range of layer thickness exemplified above.

[0059] The temperature-responsive polymer is preferably a block copolymer having a water-insoluble block segment and a temperature-responsive block segment. When the temperature-responsive polymer is such a block copolymer, mass productivity of the cell culture substrate can be improved and contamination of the produced cell aggregates with the temperature-responsive polymer can be suppressed. The ratio of the constituent units of the temperature-responsive block segment 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, because this is suitable for rapid detachment of cell aggregates from the cell culture substrate.

[0060] Furthermore, since the temperature-responsive polymer is a block copolymer having a water-insoluble block segment and a temperature-responsive block segment, temperature responsiveness can be imparted to the surface of the cell culture substrate by the simple method of dropping a solution containing the temperature-responsive polymer onto the surface of the cell culture substrate and drying it. Furthermore, since the layer formed in this manner has the preferred thickness of the temperature-responsive polymer, even if the entire surface of the layer containing the hydrophilic polymer is coated with the temperature-responsive polymer, the properties of the above-mentioned regions (A) and (B) are less impaired.

[0061] Examples of the monomer unit constituting the temperature-responsive block segment 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-t-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-ethylmethylacrylamide, and N,N-diethylacrylamide; 1-(1-oxo-2-propenyl)-pyrrolidine, 1-(1-oxo-2-propenyl)-pyrrolidine, 1-(1-oxo-2-propenyl)-pyrrolidine, 1-(1-oxo-2-propenyl)-pyrrolamide ... Examples of suitable (meth)acrylamide derivatives include (meth)acrylamide derivatives having a cyclic group such as 1-(1-oxo-2-methyl-2-propenyl)-piperidine, 4-(1-oxo-2-methyl-2-propenyl)-morpholine, 1-(1-oxo-2-methyl-2-propenyl)-pyrrolidine, 1-(1-oxo-2-methyl-2-propenyl)-piperidine, and 4-(1-oxo-2-methyl-2-propenyl)-morpholine; vinyl ethers such as methyl vinyl ether; and proline derivatives such as N-proline methyl ester acrylamide. Among these, N,N-diethylacrylamide, N-n-propylacrylamide, N-isopropylacrylamide, N-n-propylmethacrylamide, N-ethoxyethylacrylamide, N-tetrahydrofurfuryl acrylamide, and N-tetrahydrofurfurylmethacrylamide are preferred, N-n-propylacrylamide and N-isopropylacrylamide are more preferred, and N-isopropylacrylamide is particularly preferred, as they are suitable for setting the response temperature at 0 to 50°C. Furthermore, when a medium at room temperature is used during medium replacement in a culture operation, N-n-propylacrylamide and N-proline methyl ester acrylamide are preferred because they are suitable for setting the response temperature of the block copolymer to a temperature lower than room temperature.

[0062] Examples of monomer units constituting the water-insoluble block segment include n-butyl acrylate, n-butyl methacrylate, isobutyl acrylate, isobutyl methacrylate, t-butyl acrylate, t-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, those having a reactive group are preferred because they are suitable for firmly immobilizing the block copolymer on a substrate, and examples thereof include 4-azidophenyl acrylate, 4-azidophenyl methacrylate, 2-((4-azidobenzoyl)oxy)ethyl acrylate, and 2-((4-azidobenzoyl)oxy)ethyl methacrylate. Furthermore, structures having an aromatic ring are preferred because they are suitable for enhancing cell proliferation, and examples thereof include 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, and styrene.

[0063] The water-insoluble block segment may also contain a repeating unit that controls the response temperature of the block copolymer. Examples of the repeating unit that controls the response temperature of the block copolymer include hydrophilic or hydrophobic components, and are not particularly limited, including those having an amino group such as 2-dimethylaminoethyl acrylate, 2-dimethylaminoethyl methacrylate, 2-diethylaminoethyl acrylate, 2-diethylaminoethyl methacrylate, and N-[3-(dimethylamino)propyl]acrylamide; those having a betaine such as N-(3-sulfopropyl)-N-methacryloyloxyethyl-N,N-dimethylammonium betaine and N-methacryloyloxyethyl-N,N-dimethylammonium-α-N-methylcarboxybetaine; hydroxyethyl acrylate, hydroxyethyl methacrylate, N-(2-hydroxyethyl)acrylamide, polyethylene glycol monoacrylate, polyethylene glycol monomethacrylate, polypropylene glycol monoacrylate, polypropylene glycol monomethacrylate, methoxypolyethylene glycol monoacrylate, methoxypolyethylene glycol monoacrylate, methoxypolyethylene glycol monoacrylate, methoxypolyethylene glycol monoacrylate, hydroxyethyl acrylate, hydroxyethyl methacrylate, N-(2-hydroxyethyl)acrylamide, polyethylene glycol monoacrylate, polyethylene glycol monomethacrylate, polypropylene glycol monoacrylate, polypropylene glycol monometh ...hydroxyethyl acrylate, hydroxyethyl methacrylate, N-(2-hydroxyethyl)acrylamide, polyethylene glycol mono those having a polyethylene glycol group or a methoxyethyl group, such as ethylene glycol monomethacrylate, diethylene glycol monomethyl ether acrylate, diethylene glycol monomethyl ether methacrylate, diethylene glycol monoethyl ether acrylate, diethylene glycol monoethyl ether methacrylate, 2-methoxyethyl acrylate, 2-methoxyethyl methacrylate, 2-ethoxyethyl acrylate, 2-ethoxyethyl methacrylate, 3-butoxyethyl acrylate, 3-butoxyethyl methacrylate, 3-butoxyethyl acrylamide, furfuryl acrylate, furfuryl methacrylate, tetrahydrofurfuryl acrylate, and tetrahydrofurfuryl methacrylate; and those having an acrylate group, such as methoxymethyl acrylate, methoxymethyl methacrylate, 2-ethoxymethyl acrylate, 2-ethoxymethyl methacrylate, 3-butoxymethyl acrylate, 3-butoxymethyl methacrylate, and 3-butoxymethyl acrylamide;Examples of phosphorylcholine groups include those having a phosphorylcholine group, such as 2-methacryloyloxyethyl phosphorylcholine, 2-acryloyloxyethyl phosphorylcholine, 3-(meth)acryloyloxypropyl phosphorylcholine, 4-(meth)acryloyloxybutyl phosphorylcholine, 6-(meth)acryloyloxyhexyl phosphorylcholine, 10-(meth)acryloyloxydecyl phosphorylcholine, ω-(meth)acryloyl(poly)oxyethylene phosphorylcholine, 2-acrylamidoethyl phosphorylcholine, 3-acrylamidopropyl phosphorylcholine, 4-acrylamidobutyl phosphorylcholine, 6-acrylamidohexyl phosphorylcholine, 10-acrylamidodecyl phosphorylcholine, and ω-(meth)acrylamido(poly)oxyethylene phosphorylcholine;

[0064] Region (B) is adjacent to region (A) and does not have cell adhesiveness or cell proliferation properties. If region (B) is adjacent to region (A) and does not have cell proliferation properties, when cells are cultured, it is possible to form cell aggregates only in region (A), and to create a state in which no cells are present around part or all of region (A). Furthermore, since this is suitable for uniforming the size and shape of the cell aggregates produced, it is preferable that region (B) not only has no cell proliferation properties but also no cell adhesiveness.

[0065] The shape of region (B) is not limited other than being adjacent to region (A), but since this is suitable for producing cell aggregates of uniform size and shape, it is preferable that region (B) be adjacent to the boundary line of region (A) for a length of at least 20%, more preferably at least 50%, even more preferably at least 80%, and it is most preferable that region (A) is entirely surrounded by region (B).

[0066] The area ratio of region (A) to region (B) is not particularly limited, but since this is suitable for increasing the number of cell aggregates that can be produced per unit area of ​​the cell culture substrate, the area of ​​region (A) is preferably 10% or more, more preferably 30% or more, even more preferably 50% or more, and most preferably 70% or more of the total area of ​​region (A) and region (B). Furthermore, since this is suitable for providing a sufficient distance between multiple (A) regions and preventing the cell aggregates of multiple (A) regions from fusing together to form a non-uniform shape, the area of ​​region (B) is preferably 20% or more, more preferably 40% or more, even more preferably 60% or more, and most preferably 80% or more of the total area of ​​region (A) and region (B).

[0067] The cell culture substrate may optionally have a layer containing a biologically derived substance on its surface. The layer containing the biologically derived substance may be present on the entire surface of the cell culture substrate, or may be present only on the surface of region (A). The biologically derived substance is not particularly limited, and examples thereof include matrigel, laminin, fibronectin, vitronectin, collagen, etc.

[0068] These biological substances may be natural products, or may be artificially synthesized using genetic engineering techniques, or may be fragments obtained by cleavage with restriction enzymes, or synthetic proteins or peptides obtained by chemically synthesizing substances equivalent to these biological substances.

[0069] As Matrigel, commercially available products such as Matrigel (manufactured by Corning Incorporated) and Geltrex (manufactured by Thermo Fisher Scientific) can be suitably used due to their ease of availability.

[0070] The type of laminin is not particularly limited, but examples that can be used include laminin 511, laminin 521, and laminin 511-E8 fragment, which have been reported to exhibit high activity against α6β1 integrin expressed on the surface of human iPS cells. Laminin may be a natural product, may be artificially synthesized using genetic engineering techniques, or may be a synthetic protein or peptide obtained by chemically synthesizing a substance equivalent to laminin. Commercially available products such as iMatrix-511 (manufactured by Nippi Corporation) are suitable for use due to their ease of availability.

[0071] Vitronectin may be a natural product, artificially synthesized by genetic recombination technology, or a synthetic protein or peptide obtained by chemically synthesizing a substance equivalent to vitronectin. In view of availability, commercially available products such as vitronectin, human plasma-derived (manufactured by Wako Pure Chemical Industries, Ltd.), synthemax (manufactured by Corning Incorporated), and Vitronectin (VTN-N) (manufactured by Thermo Fisher Scientific) can be preferably used.

[0072] Fibronectin may be a natural product, artificially synthesized by genetic recombination technology, or a synthetic protein or peptide obtained by chemically synthesizing a substance equivalent to fibronectin. In view of availability, commercially available products such as fibronectin solution, human plasma-derived fibronectin (manufactured by Wako Pure Chemical Industries, Ltd.), and Retronectin (manufactured by Takara Bio Inc.) can be preferably used.

[0073] The type of collagen is not particularly limited, and for example, type I collagen, type IV collagen, etc. can be used. Collagen may be a natural product, may be artificially synthesized using genetic recombination technology, or may be a synthetic peptide obtained by chemically synthesizing a substance equivalent to collagen. In view of ease of availability, commercially available products such as collagen I, human (manufactured by Corning Incorporated) and collagen IV, human (manufactured by Corning Incorporated) can be preferably used.

[0074] From the viewpoint of suppressing denaturation of the biological substance and enhancing cell proliferation, the biological substance is preferably immobilized on the cell culture substrate by a non-covalent bond. Here, "non-covalent bond" refers to a bonding force other than a covalent bond derived from intermolecular forces, such as electrostatic interaction, water-insoluble interaction, hydrogen bond, π-π interaction, dipole-dipole interaction, London dispersion force, or other van der Waals interaction. The biological substance may be immobilized on the block copolymer by a single bonding force or a combination of multiple bonding forces.

[0075] The method for immobilizing biological substances is not particularly limited, but suitable methods include, for example, applying a solution of the biological substance to a cell culture substrate for a predetermined period of time to immobilize the substance, and adding the biological substance to the culture medium when culturing cells to allow the biological substance to be adsorbed onto the cell culture substrate and immobilized.

[0076] The cell culture substrate according to this embodiment may have a partition plate (for example, a partition plate having a cross-sectional area of ​​0.05 cm 2 in the in-plane direction) on the substrate, if necessary. ２ More than 100cm ２ A structure for separating each cell aggregate may be provided by providing a plate having through-holes (described below).

[0077] The cell culture substrate according to this embodiment may be sterilized. There are no particular limitations on the sterilization method, but high-pressure steam sterilization, UV sterilization, gamma-ray sterilization, ethylene oxide gas sterilization, etc. can be used. From the viewpoint of suppressing denaturation of the block copolymer, high-pressure steam sterilization, UV sterilization, and ethylene oxide gas sterilization are preferred. From the viewpoint of suppressing deformation of the substrate, UV sterilization or ethylene oxide gas sterilization is more preferred. From the viewpoint of excellent mass productivity, ethylene oxide gas sterilization is preferred.

[0078] The cells to be cultured using the cell culture substrate according to this embodiment are not particularly limited as long as they can adhere to the surface before the application of a temperature drop stimulus. Examples include various established cell lines such as Chinese hamster ovary-derived CHO cells, mouse connective tissue L929 cells, human embryonic kidney-derived HEK293 cells, and human cervical cancer-derived HeLa cells, as well as epithelial cells and endothelial cells that constitute various tissues and organs in the body, contractile skeletal muscle cells, smooth muscle cells, and cardiac muscle cells, neuronal cells, glial cells, and fibroblasts that constitute the nervous system, hepatic parenchymal cells, non-hepatic parenchymal cells, and adipocytes that are involved in the metabolism of the body, as well as stem cells present in various tissues such as mesenchymal stem cells, bone marrow cells, and Muse cells, as well as stem cells with pluripotency (pluripotent stem cells) such as ES cells and iPS cells, and cells induced to differentiate therefrom.

[0079] The cell culture substrate according to this embodiment can be produced, for example, by a production method including a coating step in which at least a portion of the surface of the substrate is coated with a composition containing a hydrophilic polymer to form a layer containing the hydrophilic polymer, and a patterning step in which part of the surface of the layer containing the hydrophilic polymer is subjected to plasma treatment to form region (A) in the plasma-treated part.

[0080] The coating step involves coating at least a portion of the surface of the substrate with a composition containing a hydrophilic polymer to form a layer containing the hydrophilic polymer. By forming a layer containing the hydrophilic polymer, the substrate can be rendered free of cell adhesiveness and cell proliferation. The method for forming the layer containing the hydrophilic polymer is not particularly limited, and examples include a method of forming the layer by applying a composition containing the hydrophilic polymer to at least a portion of the surface of the substrate. Examples of methods for applying the composition containing the hydrophilic polymer include various commonly known methods, such as painting, brush coating, dip coating, spin coating, bar coating, flow coating, spray coating, roll coating, air knife coating, blade coating, gravure coating, microgravure coating, and slot die coating.

[0081] 3 is a schematic diagram (perspective view) of a substrate having a layer containing a hydrophilic polymer formed on its surface after the coating process. In FIG. 3, a layer 2 containing a hydrophilic polymer is formed on the entire surface of a substrate 1.

[0082] When the hydrophilic polymer is reactive to active energy rays, the coating step may include irradiating the layer containing the hydrophilic polymer with active energy rays to fix the layer containing the hydrophilic polymer to the surface of the substrate. Examples of the hydrophilic polymer reactive to active energy rays include the above-mentioned hydrophilic polymers having functional groups reactive to active energy rays (e.g., UV, electron beams, etc.). Examples of active energy rays include UV, electron beams, etc.

[0083] Irradiating a hydrophilic polymer reactive to active energy rays with active energy rays causes a chemical reaction between the hydrophilic polymers or between the hydrophilic polymer and the substrate, thereby immobilizing a layer containing the hydrophilic polymer on the surface of the substrate. By immobilizing the layer containing the hydrophilic polymer on the surface of the substrate, a layer containing the hydrophilic polymer can be formed without deforming the layer containing the hydrophilic polymer when a composition containing the temperature-responsive polymer is applied in the temperature-responsive layer-forming step described below. Furthermore, by immobilizing the layer containing the hydrophilic polymer on the surface of the substrate, the shape of the region (A) formed in the patterning step described below can be maintained.

[0084] The patterning process is a process of performing plasma treatment on a portion of the surface of the layer containing a hydrophilic polymer, thereby forming 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. The gas introduced during the plasma treatment is preferably air, nitrogen, oxygen, or water vapor, and more preferably oxygen or water vapor. The gas pressure of the introduced gas is preferably 1 Pascal (Pa) or more but less than 20 Pascals, more preferably 1 Pascal to 15 Pascals, and even more preferably 1 Pascal to 10 Pascals. By not increasing the gas pressure too much, it becomes easier to keep the inclination angle of the inclined surface in region (A) within the above-mentioned preferred range. Furthermore, since it becomes even easier to keep the inclination angle of the inclined surface in region (A) within the above-mentioned preferred range, it is particularly preferable to perform the plasma treatment by reactive ion etching (RIE). RIE is also called anisotropic etching. The method for patterning the (A) region is not particularly limited, but examples thereof include a method in which a desired portion (unmasked portion) is subjected to plasma treatment while covered with a metal mask, a silicon mask, a surface protection film, or the like.

[0085] 4 is a schematic diagram (perspective view) of the cell culture substrate after the patterning step. In the cell culture substrate 10 shown in FIG. 4, circular regions (regions indicated by A) are arranged at equal intervals. For example, the area of ​​the region is 0.001 mm. ２ More than 5 mm ２ The area indicated by A corresponds to the area where plasma treatment was performed. The area indicated by A has cell adhesive properties and cell proliferation properties due to the surface of the layer 2 containing a hydrophilic polymer being modified by plasma treatment. The area other than the circular area (area indicated by B) corresponds to the area where plasma treatment was not performed, for example, because it is protected by a metal mask or the like. The area indicated by B does not have cell adhesive properties or cell proliferation properties because the surface of the layer 2 containing a hydrophilic polymer.

[0086] After the patterning step, a step of washing the hydrophilic polymer with a solvent to dissolve and remove the hydrophilic polymer not immobilized on the surface from the substrate surface may be performed. Removing the unimmobilized hydrophilic polymer exposes the substrate surface, allowing the formation of region (A). The solvent used preferably contains water and an alcohol, as this is suitable for removing compounds by-produced by the self-reaction of the hydrophilic polymer reactive to active energy rays, such as compounds containing nitrenes derived from azide groups. Washing with a mixed solvent of water and alcohol can reduce the hydrophilic polymer-derived components remaining in region (A), thereby improving the cell adhesiveness and cell proliferation of region (A). Lower alcohols are preferred, such as methanol, ethanol, 2-propanol, t-butanol, isobutanol, pentanol, and hexanol, with methanol or ethanol being more preferred. The alcohol content is preferably 50 to 95%, more preferably 50 to 90%, particularly preferably 60 to 90%, and most preferably 70 to 90%. In addition, in other embodiments, the manufacturing method may not include a patterning step, and the shape of region (A) may be formed only by the coating step. In this case, examples of application methods in the coating step include injection printing such as inkjet printing and on-demand printing, relief printing such as flexographic printing and letterpress printing, intaglio printing such as gravure printing and pad printing, lithographic printing such as offset printing, and stencil printing such as screen printing. Surface modification of region (A) to make it a region with cell adhesiveness and cell proliferation properties may be performed before or after the coating step. Surface modification can be performed, for example, by corona discharge treatment, plasma irradiation, ultraviolet irradiation, etc.

[0087] The manufacturing method according to this embodiment may further include a bonding step and a temperature responsive layer forming step, in addition to the coating step and the patterning step, as necessary.

[0088] In the lamination process, after the patterning process, the cross-sectional area in the in-plane direction is 0.05 cm ２ More than 100cm ２This is a process of laminating a plate having the following through-holes to a substrate on the side of the substrate coated with a layer containing a hydrophilic polymer. ２ More than 100cm ２ By laminating a plate having the following through-holes to a substrate, a plate having a space for containing a culture medium can be produced with high mass productivity. FIG. 5 is a schematic diagram (perspective view) of the cell culture substrate after the lamination process. The cell culture substrate 11 shown in FIG. 5 is, for example, a cell culture substrate 10 such as that shown in FIG. 4 with a partition plate 20 (with a cross-sectional area in the in-plane direction of 0.05 cm ). ２ More than 100cm ２ The cross-sectional area in the in-plane direction is 0.05 cm ２ More than 100cm ２ The plate having the following through-holes may be, for example, a partition member having a plurality of cylindrical partition walls that can partition the surface on the side where cells are cultured. ２ More than 100cm ２ The side walls of the following through-holes function as cylindrical partition walls.

[0089] The temperature-responsive layer formation step is a step in which, after the patterning step, the surface of the plasma-treated hydrophilic polymer-containing layer is coated with a composition containing a temperature-responsive polymer to form a layer containing a temperature-responsive polymer. In this case, by forming the layer containing the temperature-responsive polymer to a thickness of 100 nm or less, the surface of the formed (A) region is easily coated with sparse molecular chains of the temperature-responsive polymer, which is suitable for imparting temperature responsiveness while maintaining the functions of the (A) region (cell adhesiveness and cell proliferation). Furthermore, by forming the layer containing the temperature-responsive polymer to a thickness of 1 nm or more, sufficient temperature responsiveness can be imparted, making it possible to produce a cell culture substrate capable of rapid formation of cell aggregates, which is also suitable.

[0090] As a method for coating with a composition containing a temperature-responsive polymer, the same method as the above-mentioned method for applying a composition containing a hydrophilic polymer can be suitably used.

[0091] As a method for coating with a composition containing a temperature-responsive polymer, it is also preferable to coat the entire surface of the cell culture substrate with a temperature-responsive substance. When coating with a composition containing a temperature-responsive polymer, the mass productivity of the cell culture substrate can be improved by using a commonly used coating method without patterning. Furthermore, by coating the entire surface of the cell culture substrate with a composition containing a temperature-responsive polymer, temperature responsiveness is imparted to region (A), and region (B) is also coated with the temperature-responsive polymer. By coating region (B) with the temperature-responsive polymer, cell adhesiveness in region (B) can be reduced.

[0092] The cell culture kit according to this embodiment includes a cell culture substrate, which may be the cell culture substrate according to the present invention described above, provided with a partition member having a plurality of cylindrical partition walls that can partition the surface on which cells are cultured.

[0093] The cell culture kit according to this embodiment may include a temperature-responsive polymer or a coating agent containing a temperature-responsive polymer in addition to the cell culture substrate, which allows researchers performing culture to easily adjust the layer thickness of the temperature-responsive polymer depending on the type of cells.

[0094] The coating agent may contain a solvent. Examples of solvents that can be contained in the coating agent include water, organic solvents, and 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. A mixed solvent of water and alcohols is preferably used because it is suitable for achieving a uniform coating thickness. The content of the temperature-responsive polymer, based on the total mass of the coating agent, can be 0.1 to 50 wt %, 0.2 to 10 wt %, or 0.5 to 5 wt %.

[0095] The coating agent may contain other components in addition to the temperature-responsive polymer and the solvent. Examples of other components include components for enhancing cell adhesion, such as a polymer consisting only of a water-insoluble block segment.

[0096] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples. Unless otherwise specified, commercially available reagents were used.

[0097] Example 1 0.9 mL of an 80 wt % aqueous ethanol solution containing 0.6 wt % solids concentration of polyvinyl alcohol (BIOSURFINE®-AWP, manufactured by Toyo Gosei Co., Ltd.) having an azide group as a hydrophilic polymer was dropped onto a polycarbonate film (trade name: Panlite®, manufactured by Teijin Limited, thickness: 0.18 mm) (substrate), and the film was spin-coated (2000 rpm, 60 seconds) using a spin coater (trade name: MS-B150, manufactured by Mikasa Co., Ltd.). The film was then left to stand for 1 hour under a high-pressure mercury lamp to cure the hydrophilic polymer via UV irradiation, thereby forming a hydrophilic polymer layer (layer thickness: 75 nm). A metal mask (spot center distance 800 μm) having multiple 0.2 mm diameter circular holes (spots) was placed on the hydrophilic polymer layer, and a plasma treatment was performed on top of the metal mask using a plasma irradiation device (manufactured by Samco Corporation, product name: Aqua Plasma (registered trademark) Cleaner AQ-500), thereby forming region (A) in the plasma-treated area. Region (B) was also 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 a plasma treatment that utilizes the ion collision effect in addition to a radical-induced chemical reaction by applying a high-frequency voltage to the equipment chamber in which the substrate is placed. On the other hand, "isotropic" refers to a plasma treatment that utilizes a radical-induced chemical reaction without applying a high-frequency voltage to the equipment chamber.

[0098] Examples 2-4 Cell culture substrates were prepared in the same manner as in Example 1, except that the plasma treatment conditions were changed as shown in Table 1.

[0099] Example 5 A cell culture substrate was prepared in the same manner as in Example 1, except that a plasma irradiation device (manufactured by Samco Inc., product name: Aqua Plasma (registered trademark) Cleaner AQ-500) was replaced with a plasma irradiation device (manufactured by Samco Inc., product name: 10-NR) and the plasma treatment conditions were as shown in Table 1.

[0100] Example 6 A cell culture substrate was prepared in the same manner as in Example 1, except that an ICP plasma irradiation device was used, a metal mask (having a plurality of circular holes (spots) with a diameter of 0.1 mm and a center-to-center distance of 500 μm) was used instead of a metal mask (having a plurality of circular holes (spots) with a diameter of 0.2 mm and a center-to-center distance of 800 μm), and the plasma treatment conditions were as shown in Table 1.

[0101] Example 7 The cell culture substrate of Example 6 was used after being stored in an environment of 15-30° C. and 40-90% RH for 6 months.

[0102] Comparative Example 1 A cell culture substrate was prepared in the same manner as in Example 1, except that a plasma irradiation device (manufactured by Sakigake Semiconductor Co., Ltd., product name: Plasma Etcher CPE-400) was used instead of a plasma irradiation device (manufactured by Samco Inc., product name: Aqua Plasma (registered trademark) Cleaner AQ-500), a metal mask (having a plurality of circular holes (spots) with a diameter of 0.1 mm and a spot center-to-center distance of 500 μm) was used instead of a metal mask (having a plurality of circular holes (spots) with a diameter of 0.2 mm and a spot center-to-center distance of 800 μm), and the plasma treatment conditions were as shown in Table 1.

[0103] Comparative Example 2-4 Cell culture substrates were prepared in the same manner as in Example 1, except that no hydrophilic polymer layer was formed and the plasma treatment conditions were changed as shown in Table 1.

[0104] Comparative Example 5 The polycarbonate film (without a hydrophilic polymer layer and without plasma treatment) used in Examples 1-6 and Comparative Examples 1-4 was used.

[0105] Comparative Example 6 The polycarbonate film having a hydrophilic polymer layer (layer thickness: 75 nm, not plasma treated) used in Examples 1-6 and Comparative Examples 1-4 was used.

[0106] <Evaluation of Cell Culture Substrate> (Measurement of Hydrophilic Polymer Layer Thickness) The thickness of the hydrophilic polymer layer formed on the substrate surface was determined by calculating the coating amount per unit area from the surface area of ​​the substrate, the concentration of the hydrophilic polymer in the solution, and the volume of the solution dropped onto the substrate, assuming the specific gravity of the polymer to be 1. The results are shown in Table 1.

[0107] (Evaluation of the shape of region (A)) The shape of region (A) formed by etching using plasma treatment was measured using a stylus profiling system (manufactured by Bruker Corporation, product name: DEKTAK XT). The cell culture substrate was fixed on a glass substrate with tape, and measurements were carried out under the following conditions. The scanning direction was along the axis passing through the center (center of gravity) of the bottom surface of region (A). Z-axis measurement range: 6.5 μm X-axis measurement range: 500 μm Measurement time: 30 seconds Load force: 1 mg

[0108] From the obtained profile, the maximum depth (nm), etching gradient (μm-Z axis / mm-X axis), and gradient angle (°) were determined. The maximum depth was defined as the maximum vertical distance from the interface on the side opposite to the surface of the hydrophilic polymer layer in contact with the substrate. In other words, it is synonymous with the out-of-plane distance between the bottom surface of region (A) and the surface of region (B). The etching gradient was defined as the gradient of an approximate line obtained by linearly approximating (by least squares method) the gradient of the gradient of region (A) in the depth range of 20% to 70% when the maximum depth was defined as 100%. The gradient angle was defined as the angle between the bottom surface of region (A) and the approximate line. The results are shown in Table 2.

[0109] (Evaluation of the amount of surface functional groups in region (A)) The amount of surface functional groups in region (A) formed by plasma treatment was measured by X-ray photoelectron spectroscopy. Under the conditions in Table 1, a polycarbonate film was subjected to plasma or RIE treatment on the entire surface, followed by gas-phase chemical modification using trifluoroethanol, di-t-butylcarbodiimide, and pyridine to prepare a measurement sample. Measurement was performed using a PH15000 VersaProbe II (manufactured by ULVAC-PHI) under the following conditions. X-ray source: Monochrome Al-Kα radiation (output: 25 W) Analysis area: 1000 × 300 μm Energy resolution: Wide scan spectrum 117.40 eV High resolution spectrum 93.90 eV Charge correction: C1s main peak (284.8 eV)

[0110] From the following formula derived from the reaction formula of gas-phase chemical modification, the carbon ratio of carboxyl groups to all carbons in the (A) region (R ＣＯＯＨ ) was calculated. ＣＯＯＨ (%) = (F concentration (atom%)) / (3 × C concentration (atom%) - 2 × F concentration (atom%)) / r ＣＯＯＨ ×100 r ＣＯＯＨ : Reaction rate (calculated from polyacrylic acid (standard sample)) The results are shown in Table 3. In Comparative Examples 5 and 6, the measurement was performed on a region near the center of the film.

[0111] (Evaluation of Water Contact Angle) The water contact angle of the film surface modified by plasma treatment was evaluated using a contact angle measuring instrument (Model: DM300, manufactured by Kyowa Interface Science Co., Ltd.). A measurement sample was prepared by treating a polycarbonate film under the conditions shown in Table 1, and 1 μL of water was dropped onto the surface-modified surface, and the water contact angle (°) was measured after 20 seconds. The results are shown in Table 3. In Comparative Examples 5 and 6, the measurement was performed on a region near the center of the film.

[0112] (Evaluation of the Shape of the Openings) A solution of fluorescently labeled cell culture protein substrate (manufactured by Matrixome Co., Ltd., product name: iMatrix-511) was added to the openings (region (A)) to allow the protein substrate to adsorb. After removing the protein substrate solution and washing with PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd.), the cell culture substrate was observed using a fluorescence microscope from the hydrophilic polymer layer side in a direction perpendicular to the substrate, and a fluorescent image was taken. The major axis diameter (μm) and minor axis diameter (μm) of the fluorescent image corresponding to each opening were measured, and the aspect ratio (major axis diameter (μm) / minor axis diameter (μm)) was calculated. Measurements were performed on approximately 1200 to 1300 samples for each cell culture substrate, and the average value was calculated. CV (a value expressed as a percentage by dividing the standard deviation by the mean value) was also calculated. The results are shown in Table 2. Since the protein substrate is adsorbed to the inclined surface and bottom surface of region (A), the fluorescent image when the cell culture substrate is observed from the hydrophilic polymer layer side in a direction perpendicular to the substrate takes the shape of an opening (a planar region defined by the boundary between region (A) and region (B)).

[0113] (Cell Culture Evaluation) The cell culture substrate was attached to the bottom of a bottomless 6-well plate, which was sterilized and used as a culture vessel. Human iPS cells 201B7 strain were used, and the cell culture substrate was cultured at 15,000 cells / cm. ２ The cells were seeded in AK02N medium (Ajinomoto Co., Inc.) (2 mL / well) and incubated at 37°C and CO ２ The cells were cultured in an environment with a 5% concentration. Until 24 hours after seeding, Y-27632 (manufactured by Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) and a cell culture protein substrate (manufactured by Matricsome Co., Ltd., product name: iMatrix-511) (1.25 μg / mL) were added to the medium. The medium was replaced 1, 3, and 5 days after the start of culture. Six days after the start of culture, the shape of the cell aggregates was evaluated by observation using a phase-contrast microscope. In addition, the size of the cell aggregates (average of the major axis diameter (μm) and minor axis diameter (μm)) was measured, and the hypertrophy rate (%) was calculated. The hypertrophy rate was defined as the value (percentage) obtained by dividing the size of the cell aggregate by the diameter of the metal mask spot. For each cell culture substrate, n = 15 cell aggregate sizes were measured and the hypertrophy rate was calculated, and the average value was obtained. The results are shown in Table 2.

[0114]

[0115]

[0116] The cell culture substrates of Examples 1 to 3 and 5 to 7, in which region (A) was formed by anisotropic etching, had a large inclination angle (85.9° to 88.7°) and a clearly defined uneven shape. Furthermore, the cell culture substrate of Example 4, in which region (A) was formed by isotropic etching but at a reduced gas pressure, had a significantly larger inclination angle and a clearly defined uneven shape compared to the cell culture substrate of Comparative Example 1, in which the gas pressure was high. Furthermore, the cell culture substrates of Examples 1 to 7 had an aspect ratio of the opening shape of 1.1 or less, and the cell culture substrates of Examples 1 to 5 also had a cell aggregate expansion rate of 140% or less, while the cell culture substrates of Examples 6 and 7 also had a cell aggregate expansion rate of 165% or less. It can be seen that region (A) was formed with a size and shape close to the intended 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 to 3 and 5 to 7 had a small CV of the aspect ratio of the opening shape, and were more uniform in size and shape. Furthermore, the substrates treated with anisotropic etching and isotropic etching under low gas pressure conditions had a diameter / maximum depth ratio of the opening in region (A) within the range of 500 to 3,000, showing a clear uneven shape. On the other hand, in Comparative Example 1, the etching inclination angle was small, and the shape was unclear. Furthermore, since Comparative Examples 2 to 4 did not have region (B), cells adhered and proliferated nonspecifically even outside region (A), making size control difficult. Furthermore, since Comparative Examples 5 to 6 did not have region (A), 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 treatment conditions of Examples 1 and 5 to 7 was 0.01. ＣＯＯＨ ) was 0.25% or more, and the culture performance was also good. Furthermore, as is clear from the cell culture results, the use of these cell culture substrates makes it possible to more stably obtain cell aggregates with uniform size and shape.

[0117] A...(A) region, A1...inclined surface, A2...bottom surface, B...(B) region, H...maximum depth of (A) region, 1...substrate, 2...layer containing hydrophilic polymer, 10, 11...cell culture substrate, 20...partition plate.

Claims

1. A cell culture substrate comprising a substrate and a layer containing a hydrophilic polymer covering at least a portion of the surface of the substrate, the substrate having the following region (A) and region (B): (A) a region having cell adhesiveness and cell proliferation properties, and (B) a region adjacent to region (A) that does not have cell adhesiveness or cell proliferation properties. Region (A) consists of a recess formed in the layer containing the hydrophilic polymer, the recess having an inclined surface and a bottom surface, the inclination angle of the inclined surface being 40° or more and 110° or less.

2. The cell culture substrate according to claim 1, wherein the maximum depth of the region (A) is 1 nm or more and 500 nm or less.

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

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

5. The plane area defined by the boundary between the (A) region and the (B) region has an area of ​​0.001 mm ２ More than 5mm ２ The cell culture substrate according to claim 1 or 2, which is an ellipse having a width of 1 or less and an aspect ratio of 1 or more and 1.1 or less.

6. The cell culture substrate according to claim 5, wherein the ratio (diameter / maximum depth) of the diameter (nm) of the opening of the (A) region to the maximum depth (nm) of the (A) region is 500 or more and 3,000 or less.

7. The cell culture substrate according to claim 1 or 2, wherein the minimum distance between the (A) regions is 400 μm or more and 10,000 μm or less.

8. The cell culture substrate according to claim 1 or 2, wherein the water contact angle of the region (A) is 20° or more and 110° or less.

9. The cell culture substrate according to claim 1 or 2, wherein the region (A) is formed by reactive ion etching treatment.

10. The carbon ratio of the carboxyl group to the total carbon in the (A) region (R ＣＯＯＨ 3. The cell culture substrate according to claim 1, wherein the content of the cation exchange resin in the cell culture substrate is 0.25% or more.

11. The cell culture substrate 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 producing a cell culture substrate according to claim 1, comprising: a coating step of coating at least a portion of a surface of a substrate with a composition containing a hydrophilic polymer to form a layer containing said hydrophilic polymer; and a patterning step of subjecting a portion of the surface of said layer containing said hydrophilic polymer to a plasma treatment to form said region (A) in the portion that has been subjected to the plasma treatment.

13. The method for producing a cell culture substrate according to claim 12, wherein the hydrophilic polymer is reactive to active energy rays, and the coating step includes irradiating a layer containing the hydrophilic polymer with active energy rays to fix the layer on the surface of the substrate.

14. The method for producing a cell culture substrate according to claim 12 or 13, wherein the plasma treatment is a reactive ion etching treatment.

15. After the patterning step, the cross-sectional area in the in-plane direction is 0.05 cm ２ More than 100cm ２ The method according to claim 12 or 13, further comprising a lamination step of laminating a plate having the following through holes to the substrate on the side of the substrate coated with the layer containing the hydrophilic polymer:

16. The manufacturing method described in claim 12 or 13, further comprising a temperature-responsive layer formation step of coating the surface of the layer containing the hydrophilic polymer that has been subjected to plasma treatment after the patterning step with a composition containing a temperature-responsive polymer, thereby forming a layer containing the temperature-responsive polymer.

17. A cell culture kit comprising a cell culture substrate, the cell culture substrate being the cell culture substrate described in claim 1 or 2, and provided with a partition member having a plurality of cylindrical partitions capable of dividing the surface on which cells are cultured.

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