JP2024067168A - Cell culture substrate, and method for producing the same - Google Patents

Abstract

To provide a cell culture substrate that has a uniform particle diameter and is capable of peeling and recovering a cell aggregate without a temperature change after culture, and a method for producing the cell culture substrate.SOLUTION: A cell culture substrate has a substrate having irregularity on a surface and a hydrophilic polymer layer covering the substrate along the irregularity, where the maximum height in a reference length 10 μm of the surface irregularity that the cell culture substrate has is 0.1 to 2 μm, the cell culture substrate has the following two regions (A) and (B), and the regions (A) are plasma treatment regions. (A) island-shaped regions having an area of 0.001 to 5 mm2 which has cell adhesiveness and cell proliferating property. (B) a region which is adjacent to the regions (A) and has no cell proliferating property.SELECTED DRAWING: Figure 4

Description

The present invention relates to a cell culture substrate and a method for producing the same.

Pluripotent stem cells, such as embryonic stem cells (ES cells) and artificial cells (iPS cells), have the ability to differentiate into various tissues in the body (pluripotency), and have attracted much attention as a cell source for regenerative medicine and drug screening. To apply pluripotent stem cells to regenerative medicine and drug screening, it is necessary to differentiate the pluripotent stem cells into the desired cells, and at that time, it is necessary to form cell aggregates of pluripotent stem cells. In addition, pluripotent stem cells can differentiate into various cells, but it is known that the optimal size of the cell aggregate varies depending on the type of cell after differentiation, and it is desirable to control the size and create cell aggregates of uniform size.

A method for forming cell aggregates has been known in the past in which pluripotent stem cells are made to spontaneously form aggregates by using a substrate to which the pluripotent stem cells do not adhere (see, for example, Patent Document 1). Although this method is excellent for mass-producing cell aggregates, it has the problem that it is not possible to obtain cell aggregates of uniform size.

As a method for forming cell aggregates of uniform size, a method is known in which a cell culture substrate is treated to prevent cells from adhering and has depressions on its surface with a diameter larger than the size of the cells (see, for example, Patent Document 2). However, when using a cell culture substrate with such depressions, since the cells are not fixed to the cell culture substrate, the cell aggregates tend to pop out of the depressions during culture, and the popped cell aggregates tend to fuse with each other, resulting in a problem that the size of the cell aggregates tends to be non-uniform.

In response to the above problem, a method has been proposed in which a cell culture substrate having two regions, a cell adhesive region and a cell non-adhesive region, is used to form cell aggregates in a state in which the cells are adhered to and fixed on the cell culture substrate (see, for example, Patent Document 3). In this method, the cell aggregates are fixed to the cell culture substrate, so that fusion of the cell aggregates with each other during culture can be prevented, and cell aggregates of uniform size can be obtained. However, after culturing the cell aggregates, it is necessary to detach the cell aggregates that are firmly adhered to the cell culture substrate, and the detachment is likely to damage the cells, resulting in a decrease in cell quality, such as a decrease in cell viability and a loss of pluripotency.

In response to this problem, a cell culture substrate coated with a polymer that can control cell adhesion by temperature change has been proposed (see, for example, Patent Document 4). In this method, after culturing cell aggregates, the temperature of the cell culture substrate is lowered to room temperature or lower to detach and recover the cell aggregates. However, there is a problem in that cells generally survive at an optimal temperature of 37°C, and a drop in temperature can easily cause deterioration in cell quality.

Japanese Patent Application Laid-Open No. 8-140673 JP 2015-073520 A International Publication No. 2010/134606 JP 2015-211688 A

The object of the present invention is to provide a cell culture substrate that has a uniform particle size and enables cell aggregates to be detached and recovered without temperature change after culture, and a method for producing the cell culture substrate.

As a result of intensive research conducted in consideration of the above points, the inventors discovered that the above problems could be solved by a cell culture substrate that has a specific surface roughness and forms a specific size of cell adhesion and cell proliferation area, and thus completed the present invention.

That is, one aspect of the present invention relates to the following [1] to [7].
[1] A cell culture substrate comprising a substrate having an uneven surface and a hydrophilic polymer layer covering the substrate along the uneven surface, the maximum height of the uneven surface of the cell culture substrate being 0.1 to 2 μm over a reference length of 10 μm, the cell culture substrate having the following two regions (A) and (B), the region (A) being a plasma-treated region:
(A) an island-like region having an area of 0.001 to ⁵ mm2 and having cell adhesiveness and cell proliferation properties; (B) a region adjacent to the region (A) and not having cell proliferation properties. [2] The cell culture substrate according to [1], wherein the substrate has a substrate sheet and a three-dimensional element having a vertical length of 0.1 to 3 μm that is arranged on or adhered to the substrate sheet.
[3] The cell culture substrate according to [2], wherein the number density of the three-dimensional elements is 0.1 to 10 pieces/ ^μm2 .
[4] The cell culture substrate according to any one of [1] to [3], wherein the coefficient of variation of the distance between convex portions of the surface irregularities is 3 to 50%.
[5] The cell culture substrate according to any one of [1] to [4], wherein the hydrophilic polymer contains a compound represented by the following general formula (1) or (2):

[In formula (1), ^R1 and ^R2 each independently represent a hydrogen atom or a methyl group, ^R3 represents an arbitrary substituent, and m and n each independently represent a positive integer.]

[In formula (2), R ⁴ , R ⁵ and R ⁶ each independently represent a hydrogen atom or a methyl group, R ⁷ represents an arbitrary substituent, and x, y and z each independently represent a positive integer.]
[6] The cell culture substrate according to any one of [1] to [5], wherein the region (A) has a carboxyphenyl group.
[7] A method for producing a cell culture substrate according to any one of [1] to [6], comprising the following steps (1) and (2):
(1) A step of forming a hydrophilic polymer layer on a substrate having an uneven surface along the unevenness, the maximum height of the surface unevenness of the hydrophilic polymer layer being 0.1 to 2 μm at a reference length of 10 μm; (2) A step of performing a plasma treatment on only a part of the surface of the hydrophilic polymer layer to form two regions (A) and (B) as follows: (A) an island-like region having an area of 0.001 to ⁵ mm2 and having cell adhesiveness and cell proliferation properties; and (B) a region adjacent to the region (A) and not having cell proliferation properties.

The present invention provides a cell culture substrate that has a uniform particle size and enables cell aggregates to be detached and recovered without temperature change after culture, and a method for producing the cell culture substrate.

FIG. 1 is a schematic diagram (cross-sectional view) of a cell culture substrate of the present invention. FIG. 2 is a schematic diagram (perspective view) of the substrate. FIG. 3 is a schematic diagram (perspective view) of a substrate provided with a hydrophilic polymer layer. FIG. 4 is a schematic diagram (perspective view) of the cell culture substrate of the present invention. FIG. 5 is a schematic diagram (perspective view) of a cell culture dish in which a plate having through-holes is attached to the cell culture substrate of the present invention. FIG. 6 is an electron microscope image of the surface irregularities of the cell culture substrate of Example 1. FIG. 7 shows laser microscope images of areas (A) and (B) in the cell culture substrate of Example 1. FIG. 8 is an electron microscope image of the surface irregularities in the cell culture substrate of Example 4. FIG. 9 is an electron microscope image of the surface irregularities in the cell culture substrate of Example 5. FIG. 10 is an electron microscope image of the surface irregularities in the cell culture substrate of Example 6.

The following describes in detail the form for carrying out the present invention (hereinafter, simply referred to as the "present embodiment"). The following present embodiment is an example for explaining the present invention, and is not intended to limit the present invention to the following content. The present invention can be carried out with appropriate modifications within the scope of its intent.

In the present invention, a "cell aggregate" refers to a three-dimensional aggregate of cells formed by the gathering of multiple cells, and may be spherical with gaps or hollow.

In addition, in the present invention, "temperature responsiveness" refers to the degree of hydrophilicity/hydrophobicity changing with temperature change. Furthermore, the boundary temperature at which the degree of hydrophilicity/hydrophobicity changes is referred to as the "response temperature."

In the present invention, a "biological substance" refers to a substance present in the body of a living organism, and may be a natural product, may be artificially synthesized using recombinant gene technology, or may be a substance chemically synthesized based on the above-mentioned biological substance. There are no particular limitations on biological substances, but examples include nucleic acids, proteins, and polysaccharides, which are the basic materials that make up living organisms, and their component elements, such as nucleotides, nucleosides, amino acids, and various sugars, as well as lipids, vitamins, and hormones.

In the present invention, "cell adhesiveness" refers to the ease with which cells adhere to a cell culture substrate at the culture temperature, and "having cell adhesiveness" refers to the ability of cells to adhere to a substrate or cell culture substrate directly or via a biological substance at the culture temperature. In addition, "not having cell adhesiveness" refers to the inability of cells to adhere to a substrate or cell culture substrate at the culture temperature.

In the present invention, "cell proliferation" refers to the ease with which cells proliferate at the culture temperature, and "having cell proliferation" refers to the ability of cells to adhere to a substrate or cell culture substrate directly or via a biological substance at the culture temperature and further proliferate. Furthermore, "not having cell proliferation" refers to the ability of cells to not adhere to a substrate or cell culture substrate at the culture temperature, or to adhere but not proliferate. Furthermore, "high cell proliferation" refers to the ability to proliferate into more cells when compared over the same culture period.

In the present invention, the hydrophilic polymer layer "coating the substrate along the unevenness" means that the surface unevenness resulting from the unevenness of the substrate is formed so that the maximum height at a reference length of 10 μm is 0.1 to 2 μm, and does not include a state in which the unevenness of the substrate surface is filled with the hydrophilic polymer layer, resulting in a maximum height of less than 0.1 μm at a reference length of 10 μm.

A cell culture substrate according to one embodiment of the present invention comprises a substrate having an uneven surface, and a hydrophilic polymer layer that covers the substrate along the uneven surface. The uneven surface of the substrate causes the hydrophilic polymer layer to have uneven surfaces. The uneven surface of the hydrophilic polymer layer is also the uneven surface of the cell culture substrate, and is referred to as "surface unevenness" in the present invention. Hereinafter, when the term "unevenness" is used, it means "uneven surface of the substrate" and is clearly distinguished from "surface unevenness". The maximum height of the surface unevenness at a reference length of 10 μm of the cell culture substrate is 0.1 to 2 μm, and the substrate has the following two regions (A) and (B). Region (A) is a plasma-treated region.
(A) an island-like region having an area of 0.001 to ⁵ mm2 and having cell adhesiveness and cell proliferation properties; (B) a region adjacent to the region (A) and not having cell proliferation properties;

In the present invention, a clear distinction is made between the "substrate" and the "cell culture substrate." Explaining with reference to FIG. 1, the substrate 1 is a member that comes into contact with the hydrophilic polymer layer 2, and the cell culture substrate 10 is the entire article for forming cell aggregates.

The material of the substrate is preferably at least one selected from the group consisting of glass, polystyrene, polyethylene, polyethylene terephthalate, polycarbonate, cellulose acetate, nitrocellulose, and polyvinylidene fluoride, more preferably at least one selected from the group consisting of glass, polystyrene, polyethylene, polyethylene terephthalate, and polycarbonate, particularly preferably at least one selected from glass, polystyrene, polyethylene terephthalate, and polycarbonate, and most preferably glass or polyethylene terephthalate.

The shape of the substrate, excluding the uneven portions, may be a planar shape such as a plate or film, a fiber, a porous particle, a porous membrane, a hollow fiber, etc.

The substrate may be a container generally used for cell culture (cell culture dishes such as petri dishes, flasks, well plates, bags, etc.) that has an uneven surface (cell culture surface). For ease of culture operations, a substrate with a planar shape such as a plate or film, or a flat porous membrane is preferable. If necessary, a partition plate may be provided on the substrate to provide a structure for separating each cell aggregate.

The structure of the convex portion present on the surface of the substrate may be at least one selected from the group consisting of a pyramidal structure, a frustum structure, a columnar structure, a columnar structure lying on its side, a spherical structure, a hemispherical structure, and a mountain-shaped structure.

Examples of the pyramidal structure include triangular pyramids, square pyramids, polygonal pyramids, and cones. When the convex portion is a pyramidal structure, the apex of the pyramid can contact the cell. The area of the base of the pyramidal structure may be 0.01 to 100 μm ² , 0.1 to 50 μm ² , or 1 to 10 μm ² .

Examples of the frustum structure include a triangular frustum, a square frustum, a polygonal frustum, a circular frustum, etc. When the convex portion has a frustum structure, the smaller of the two base surfaces may contact the cell. The area of the smaller of the two base surfaces of the frustum structure may be 0.01 to 100 μm ² , 0.1 to 50 μm ² , or 1 to 10 μm ² .

Examples of the columnar structure include triangular prisms, quadrangular prisms, polygonal prisms, and cylinders. When the convex portion is a columnar structure, one of the base surfaces can contact a cell. The area of the base surface of the columnar structure may be 0.01 to 100 μm ² , 0.1 to 50 μm ² , or 1 to 10 μm ² .

Examples of the columnar structure lying on its side include a triangular columnar structure and a cylindrical columnar structure. By lying the column on its side, linear projections and recesses can be obtained. When the convex portion is a columnar structure lying on its side, one side or one edge can contact the cell. The length of the columnar body lying on its side in the in-plane direction of the substrate may be 1 to 100 μm, 10 to 90 μm, 20 to 80 μm, 30 to 70 μm, or 40 to 60 μm.

When the convex portion has a spherical or hemispherical structure, the diameter of the sphere may be 0.1 to 10 μm, 0.2 to 5 μm, or 0.5 to 1 μm. When the convex portion has a hemispherical structure, the apex of the sphere may contact the cell. The diameter of the sphere and hemisphere may be 0.1 to 10 μm, 0.2 to 5 μm, or 0.5 to 1 μm.

When the convex portion has a mountain-shaped structure, the apex of the mountain may contact the cell. The long diameter of the mountain-shaped structure in the in-plane direction of the substrate may be 0.1 to 10 μm, 0.2 to 5 μm, or 0.5 to 1 μm.

The unevenness of the substrate may be a structure obtained by drilling a plurality of holes in a flat substrate sheet (hereinafter also referred to as a hole structure). In this case, the non-hole portion of the substrate sheet may be in contact with the cells. The holes may have the following shapes: a pyramid such as a triangular pyramid, a square pyramid, a polygonal pyramid, or a cone; a frustum such as a truncated triangular pyramid, a square pyramid, a polygonal pyramid, or a cone; a cylinder such as a triangular prism, a square prism, a polygonal prism, or a cylinder; a hemisphere; a valley shape, or the like. Examples of materials for the substrate sheet include the same materials as those listed as materials for the substrate.

The unevenness of the substrate can be formed by nanoimprinting, reactive etching, electron beam processing, etc. The unevenness of the substrate can also be formed by applying three-dimensional elements such as particles having the above-mentioned structure (at least one selected from the group consisting of a pyramidal structure, a frustum structure, a structure of a column lying on its side, a spherical structure, a hemispherical structure, and a mountain-shaped structure) to a flat substrate sheet.

The unevenness of the substrate is suitable for making the maximum height of the cell culture substrate after the hydrophilic polymer layer is formed 0.1 to 2 μm, so it is preferable that the maximum height at a reference length of 10 μm is 0.1 to 3 μm. The maximum height of the unevenness of the substrate at a reference length of 10 μm may be 0.1 μm or more, 0.2 μm or more, 0.3 μm or more, 0.4 μm, 0.8 μm or more, 1 μm or more, or 1.4 or more. The maximum height of the unevenness of the substrate at a reference length of 10 μm may be 3 μm or less, 2 μm or less, 1.6 μm or less, 1.5 μm or less, 1.2 μm or less, 1.0 μm or less, or 0.5 μm or less. The unevenness can be formed so that the maximum height is 0.1 to 3 μm by nanoimprinting, reactive etching, electron beam processing, etc. By applying a three-dimensional element to the substrate sheet, with a height of 0.1 to 3 μm from the surface in contact with the substrate sheet to the point or surface that can come into contact with the cell, it is possible to form irregularities with a maximum height of 0.1 to 3 μm.

The unevenness of the substrate is suitable for making it easier to peel off and recover cell aggregates, so a columnar structure with a height of 0.1 to 3 μm or a hole structure with a depth of 0.1 to 3 μm is preferred, with a columnar structure with a depth of 0.1 to 3 μm being even more preferred.

The number density of the three-dimensional elements is preferably 0.1 to 10 pieces/ ^μm2 , since this is suitable for both facilitating the detachment and recovery of cell aggregates and cell proliferation. In addition, in order to facilitate the detachment and recovery of cell aggregates, 0.5 pieces/ ^μm2 or more is more preferable, 1 piece/ ^μm2 or more is particularly preferable, and 5 pieces/ ^μm2 or more is most preferable. In addition, in order to enhance cell proliferation, 8 pieces/μm2 or ^less is more preferable, 5 pieces/ ^μm2 or less is particularly preferable, and 2 pieces/ ^μm2 or less is most preferable.

As the substrate on which the three-dimensional elements are applied, a substrate on which spherical particles having a diameter of 0.1 to 3 μm are fixed to the surface is preferred. Examples of materials for the three-dimensional elements include silica, polymethyl (meth)acrylate, polystyrene, etc. For three-dimensional objects, silica particles are preferred. As the silica particles, those which have been surface-treated with a silane coupling agent are preferred, since they are suitable for increasing the scratch resistance of the surface of the cell culture substrate, which makes it difficult to scratch even when the surface is rubbed with tweezers or a pipette, and those which have been surface-treated with a silane coupling agent having a reactive group are even more preferred. As the silane coupling agent having a reactive group, there are no particular limitations, but a silane coupling agent having a reactive double bond is even more preferred, a silane coupling agent having a vinyl group or a (meth)acrylic group is particularly preferred, and a silane coupling agent having a (meth)acrylic group is most preferred. In addition, a polymer (resin) for fixing the three-dimensional elements may be applied together.

Examples of the silane coupling agents include vinyltrimethoxysilane, vinyltriethoxysilane, 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, 3-glycidoxypropyltrimethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, Examples include silane, N-2-(aminoethyl)-3-aminopropylmethyldimethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, tris-(trimethoxysilylpropyl)isocyanurate, 3-ureidopropyltrialkoxysilane, 3-mercaptopropylmethyldimethoxysilane, bis(triethoxysilylpropyl)tetrasulfide, 3-isocyanatopropyltriethoxysilane, and 3-glycidoxypropylmethyldimethoxysilane.

The three-dimensional elements are preferably coated with a polymer (resin). The polymer (resin) for fixing the three-dimensional elements is not particularly limited, and examples thereof include at least one selected from the group consisting of polystyrene, polyethylene, polyethylene terephthalate, polycarbonate, cellulose acetate, nitrocellulose, and polyvinylidene fluoride resin, active energy ray crosslinkable resin, and thermal crosslinkable resin. In the case of an active energy ray crosslinkable resin, the resulting substrate has excellent scratch resistance. Here, in the present invention, "active energy rays" refers to ionizing radiation such as ultraviolet rays, electron beams, α rays, β rays, and γ rays. Examples of the above active energy ray crosslinkable resin include compounds having crosslinkable groups such as acrylic groups, methacrylic groups, oxetane groups, alicyclic epoxy groups, glycidyl groups, vinyl ether groups, maleimide groups, and acrylamide groups in the molecule. Crosslinkable resins having acrylic or methacrylic groups are preferred because they have better scratch resistance.

Examples of the crosslinkable resins having an acrylic or methacrylic group include monofunctional acrylates such as (meth)acrylate, methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, n-hexyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, cyclohexyl (meth)acrylate, 2-methoxyethyl (meth)acrylate, and p-methoxycyclohexyl (meth)acrylate; trimethylolpropane ethoxy triacrylate, trimethylolpropane propane ethoxy triacrylate, and the like. Polyfunctional (meth)acrylates such as methoxytriacrylate, pentaerythritol ethoxytetraacrylate, pentaerythritol triacrylate, pentaerythritol tetraacrylate, dipentaerythritol hexaacrylate, polyethylene glycol diacrylate, polypropylene glycol diacrylate, tricyclodecane dimethanol diacrylate, and ethoxylated phenyl acrylate; U-4HA, U-6HA, U-6LPA, UA-5300H, UA-122P, U-200PA, and UA-7100. (2-15 functional urethane acrylates manufactured by Shin-Nakamura Chemical Co., Ltd.); epoxy (meth)acrylates such as EBECRYL600, EBECRYL860, and EBECRYL373 (manufactured by Daicel-Allnex Corporation); polyester (meth)acrylates such as EBECRYL853 and EBECRYL1830 (manufactured by Daicel-Allnex Corporation); polymers having an acrylic group or a methacrylic group on the side chain (for example, NK Polymer AP-2500, NK Polymer GH-1203, etc., manufactured by Shin-Nakamura Chemical Co., Ltd.); LINC-3 Examples of such resins include monofunctional or polyfunctional (meth)acrylates containing fluorine such as 1,6-bis(acryloyloxy)hexane, ...

In the present invention, the hydrophilic polymer in the hydrophilic polymer layer may have either a phosphorylcholine group or a hydroxyl group. By having the hydrophilic polymer have a phosphorylcholine group or a hydroxyl group, it is possible to make the area coated with the hydrophilic polymer an area to which cells do not adhere. In addition, by having the hydrophilic polymer have a phosphorylcholine group or a hydroxyl group, the area can be made into an area having cell adhesiveness and cell proliferation properties by simply performing a short, weak plasma treatment. There are no particular limitations on the type of hydrophilic polymer other than having either a phosphorylcholine group or a hydroxyl group, but examples of commercially available products include Lipidure(R) CM5206 (manufactured by NOF Corp.), Lipidure(R) CM2001 (manufactured by NOF Corp.), and BIOSURFINE(R)-AWP (manufactured by Toyo Gosei Co., Ltd.). In addition, commercially available substrates coated with hydrophilic polymers such as PrimeSurface(R) (manufactured by Sumitomo Bakelite Co., Ltd.), EZ-BindShut(R) (manufactured by AGC Technoglass Co., Ltd.), and EZ-BindShutII(R) (manufactured by AGC Technoglass Co., Ltd.) can be suitably used.

The hydrophilic polymer layer preferably contains a compound represented by the above general formula (1) or (2). When the hydrophilic polymer layer contains a compound represented by the above general formula (1) or (2), cells are more likely to adhere and grow only in the (A) region, which is suitable for forming cell aggregates with a more uniform shape.

As ^R3 or ^R7 in the above general formula (1) or (2), a hydrophobic group or a UV-reactive functional group is preferable because it is suitable for immobilizing a hydrophilic polymer on a substrate. As the hydrophobic group, a linear or cyclic alkyl group such as a methyl group, an ethyl group, a propyl group, a butyl group, or a cyclohexyl group can be preferably used. In addition, as the UV-reactive functional group, an azide group can be preferably used. ^R3 or ^R7 may be a phenyl group or a phenylene group that may have a substituent such as an azide group.

The thickness of the hydrophilic polymer layer is preferably 5 to 2000 nm. If the thickness of the hydrophilic polymer layer is 5 to 2000 nm, the substrate can be easily coated along the unevenness of the substrate. In other words, the unevenness of the substrate is less likely to be filled with the hydrophilic polymer layer. By making the thickness of the hydrophilic polymer layer 5 to 2000 nm, it becomes easier to adhere and grow cells only in the (A) region, and also easier to collect cells in the (A) region by cell migration, thereby increasing the cell viability of the cell aggregate. Here, in the present invention, the "layer thickness" of the hydrophilic polymer layer refers to the length in the out-of-plane direction from the interface between the substrate and the hydrophilic polymer layer to the interface of the hydrophilic polymer layer on the opposite side to the substrate. In the range where the layer thickness exceeds 10 nm, the average layer thickness of the hydrophilic polymer layer can be obtained using ultrathin sections of the cell culture substrate prepared by a microtome. Specifically, the average layer thickness can be calculated by observing the cross-sectional image of the ultrathin section with a transmission electron microscope, measuring the thickness at 10 randomly selected points, and averaging the measured values. In addition, when the layer thickness is in the range of 10 nm or less, it can be measured using an ellipsometer. Since it is suitable for suppressing adhesion of cells to the (B) region, a layer thickness of 10 nm or more is more preferable, 50 nm or more is particularly preferable, and 100 nm or more is most preferable. In addition, since it is suitable for increasing the cell survival rate of the cell aggregate by collecting cells in the (A) region through cell migration, a layer thickness of 1000 nm or less is more preferable, 500 nm or less is particularly preferable, and 200 nm or less is most preferable.

The cell culture substrate of the present invention has a maximum height of 0.1 to 2 μm at a reference length of 10 μm of the surface irregularities. Since the maximum height is 0.1 μm or more, the cultured cell aggregates are weakly attached to the cell culture substrate, and the cell aggregates can be easily peeled off and collected without temperature change after culture. Furthermore, since the maximum height is 0.1 μm or more, the cells seeded on the cell culture substrate can be promoted to spontaneously associate by migration, and a cell aggregate composed of cells with a high cell survival rate can be formed. Since the cell aggregates can be more easily collected and are suitable for forming a cell aggregate composed of cells with a high cell survival rate, the maximum height may be 0.2 μm or more, 0.3 μm or more, 0.4 μm, 0.8 μm or more, 1 μm or more, or 1.4 μm or more. Since the maximum height is 2 μm or less, cell adhesiveness and cell proliferation properties can be imparted, and the cell aggregates can be cultured in a short time. Since it is suitable for culturing cell aggregates in a short time, the maximum height may be 1.5 μm or less, 1.2 μm or less, 1.0 μm or less, or 0.5 μm or less. Here, the "maximum height" of the cell culture substrate in the present invention refers to the maximum height Rz of the contour curve at a reference length of 10 μm determined according to JIS B0601:2013 (for example, the length indicated by symbol H in FIG. 1), and can be calculated by measuring an atomic force microscope image of a representative surface of the cell culture substrate and determining the maximum height at a reference length of 10 μm for the contour curve at 10 randomly selected points.

In the cell culture substrate of the present invention, the distance between the convex portions of the surface irregularities is preferably 0.1 to 10 μm. By having the distance between the convex portions be 0.1 to 2 μm, cell proliferation is enhanced, cell aggregates are formed in a short time, and the cell aggregates can be more easily peeled off without temperature change, thereby increasing the recovery rate of the cell aggregates. Since it is more suitable for forming cell aggregates in a short time, the distance between the convex portions may be 0.5 μm or more, 0.6 μm or more, 0.7 μm or more, 0.8 μm or more, 0.9 μm or more, 1 μm or more, 1.5 μm or more, or 3 μm or more. Since it is more suitable for increasing the recovery rate of the cell aggregates, the distance between the convex portions may be 5 μm or less, 2.5 μm or less, 2 μm or less, or 1 μm or less. Here, the "distance between convexities" of the cell culture substrate in the present invention refers to the length of the contour element determined in accordance with JIS B0601:2013 (for example, the length indicated by the symbol P in FIG. 1), and can be calculated by measuring an atomic force microscope image of a representative surface of the cell culture substrate and determining the length between 20 or more convexities on a contour curve randomly selected with a reference length of 10 μm.

The cell culture substrate of the present invention preferably has a coefficient of variation of the distance between the convex portions of the surface unevenness of 3 to 50%. By having a coefficient of variation of the distance between the convex portions of 3 to 50%, a uniform surface can be provided to all cells adhering to the (A) region, and thus the cultured cell aggregates can be homogeneously peeled off. By homogeneously peeling off the cultured cell aggregates, it is possible to suppress the cell aggregates from being partially damaged during peeling, which causes variation in particle size. Since this is suitable for homogeneously peeling off the cultured cell aggregates, it is more preferable that the coefficient of variation of the distance between the convex portions of the surface unevenness is 40% or less, particularly preferably 30% or less, and most preferably 20% or less. However, in the case of a completely uniform structure, when light is applied to the cell culture substrate, moire (interference fringes) are generated, which may make it difficult to observe the cells using a microscope that is normally used for observing the cells. Therefore, in order to facilitate microscope observation, it is more preferable that the coefficient of variation of the distance between the convex portions of the surface unevenness is 5% or more, particularly preferably 7% or more, and most preferably 10% or more. Here, the coefficient of variation of the distance between convex portions can be calculated by determining the distance between convex portions described above and calculating the coefficient of variation (standard deviation/average value) for the 20 measured values.

The (A) region has cell adhesiveness and cell proliferation properties. By having the (A) region have cell adhesiveness and cell proliferation properties, the cell culture substrate of the present invention can form cell aggregates by adhesion culture of cells. As a result, it becomes easier to prevent cell aggregates from floating and bonding, resulting in uneven sizes. If the (A) region does not have cell adhesiveness or cell proliferation properties, cells cannot be cultured to form cell aggregates.

The (A) region is an island-shaped region having an area of 0.001 to ⁵ mm2. By having an island-shaped region having an area of 0.001 to ⁵ mm2, it becomes easier to form a cell aggregate having a uniform particle size when cells are cultured. Since it is more suitable for forming a cell aggregate suitable for applications such as differentiation induction of pluripotent stem cells, the area of the (A) region may be 0.005 ^mm2 or more, 0.01 ^mm2 or more, or 0.02 ^mm2 or more. Since it is more suitable for forming a cell aggregate suitable for applications such as differentiation induction of pluripotent stem cells, the area of the (A) region may be 1 ^mm2 or less, 0.5 ^mm2 or less, 0.2 ^mm2 or less, 0.1 ^mm2 or less, or 0.05 ^mm2 or less.

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

In the present invention, "island-like" refers to an independent region arranged on a plane like an island structure in a sea-island structure. Since the (A) region is an island-like region having cell adhesiveness and cell proliferation properties, the cell culture substrate of the present invention can produce cell aggregates of more uniform size. If the (A) region is not island-like, for example, if it has a striped structure, cell aggregates cannot be produced. There is no particular limitation on the shape of the island, which can be set appropriately depending on the shape of the desired cell aggregate. Examples of the island shape include a circle, an ellipse, a polygon, and a closed shape consisting of an indefinite straight line or curve. In addition, since it is suitable for producing cell aggregates with a shape close to a sphere, the island shape is preferably a circle, an ellipse, or a polygon, more preferably a circle, an ellipse, or a rectangle, particularly preferably a circle, an ellipse, or a square, and most preferably a circle or an ellipse.

In addition, since the present invention is suitable for producing cell aggregates having a shape close to a sphere, the aspect ratio of the island shape is preferably 5 or less, more preferably 2 or less, particularly preferably 1.5 or less, and most preferably 1.1 or less. Here, in the present invention, "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.

The (B) region is adjacent to the (A) region and does not have cell adhesion or cell proliferation properties. By being adjacent to the (A) region and not having cell proliferation properties, when cells are cultured, it is possible to form cell aggregates only in the (A) region, and to create a state in which no cells are present around the (A) region. In addition, since this is more suitable for uniforming the size and shape of the cell aggregates produced, it is preferable that the (B) region not only has no cell proliferation properties but also no cell adhesion properties.

There are no limitations on the shape of the (B) region other than that it is adjacent to the (A) region, but since this is more suitable for producing cell aggregates of uniform size and shape, it is preferable that the (B) region is adjacent to the (A) region for a length of at least 20% of the boundary line, more preferably at least 50%, particularly preferably at least 80%, and it is most preferable that the (A) region is entirely surrounded by the (B) region. In addition, since this is suitable for increasing the mass productivity of the cell culture substrate, it is preferable that the (A) region is an island-like structure and the (B) region is an ocean-like structure.

There is no particular limitation on the area ratio of the above (A) region and (B) region, but since this is suitable for increasing the number of cell aggregates that can be produced per unit area of the culture substrate, the area of the (A) region is preferably 10% or more of the entire substrate, more preferably 30% or more, particularly preferably 50% or more, and most preferably 70% or more. In addition, since it 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 an uneven shape, the area of the (B) region is preferably 20% or more of the entire substrate, more preferably 40% or more, particularly preferably 60% or more, and most preferably 80% or more.

The step at the boundary between the above (A) and (B) regions is preferably 1 nm or more, more preferably 10 nm or more, particularly preferably 50 nm or more, and most preferably 100 nm or more, because it is suitable for suppressing air bubble adhesion when a medium is added to a cell culture substrate, and is also suitable for suppressing air bubble adhesion, so the step is preferably 10,000 nm or less, more preferably 1000 nm or less, particularly preferably 500 nm or less, and most preferably 200 nm or less. If air bubbles adhere, it is necessary to perform degassing or repeatedly discharge and aspirate the medium using a pipette to remove the air bubbles, which impairs the mass productivity of the cell aggregates, so it is desirable that air bubbles do not adhere. Here, in the present invention, the "step" at the boundary between region (A) and region (B) refers to the difference in length of the cell culture substrate at the boundary between region (A) and region (B) in the out-of-plane direction of the substrate, and when the step is less than 10 nm, it can be measured using an ellipsometer. When the step is in the range of 10 to 500 nm, a cross-sectional image can be measured using a transmission electron microscope using an ultrathin section of the cell culture substrate created with a microtome, and the distance can be calculated by measuring and averaging the distance at 10 randomly selected points. Furthermore, when the step is in the range of more than 500 nm, it can be measured using a stylus-type step gauge.

Furthermore, since this is suitable for forming a cell aggregate suitable for culturing pluripotent stem cells and inducing differentiation into endodermal or ectodermal cells, it is preferable that the area of the (A) region is 0.005 to ^0.2 mm2 and the number per unit area of the (A) region is 200 to 1000 cells/ ^cm2 , it is more preferable that the area of the (A) region is 0.01 to 0.15 ^mm2 and the number per unit area of the (A) region is 250 to 800 cells/ ^cm2 , it is particularly preferable that the area of the (A) region is 0.02 to 0.1 ^mm2 and the number per unit area of the (A) region is 300 to 600 cells/ ^cm2 , and it is most preferable that the area of the (A) region is 0.03 to 0.05 ^mm2 and the number per unit area of the (A) region is 300 to 400 cells/ ^cm2 .

Furthermore, since this is suitable for forming a cell aggregate suitable for culturing pluripotent stem cells and inducing differentiation into mesodermal cells, it is preferable that the area of the (A) region is 0.2 to ² mm2 and the number per unit area of the (A) region is 3 to 15 cells/ ^cm2 , it is more preferable that the area of the (A) region is 0.3 to ^1.5 mm2 and the number per unit area of the (A) region is 4 to 12 cells/ ^cm2 , it is particularly preferable that the area of the (A) region is 0.4 to ¹ mm2 and the number per unit area of the (A) region is 5 to 10 cells/ ^cm2 , and it is most preferable that the area of the (A) region is 0.5 to 0.7 ^mm2 and the number per unit area of the (A) region is 6 to 8 cells/ ^cm2 .

In addition, 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 above (A) regions is preferably 500 to 10,000 μm, more preferably 1,000 to 8,000 μm, particularly preferably 2,000 to 5,000 μm, and most preferably 3,000 to 4,000 μm.

In the present invention, the above-mentioned (A) region is a region in which the hydrophilic polymer layer has been modified by plasma treatment. By using the (A) region as a plasma-treated region, cells can adhere and grow only in the (A) region. Since the plasma treatment of the hydrophilic polymer layer can be performed in a short time, the mass productivity of the cell culture substrate can be improved.

It is preferable that the ratio of the peak intensity at 287 eV/peak intensity at 285 eV in the C1s spectrum of the XPS measurement is 0.05 or more higher in the (A) region than in the (B) region. Since the ratio of the peak intensity at 287 eV/peak intensity at 285 eV in the C1s spectrum of the XPS measurement is 0.05 or more higher in the (A) region than in the (B) region, the difference in cell proliferation between the (A) region and the (B) region can be increased. As a result, it is possible to make it easier for cells to proliferate only in the (A) region, and to form a more uniform cell aggregate. Since it is suitable for forming a more uniform cell aggregate, the ratio of the peak intensity at 287 eV/peak intensity at 285 eV in the C1s spectrum of the XPS measurement is more preferably 0.07 or more, particularly preferably 0.1 or more, and most preferably 0.15 or more.

The (A) region preferably has a carboxyphenyl group because it is suitable for enhancing cell proliferation and forming cell aggregates in a short time. The type of carboxyphenyl group is not particularly limited, but a phenyl group having 1 to 3 carboxy groups bonded thereto is preferred. For example, when the hydrophilic polymer contains a compound represented by formula (1) or (2), and R ³ or R ⁷ has a phenyl group or a phenylene group to which a substituent such as an azide group may be bonded, a carboxyphenyl group can be introduced into the (A) region by subjecting the hydrophilic polymer layer to a plasma treatment.

The above (A) region is not prevented from having temperature responsiveness. When culturing cells on a cell culture substrate, the cells can be cultured at a temperature close to body temperature, so the response temperature is preferably 50°C or lower, and more preferably 35°C or lower. Since this makes it easier to prevent cells from detaching when performing operations such as changing the medium during cell culture, the response temperature may be 25°C or lower or 15°C or lower. Furthermore, since it is possible to form cell aggregates by cooling operations 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 particularly preferably 15°C or higher.

The cell culture substrate according to one embodiment of the present invention may be formed by bonding a plate having a through hole with a cross-sectional area in the in-plane direction of 0.05 to ¹⁰⁰ cm2.

In the present invention, the cell culture substrate may contain a biological substance on the surface as necessary. There is no particular limitation on the biological substance, but examples of the biological substance include matrigel, laminin, fibronectin, vitronectin, collagen, etc.

These biological substances may be natural products, or may be artificially synthesized using recombinant gene technology, or may be fragments obtained by cleavage with restriction enzymes, or may be synthetic proteins or peptides based on these biological substances.

In the present invention, as the above-mentioned Matrigel, commercially available products such as Matrigel (manufactured by Corning Incorporated) and Geltrex (manufactured by Thermo Fisher Scientific) can be preferably used due to their ease of availability.

The type of laminin is not particularly limited, but for example, laminin 511, laminin 521, or laminin 511-E8 fragment, which have been reported to exhibit high activity against α6β1 integrin expressed on the surface of human iPS cells, can be used. The laminin may be a natural product, may be artificially synthesized using recombinant gene technology, or may be a synthetic protein or peptide based on the laminin. In view of ease of availability, a commercially available product such as iMatrix-511 (manufactured by Nippi Corporation) can be preferably used.

The vitronectin may be a natural product, may be artificially synthesized by recombinant gene technology, or may be a synthetic protein or peptide based on the 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 suitably used.

The fibronectin may be a natural product, may be artificially synthesized by recombinant gene technology, or may be a synthetic protein or peptide based on the fibronectin. In view of ease of availability, commercially available products such as fibronectin solution, human plasma-derived (manufactured by Wako Pure Chemical Industries, Ltd.) and Retronectin (manufactured by Takara Bio Inc.) can be suitably used.

The type of collagen is not particularly limited, but for example, type I collagen or type IV collagen can be used. The collagen may be a natural product, may be artificially synthesized using recombinant gene technology, or may be a synthetic peptide based on the 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.

In the present invention, it is preferable that the biological substance is immobilized on the cell culture substrate by a non-covalent bond rather than a covalent bond, since this can suppress the denaturation of the biological substance and further enhance cell proliferation. Here, in the present invention, "non-covalent bond" refers to a bonding force other than a covalent bond that is derived from intermolecular forces, such as electrostatic interactions, water-insoluble interactions, hydrogen bonds, π-π interactions, dipole-dipole interactions, London dispersion forces, and other van der Waals interactions. The biological substance may be immobilized on the block copolymer by a single bonding force or a combination of multiple bonding forces.

In the present invention, the method for immobilizing a biological substance is not particularly limited, but for example, a method of immobilizing the biological substance by applying a solution of the biological substance to a cell culture substrate for a predetermined period of time, or a method of adding the biological substance to the culture medium during cell culture to adsorb the biological substance to the cell culture substrate and immobilize it can be suitably used.

The cell culture substrate of the present invention 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. High-pressure steam sterilization, UV sterilization, and ethylene oxide gas sterilization are preferred because they are suitable for suppressing denaturation of the block copolymer, UV sterilization and ethylene oxide gas sterilization are more preferred because they are suitable for suppressing deformation of the substrate, and ethylene oxide gas sterilization is particularly preferred because of its excellent mass productivity.

The cells to be cultured using the cell culture substrate of the present invention are not particularly limited as long as they can adhere to the surface before the stimulation by temperature drop is applied. For example, in addition to various established cell lines such as Chinese hamster ovary-derived CHO cells, mouse connective tissue L929, human embryonic kidney-derived HEK293 cells, and human cervical cancer-derived HeLa cells, for example, epithelial cells and endothelial cells constituting various tissues and organs in the body, skeletal muscle cells exhibiting contractility, smooth muscle cells, cardiac muscle cells, neuron cells constituting the nervous system, glial cells, fibroblasts, hepatic parenchymal cells involved in the metabolism of the body, non-parenchymal hepatic cells, and adipocytes, as cells having differentiation ability, stem cells present in various tissues such as mesenchymal stem cells, bone marrow cells, and Muse cells, as well as stem cells having differentiation pluripotency such as ES cells and iPS cells (pluripotent stem cells), and cells induced to differentiate from them, can be used. From the viewpoint of cell proliferation and detachment in the culture substrate of the present invention, stem cells or pluripotent stem cells are preferred, mesenchymal stem cells or pluripotent stem cells are more preferred, pluripotent stem cells are particularly preferred, and iPS cells are most preferred.

A method for producing a cell culture substrate according to another embodiment of the present invention includes the following steps (1) and (2).
(1) A step of forming a hydrophilic polymer layer on a substrate having an uneven surface along the unevenness, the maximum height of the surface unevenness of the hydrophilic polymer layer being 0.1 to 2 μm at a reference length of 10 μm; (2) A step of performing a plasma treatment on only a part of the surface of the hydrophilic polymer layer to form the two regions (A) and (B).

The substrate having the uneven surface in the step (1) can be obtained by nanoimprinting, reactive etching, electron beam processing, etc. The step (1) may include the steps (1-1) and (1-2).
(1-1) A step of applying three-dimensional elements having a major axis of 0.1 to 3 μm, which may be surface-coated with a polymer, to a substrate sheet, and arranging the three-dimensional elements on the substrate sheet; or, a step of applying three-dimensional elements having a major axis of 0.1 to 3 μm, which are surface-coated with a polymer, to a substrate sheet, and then fusing the three-dimensional elements to the substrate sheet by heating. (1-2) A step of coating a hydrophilic polymer with a layer thickness of 5 to 200 nm on the surface of the substrate sheet on which the three-dimensional elements are arranged or fixed (bonded).

In step (1-1), when a three-dimensional element whose surface is coated with a polymer is used and fused to the substrate sheet by heating, the three-dimensional element is fixed to the substrate sheet, so that the three-dimensional element is less likely to move or aggregate in steps (1-2) and after, and it becomes easier to form a uniform surface structure over the entire surface of the cell culture substrate. The polymer is a polymer (resin) capable of fixing the three-dimensional elements such as the particles described above.

In step (1-2), a UV-reactive hydrophilic polymer is coated on the surface of the substrate sheet to form a hydrophilic polymer layer. By forming the hydrophilic polymer layer, the surface of the cell culture substrate can be rendered in a state in which it has no cell adhesiveness or cell proliferation properties. There are no particular limitations on the method for forming the hydrophilic polymer layer, but various commonly known methods can be used, such as coating, brush coating, dip coating, spin coating, bar coding, flow coating, spray coating, roll coating, air knife coating, blade coating, gravure coating, microgravure coating, and slot die coating.

The step (1) may include a step of irradiating the hydrophilic polymer layer with UV light to chemically react the hydrophilic polymer layer and fix it to the substrate surface. By irradiating the UV-reactive hydrophilic polymer with UV light, a chemical reaction occurs between the hydrophilic polymers or between the hydrophilic polymer and the substrate, and the hydrophilic polymer layer is fixed to the substrate surface. Fixing the hydrophilic polymer layer to the substrate surface can increase the scratch resistance of the cell culture substrate. A schematic diagram of a substrate having an uneven surface used in the step (1) is shown in Figure 2. The substrate coated with the hydrophilic polymer layer obtained in the step (1) is shown in Figure 3.

In step (2), the surface of the immobilized hydrophilic polymer layer is subjected to plasma treatment to form an area having cell adhesiveness and cell proliferation properties and having an area of 0.001 to ⁵ mm2. A suitable plasma treatment method can be appropriately adjusted depending on the type of hydrophilic polymer used and the layer thickness, but is preferably 5 seconds to 30 minutes, more preferably 5 seconds to 10 minutes, particularly preferably 10 seconds to 5 minutes, and most preferably 15 seconds to 1 minute. In addition, the patterning method is not particularly limited, but includes a method in which the surface is treated with plasma in a state covered with a metal mask, a silicon mask, a surface protection film, or the like to form a desired shape. The cell culture substrate according to one embodiment of the present invention obtained by steps (1) and (2) is shown in FIG. 4.

The method for producing a cell culture substrate according to one embodiment of the present invention may further include step (3). The surface of the plasma-treated hydrophilic polymer is coated with a temperature-responsive polymer to form a layer of the temperature-responsive polymer. In this case, by forming a layer of the temperature-responsive substance on the surface of the substrate with a layer thickness of 100 nm or less, even when step (3) is performed after step (2), the molecular chains of the temperature-responsive substance are easily coated in a sparse state on the cell-proliferative surface formed in the patterning step, which is suitable for imparting temperature responsiveness while maintaining the effect of patterning. In addition, by making the layer thickness 1 nm or more, it is possible to produce a cell culture substrate that is capable of imparting sufficient temperature responsiveness and quickly performing the cell aggregate formation step, which is suitable.

The method for coating the temperature-responsive substance can be the same as the above-mentioned hydrophilic polymer coating method.

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

The method for producing a cell culture substrate according to one aspect of the present invention may further include a step (4).
(4) A step of bonding a plate having a through hole with a cross-sectional area of 0.05 to ¹⁰⁰ cm2 in the in-plane direction to the substrate.

In step (4), a plate having through holes with a cross-sectional area of 0.05 to ¹⁰⁰ cm2 in the in-plane direction is attached to the substrate, thereby making it possible to produce cell culture dishes having a space for placing a culture medium with high mass productivity. A cell culture substrate according to one embodiment of the present invention obtained by steps (1), (2), and (4) is shown in Figure 5. In the cell culture substrate of Figure 5, step (3) may or may not be performed.

Yet another aspect of the present invention relates to a method for producing a cell aggregate using the above-mentioned cell culture substrate. The method for producing a cell aggregate includes the following steps (i) and (ii):
(i) seeding pluripotent stem cells on the cell culture substrate; and (ii) culturing the seeded pluripotent stem cells to form hemispherical cell aggregates having an average substrate height in the out-of-plane direction/diameter in the in-plane direction of the substrate of 0.2 to 0.8.

Yet another aspect of the present invention relates to a method for inducing differentiation of pluripotent stem cells, comprising the above-mentioned method for producing a cell aggregate, further comprising the following step (iii):
(iii) A step of inducing differentiation of the cell aggregate to form a cell aggregate of three germ layers cells.

Step (i) is a step of seeding cells on the cell culture substrate using the cell culture substrate. In the present invention, "seeding cells" refers to contacting the cell suspension with the cell culture substrate by applying a medium in which cells are dispersed (hereinafter referred to as a "cell suspension") onto the cell culture substrate or by injecting the medium into the cell culture substrate. By using a cell culture substrate having a cell proliferation region, cells can be cultured in step (ii) described below. If the cell culture substrate does not have a cell proliferation region, cells cannot be cultured in step (ii).

In the above step (i), the culture is performed under conditions effective for maintaining the undifferentiated state of the cells. There are no particular limitations on the conditions effective for maintaining the undifferentiated state, but examples include setting the cell density at the start of the culture to the preferred range described below as the cell density at the time of seeding, and carrying out the culture in the presence of an appropriate liquid medium. As a medium effective for maintaining the undifferentiated state of the cells, for example, a medium containing one or more of insulin, transferrin, selenium, ascorbic acid, sodium bicarbonate, basic fibroblast growth factor, transforming growth factor β (TGFβ), CCL2, activin, and 2-mercaptomethanol, which are known to be factors for maintaining the undifferentiated state of the cells, can be preferably used. Since it is particularly suitable for maintaining the undifferentiated state of the cells, it is more preferable to use a medium containing insulin, transferrin, selenium, ascorbic acid, sodium bicarbonate, basic fibroblast growth factor, and transforming growth factor β (TGFβ), and it is most preferable to use a medium containing basic fibroblast growth factor.

There are no particular limitations on the type of medium to which the basic fibroblast growth factor is added. For example, commercially available products include DMEM (Sigma-Aldrich Co. LLC), Ham's F12 (Sigma-Aldrich Co. LLC), D-MEM/Ham's F12 (Sigma-Aldrich Co. LLC), Primate ES Cell Medium (REPROCELL Co., Ltd.), StemFit AK02N (Ajinomoto Co., Inc.), StemFit AK03 (Ajinomoto Co., Inc.), mTeSR1 (STEMCELL TECHNOLOGIES), TeSR-E8 (STEMCELL TECHNOLOGIES), ReproNaive (manufactured by REPROCELL Co., Ltd.), ReproXF (manufactured by REPROCELL Co., Ltd.), ReproFF (manufactured by REPROCELL Co., Ltd.), ReproFF2 (manufactured by REPROCELL Co., Ltd.), NutriStem (manufactured by Biological Industries, Inc.), iSTEM (manufactured by Takara Bio Inc.), GS2-M (manufactured by Takara Bio Inc.), hPSC Growth Medium DXF (manufactured by PromoCell Co., Ltd.), and the like. Primate ES Cell Medium (manufactured by REPROCELL Co., Ltd.), StemFit AK02N (manufactured by Ajinomoto Co., Ltd.) or StemFit AK03 (manufactured by Ajinomoto Co., Ltd.) are preferred because they are suitable for maintaining the undifferentiated state of cells, with StemFit AK02N (manufactured by Ajinomoto Co., Ltd.) or StemFit AK03 (manufactured by Ajinomoto Co., Ltd.) being more preferred, and StemFit AK02N (manufactured by Ajinomoto Co., Ltd.) being particularly preferred.

In the above step (i), the method of seeding the cells is not particularly limited, but for example, the cell suspension can be injected into the cell culture substrate. The cell density at the time of seeding is not particularly limited, but in order to maintain and proliferate the cells, 1.0×10 ² to 1.0×10 ⁶ cells/cm ² is preferable, 5.0×10 ² to 5.0×10 ⁵ cells/cm ² is more preferable, 1.0×10 ³ to 2.0×10 ⁵ cells/cm ² is particularly preferable, and 1.2×10 ³ to 1.0×10 ⁵ cells/cm ² is most preferable.

The medium used in the above step (i) is preferably a medium containing the above basic fibroblast growth factor and a Rho-binding kinase inhibitor, since this is suitable for maintaining cell survival. In particular, when human cells are used and the cell density of the human cells is low, the addition of a Rho-binding kinase inhibitor may be effective in maintaining the survival of the human cells. Examples of the Rho-binding kinase inhibitor that can be used include (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide.2HCl.H ₂ O (Y-27632, manufactured by Wako Pure Chemical Industries, Ltd.) and 1-(5-Isoquinolinesulfonyl) homopiperazine hydrochloride (HA1077, manufactured by Wako Pure Chemical Industries, Ltd.). The concentration of the Rho-binding kinase inhibitor added to the medium is within a range that is effective for maintaining the survival of human cells and does not affect the undifferentiated state of human cells, and is preferably 1 μM to 50 μM, more preferably 3 μM to 20 μM, even more preferably 5 μM to 15 μM, and most preferably 8 μM to 12 μM.

Shortly after starting step (i) above, the cells begin to adhere to the cell culture substrate.

In step (ii), the seeded pluripotent stem cells are cultured to form hemispherical cell aggregates with an average height outside the substrate plane/diameter inside the substrate plane of 0.2 to 0.8. The culture temperature is preferably 30 to 42°C, more preferably 32 to 40°C, particularly preferably 36 to 38°C, and most preferably 37°C, as this is suitable for maintaining the proliferation ability, physiological activity, and function of the cells.

The first medium change is preferably performed 22 to 26 hours after the start of the above step (ii). The second medium change is preferably performed 48 to 72 hours later, and thereafter, the medium change is preferably performed every 24 to 48 hours. During this time, the cells grow and form flat cell clusters called colonies. A colony is a state in which cells are two-dimensionally attached to a cell culture substrate. The culture is continued until the size of the colony becomes about the size of region (A), and by continuing the culture further, a three-dimensional cell aggregate is formed. The shape of the three-dimensional cell aggregate is preferably a hemisphere with an average height in the outward direction of the substrate surface/diameter in the inward direction of the substrate surface of 0.2 to 0.8. The height in the outward direction of the substrate surface/diameter in the inward direction of the substrate surface are calculated by observing multiple cell aggregates under a microscope and averaging the values. By forming a hemispherical cell aggregate with an average height in the outward direction of the substrate surface/diameter in the inward direction of the substrate surface of 0.2 to 0.8, differentiation of pluripotent stem cells into three germ layers can be efficiently induced. Furthermore, since this is suitable for increasing the efficiency of inducing differentiation into three germ layer cells, it is more preferable that the average of the height in the outward direction of the substrate surface/diameter in the inward direction of the substrate surface is 0.3 to 0.7, and 0.4 to 0.6 is particularly preferable.

In the step (iii) of the present invention, the cell aggregate is induced to differentiate to form a cell aggregate of three germ layers. The step (iii) of the present invention may be performed after the step (ii) or in parallel with the step (ii). That is, the step (iii) may be started immediately after the seeded pluripotent stem cells adhere to the cell culture substrate, and differentiation may be induced while forming a cell aggregate. In the present invention, the three germ layers refer to any of endodermal cells, mesodermal cells, and ectodermal cells. In the step (iii), the cells are cultured in a medium containing a differentiation-inducing factor.

The differentiation induction factor in the above step (iii) preferably contains an endoderm induction factor, since this is suitable for increasing the efficiency of differentiation induction into endoderm cells. As the endoderm induction factor, since this is suitable for increasing the efficiency of differentiation induction into embryoid bodies, it is preferable that the differentiation induction factor is a single or multiple differentiation induction factors selected from the group consisting of Wnt protein, bone morphogenetic protein (BMP), insulin-like growth factor, and activin, and it is particularly preferable that the differentiation induction factor contains any one of Wnt3a, BMP4, IGF1, and activin A.

The differentiation induction factor in the above step (iii) preferably contains a mesoderm induction factor, since it is suitable for increasing the efficiency of differentiation induction into mesoderm cells. As the above mesoderm induction factor, since it is suitable for increasing the efficiency of differentiation induction into embryoid bodies, it is preferable that the differentiation induction factor is a single or multiple differentiation induction factors selected from the group consisting of a GSK3β inhibitor, bone morphogenetic protein (BMP) and activin, and it is particularly preferable that the differentiation induction factor contains any one of activin A, CHIR99021 (GSK3β inhibitor) and BMP4.

The differentiation induction factor in the above step (iii) preferably contains an ectodermal induction factor, since this is suitable for increasing the efficiency of differentiation induction into ectodermal cells. The above ectodermal induction factor preferably contains any one of Noggin (BMP inhibitor), dorsomorphin (BMP inhibitor), SB431542 (TGF-β inhibitor), and an activin inhibitor, and more preferably contains a BMP inhibitor and a TGF-β inhibitor.

The concentration of the differentiation-inducing factor added to the medium is not particularly limited, but is preferably 1000 to 500 ng/mL, more preferably 500 to 100 ng/mL, and most preferably 100 ng/mL or less, in order to increase the efficiency of inducing differentiation into embryoid bodies.

The medium containing the differentiation-inducing factors is preferably changed every 24 hours during culture in order to ensure that differentiation induction progresses sufficiently. By changing the medium within 24 hours, it is possible to prevent a shortage of differentiation-inducing factors in the medium and to differentiate all cells uniformly.

The method of the present invention for inducing differentiation of pluripotent stem cells may further include the following step (iv).
(iv) culturing the cell aggregate in a medium containing one or more differentiation-inducing factors selected from the group consisting of Wnt protein, bone morphogenetic protein (BMP), insulin-like growth factor, and activin, to express a marker possessed by intestinal epithelial cells;

The intestinal epithelial cells preferably include any one or more of intestinal cells, goblet cells, enteroendocrine cells, Paneth cells, and intestinal epithelial stem cells, and more preferably include all of intestinal cells, goblet cells, enteroendocrine cells, Paneth cells, and intestinal epithelial stem cells. Intestinal epithelial cells have specific markers. For example, CDX2 and VIL1 are intestinal cell markers, MUC2 is a goblet cell marker, CGA is an enteroendocrine cell marker, DEFA6 is a Paneth cell marker, and LGR5 is an intestinal epithelial stem cell marker.

The cell culture substrate of the present invention does not have a surface with a high uneven structure with a height of more than 2 μm in the outward direction of the substrate, so air bubbles are unlikely to adhere to the substrate surface when the substrate is brought into contact with a culture medium. The number of air bubbles adhering to the substrate surface is preferably 10% or less of the number of regions (A), more preferably 5% or less, particularly preferably 3% or less, and most preferably 1% or less.

Compared to cell aggregates formed using a conventional fine uneven structure (having a high uneven structure with a height of more than 2 μm in the out-of-plane direction of the substrate), the cell aggregates formed using the cell culture substrate of the present invention have a high cell viability because the attached cells spontaneously gather. For example, when cells with a viability of 50% are used, the cell viability of the formed cell aggregates is preferably 70% or more, more preferably 80% or more, particularly preferably 85% or more, and most preferably 90% or more.

The present invention will be described in detail below with reference to embodiments for carrying out the invention. However, these are merely examples for explaining the present invention, and are not intended to limit the present invention to the following contents. Furthermore, the present invention can be practiced with appropriate modifications within the scope of the gist of the invention. Unless otherwise specified, commercially available reagents were used.

<Thickness of Hydrophilic Polymer Layer>
The layer thickness of the hydrophilic polymer coated on the substrate was determined by calculating the coating amount per unit area from the thickness of the liquid film under spin coating conditions and the weight concentration of the polymer in the solution, assuming the specific gravity of the polymer to be 1.

<(A) Area of Region>
The surface was observed using a laser microscope (VK-X200, manufactured by Keyence Corporation), and the area was calculated.

<Maximum height of surface irregularities>
The surface of the cell culture surface of the cell culture substrate was measured with an atomic force microscope (AFM5100, manufactured by Hitachi High-Technologies Corporation), and the maximum height Rz was calculated from a cross-sectional shape curve having a reference length of 10 μm. The maximum heights at 10 points were averaged.

<Distance between convex portions of surface irregularities>
The length between the convex portions was determined in an image of the cell culture surface of the cell culture substrate measured with an electron microscope (Miniscope TM3030Plus, Hitachi High-Technologies Corporation). The lengths between the convex portions at 20 points were determined and averaged.

<Coefficient of variation of distance between convex portions of surface unevenness>
The standard deviation was calculated for the distances between the 20 convex portions, and the standard deviation was divided by the average to obtain a coefficient of variation.

<Evaluation of cell aggregates>
Cultivation of cell aggregates:
The medium StemFitAK02N (Ajinomoto Co., Inc.) was added to the substrate at 0.2 mL/ ^cm2 , and iMatrix-511 solution (Nippi Co., Ltd.) was added at a concentration of 2.5 μL/mL. Human iPS cells 201B7 strain, which had been adjusted to a ratio of viable cells to total cells (cell viability) of 50% by mixing dead cells, were used as seeding cells and cultured at 15,000 cells/ ^cm2 , 37°C, and _CO2 concentration of 5%. In addition, Y-27632 (Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) was added to the medium until 24 hours after cell seeding.
Detachment and collection of cell aggregates:
The medium was pipetted 10 times to detach the cell aggregates from the cell culture substrate. The collected cell aggregates were dissociated into single cells with TrypLE-EDTA solution, and the cell count was measured with an automatic cell counter (Luna, manufactured by Shoshin EM Co., Ltd.) (referred to as cell count A). Next, the cells remaining on the cell culture substrate without being detached were treated with TrypLE-EDTA solution, and then detached with a cell scraper, and the cell count was similarly measured with an automatic cell counter (referred to as cell count B). The cell count A/(A+B) was calculated as the detachment recovery rate.
Assessment of cell aggregate size variation:
The medium was pipetted 10 times to detach the cell aggregates from the cell culture substrate. The detached cell aggregates were observed under a phase contrast microscope and the particle size was measured. The coefficient of variation (standard deviation of particle size/average particle size) was calculated from the particle sizes of the 20 cell aggregates to determine the particle size variation.

<Checking interference fringes>
The cell culture substrate was exposed to fluorescent light, and the presence or absence of interference fringes was confirmed visually.

[Example 1]
0.5 g of a 40 wt% aqueous dispersion of an anionic silica colloid (Snowtech MP-4540M, manufactured by Nissan Chemical Industries, Ltd.) having a particle size of 0.45 μm was mixed with 18 g of ion-exchanged water, and the mixture was degassed for 30 minutes while bubbling with nitrogen. 0.02 g of 3-(trimethoxysilyl)propyl methacrylate was added, and the mixture was stirred for 30 minutes at 300 rpm using a magnetic stirrer under a nitrogen atmosphere. Then, 0.08 g of sodium p-styrenesulfonate and 0.8 g of styrene dissolved in 10 g of ion-exchanged water were added, and the mixture was stirred at 300 rpm for 2 hours, and then heated to 65° C., and 0.02 g of potassium persulfate dissolved in 10 g of ion-exchanged water was added. The mixture was reacted at 65° C. for 3 hours while stirring at 300 rpm under a nitrogen atmosphere. After the reaction, all particles were precipitated by centrifugation at 4000 rpm for 10 minutes. The supernatant was removed by decantation, and the colloid was redispersed in ion-exchanged water. This purification was repeated twice to obtain polystyrene-coated silica particles.

A glass substrate was immersed in a 1 mg/mL aqueous solution of polydiallyldimethylammonium chloride for 10 seconds, then washed with ion-exchanged water and dried with an air blower. A 1 wt % dispersion of the polystyrene-coated silica particles was dropped onto the glass substrate. After 10 seconds, the substrate was rinsed with ion-exchanged water and then immersed in boiling water for 5 minutes. It was then cooled to room temperature and dried.

An ethanol solution containing 0.8 wt% solids of polyvinyl alcohol (BIOSURFINE®-AWP, manufactured by Toyo Gosei Co., Ltd.) having an azide group as a hydrophilic polymer was dropped onto the glass substrate covered with the polystyrene-coated silica particles, and spin-coated at 2000 rpm. The film was cured by UV irradiation. The thickness of the hydrophilic polymer layer was about 40 nm. A metal mask with multiple circular holes with a diameter of 0.2 mm was placed, and a plasma irradiation device (manufactured by Vacuum Device Co., Ltd., product name Plasma Ion Bombarder PIB-20) was used to perform plasma treatment (gas pressure of 20 Pa, conductive current of 20 mA, irradiation time of 1 minute) from above the metal mask to form cell adhesive and cell proliferation regions. The above-mentioned patterned glass substrate was attached to the bottom of a well plate with through-holes.

The surface irregularities of the prepared cell culture substrate are shown in FIG. 6, and laser microscope images of the (A) and (B) regions are shown in FIG. 7. The configuration and evaluation results of the prepared cell culture substrate are shown in Table 1. The prepared cell culture substrate had an area of about 0.03 mm ² for region (A), and the maximum height of the surface irregularities at a reference length of 10 μm of the cell culture substrate was 0.39 μm. Culture evaluation of human iPS cells was performed on this cell culture substrate, and the formation of cell aggregates was confirmed 3 days after the start of culture. The detachment recovery rate of the cell aggregates was 82.5%, and the particle size variation was 18.1%. No interference fringes were generated on the cell culture substrate.

[Example 2]
Instead of anionic silica colloid with a particle size of 0.45 μm (Snowtech MP-4540M, Nissan Chemical Industries, Ltd.), anionic silica colloid with a particle size of 0.2 μm (Snowtech MP-2040M, Nissan Chemical Industries, Ltd.) was used, and the rest of the procedure was the same as in Example 1 to prepare a cell culture substrate. The prepared cell culture substrate had an area of region (A) of about 0.03 mm ² , and the maximum height of the surface irregularities at a reference length of 10 μm of the cell culture substrate was 0.18 μm. Culture evaluation of human iPS cells was performed on this cell culture substrate, and the formation of cell aggregates was confirmed 3 days after the start of culture. The detachment recovery rate of the cell aggregates was 73.4%, and the particle size variation was 25.2%. No interference fringes were generated on the cell culture substrate.

[Example 3]
0.2 g of silica colloid powder (Sea Hoster KEP-150, manufactured by Nippon Shokubai Co., Ltd.) with a particle size of 1.5 μm was dispersed in ethanol, and then centrifuged to replace the solvent with pure water. The mixture was degassed for 30 minutes while bubbling with nitrogen. 0.02 g of 3-(trimethoxysilyl)propyl methacrylate was added, and the mixture was stirred for 30 minutes at 300 rpm using a magnetic stirrer under a nitrogen atmosphere. Then, 0.08 g of sodium p-styrenesulfonate and 1.6 g of styrene dissolved in 10 g of ion-exchanged water were added, and the mixture was stirred for 2 hours at 300 rpm, and then heated to 65° C., and 0.02 g of potassium persulfate dissolved in 10 g of ion-exchanged water was added. The mixture was reacted for 3 hours at 65° C. while stirring at 300 rpm under a nitrogen atmosphere. After the reaction, all particles were precipitated by centrifugation at 4000 rpm for 10 minutes. The supernatant was removed by decantation, and the colloid was redispersed in ion-exchanged water. This purification was carried out twice to obtain polystyrene-coated silica particles.

A glass substrate was immersed in a 1 mg/mL aqueous solution of polydiallyldimethylammonium chloride for 10 seconds, then washed with ion-exchanged water and dried with an air blower. A 1 wt % dispersion of the polystyrene-coated silica particles was dropped onto the glass substrate. After 10 seconds, the substrate was rinsed with ion-exchanged water and then immersed in boiling water for 5 minutes. It was then cooled to room temperature and dried.

An ethanol solution containing 0.8 wt% solids of polyvinyl alcohol (BIOSURFINE®-AWP, manufactured by Toyo Gosei Co., Ltd.) having an azide group as a hydrophilic polymer was dropped onto the glass substrate covered with the polystyrene-coated silica particles, and spin-coated at 2000 rpm. The film was cured by UV irradiation. The thickness of the hydrophilic polymer layer was about 40 nm. A metal mask with multiple circular holes with a diameter of 0.2 mm was placed, and a plasma irradiation device (manufactured by Vacuum Device Co., Ltd., product name Plasma Ion Bombarder PIB-20) was used to perform plasma treatment (gas pressure of 20 Pa, conductive current of 20 mA, irradiation time of 1 minute) from above the metal mask to form cell adhesive and cell proliferation regions. The above-mentioned patterned glass substrate was attached to the bottom of a well plate with through-holes.

The area of the (A) region of the prepared cell culture substrate was approximately 0.03 ^mm2 , and the maximum height of the surface irregularities at a reference length of 10 μm of the cell culture substrate was 1.45 μm. Human iPS cells were cultured on this cell culture substrate, and the formation of cell aggregates was confirmed 3 days after the start of culture. The detachment recovery rate of the cell aggregates was 88.9%, and the particle size variation was 17.3%. No interference fringes were generated on the cell culture substrate.

[Example 4]
An ethanol solution containing 0.8 wt% solids of polyvinyl alcohol (BIOSURFINE®-AWP, manufactured by Toyo Gosei Co., Ltd.) having an azide group as a hydrophilic polymer was dropped onto a resin film (FLP230/200-120, manufactured by SCIVAX Co., Ltd.) on which protrusions of about 0.2 μm had been formed by nanoimprinting, and spin-coated at 2000 rpm. The film was cured by UV irradiation. The layer thickness of the hydrophilic polymer layer was about 40 nm. A metal mask having a plurality of circular holes with a diameter of 0.2 mm was placed, and a plasma irradiation device (manufactured by Vacuum Device Co., Ltd., product name Plasma Ion Bombarder PIB-20) was used to perform plasma treatment (20 Pa gas pressure, conductive current 20 mA, irradiation time 1 minute) from above the metal mask to form a cell adhesive and cell proliferation region. The above-mentioned patterned resin film was attached to the bottom surface of a well plate having through-holes.

The surface irregularities of the cell culture substrate produced are shown in Figure 8. The area of the (A) region of the cell culture substrate produced was approximately 0.03 ^mm2 , and the maximum height of the surface irregularities at a reference length of 10 μm of the cell culture substrate was 0.18 μm. Human iPS cells were cultured on this cell culture substrate and the formation of cell aggregates was confirmed 3 days after the start of culture. The detachment recovery rate of the cell aggregates was 71.5%, and the particle size variation was 17.1%. Interference fringes were generated on the cell culture substrate.

[Example 5]
Instead of a resin film (FLP230/200-120, manufactured by SCIVAX Corp.) on which protrusions of about 0.2 μm were formed by nanoimprinting, a resin film (FLP500/500-50×50, manufactured by SCIVAX Corp.) on which protrusions of about 0.5 μm were formed by nanoimprinting was used. Also, instead of a metal mask having a plurality of circular holes with a diameter of 0.2 mm, a metal mask having a plurality of circular holes with a diameter of 0.5 mm was used. Otherwise, a cell culture substrate was produced in the same manner as in Example 4.

The surface irregularities of the cell culture substrate produced are shown in Figure 9. The area of the (A) region of the cell culture substrate produced was approximately 0.2 ^mm2 , and the maximum height of the surface irregularities at a reference length of 10 μm of the cell culture substrate was 0.48 μm. Human iPS cells were cultured on this cell culture substrate and the formation of cell aggregates was confirmed 5 days after the start of culture. The detachment recovery rate of the cell aggregates was 81.7%, and the particle size variation was 19.2%. Interference fringes were generated on the cell culture substrate.

[Example 6]
Instead of a resin film (FLP230/200-120, manufactured by SCIVAX Corp.) on which protrusions of about 0.2 μm were formed by nanoimprinting, a resin film (FLH230/200-120, manufactured by SCIVAX Corp.) on which a hole structure of about 0.5 μm was formed by nanoimprinting was used. The rest of the procedure was the same as in Example 4 to prepare a cell culture substrate.

The surface irregularities of the cell culture substrate produced are shown in Figure 10. The area of the (A) region of the cell culture substrate produced was approximately 0.03 ^mm2 , and the maximum height of the surface irregularities at a reference length of 10 μm of the cell culture substrate was 0.16 μm. Human iPS cells were cultured on this cell culture substrate and the formation of cell aggregates was confirmed 3 days after the start of culture. The detachment recovery rate of the cell aggregates was 71.9%, and the particle size variation was 22.5%. Interference fringes were generated on the cell culture substrate.

[Example 7]
A cell culture substrate was prepared in the same manner as in Example 1, except that a polymer having a phosphorylcholine group (Lipidure CR-2001, NOF Corp.) was used as the hydrophilic polymer instead of polyvinyl alcohol having an azide group (BIOSURFINE®-AWP, Toyo Gosei Co., Ltd.). The thickness of the hydrophilic polymer layer was about 30 nm.

The area of the (A) region of the prepared cell culture substrate was approximately 0.03 ^mm2 , and the maximum height of the surface irregularities at a reference length of 10 μm of the cell culture substrate was 0.42 μm. Human iPS cells were cultured on this cell culture substrate, and the formation of cell aggregates was confirmed 3 days after the start of culture. The detachment recovery rate of the cell aggregates was 84.5%, and the particle size variation was 19.9%. No interference fringes were generated on the cell culture substrate.

[Example 8]
A cell culture substrate was prepared in the same manner as in Example 1 except that a metal mask having a plurality of circular holes with a diameter of 1.5 mm was used instead of the metal mask having a plurality of circular holes with a diameter of 0.2 mm.

The area of the (A) region of the prepared cell culture substrate was approximately 1.76 mm ² , and the maximum height of the surface irregularities at a reference length of 10 μm of the cell culture substrate was 0.34 μm. Human iPS cells were cultured on this cell culture substrate, and the formation of cell aggregates was confirmed 14 days after the start of culture. The detachment recovery rate of the cell aggregates was 86.7%, and the particle size variation was 28.2%. No interference fringes were generated on the cell culture substrate.

[Comparative Example 1]
A cell culture substrate was prepared in the same manner as in Example 1, except that an anionic silica colloid having a particle size of 0.06 μm (Snowtech ST-YL, Nissan Chemical Industries, Ltd.) was used instead of an anionic silica colloid having a particle size of 0.45 μm (Snowtech MP-4540M, Nissan Chemical Industries, Ltd.).

The cell culture substrate thus prepared had an area of region (A) of approximately 0.03 mm ² , and a maximum height of the surface irregularities of 0.01 μm at a reference length of 10 μm. Human iPS cells were cultured on the cell culture substrate, and the formation of cell aggregates was confirmed 3 days after the start of culture. The detachment recovery rate of the cell aggregates was 20.2%, which was low. In addition, the particle size variation of the cell aggregates was 65.6%, which was a large variation.

[Comparative Example 2]
0.2 g of silica colloid powder (Sea Hoster KEP-250, manufactured by Nippon Shokubai Co., Ltd.) with a particle size of 2.5 μm was dispersed in ethanol, and then centrifuged to replace the solvent with pure water. The mixture was degassed for 30 minutes while bubbling with nitrogen. 0.02 g of 3-(trimethoxysilyl)propyl methacrylate was added, and the mixture was stirred for 30 minutes at 300 rpm using a magnetic stirrer under a nitrogen atmosphere. Then, 0.08 g of sodium p-styrenesulfonate and 0.06 g of styrene dissolved in 10 g of ion-exchanged water were added, and the mixture was stirred for 2 hours at 300 rpm, and then heated to 65° C., and 0.02 g of potassium persulfate dissolved in 10 g of ion-exchanged water was added. The mixture was reacted for 3 hours at 65° C. while stirring at 300 rpm under a nitrogen atmosphere. After the reaction, all particles were precipitated by centrifugation at 4000 rpm for 10 minutes. The supernatant was removed by decantation, and the colloid was redispersed in ion-exchanged water. This purification was carried out twice to obtain polystyrene-coated silica particles.

A glass substrate was immersed in a 1 mg/mL aqueous solution of polydiallyldimethylammonium chloride for 10 seconds, then washed with ion-exchanged water and dried with an air blower. A 1 wt % dispersion of the polystyrene-coated silica particles was dropped onto the glass substrate. After 10 seconds, the substrate was rinsed with ion-exchanged water and then immersed in boiling water for 5 minutes. It was then cooled to room temperature and dried.

An ethanol solution containing 0.8 wt% solids of polyvinyl alcohol (BIOSURFINE®-AWP, Toyo Gosei Co., Ltd.) having an azide group as a hydrophilic polymer was dropped onto the glass substrate covered with the polystyrene-coated silica particles, and spin-coated at 2000 rpm. The film was cured by UV irradiation. A metal mask with multiple circular holes with a diameter of 0.2 mm was placed, and a plasma irradiation device (Plasma Ion Bombarder PIB-20, Vacuum Device Co., Ltd.) was used to perform plasma treatment (20 Pa gas pressure, 20 mA conductive current, irradiation time 1 minute) from above the metal mask to form cell adhesive and cell proliferation regions. The above-mentioned patterned glass substrate was attached to the bottom of a well plate with through-holes.

The area of the (A) region of the prepared cell culture substrate was approximately 0.03 ^mm2 , and the maximum height of the surface irregularities at a reference length of 10 μm of the cell culture substrate was 2.41 μm. Human iPS cell culture was evaluated on this cell culture substrate, but the cells did not proliferate and no aggregates were formed.

[Comparative Example 3]
A cell culture substrate was prepared in the same manner as in Example 1, except that the hydrophilic polymer layer in Example 1 was not formed.

The cell culture substrate produced had a maximum height of surface irregularities of 0.46 μm at a reference length of 10 μm. Human iPS cells were cultured and evaluated on this cell culture substrate, but the cells proliferated in a sheet-like form and no cell aggregates were formed. When the sheet-like cells were peeled off, the particle size variation was 62.3%, which was a large variation.

[Comparative Example 4]
A cell culture substrate was prepared in the same manner as in Example 1, except that the plasma treatment in Example 1 was not carried out.

The cell culture substrate produced had a maximum height of surface irregularities of 0.4 μm at a reference length of 10 μm. Human iPS cell culture was evaluated on this cell culture substrate, but no cell aggregates were formed.

[Comparative Example 5]
A cell culture substrate was prepared in the same manner as in Example 1 except that a metal mask having a plurality of circular holes with a diameter of 3 mm was used instead of the metal mask having a plurality of circular holes with a diameter of 0.2 mm.

The area of the (A) region of the cell culture substrate thus prepared was approximately 7.1 ^mm2 , and the maximum height of the surface irregularities at a reference length of 10 μm of the cell culture substrate was 0.38 μm. Human iPS cell culture evaluation was performed on this cell culture substrate, but the cells proliferated in a sheet shape and no cell aggregates were formed. When the sheet-like cells were peeled off, the particle size variation was 48.5%, which was a large variation.

The configurations of Examples 1 to 8 and Comparative Examples 1 to 5 are shown in Table 1. The evaluation results of the cell aggregates produced using the cell culture substrates of Examples 1 to 8 and Comparative Examples 1 to 5 are shown in Table 2.

A...(A) region, B...(B) region, H...maximum height of surface irregularities, P...distance between convex portions of surface irregularities, 1...substrate, 2...hydrophilic polymer layer, 3...plate having through holes.

Claims (7)

A cell culture substrate comprising a substrate having an uneven surface and a hydrophilic polymer layer covering the substrate along the uneven surface, the maximum height of the uneven surface of the cell culture substrate being 0.1 to 2 μm over a reference length of 10 μm, the cell culture substrate having the following two regions (A) and (B), the region (A) being a plasma-treated region:
(A) an island-like region having an area of 0.001 to ⁵ mm2 and having cell adhesiveness and cell proliferation properties; (B) a region adjacent to the region (A) and not having cell proliferation properties; The cell culture substrate according to claim 1, wherein the substrate has a substrate sheet and a three-dimensional element having a vertical length of 0.1 to 3 μm arranged on the substrate sheet. The cell culture substrate according to claim 2, wherein the number density of the three-dimensional elements is 0.1 to 10 pieces/ ^μm2 . The cell culture substrate according to claim 1, wherein the coefficient of variation of the distance between the convex portions of the surface irregularities is 3 to 50%. The cell culture substrate according to claim 1 , wherein the hydrophilic polymer contains a compound represented by the following general formula (1) or (2):

[In formula (1), ^R1 and ^R2 each independently represent a hydrogen atom or a methyl group, ^R3 represents an arbitrary substituent, and m and n each independently represent a positive integer.]

[In formula (2), R ⁴ , R ⁵ and R ⁶ each independently represent a hydrogen atom or a methyl group, R ⁷ represents an arbitrary substituent, and x, y and z each independently represent a positive integer.] The cell culture substrate according to claim 1, wherein the (A) region has a carboxyphenyl group. The method for producing a cell culture substrate according to any one of claims 1 to 6, comprising the following steps (1) and (2):
(1) A step of forming a hydrophilic polymer layer on a substrate having an uneven surface along the unevenness, the maximum height of the surface unevenness of the hydrophilic polymer layer being 0.1 to 2 μm at a reference length of 10 μm; (2) A step of performing a plasma treatment on only a part of the surface of the hydrophilic polymer layer to form two regions (A) and (B) as follows: (A) an island-like region having an area of 0.001 to ⁵ mm2 and having cell adhesiveness and cell proliferation properties; and (B) a region adjacent to the region (A) and not having cell proliferation properties.

Images