Cell culture substrate and method for producing same, method for inducing differentiation of pluripotent stem cells, and cell culture kit

Abstract

Provided is a cell culture substrate comprising a substrate and a layer that covers at least a portion of the surface of the substrate, has a layer thickness of 5 to 2000 nm and contains a hydrophilic polymer, in which the hydrophilic polymer contains a phosphorylcholine group or a hydroxyl group, the cell culture substrate has a region (A) and a region (B) as mentioned below, and the bump height in a boundary region between the region (A) and the region (B) is 1 to 500 nm. (A) An island-like region which has cell adhesiveness and cell proliferating properties and has an area of 0.001 to 5 mm2. (B) A region which is located adjacent to the region (A) and does not have cell adhesiveness or cell proliferating properties.

Description

[Technical field]

[0001] The present invention relates to a cell culture substrate and a method for producing the same, a method for inducing differentiation of pluripotent stem cells, and a cell culture kit. [Background technology]

[0002] Pluripotent stem cells, such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), have the ability to differentiate into various tissues in the body (pluripotency), and have attracted 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 pluripotent stem cells into the target 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, so it is desirable to control the size and create cell aggregates of uniform size.

[0003] Conventionally, a method for forming cell aggregates has been known 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 in terms of mass production of cell aggregates, it has the problem that it is not possible to obtain cell aggregates of uniform size.

[0004] As a method for forming cell aggregates of uniform size, a method using a cell culture substrate having a finely irregular surface is known (see, for example, Patent Document 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 8-140673 [Patent Document 2] JP 2015-073520 A Summary of the Invention [Problem to be solved by the invention]

[0006] The cell culture substrate with fine irregularities as disclosed in Patent Document 2 has a problem that it is poor in mass production and is not suitable for forming a large amount of cell aggregates. In addition, since cells are forcibly aggregated within the fine irregularities, there is a problem that dead cells are likely to be mixed into the cell aggregates. In addition, the present inventors have found that when a culture medium is brought into contact with a cell culture substrate having fine irregularities, air bubbles are likely to be captured in the fine irregularities on the surface, and the air bubbles must be removed before starting culture. In order to remove the air bubbles, it is usually necessary to repeatedly suck and discharge the culture medium using a pipette, which is a problem of poor workability.

[0007] An object of the present invention is to provide a cell culture substrate capable of forming cell aggregates, increasing the survival rate of cells in the cell aggregates, and eliminating the need for removing air bubbles during culture, and a method for producing the same. Another object of the present invention is to provide a method for inducing differentiation of pluripotent stem cells using the cell culture substrate, which has excellent efficiency in inducing differentiation into three germ layer cells. [Means for solving the problem]

[0008] The present invention relates to a cell culture substrate comprising a substrate and a layer containing a hydrophilic polymer and having a layer thickness of 5 to 2000 nm covering at least a portion of the surface of the substrate, the hydrophilic polymer containing a phosphorylcholine group or a hydroxyl group and having the following region (A) and region (B), wherein the height of the irregularities at the boundary between region (A) and region (B) is 1 to 500 nm: (A) Area with cell adhesion and cell proliferation: 0.001 to 5 mm 2 Island-like region of (B) A region adjacent to the region (A) that does not have cell adhesiveness or cell proliferation properties.

[0009] The present invention also relates to a method for producing the above-mentioned cell culture substrate, comprising the steps of: (1) coating at least a portion of the surface of the substrate with a composition containing a UV-reactive hydrophilic polymer to form a layer containing the hydrophilic polymer; (2) irradiating the layer containing the hydrophilic polymer with UV light to immobilize the layer containing the hydrophilic polymer on the surface of the substrate; and (3) performing a plasma treatment on a portion of the surface of the immobilized hydrophilic polymer-containing layer to form an (A) region in the plasma-treated portion.

[0010] The present invention also relates to a method for producing the above-mentioned cell culture substrate, comprising the steps of: (1') using a substrate formed from a polymer containing an aromatic hydrocarbon group in a repeating unit, performing a plasma treatment on the surface of the substrate, and forming the above-mentioned (A) region in the plasma-treated portion; (2') coating at least a portion of the surface of the substrate with a composition containing a UV-reactive hydrophilic polymer to form a layer containing the hydrophilic polymer; (3') irradiating a portion of the layer containing the hydrophilic polymer with UV light to immobilize the portion of the layer containing the hydrophilic polymer on the surface of the substrate; and (4') washing the hydrophilic polymer with a solvent to dissolve the hydrophilic polymer that is not immobilized on the surface and remove it from the surface of the substrate.

[0011] The present invention further relates to a method for inducing differentiation of pluripotent stem cells, comprising the steps of: (i) seeding pluripotent stem cells on the cell culture substrate; (ii) culturing the pluripotent stem cells to form hemispherical cell aggregates having a height / diameter ratio of 0.2 to 0.8; and (iii) inducing differentiation of the cell aggregates to form cell aggregates of three germ layer cells.

[0012] The present invention also relates to a cell culture kit comprising the above-mentioned cell culture substrate and a block copolymer containing a water-insoluble block segment and a temperature-responsive block segment, or a coating agent containing the above-mentioned block copolymer. Effect of the Invention

[0013] According to the present invention, it is possible to provide a cell culture substrate capable of forming a cell aggregate, increasing the survival rate of cells in the cell aggregate, and eliminating the need for removing air bubbles during culture, and a method for producing the same. According to the present invention, it is also possible to provide a method for inducing differentiation of pluripotent stem cells using the cell culture substrate. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a schematic diagram (cross-sectional view) of a cell culture substrate according to one embodiment. [Diagram 2] FIG. 1 is a schematic diagram (perspective view) of a substrate having a layer containing a hydrophilic polymer formed on its surface after step (1) in a manufacturing method according to one embodiment. [Diagram 3] FIG. 2 is a schematic diagram (perspective view) of the cell culture substrate after step (3) in the production method according to one embodiment. [Figure 4] FIG. 2 is a schematic diagram (perspective view) of the cell culture substrate after step (4) in the production method according to one embodiment. [Diagram 5] 1 shows phase-contrast microscope images of cells on "day 0 of differentiation," "day 43 of differentiation," and "day 64 of differentiation" in a test of inducing differentiation of human iPS cells into intestinal epithelial cells (Example 8). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Hereinafter, the embodiments of the present invention will be described in detail. Note that the present invention is not limited to the following embodiments, and the following embodiments can be modified and implemented within the scope in which the above-mentioned effects can be obtained.

[0016] As used herein, the term "cell aggregate" refers to a three-dimensional aggregate of cells formed by the gathering of multiple cells. The shape of the three-dimensional aggregate may be an ellipsoid such as a sphere, or a hemisphere. These shapes may have gaps formed by folding a sheet of cells, or may be hollow.

[0017] In this specification, the term "temperature responsive" refers to a change in the degree of hydrophilicity / hydrophobicity due to a change in temperature. Furthermore, the boundary temperature at which the degree of hydrophilicity / hydrophobicity changes is referred to as a "response temperature."

[0018] In this specification, the term "biologically derived substance" refers to a substance present in the body of a living organism, and a chemically synthesized substance that is equivalent to the substance. The substance present in the body of a living organism may be a natural product, or may be artificially synthesized using recombinant gene technology or the like. There is no particular limitation on the biologically derived substance, and examples thereof include nucleic acids, proteins, and polysaccharides, which are basic materials that constitute living organisms, and their constituent elements, such as nucleotides, nucleosides, amino acids, and various sugars, as well as lipids, vitamins, and hormones.

[0019] As used herein, "cell adhesiveness" refers to the ease with which cells adhere to a substrate or cell culture substrate at culture temperature, "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 culture temperature, and "not having cell adhesiveness" refers to the inability of cells to adhere to a substrate or cell culture substrate at culture temperature.

[0020] As used herein, "cell proliferation" refers to the ease with which cells proliferate at the culture temperature, "having cell proliferation" refers to the ability of cells to proliferate at the culture temperature, and "not having cell proliferation" refers to the inability of cells to proliferate at the culture temperature. "High cell proliferation" refers to proliferation into more cells when compared over the same culture period.

[0021] As used herein, the term "tridermal cells" refers to at least one type selected from the group consisting of endodermal cells, ectodermal cells, and mesodermal cells.

[0022] The cell culture substrate according to the present embodiment includes a substrate and a layer containing a hydrophilic polymer having a thickness of 5 to 2000 nm, which covers at least a part of the surface of the substrate. Here, the hydrophilic polymer contains a phosphorylcholine group or a hydroxyl group. The cell culture substrate according to the present embodiment also includes the following region (A) and region (B), and the height of the irregularities at the boundary between region (A) and region (B) is 1 to 500 nm. (A) Area with cell adhesion and cell proliferation: 0.001 to 5 mm 2 Island-like region of (B) A region adjacent to region (A) that does not have cell proliferation properties

[0023] FIG. 1 is a schematic diagram (cross-sectional view) of a cell culture substrate according to one embodiment. The cell culture substrate 10 shown in FIG. 1 includes a substrate 1 and a layer 2 (having a layer thickness of, for example, 5 to 2000 nm) containing a hydrophilic polymer that covers the surface of the substrate 1. In FIG. 1, A and B indicate region (A) and region (B), respectively. In addition, in FIG. 1, H indicates the height of the unevenness at the boundary between region (A) and region (B). The layer thickness of the layer 2 containing a hydrophilic polymer means the distance from the surface where the substrate 1 and the layer 2 containing a hydrophilic polymer contact each other to the surface of region (B) (region indicated by B in FIG. 1). In addition, in the cell culture substrate 10 shown in FIG. 1, the surface of region (A) is a layer containing a hydrophilic polymer, but is not limited thereto. For example, the surface of region (A) may be substrate 1.

[0024] The substrate used for the cell culture substrate according to the present embodiment is not particularly limited, but is preferably formed from at least one selected from the group consisting of polystyrene, polyethylene, polyethylene terephthalate, polycarbonate, cycloolefin polymer, cellulose acetate, nitrocellulose and polyvinylidene fluoride, more preferably formed from at least one selected from the group consisting of polystyrene, polyethylene terephthalate, polycarbonate and cycloolefin polymer, further preferably formed from at least one selected from polystyrene, polyethylene terephthalate and polycarbonate, and most preferably formed from polystyrene or polycarbonate. Commercially available cycloolefin polymers include ZEONEX (manufactured by Nippon Zeon Co., Ltd.), ZEONOR (manufactured by Nippon Zeon Co., Ltd.), and ARTON (manufactured by JSR Co., Ltd.).

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

[0026] The refractive index and thickness of the substrate measured with D-line (wavelength 589 nm) are preferably 1.4 to 1.6 and 0.01 mm or more and 0.5 mm or less, respectively, more preferably 1.45 to 1.6 and 0.05 mm or more and 0.4 mm or less, respectively, even more preferably 1.45 to 1.55 and 0.1 mm or more and 0.3 mm or less, respectively, and particularly preferably 1.5 to 1.55 and 0.15 mm or more and 0.2 mm or less. When the refractive index and thickness of the substrate are within these ranges, the phase contrast image of the cells becomes clearer.

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

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

[0029] The layer containing the hydrophilic polymer has a thickness of 5 to 2000 nm. By having a layer thickness of 5 to 2000 nm, it is possible to adhere and grow cells only in the (A) region, and also to easily collect cells in the (A) region by cell migration, thereby increasing the cell viability of the cell aggregate. If the layer thickness is less than 5 nm, cells also grow in the (B) region, so that a cell aggregate cannot be formed. If the layer thickness exceeds 2000 nm, cells cannot migrate, so that the viability of the cells contained in the cell aggregate decreases. Here, 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 (excluding the (A) region) of the hydrophilic polymer layer on the opposite side to the substrate. In the range where the layer thickness exceeds 10 nm, the cross-sectional image is measured by a transmission electron microscope using an ultrathin section of the cell culture substrate prepared by a microtome, and the distance is measured at 10 randomly selected points, and the average is calculated. 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, the layer thickness is more preferably 10 nm or more, even more preferably 50 nm or more, and most preferably 100 nm or more. Furthermore, since it is suitable for increasing the cell survival rate of the cell aggregate by collecting cells in the (A) region through cell migration, the layer thickness is more preferably 1000 nm or less, even more preferably 500 nm or less, and most preferably 200 nm or less.

[0030] The hydrophilic polymer contains a phosphorylcholine group or a hydroxyl group. By containing a phosphorylcholine group or a hydroxyl group, it is possible to make the area covered with the hydrophilic polymer into an area to which cells do not adhere. In addition, since such a hydrophilic polymer does not need to be completely decomposed and removed, the area can be made into an area having cell adhesiveness and cell proliferation properties by only performing a short-term weak plasma treatment, and cells are unlikely to be contaminated with decomposition products of the hydrophilic polymer. Other than containing a phosphorylcholine group or a hydroxyl group, the type of hydrophilic polymer is not particularly limited, and 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, examples of commercially available substrates that are already coated with a hydrophilic polymer include 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.).

[0031] The hydrophilic polymer preferably contains a compound represented by the following general formula (1), a compound represented by the following general formula (2), or a compound represented by the following general formula (3).

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

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

[0034] When the hydrophilic polymer contains a compound represented by the above general formula (1), a compound represented by the above general formula (2), or a compound represented by the above general formula (3), cells can be easily attached and proliferated, and it is suitable for forming a cell aggregate having a uniform shape in the (A) region. 3 R 7 , and R 10 is preferably a hydrophobic group or a UV-reactive functional group, since 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, an acrylate group, a methacrylate group, an epoxy group, or the like can be used, and an azide group can be preferably used.

[0035] (A) Area 0.001 to 5 mm2 has cell adhesion and cell proliferation properties. 2 It is an island-like region with an area of ​​0.001 to 5 mm 2 Since the island-like regions are uniform in size, cell aggregates of uniform particle size can be formed when cells are cultured. In addition, the island-like regions are suitable for forming cell aggregates suitable for applications such as differentiation induction of pluripotent stem cells, and therefore the area of ​​the island-like regions is preferably 0.005 to 1 mm. 2 is preferable, and the area is 0.01 to 0.5 mm 2 More preferably, the area is 0.015 to 0.25 mm 2 More preferably, the area is 0.02 to 0.2 mm 2 is most preferred.

[0036] The (A) region being an island-like region means that the (A) region exists in a state independent from regions other than the (A) region. Since the (A) region is an island-like region having cell adhesiveness and cell proliferation properties, living cells are concentrated in the (A) region, and therefore a cell aggregate can be produced. When the (A) region is not island-like, for example, when it has a striped structure, a cell aggregate cannot be produced. The shape of the island is not particularly limited and can be appropriately set according to the shape of the target cell aggregate, and examples thereof include a circle, an ellipse, a polygon, or a closed shape formed by straight lines and curves. In addition, since it is suitable for producing a cell aggregate having a shape close to a sphere, the shape of the island is preferably a circle, an ellipse, or a polygon, more preferably a circle, an ellipse, or a rectangle, even more preferably a circle, an ellipse, or a square, and most preferably a circle or an ellipse.

[0037] Since this is suitable for producing cell aggregates of 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, even more preferably 20% or less, and most preferably 5% or less.

[0038] Since it 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, even more preferably 1.5 or less, and most preferably 1.1 or less. Here, the "aspect ratio" refers to the ratio of the maximum diameter (major diameter) to the minimum diameter (minor diameter) of the shape, that is, major diameter / minor diameter.

[0039] In addition, since it is suitable for forming a cell aggregate suitable for culturing pluripotent stem cells and inducing their differentiation into endodermal cells or ectodermal cells, the area of ​​region (A) is 0.005 to 0.2 mm 2 and the number of the (A) regions is 200 to 1000 / cm based on the total area of ​​the (A) regions and the (B) regions. 2 It is preferable that the area of ​​the (A) region is 0.01 to 0.15 mm 2and the number of the (A) regions is 250 to 800 / cm based on the total area of ​​the (A) regions and the (B) regions. 2 It is more preferable that the area of ​​the (A) region is 0.02 to 0.1 mm 2 and the number of (A) regions is 300 to 600 / cm based on the total area of ​​(A) region and (B) region. 2 It is more preferable that the area of ​​the (A) region is 0.03 to 0.05 mm 2 and the number of the (A) regions is 300 to 400 / cm based on the total area of ​​the (A) regions and the (B) regions. 2 It is most preferable that:

[0040] Since it is suitable for forming a cell aggregate suitable for culturing pluripotent stem cells and inducing their differentiation into mesodermal cells, the area of ​​region (A) is 0.2 to 2 mm 2 and the number of (A) regions is 3 to 15 / cm based on the total area of ​​(A) region and (B) region. 2 It is preferable that the area of ​​the (A) region is 0.3 to 1.5 mm 2 and the number of (A) regions is 4 to 12 / cm based on the total area of ​​(A) region and (B) region. 2 More preferably, the area of ​​region (A) is 0.4 to 1 mm 2 and the number of (A) regions is 5 to 10 / cm based on the total area of ​​(A) region and (B) region. 2 It is more preferable that the area of ​​the (A) region is 0.5 to 0.7 mm 2 and the number of (A) regions is 6 to 8 / cm based on the total area of ​​(A) region and (B) region. 2 It is most preferable that:

[0041] Furthermore, since this is suitable for increasing the oxygen concentration around the cells and increasing the survival rate of the cell aggregates, the minimum distance between the (A) regions is preferably 500 to 10,000 μm, more preferably 1,000 to 8,000 μm, even more preferably 2,000 to 5,000 μm, and most preferably 3,000 to 4,000 μm.

[0042] Region (A) can be formed, for example, by forming a layer containing a hydrophilic polymer on the surface of a substrate, and then modifying a part of the surface of the layer containing a hydrophilic polymer by corona treatment, UV treatment, or plasma treatment. The modification is preferably carried out by plasma treatment. Region (A) is a region in which the surface of the layer containing a hydrophilic polymer has been modified by plasma treatment or the like, so that region (A) has cell adhesiveness and cell proliferation properties. Region (A) can be patterned by a short-term plasma treatment or the like, which can increase the mass productivity of cell culture substrates.

[0043] In addition, it is preferable that the ratio of the peak intensity at 287 eV / peak intensity at 285 eV in the C1s spectrum measured by XPS (X-ray photoelectron spectroscopy) for region (A) is 0.05 or more larger than the ratio of the peak intensity at 287 eV / peak intensity at 285 eV in the C1s spectrum measured by XPS for region (B). In this case, the difference in cell proliferation between region (A) and region (B) can be increased, so that many cells can be proliferated in region (A), and uniform cell aggregates can be easily formed in region (A). Because this is more suitable for forming uniform cell aggregates, it is more preferable that the ratio of the peak intensity at 287 eV to the peak intensity at 285 eV in the C1s spectrum of XPS measurement for region (A) is 0.07 or more greater than the ratio of the peak intensity at 287 eV to the peak intensity at 285 eV in the C1s spectrum of XPS measurement for region (B), more preferably 0.1 or more greater, and most preferably 0.15 or more greater.

[0044] The method for adjusting the ratio of the 287 eV peak intensity / 285 eV peak intensity in the C1s spectrum of the XPS measurement to the above-mentioned range is not particularly limited, but a method of subjecting only a part (the part to become the (A) region) of the layer containing a hydrophilic polymer formed on the surface of the substrate to plasma treatment, corona treatment, UV treatment, etc. is preferred, a method of subjecting a plasma treatment or corona treatment is more preferred, and a method of subjecting a plasma treatment is particularly preferred. As a method for subjecting only the part to become the (A) region, a method of covering the layer containing a hydrophilic polymer formed on the surface of the substrate with a mask made by laser processing and subjecting the layer to plasma treatment, etc. from above the mask can be mentioned. Conditions such as plasma intensity and plasma exposure time can be appropriately adjusted so that the difference between the ratio of the peak intensity at 287 eV / the peak intensity at 285 eV in the C1s spectrum of XPS measurement of region (A) and the ratio of the peak intensity at 287 eV / the peak intensity at 285 eV in the C1s spectrum of XPS measurement of region (B) falls within the above-mentioned range. In the case of plasma treatment, however, it is easier to fall within the above-mentioned range by using a gas containing oxygen and nitrogen as the introduced gas.

[0045] The (A) region may be temperature responsive since it is suitable for detaching cultured cell aggregates. When the (A) region is temperature responsive, since it is possible to culture cells at a temperature close to body temperature when culturing cells on the cell culture substrate, the response temperature is preferably 50°C or less, and more preferably 35°C or less. Furthermore, since it is suitable for preventing cells from detaching when performing operations such as changing the medium during culture, the response temperature is particularly preferably 25°C or less. Furthermore, since it is possible to form cell aggregates by a cooling operation at a temperature that does not damage the cells, the response temperature is preferably 4°C or more, more preferably 10°C or more, and even more preferably 15°C or more.

[0046] When the (A) region has temperature responsiveness, for example, a layer containing a temperature responsive polymer having a layer thickness of 1 to 100 nm may be further provided on the surface of the layer containing a hydrophilic polymer (the surface including the (A) region and the (B) region). By having a layer containing a temperature responsive polymer with a layer thickness of 1 to 100 nm, it is possible to impart temperature responsiveness to the (A) region without impairing the respective characteristics of the (A) region and the (B) region formed on the surface of the layer containing a hydrophilic polymer. In order to be suitable for imparting temperature responsiveness to the (A) region without impairing the respective characteristics of the (A) region and the (B) region, the layer thickness of the layer containing a temperature responsive polymer is more preferably 3 to 50 nm, further preferably 5 to 40 nm, and most preferably 10 to 35 nm. The layer thickness of the temperature responsive polymer suitable for not impairing cell proliferation and for allowing cells to be peeled off and recovered by temperature responsiveness after culture varies depending on the cells to be cultured, and can be appropriately adjusted within the range of layer thickness exemplified above.

[0047] The temperature-responsive polymer is preferably a block copolymer having a water-insoluble block segment and a temperature-responsive block segment. When the temperature-responsive polymer is such a block copolymer, the mass productivity of the cell culture substrate can be improved and the contamination of the produced cell aggregates with the temperature-responsive polymer can be suppressed. The ratio of the constituent units of the temperature-responsive block segment contained in the temperature-responsive polymer is preferably 70 wt% or more, more preferably 80 wt% or more, particularly preferably 90 wt% or more, and most preferably 92 wt% or more, since this is suitable for quickly peeling off the cell aggregates from the cell culture substrate.

[0048] In addition, since the temperature-responsive polymer is a block copolymer having a water-insoluble block segment and a temperature-responsive block segment, the surface of the cell culture substrate can be provided with temperature responsiveness by a simple method of dropping a solution containing the temperature-responsive polymer onto the surface of the cell culture substrate and drying it. In addition, since the layer formed at this time has the above-mentioned preferred layer thickness of the temperature-responsive polymer, even if the entire surface of the layer containing the hydrophilic polymer is covered with the temperature-responsive polymer, the properties of the above-mentioned regions (A) and (B) are less likely to be impaired. When using a cell culture kit having the cell culture substrate of the present invention and a temperature-responsive polymer that is a block copolymer or a coating agent containing the above-mentioned block copolymer, a researcher who performs the culture can easily adjust the layer thickness of the temperature-responsive polymer depending on the type of cells.

[0049] The coating agent may contain a solvent. Examples of the solvent that may be contained in the coating agent include water, an organic solvent, or a mixture thereof. Examples of the organic solvent include alcohols such as methanol, ethanol, 1-propanol, 2-propanol, and 1-butanol; acetonitrile, formamide, N,N-dimethylformamide, dimethylsulfoxide, tetrahydrofuran, 1,4-dioxane, and methyl ethyl ketone. It is preferable to use a mixed solvent of water and alcohols because it is suitable for making the coating thickness uniform. The content of the block copolymer having a water-insoluble block segment and a temperature-responsive block segment based on the total mass of the coating agent can be 0.1 to 50% by weight, 0.2 to 10% by weight, or 0.5 to 5% by weight.

[0050] The coating agent may contain other components in addition to the block copolymer having a water-insoluble block segment and a temperature-responsive block segment and the solvent. Examples of the other components include components for enhancing cell adhesion, such as a polymer consisting of only a water-insoluble block segment.

[0051] Examples of monomer units constituting the temperature-responsive block segment include (meth)acrylamide compounds such as acrylamide and methacrylamide; N-alkyl-substituted (meth)acrylamide derivatives such as N,N-diethylacrylamide, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, Nt-butylacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, and N-tetrahydrofurfurylmethacrylamide; N,N-dialkyl-substituted (meth)acrylamide derivatives such as N,N-dimethyl(meth)acrylamide, N,N-ethylmethylacrylamide, and N,N-diethylacrylamide; 1-(1-oxo-2-propenyl)-pyrrolidine, 1-(1-oxo-2-propenyl)-pyrrolidine, 1-(1-oxo-2-propenyl)-pyrrolamide ... Examples of the cyclic group-containing (meth)acrylamide derivatives include cyclic group-containing (meth)acrylamide derivatives such as 1-(1-oxo-2-methyl-2-propenyl)-piperidine, 4-(1-oxo-2-methyl-2-propenyl)-morpholine, 1-(1-oxo-2-methyl-2-propenyl)-pyrrolidine, 1-(1-oxo-2-methyl-2-propenyl)-piperidine, and 4-(1-oxo-2-methyl-2-propenyl)-morpholine; vinyl ethers such as methyl vinyl ether; and proline derivatives such as N-proline methyl ester acrylamide. Since they are suitable for setting the response temperature at 0 to 50° C., N,N-diethylacrylamide, Nn-propylacrylamide, N-isopropylacrylamide, Nn-propylmethacrylamide, N-ethoxyethylacrylamide, N-tetrahydrofurfuryl acrylamide, and N-tetrahydrofurfurylmethacrylamide are preferred, Nn-propylacrylamide and N-isopropylacrylamide are more preferred, and N-isopropylacrylamide is particularly preferred. Furthermore, when a room temperature medium is used for medium replacement during culture operations, Nn-propylacrylamide and N-proline methyl ester acrylamide are preferred because they are suitable for setting the response temperature of the block copolymer at a temperature lower than room temperature.

[0052] Examples of the monomer units constituting the water-insoluble block segment include n-butyl acrylate, n-butyl methacrylate, isobutyl acrylate, isobutyl methacrylate, t-butyl acrylate, t-butyl methacrylate, n-hexyl acrylate, n-hexyl methacrylate, n-octyl acrylate, n-octyl methacrylate, n-decyl acrylate, n-decyl methacrylate, n-dodecyl acrylate, n-dodecyl methacrylate, n-tetradecyl acrylate, and n-tetradecyl methacrylate. In addition, since it is suitable for firmly fixing the block copolymer to the substrate, those having a reactive group are preferable, and examples thereof include 4-azidophenyl acrylate, 4-azidophenyl methacrylate, 2-((4-azidobenzoyl)oxy)ethyl acrylate, and 2-((4-azidobenzoyl)oxy)ethyl methacrylate. Furthermore, structures having an aromatic ring are preferred because they are suitable for enhancing cell proliferation, and examples thereof include 2-hydroxyphenyl acrylate, 2-hydroxyphenyl methacrylate, 3-hydroxyphenyl acrylate, 3-hydroxyphenyl methacrylate, 4-hydroxyphenyl acrylate, 4-hydroxyphenyl methacrylate, N-(2-hydroxyphenyl)acrylamide, N-(2-hydroxyphenyl)methacrylamide, N-(3-hydroxyphenyl)acrylamide, N-(3-hydroxyphenyl)methacrylamide, N-(4-hydroxyphenyl)acrylamide, N-(4-hydroxyphenyl)methacrylamide, styrene, and the like.

[0053] The water-insoluble block segment may also contain a repeating unit that controls the response temperature of the block copolymer. Examples of the repeating unit that controls the response temperature of the block copolymer include hydrophilic or hydrophobic components, and are not particularly limited. For example, those having an amino group such as 2-dimethylaminoethyl acrylate, 2-dimethylaminoethyl methacrylate, 2-diethylaminoethyl acrylate, 2-diethylaminoethyl methacrylate, and N-[3-(dimethylamino)propyl]acrylamide; those having a betaine such as N-(3-sulfopropyl)-N-methacryloyloxyethyl-N,N-dimethylammonium betaine and N-methacryloyloxyethyl-N,N-dimethylammonium-α-N-methylcarboxybetaine; hydroxyethyl acrylate, hydroxyethyl methacrylate, N-(2-hydroxyethyl)acrylamide, polyethylene glycol monoacrylate, polyethylene glycol monomethacrylate, polypropylene glycol monoacrylate, polypropylene glycol monomethacrylate, methoxypolyethylene glycol mono ... those having a polyethylene glycol group or a methoxyethyl group, such as polyethylene glycol monomethacrylate, diethylene glycol monomethyl ether acrylate, diethylene glycol monomethyl ether methacrylate, diethylene glycol monoethyl ether acrylate, diethylene glycol monoethyl ether methacrylate, 2-methoxyethyl acrylate, 2-methoxyethyl methacrylate, 2-ethoxyethyl acrylate, 2-ethoxyethyl methacrylate, 3-butoxyethyl acrylate, 3-butoxyethyl methacrylate, 3-butoxyethyl acrylamide, furfuryl acrylate, furfuryl methacrylate, tetrahydrofurfuryl acrylate, and tetrahydrofurfuryl methacrylate; those having an acrylate group, such as methoxymethyl acrylate, methoxymethyl methacrylate, 2-ethoxymethyl acrylate, 2-ethoxymethyl methacrylate, 3-butoxymethyl acrylate, 3-butoxymethyl methacrylate, and 3-butoxymethyl acrylamide;Examples of phosphorylcholine groups include 2-methacryloyloxyethyl phosphorylcholine, 2-acryloyloxyethyl phosphorylcholine, 3-(meth)acryloyloxypropyl phosphorylcholine, 4-(meth)acryloyloxybutyl phosphorylcholine, 6-(meth)acryloyloxyhexyl phosphorylcholine, 10-(meth)acryloyloxydecyl phosphorylcholine, ω-(meth)acryloyl(poly)oxyethylene phosphorylcholine, 2-acrylamidoethyl phosphorylcholine, 3-acrylamidopropyl phosphorylcholine, 4-acrylamidobutyl phosphorylcholine, 6-acrylamidohexyl phosphorylcholine, 10-acrylamidodecyl phosphorylcholine, and ω-(meth)acrylamido(poly)oxyethylene phosphorylcholine.

[0054] Region (B) is adjacent to region (A) and does not have cell adhesiveness or cell proliferation properties. When region (B) is adjacent to region (A) and does not have cell proliferation properties, it is possible to form a cell aggregate only in region (A) when cells are cultured, and to create a state in which no cells are present around part or all of region (A). In addition, it is preferable that region (B) does not have cell adhesiveness as well as cell proliferation properties, since this is suitable for uniforming the size and shape of the cell aggregates produced.

[0055] The shape of region (B) is not limited other than that it is adjacent to region (A), but since this is suitable for producing cell aggregates of uniform size and shape, it is preferable that region (B) is adjacent to region (A) over a length of at least 20% of the boundary line, more preferably at least 50%, even more preferably at least 80%, and it is most preferable that region (A) is entirely surrounded by region (B). Also, since this is suitable for increasing the mass productivity of the cell culture substrate, it is preferable that region (A) is an island-like structure and region (B) is an ocean-like structure.

[0056] The area ratio of the (A) region and the (B) region is not particularly limited, but since it is suitable for increasing the number of cell aggregates that can be produced per unit area of ​​the cell culture substrate, the area of ​​the (A) region is preferably 10% or more, more preferably 30% or more, even more preferably 50% or more, and most preferably 70% or more relative to the total area of ​​the (A) region and the (B) region. In addition, since it is suitable for providing a sufficient distance between the multiple (A) regions and suppressing the cell aggregates of the multiple (A) regions from fusing to form a non-uniform shape, the area of ​​the (B) region is preferably 20% or more, more preferably 40% or more, even more preferably 60% or more, and most preferably 80% or more relative to the total area of ​​the (A) region and the (B) region.

[0057] The unevenness height (the distance in the out-of-plane direction between the surface of the (A) region and the surface of the (B) region) at the boundary between the (A) region and the (B) region is 1 to 500 nm. When the unevenness height is 500 nm or less, the number of dead cells captured by the unevenness can be suppressed, and the number of dead cells mixed into the cell aggregate can be reduced. This makes it possible to increase the cell viability of the cell aggregate. In addition, when the unevenness height is 500 nm or less, air bubbles are less likely to adhere to the unevenness. This eliminates the need to perform degassing to remove air bubbles or to repeatedly discharge and aspirate the medium using a pipetter, improving operability. When the unevenness height is 1 nm or more, live cells that have spontaneously moved (migrated) on the cell culture substrate gather in the (A) region, making it possible to increase the cell viability of the cell aggregate. Since this is suitable for increasing the cell viability of the formed cell aggregate, the unevenness height is more preferably 400 nm or less, even more preferably 100 nm or less, and most preferably 50 nm or less.

[0058] The cell culture substrate may have a layer containing a biological substance on the surface as necessary. The layer containing the biological substance may be present on the entire surface of the cell culture substrate, or may be present only on the surface of the (A) region. There is no particular limitation on the biological substance, and examples thereof include matrigel, laminin, fibronectin, vitronectin, collagen, etc.

[0059] These biological substances may be natural products, or may be artificially synthesized using recombinant gene technology or the like. They may also be fragments obtained by cleavage with restriction enzymes or the like, or synthetic proteins or peptides obtained by chemically synthesizing substances equivalent to these biological substances.

[0060] As Matrigel, commercially available products such as Matrigel (manufactured by Corning Incorporated) and Geltrex (manufactured by Thermo Fisher Scientific) can be suitably used due to their ease of availability.

[0061] The type of laminin is not particularly limited, but for example, laminin 511, laminin 521, laminin 511-E8 fragment, etc., which have been reported to exhibit high activity against α6β1 integrin expressed on the surface of human iPS cells, can be used. Laminin may be a natural product, may be artificially synthesized by recombinant gene technology, etc., or may be a synthetic protein or synthetic peptide obtained by chemically synthesizing a substance equivalent to laminin. In view of ease of availability, a commercially available product such as iMatrix-511 (manufactured by Nippi Corporation) can be preferably used.

[0062] Vitronectin may be a natural product, may be artificially synthesized by recombinant gene technology, or may be a synthetic protein or peptide obtained by chemically synthesizing a substance equivalent to vitronectin. In view of availability, commercially available products such as vitronectin, human plasma derived (Wako Pure Chemical Industries, Ltd.), synthemax (Corning Incorporated), and Vitronectin (VTN-N) (Thermo Fisher Scientific) can be preferably used.

[0063] Fibronectin may be a natural product, may be artificially synthesized by recombinant gene technology, or may be a synthetic protein or peptide obtained by chemically synthesizing a substance equivalent to fibronectin. In view of availability, commercially available products such as fibronectin solution, human plasma-derived (manufactured by Wako Pure Chemical Industries, Ltd.), and Retronectin (manufactured by Takara Bio Inc.) can be preferably used.

[0064] The type of collagen is not particularly limited, and for example, type I collagen, type IV collagen, etc. can be used. Collagen may be a natural product, may be artificially synthesized by gene recombination technology, etc., or may be a synthetic peptide obtained by chemically synthesizing a substance equivalent to collagen. In view of ease of availability, commercially available products such as collagen I, human (manufactured by Corning Incorporated) and collagen IV, human (manufactured by Corning Incorporated) can be preferably used.

[0065] From the viewpoint of suppressing the denaturation of the biological substance and enhancing cell proliferation, it is preferable that the biological substance is immobilized on the cell culture substrate by a non-covalent bond. Here, "non-covalent bond" refers to a bonding force other than a covalent bond derived from an intermolecular force, such as electrostatic interaction, water-insoluble interaction, hydrogen bond, π-π interaction, dipole-dipole interaction, London dispersion force, other van der Waals interaction, etc. The biological substance may be immobilized on the block copolymer by a single bonding force or a combination of multiple bonding forces.

[0066] The method for immobilizing the biological substance is not particularly limited. For example, a method in which a solution of the biological substance is applied to a cell culture substrate for a predetermined period of time to immobilize the biological substance, or a method in which the biological substance is added to the culture medium when culturing cells, and the biological substance is adsorbed onto the cell culture substrate and immobilized thereon can be suitably used.

[0067] The cell culture substrate according to the present embodiment may, if necessary, have a partition plate (for example, a partition plate having a cross-sectional area in the in-plane direction of 0.05 to 100 cm 2 A structure for separating each cell aggregate may be provided, for example, by providing a plate having a through hole.

[0068] The cell culture substrate according to the present embodiment may be sterilized. There is no particular limitation on the sterilization method, but high-pressure steam sterilization, UV sterilization, gamma ray sterilization, ethylene oxide gas sterilization, etc. can be used. From the viewpoint of suppressing denaturation of the block copolymer, high-pressure steam sterilization, UV sterilization, and ethylene oxide gas sterilization are preferred. From the viewpoint of suppressing deformation of the substrate, UV sterilization or ethylene oxide gas sterilization is more preferred. From the viewpoint of excellent mass productivity, ethylene oxide gas sterilization is preferred.

[0069] The cells to be cultured using the cell culture substrate according to the present embodiment 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 fetal kidney-derived HEK293 cells, and human cervical cancer-derived HeLa cells, for example, epithelial cells and endothelial cells constituting each tissue and organ in the living 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 living body, non-parenchymal liver cells and adipocytes, and cells having differentiation potential such as mesenchymal stem cells, bone marrow cells, and Muse cells present in various tissues, as well as stem cells (pluripotent stem cells) having differentiation potential such as ES cells and iPS cells, and cells induced to differentiate therefrom. From the viewpoint of cell proliferation and detachment in the cell culture substrate according to one embodiment, stem cells or pluripotent stem cells are preferred, mesenchymal stem cells or pluripotent stem cells are more preferred, pluripotent stem cells are even more preferred, and iPS cells are most preferred.

[0070] As described in the Examples below, the cell culture substrate according to this embodiment can be suitably used for inducing differentiation from pluripotent stem cells into three germ layer cells. The cell culture substrate according to this embodiment can also be suitably used for inducing differentiation from pluripotent stem cells into intestinal epithelial cells. Thus, the cell culture substrate according to this embodiment may be for inducing differentiation from pluripotent stem cells into three germ layer cells, or for inducing differentiation from pluripotent stem cells into intestinal epithelial cells.

[0071] The cell culture substrate according to the present embodiment can be produced, for example, by a production method including the following steps (1), (2), and (3). This production method is excellent in mass productivity. In step (3), corona treatment or UV treatment can be performed instead of plasma treatment. Step (1) is a step of coating at least a part of the surface of a substrate with a composition containing a UV-reactive hydrophilic polymer to form a layer containing a hydrophilic polymer. Step (2) is a step of irradiating the layer containing the hydrophilic polymer with UV light, thereby fixing the layer containing the hydrophilic polymer to the surface of the substrate through a chemical reaction. Step (3) is a step of subjecting a part of the surface of the layer containing the immobilized hydrophilic polymer to a plasma treatment, thereby forming an (A) region in the plasma-treated part.

[0072] The cell culture substrate according to this embodiment can also be produced by a production method including the following steps (1'), (2'), (3') and (4'). Step (1') A step of using a substrate formed from a polymer containing an alicyclic hydrocarbon group or an aromatic hydrocarbon group in a repeating unit, subjecting the surface of the substrate to a plasma treatment, and forming the above-mentioned (A) region in the plasma-treated portion. Step (2') A step of coating at least a part of the surface of a substrate with a composition containing a UV-reactive hydrophilic polymer to form a layer containing the hydrophilic polymer. Step (3'): A step of irradiating a part of the layer containing the hydrophilic polymer with UV light to fix the part of the layer containing the hydrophilic polymer on the surface of the base material. Step (4'): A step of washing the hydrophilic polymer with a solvent to dissolve and remove the hydrophilic polymer that is not immobilized on the surface from the surface of the substrate.

[0073] The production method according to the present embodiment may further include the following step (4), in addition to steps (1), (2) and (3), or steps (1′), (2′), (3′) and (4′), as necessary. Step (4) After step (3) or (4'), a cross-sectional area in the in-plane direction is 0.05 to 100 cm 2 A step of bonding the plate having the through holes to a substrate on the side of the substrate that is coated with the layer containing a hydrophilic polymer.

[0074] Moreover, the production method according to one embodiment may further include the following step (5) after step (3) or (4'). When step (4) is carried out, step (5) is preferably carried out after step (3) or (4') and before step (4). Step (5): After step (3) or (4'), a step of coating the surface of the layer containing a hydrophilic polymer that has been subjected to plasma treatment with a composition containing a temperature-responsive polymer to form a layer containing a temperature-responsive polymer.

[0075] In step (1), at least a part of the surface of the substrate is coated with a composition containing a UV-reactive hydrophilic polymer to form a layer containing a hydrophilic polymer. By forming a layer containing a hydrophilic polymer, it is possible to make the substrate free of cell adhesiveness and cell proliferation properties. The method for forming the layer containing a hydrophilic polymer is not particularly limited, and examples thereof include a method for forming the layer by applying a composition containing a hydrophilic polymer to at least a part of the surface of the substrate. Examples of methods for applying the composition containing a hydrophilic polymer include various commonly known methods 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.

[0076] Fig. 2 is a schematic diagram (perspective view) of a substrate having a layer containing a hydrophilic polymer formed on its surface after step (1). In Fig. 2, a layer 2 containing a hydrophilic polymer is formed on the entire surface of a substrate 1.

[0077] In step (2), the layer 2 containing the hydrophilic polymer is irradiated with UV light, and the layer containing the hydrophilic polymer is fixed to the surface of the substrate. By irradiating the UV-reactive hydrophilic polymer with UV light, a chemical reaction occurs between the hydrophilic polymers themselves or between the hydrophilic polymer and the substrate, and the layer containing the hydrophilic polymer is fixed to the surface of the substrate. By fixing the layer containing the hydrophilic polymer to the surface of the substrate, a layer containing the temperature-responsive polymer can be formed without deforming the layer containing the hydrophilic polymer when a composition containing the temperature-responsive polymer is applied in step (5) described later. In addition, by fixing the layer containing the hydrophilic polymer to the surface of the substrate, the shape of the region (A) formed on a part of the surface of the layer containing the hydrophilic polymer in step (3) described later can be maintained.

[0078] In step (3), a part of the surface of the layer containing the immobilized hydrophilic polymer is subjected to plasma treatment, and the (A) region is formed in the plasma-treated part. A suitable plasma treatment method can be appropriately adjusted depending on the type of hydrophilic polymer used and the layer thickness, but the plasma exposure time 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 method of patterning the (A) region is not particularly limited, but examples include a method in which the desired part (unmasked part) is subjected to plasma treatment while covered with a metal mask, a silicon mask, a surface protection film, or the like.

[0079] 3 is a schematic diagram (perspective view) of the cell culture substrate after step (3). In the cell culture substrate 10 shown in FIG. 3, circular regions (regions indicated by A) are arranged at equal intervals. For example, the area of ​​the region is 0.001 to 5 mm. 2) corresponds to the area where plasma treatment was performed. The area indicated by A has cell adhesiveness and cell proliferation properties as a result of the surface of layer 2 containing a hydrophilic polymer being modified by plasma treatment. The area other than the circular area (area indicated by B) corresponds to the area where plasma treatment was not performed as it is protected by, for example, a metal mask. The area indicated by B does not have cell adhesiveness or cell proliferation properties as the surface of layer 2 containing a hydrophilic polymer is

[0080] In step (1'), a polymer substrate containing an alicyclic hydrocarbon group or an aromatic hydrocarbon group in the repeating unit is used, and the surface of the substrate is subjected to plasma treatment to form the above-mentioned (A) region in the plasma-treated portion. By subjecting the surface of the polymer substrate containing an alicyclic hydrocarbon group or an aromatic hydrocarbon group to plasma treatment, the substrate surface can be changed to a structure suitable for cell culture. The conditions for the plasma treatment are preferably 1 second to 10 minutes, more preferably 1 second to 5 minutes, and particularly preferably 1 second to 1 minute, at a gas pressure of 1 to 30 Pascals using air, nitrogen, or oxygen as the introduced gas. Examples of the alicyclic hydrocarbon group include cycloalkyl groups, and more specifically, examples thereof include cyclopropyl groups, cyclobutyl groups, cyclopentyl groups, cyclohexyl groups, cycloheptyl groups, cyclooctyl groups, and cyclononyl groups. The aromatic hydrocarbon group refers to a group obtained by removing one or more hydrogen atoms directly bonded to carbon atoms constituting a ring from an aromatic hydrocarbon. Examples of aromatic hydrocarbon groups include groups in which one or more hydrogen atoms directly bonded to carbon atoms constituting a ring have been removed from benzene, which is a monocyclic aromatic hydrocarbon, or naphthalene, which is a polycyclic aromatic hydrocarbon.

[0081] Alternatively, instead of the above step (1'), the surface of the substrate may be coated with a component suitable for cell culture. As the component suitable for cell culture, a polymer material such as polycarboxystyrene can be suitably used.

[0082] In step (2'), at least a part of the surface of the substrate is coated with a composition containing a UV-reactive hydrophilic polymer to form a layer containing the hydrophilic polymer. As a coating method, the same method as in step (1) described above can be suitably used.

[0083] In step (3'), a part of the layer containing the hydrophilic polymer is irradiated with UV light, and the part of the layer containing the hydrophilic polymer is fixed to the surface of the substrate. By irradiating a part of the layer with UV light, only the UV-irradiated part of the layer containing the hydrophilic polymer is fixed to the surface of the substrate. As a method for irradiating a part of the layer containing the hydrophilic polymer with UV light, a method of placing a quartz mask or the like on the substrate, in which a UV-non-transparent region is formed by chrome deposition or the like in a desired pattern, and irradiating the mask with UV light from above can be mentioned.

[0084] In step (4'), the hydrophilic polymer is washed with a solvent, and the hydrophilic polymer not immobilized on the surface is dissolved and removed from the surface of the substrate. By removing the hydrophilic polymer not immobilized, the substrate surface is exposed, and the (A) region can be formed. The solvent used in step (4') preferably contains water and alcohol, since it is suitable for removing compounds by-produced by the self-reaction of the UV-reactive hydrophilic polymer, such as compounds containing nitrene derived from an azide group. By washing with a mixed solvent of water and alcohol, it is possible to reduce the hydrophilic polymer-derived components remaining in the (A) region, and the cell adhesiveness and cell proliferation of the (A) region can be improved. The alcohol is preferably a lower alcohol, and for example, methanol, ethanol, 2-propanol, t-butanol, isobutanol, pentanol, and hexanol are preferable, and methanol or ethanol is more preferable. The content of the alcohol is preferably 50 to 95%, more preferably 50 to 90%, particularly preferably 60 to 90%, and most preferably 70 to 90%.

[0085] In step (4), after step (3) or (4'), the cross-sectional area in the in-plane direction is 0.05 to 100 cm 2The plate having the through hole is attached to the substrate on the side of the substrate coated with the layer containing a hydrophilic polymer. The cross-sectional area in the in-plane direction is 0.05 to 100 cm 2 By bonding a plate having such through-holes to a substrate, a plate having a space for placing a culture medium can be produced with high mass productivity. FIG. 4 is a schematic diagram (perspective view) of the cell culture substrate after step (4). The cell culture substrate 11 shown in FIG. 4 is, for example, a cell culture substrate 10 shown in FIG. 3, etc., with a partition plate 20 (with a cross-sectional area in the in-plane direction of 0.05 to 100 cm2). 2 The plate has a through hole.

[0086] In step (5), after step (3) or (4'), the surface of the layer containing the hydrophilic polymer that has been subjected to the plasma treatment is coated with a composition containing a temperature-responsive polymer to form a layer containing a temperature-responsive polymer. In this case, by forming the layer containing the temperature-responsive polymer to a thickness of 100 nm or less, the molecular chains of the temperature-responsive polymer are easily coated in a sparse state on the surface of the (A) region formed in step (3) or (4'), which is suitable for imparting temperature responsiveness while maintaining the functions of the (A) region (having cell adhesiveness and cell proliferation properties). In addition, by making the layer containing the temperature-responsive polymer to a thickness of 1 nm or more, it is possible to produce a cell culture substrate that is capable of imparting sufficient temperature responsiveness and rapidly forming cell aggregates, which is suitable.

[0087] As a method for coating with a composition containing a temperature-responsive polymer, a method similar to the above-mentioned method for applying a composition containing a hydrophilic polymer can be suitably used.

[0088] As a method for coating with a composition containing a temperature-responsive polymer, it is also preferable to coat the entire surface of the cell culture substrate with a temperature-responsive substance. When coating with a composition containing a temperature-responsive polymer, the mass productivity of the cell culture substrate can be improved by coating using a commonly used coating method without performing patterning. Furthermore, by coating the entire surface of the cell culture substrate with a composition containing a temperature-responsive polymer, temperature responsiveness is imparted to region (A), and region (B) is also coated with the temperature-responsive polymer. By coating region (B) with a temperature-responsive polymer, the cell adhesiveness of region (B) can be reduced.

[0089] The present invention also relates to a method for inducing differentiation of pluripotent stem cells. The differentiation induction method according to this embodiment includes the following steps (i) to (iii): The differentiation induction method according to this embodiment is suitably used for inducing differentiation from pluripotent stem cells to three germ layer cells. (i) seeding pluripotent stem cells onto the cell culture substrate of the present invention. (ii) Culturing the seeded pluripotent stem cells to form hemispherical cell aggregates with a height / diameter ratio of 0.2 to 0.8. (iii) A step of inducing differentiation of the cell aggregate to form a cell aggregate of three germ layer cells.

[0090] Step (i) uses the cell culture substrate according to the present invention described above, and is a step of seeding pluripotent stem cells onto the cell culture substrate. "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 "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).

[0091] In the above step (i), the culture is carried out under conditions effective for maintaining the undifferentiated state of the pluripotent stem cells. There are no particular limitations on the conditions effective for maintaining the undifferentiated state, but examples include setting the density of the pluripotent stem cells 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 pluripotent stem 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 pluripotent stem cells, can be suitably used. 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), as this is particularly suitable for maintaining the undifferentiated state of pluripotent stem cells, and it is most preferable to use a medium supplemented with basic fibroblast growth factor.

[0092] The type of medium to which the basic fibroblast growth factor is added is not particularly limited, and examples of commercially available products include DMEM (manufactured by Sigma-Aldrich Co. LLC), Ham's F12 (manufactured by Sigma-Aldrich Co. LLC), D-MEM / Ham's F12 (manufactured by Sigma-Aldrich Co. LLC), Primate ES Cell Medium (manufactured by REPROCELL Co., Ltd.), StemFit AK02N (manufactured by Ajinomoto Co., Inc.), StemFit AK03 (manufactured by Ajinomoto Co., Inc.), mTeSR1 (manufactured by STEMCELL TECHNOLOGIES), TeSR-E8 (manufactured by STEMCELL TECHNOLOGIES), and ES Cell Medium (manufactured by REPROCELL Co., Ltd.). TECHNOLOGIES), ReproNaive (REPROCELL Inc.), ReproXF (REPROCELL Inc.), ReproFF (REPROCELL Inc.), ReproFF2 (REPROCELL Inc.), NutriStem (Biological Industries Inc.), iSTEM (Takara Bio Inc.), GS2-M (Takara Bio Inc.), and hPSC Growth Medium DXF (PromoCell Inc.). 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, StemFit AK02N (manufactured by Ajinomoto Co., Ltd.) or StemFit AK03 (manufactured by Ajinomoto Co., Ltd.) are more preferred, and StemFit AK02N (manufactured by Ajinomoto Co., Ltd.) is particularly preferred, as they are suitable for maintaining the undifferentiated state of pluripotent stem cells.

[0093] In the above step (i), the seeding method of the pluripotent stem cells is not particularly limited, but for example, the seeding can be performed by injecting a cell suspension into a cell culture substrate. The cell density at the time of seeding is not particularly limited, but it is preferable to use a cell density of 1.0×10 so that the pluripotent stem cells can be maintained and proliferated. 2 ~1.0×10 6 cells / cm 2 is preferred, and 5.0×10 2 ~5.0×10 5 cells / cm 2is more preferable, and 1.0×10 3 ~2.0×10 5 cells / cm 2 More preferably, 1.2×10 3 ~1.0×10 5 cells / cm 2 is most preferred.

[0094] The medium used in the above step (i) is also suitable for maintaining the survival of pluripotent stem cells, so it is preferable to use a medium containing the above basic fibroblast growth factor and a Rho-binding kinase inhibitor. In particular, when human pluripotent stem cells are used and the cell density of human pluripotent stem cells is low, the addition of a Rho-binding kinase inhibitor may be effective in maintaining the survival of human pluripotent stem cells. Examples of Rho-binding kinase inhibitors that can be used include (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide·2HCl·H2O (Y-27632, Wako Pure Chemical Industries, Ltd.) and 1-(5-Isoquinolinesulfonyl)homopiperazine Hydrochloride (HA1077, Wako Pure Chemical Industries, Ltd.). The concentration of the Rho-binding kinase inhibitor added to the culture medium is within a range that is effective for maintaining the survival of human pluripotent stem cells and does not affect the undifferentiated state of human pluripotent stem 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.

[0095] Shortly after initiating step (i) above, the pluripotent stem cells begin to adhere to the cell culture substrate.

[0096] In step (ii), the seeded pluripotent stem cells are cultured to form hemispherical cell aggregates with a height / diameter ratio of 0.2 to 0.8. The "height" may be, for example, the maximum diameter of the cells in the out-of-plane direction of the cell culture substrate. The "diameter" may be, for example, the maximum diameter of the cells in the in-plane direction of the cell culture substrate.

[0097] Since the culture in step (ii) is suitable for maintaining the proliferation ability, physiological activity, and function of pluripotent stem cells, the culture temperature is preferably 30 to 42°C, more preferably 32 to 40°C, even more preferably 36 to 38°C, and most preferably 37°C.

[0098] (ii) The first medium change is preferably performed 22 to 26 hours after the start of the step. 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 pluripotent stem cells grow and form flat cell masses 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 the (A) region, 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 a height / diameter ratio of 0.2 to 0.8. The height / diameter ratio is obtained by observing multiple cell aggregates under a microscope, calculating the values, and averaging them. By forming a hemispherical cell aggregate with a height / diameter ratio of 0.2 to 0.8, it is possible to efficiently induce differentiation of the pluripotent stem cells into three germ layer cells. Moreover, since this is suitable for increasing the efficiency of inducing differentiation into three germ layer cells, the height / diameter ratio is more preferably 0.3 to 0.7, and further preferably 0.4 to 0.6.

[0099] In step (iii), the cell aggregate (cell aggregate of pluripotent stem cells) is induced to differentiate to form a cell aggregate of three germ layer cells. Step (iii) may be performed after step (ii) or in parallel with step (ii). That is, steps (ii) and (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. The three germ layer cells refer to any of endodermal cells, mesodermal cells, and ectodermal cells. In step (iii), the cells are cultured in a medium containing a differentiation-inducing factor.

[0100] In order to increase the efficiency of differentiation induction into endoderm cells, the differentiation induction factor in step (iii) preferably contains an endoderm induction factor. In order to increase the efficiency of differentiation induction into embryoid bodies, the endoderm induction factor is preferably 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 particularly preferably contains any one of Wnt3a, BMP4, IGFI, and activin A.

[0101] The differentiation-inducing factor in step (iii) preferably contains a mesoderm-inducing factor, since it is suitable for increasing the efficiency of differentiation-inducing into mesoderm cells. The mesoderm-inducing factor is preferably a single or multiple differentiation-inducing factors selected from the group consisting of a GSK3β inhibitor, bone morphogenetic protein (BMP) and activin, since it is suitable for increasing the efficiency of differentiation-inducing into embryoid bodies, and it is particularly preferable for the mesoderm-inducing factor to contain any one of activin A, CHIR99021 (GSK3β inhibitor) and BMP4.

[0102] In order to enhance the efficiency of differentiation induction into ectodermal cells, the differentiation induction factor in step (iii) preferably contains an ectodermal induction factor. The 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.

[0103] The concentration of the differentiation-inducing factor added to the culture 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 differentiation induction into three germ layer cells.

[0104] In order to ensure that differentiation induction proceeds sufficiently, the medium containing the differentiation induction factor is preferably changed every 24 hours during culture. By changing the medium within 24 hours, it is possible to prevent a shortage of the differentiation induction factor in the medium and to differentiate all pluripotent stem cells uniformly.

[0105] The method for inducing differentiation of pluripotent stem cells according to this embodiment may further include the following step (iv). When the method for inducing differentiation includes step (iv), it is preferably used for inducing differentiation from pluripotent stem cells to intestinal epithelial cells. (iv) Culturing the cell aggregates in a medium containing at least one differentiation-inducing factor selected from the group consisting of Wnt protein, bone morphogenetic protein (BMP), insulin-like growth factor, and activin, thereby expressing a marker possessed by intestinal epithelial cells.

[0106] 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. The 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.

[0107] The cell culture substrate according to the present invention does not have a surface uneven structure with a high height in the outward direction of the substrate, and therefore air bubbles are less likely to adhere to the surface of the cell culture substrate when a culture medium is brought into contact with the surface. For example, the cell culture substrate according to the present invention has an air bubble adhesion rate of preferably 10% or less, more preferably 5% or less, even more preferably 3% or less, and most preferably 1% or less, relative to the number of regions (A).

[0108] The cell aggregates formed using the cell culture substrate according to the present invention have a high cell viability, because the attached cells spontaneously gather, compared to the cell aggregates formed using the conventional microrelief structure. 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, even more preferably 85% or more, and most preferably 90% or more. EXAMPLES

[0109] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples. Unless otherwise specified, commercially available reagents were used.

[0110] <(A) Measurement of the area of ​​the region> An image of the surface of the cell culture substrate was obtained using a laser microscope (Keyence Corporation, product name VK-X200). Using the obtained image, the area of ​​the (A) region at 20 points was calculated on the analysis software VK-X Viewer, and the area of ​​these areas was averaged to obtain the area of ​​the (A) region.

[0111] <Measurement of unevenness height at the boundary between area (A) and area (B)> The unevenness height at the boundary was measured by measuring the thickness of the cell culture substrate in the out-of-plane direction using a laser microscope (Keyence Corporation, product name VK-X200) in laser scanning mode.

[0112] <Evaluation of air bubble adhesion> The medium StemFitAK02N (Ajinomoto Co., Inc.) was added to the cell culture substrate, and the presence or absence of air bubbles was confirmed by observing under a microscope.

[0113] <Evaluation of cell aggregates> (Culture of cell aggregates) The cell culture substrate was filled with StemFitAK02N medium (Ajinomoto Co., Inc.) at a rate of 0.2 mL / cm 2Then, iMatrix-511 solution (manufactured by Nippi Corporation) was added at a concentration of 2.5 μL / mL. Human iPS cell line 201B7, which had been adjusted to a ratio of live cells to total cells (cell viability) of 50% by mixing dead cells, was used, and the cells were cultured at 15,000 cells (total number of live and dead cells) / cm. 2 The cells were seeded so that the cells would be in a 100% sieve size and cultured at 37°C with a CO2 concentration of 5%. In addition, Y-27632 (manufactured by Wako Pure Chemical Industries, Ltd.) (concentration: 10 μM) was added to the medium until 24 hours after the cells were seeded. The presence or absence of cell aggregate formation was confirmed 1 or 2 days after the start of culture.

[0114] (Detachment of cell aggregates by cooling) After confirming the formation of cell aggregates, the cells were cooled at room temperature for 30 minutes, and detachment of the cell aggregates was confirmed.

[0115] (Assessment of cell viability) After confirming the formation of cell aggregates, the cells were treated with TrypLE-EDTA solution (a 1:1 mixture of TrypLE select (ThermoFisher) and 0.5 mM EDTA solution (Invitrogen)), then the cells were detached and collected as single cells using a cell scraper, and cell viability was measured by staining with trypan blue.

[0116] <Measurement of layer thickness of hydrophilic polymer and temperature-responsive polymer> The layer thickness of the polymers (hydrophilic polymer and temperature-responsive polymer) coated on the substrate was determined by calculating the amount of coating per unit area from the surface area of ​​the substrate, the concentration of the polymers in the solution, and the volume of the solution dropped onto the substrate, assuming the specific gravity of the polymers to be 1.

[0117] <Measurement of the composition of temperature-responsive polymers> The composition of the temperature-responsive polymer was determined by proton nuclear magnetic resonance spectroscopy ( 1 H-NMR) spectrum analysis, or carbon nuclear magnetic resonance spectroscopy using a nuclear magnetic resonance measurement device (Bruker, product name AVANCEIIIHD500) 13The carbonyl group was determined by C-NMR spectroscopy.

[0118] <Measurement of molecular weight and molecular weight distribution of temperature-responsive polymer> Weight average molecular weight (Mw), number average molecular weight (Mn) and molecular weight distribution (Mw / Mn) were measured by gel permeation chromatography (GPC). The GPC apparatus used was a Tosoh Corporation HLC-8320GPC, and two Tosoh Corporation TSKgel Super AWM-H columns were used. The column temperature was set to 40°C, and the eluent was 1,1,1,3,3,3-hexafluoro-2-isopropanol containing 10 mM sodium trifluoroacetate or N,N-dimethylformamide containing 10 mM lithium bromide. The measurement sample was prepared at 1.0 mg / mL and measured. The molecular weight calibration curve was made using polymethylmethacrylate (Polymer Laboratories Ltd.) with known molecular weight.

[0119] <Evaluation of autofluorescence intensity of substrate> The substrate was irradiated with light having excitation wavelengths of 350 nm, 488 nm, and 647 nm with an exposure time of 0.4 seconds, and a fluorescent image was taken. The RGB values ​​of these images were calculated, and the R value at an excitation wavelength of 350 nm, the G value at an excitation wavelength of 488 nm, and the B value at an excitation wavelength of 647 nm were taken as the fluorescence intensity. Similar measurements were performed on a 1.2 mm thick polystyrene plate, and the relative values ​​to the value for the 1.2 mm thick polystyrene plate are shown in Table 4 as the fluorescence intensity of each substrate.

[0120] [Example 1] A water / ethanol solution containing 0.06 wt% solids of azide-containing polyvinyl alcohol (BIOSURFINE®-AWP, Toyo Gosei Co., Ltd.) was dropped onto a 3.5 cm diameter polystyrene (PS) dish (substrate), 0.8 mL of which was dried under reduced pressure at room temperature, and then irradiated with UV light to harden the hydrophilic polymer, forming a hydrophilic polymer layer. A metal mask with multiple circular holes with a diameter of 0.2 mm was placed on the hydrophilic polymer layer, and plasma treatment (20 Pa gas pressure, 20 mA conductive current, 30 seconds irradiation time) was performed on top of the metal mask using a plasma irradiation device (Vacuum Device Co., Ltd., product name Plasma Ion Bombarder PIB-20) to form a region (region (A)) with cell adhesiveness and cell proliferation properties in the plasma-treated area. In addition, region (B) was formed in the area masked by the metal mask.

[0121] The composition of the cell culture substrate and the evaluation results are shown in Table 2. The cell culture substrate had a hydrophilic polymer layer thickness of 600 nm and an area of ​​region (A) of 0.03 mm. 2 The height of the unevenness at the boundary between region (A) and region (B) was 32 nm.

[0122] Human iPS cells were cultured on this cell culture substrate, and the formation of cell aggregates was confirmed two days after the start of culture. Visual inspection revealed that the size of the formed cell aggregates was uniform. The height / diameter ratio of the cell aggregates was estimated to be 0.5. The survival rate of the cells contained in the cell aggregates was 89%, indicating a high cell survival rate. Furthermore, no air bubbles were attached to the cell culture substrate when the medium was added.

[0123] [Example 2] A cell culture substrate was prepared in the same manner as in Example 1, except that the plasma irradiation time in Example 1 was changed to 10 minutes.

[0124] The composition of the cell culture substrate and the evaluation results are shown in Table 2. The cell culture substrate was prepared with a hydrophilic polymer layer thickness of 600 nm and an area of ​​region (A) of 0.03 mm. 2The height of the unevenness at the boundary between region (A) and region (B) was 85 nm.

[0125] Human iPS cells were cultured on this cell culture substrate, and the formation of cell aggregates was confirmed two days after the start of culture. Visual inspection revealed that the size of the formed cell aggregates was uniform. The height / diameter ratio of the cell aggregates was estimated to be 0.5. The survival rate of the cells contained in the cell aggregates was 91%, indicating a high cell survival rate. Furthermore, no air bubbles were attached to the cell culture substrate when the medium was added.

[0126] [Example 3] A cell culture substrate was prepared in the same manner as in Example 1, except that the gas pressure in the plasma treatment in Example 1 was changed to 13 Pa and the plasma exposure time was changed to 20 minutes.

[0127] The composition of the cell culture substrate and the evaluation results are shown in Table 2. The cell culture substrate was prepared with a hydrophilic polymer layer thickness of 600 nm and an area of ​​region (A) of 0.03 mm. 2 The height of the unevenness at the boundary between region (A) and region (B) was 452 nm.

[0128] Human iPS cells were cultured on this cell culture substrate, and the formation of cell aggregates was confirmed two days after the start of culture. Visual inspection revealed that the size of the formed cell aggregates was uniform. The height / diameter ratio of the cell aggregates was estimated to be 0.5. The survival rate of the cells contained in the cell aggregates was 78%, indicating a high cell survival rate. Furthermore, no air bubbles were attached to the cell culture substrate when the medium was added.

[0129] [Example 4] A 0.08 mL ethanol solution containing a polymer (Lipidure-CM5210, NOF Corp.) containing phosphorylcholine groups as a hydrophilic polymer at a solid content concentration of 0.01 wt% was dropped onto a polystyrene dish (substrate) with a diameter of 3.5 cm, and dried under reduced pressure at room temperature to form a hydrophilic polymer layer. A metal mask with multiple circular holes with a diameter of 0.2 mm was placed on the hydrophilic polymer layer, and a plasma irradiation device (Vacuum Device Corp., product name Plasma Ion Bombarder PIB-20) was used to perform plasma treatment (20 Pa gas pressure, 20 mA conductive current, irradiation time 30 seconds) from above the metal mask to form a region (region (A)) with cell adhesiveness and cell proliferation properties in the plasma-treated area. In addition, a region (B) was formed in the area masked by the metal mask.

[0130] The composition of the cell culture substrate and the evaluation results are shown in Table 2. The cell culture substrate was fabricated with a hydrophilic polymer layer thickness of 10 nm and an area of ​​region (A) of 0.03 mm. 2 The height of the unevenness at the boundary between region (A) and region (B) was 23 nm.

[0131] Human iPS cells were cultured on this cell culture substrate, and the formation of cell aggregates was confirmed two days after the start of culture. Visual inspection revealed that the size of the formed cell aggregates was uniform. The height / diameter ratio of the cell aggregates was estimated to be 0.5. The survival rate of the cells contained in the cell aggregates was 87%, indicating a high cell survival rate. Furthermore, no air bubbles were attached to the cell culture substrate when the medium was added.

[0132] [Example 5] A cell culture substrate was prepared in the same manner as in Example 4, except that the solid content concentration of the hydrophilic polymer was changed to 0.05 wt %.

[0133] The composition of the cell culture substrate and the evaluation results are shown in Table 2. The cell culture substrate was prepared with a hydrophilic polymer layer thickness of 50 nm and an area of ​​region (A) of 0.03 mm. 2 The height of the unevenness at the boundary between region (A) and region (B) was 21 nm.

[0134] Human iPS cells were cultured on this cell culture substrate, and the formation of cell aggregates was confirmed two days after the start of culture. Visual inspection revealed that the size of the formed cell aggregates was uniform. The height / diameter ratio of the cell aggregates was estimated to be 0.5. The survival rate of the cells contained in the cell aggregates was 92%, indicating a high cell survival rate. Furthermore, no air bubbles were attached to the cell culture substrate when the medium was added.

[0135] [Example 6] A cell culture substrate was prepared in the same manner as in Example 1, except that the solid content concentration of the hydrophilic polymer was changed to 0.15 wt %.

[0136] The composition of the cell culture substrate and the evaluation results are shown in Table 2. The cell culture substrate had a hydrophilic polymer layer thickness of 1500 nm and an area of ​​region (A) of 0.03 mm. 2 The height of the unevenness at the boundary between region (A) and region (B) was 34 nm.

[0137] Human iPS cells were cultured on this cell culture substrate, and the formation of cell aggregates was confirmed two days after the start of culture. Visual inspection revealed that the size of the formed cell aggregates was uniform. The height / diameter ratio of the cell aggregates was estimated to be 0.5. The survival rate of the cells contained in the cell aggregates was 85%, indicating a high cell survival rate. Furthermore, no air bubbles were attached to the cell culture substrate when the medium was added.

[0138] [Example 7] A cell culture substrate was prepared in the same manner as in Example 1, except that a 188 μm thick polyethylene terephthalate (PET) film (Lumirror, manufactured by Toray Industries, Inc.) (substrate) was used instead of a 3.5 cm diameter polystyrene (PS) dish. The culture was carried out by immobilizing a cut-out piece of the prepared cell culture substrate in a petri dish.

[0139] The composition of the cell culture substrate and the evaluation results are shown in Table 2. The cell culture substrate had a hydrophilic polymer layer thickness of 600 nm and an area of ​​region (A) of 0.03 mm. 2The height of the unevenness at the boundary between region (A) and region (B) was 38 nm.

[0140] Human iPS cells were cultured on this cell culture substrate, and the formation of cell aggregates was confirmed two days after the start of culture. Visual inspection revealed that the size of the formed cell aggregates was uniform. The height / diameter ratio of the cell aggregates was estimated to be 0.5. The survival rate of the cells contained in the cell aggregates was 84%, indicating a high cell survival rate. Furthermore, no air bubbles were attached to the cell culture substrate when the medium was added.

[0141] [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.

[0142] The composition of the cell culture substrate and the evaluation results are shown in Table 2. The cell culture substrate had a hydrophilic polymer layer thickness of 600 nm and an area of ​​region (A) of 1.76 mm 2 The height of the unevenness at the boundary between region (A) and region (B) was 35 nm.

[0143] Human iPS cells were cultured on this cell culture substrate, and the formation of cell aggregates was confirmed two days after the start of culture. Visual inspection revealed that the size of the formed cell aggregates was uniform. The height / diameter ratio of the cell aggregates was estimated to be 0.5. The survival rate of the cells contained in the cell aggregates was 89%, indicating a high cell survival rate. Furthermore, no air bubbles were attached to the cell culture substrate when the medium was added.

[0144] Using this cell culture substrate, human iPS cells were induced to differentiate into intestinal epithelial cells. 1.0 mL / dish of 1% Vitronectin (VTN-N) Recombinant Human Protein solution (Gibco) was added to the cell culture substrate, and the substrate was left to stand at 25°C for 1 hour. After 1 hour, the 1% VTN solution was removed, and 2.0 mL / dish of StemFitAK02N (Ajinomoto Co., Inc.), an undifferentiated maintenance medium, was added, and human iPS cells 201B7 strain were cultured at 3900 cells / cm. 2 The cells were seeded so that the cells were in a 37°C, 5% CO2 environment. Y-27632 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) was added to the medium until 24 hours after cell seeding. Multiple circular embryoid bodies attached to the cell culture substrate were confirmed by observation under a phase contrast microscope.

[0145] 24 hours after the cells were seeded, 2.0 mL / dish of the differentiation induction medium described below was added to initiate differentiation induction. The composition of the differentiation medium for intestinal epithelial cells was 85% KnockOut DMEM (ThermoFisher), 15% KnockOut Serum Replacement XenoFree (ThermoFisher), 0.1 mM non-essential amino acids (Sigma-Aldrich), 2 mM Gluta Max-I Supplement (ThermoFisher), 20 ng / mL basic fibroblast growth factor (bFGF, PEPRO TECH), 50 μg / mL L(+)-ascorbic acid (Fujifilm Wako Pure Chemical Industries, Ltd.), 10 ng / mL heregulin-β-1 (Fujifilm Wako Pure Chemical Industries, Ltd.), 200 ng / mL Long(R)R3IGF-I (Sigma-Aldrich), and 1% penicillin-streptomycin solution (×100) (Fujifilm Wako Pure Chemical Industries, Ltd.). The time point when the differentiation-inducing medium was added was designated as “differentiation day 0.” Thereafter, the differentiation-inducing medium was replaced every 72 hours of culture until “differentiation day 64.”

[0146] Figure 5 shows phase contrast microscope images of cells on "differentiation day 0", "differentiation day 43", and "differentiation day 64". As shown in Figure 5, the formation of small intestine-derived cysts was confirmed on "differentiation day 43", and the cysts were detached from the cell culture substrate on "differentiation day 64".

[0147] On the "64th day of differentiation", the differentiation induction medium in the dishes was removed, and 2.0 mL / dish of PBS(-) was added and washed. After washing, 1.0 mL / dish of cell detachment solution was added to the dishes, and the dishes were allowed to stand for 5 minutes at 37°C and a CO2 concentration of 5%. After allowing to stand for a predetermined time, the cell detachment solution was removed, and 1.0 mL / dish of differentiation induction medium was added to disperse the cells. The obtained cell suspension was collected, and the number of cells was counted using a Luna automatic cell counter (manufactured by Logos Biosystems).

[0148] To evaluate the induction of differentiation into intestinal epithelial cells, we employed housekeeping gene markers (GAPDH), enterocyte markers (CDX2), absorptive epithelium markers (VIL1), and intestinal stem cell markers (LGR5), and quantified the relative expression levels of the mRNAs of enterocyte markers and intestinal stem cell markers. Small intestinal tissue from healthy adults (R1234226-50, BioChain Institute) was used as a positive control.

[0149] The cells collected on "differentiation day 64" from the cell count were placed in a 1.5 mL sample tube at 1.0 × 10 6The cells were separated into 1 / tube. The cells were centrifuged (room temperature, 800×g, 5 min) and the supernatant was removed. RNA was extracted from the cells using RNeasy Plus Mini Kit (QIAGEN). The concentration of the extracted RNA was measured using Qubit4 Fluorometer and adjusted to 1 μg / 6 μL with RNase-free Water (Takara Bio Inc.). ReverTra Ace qRT Master Mix with gDNA Remover (Toyobo Co., Ltd.) was used for the reverse transcription reaction. Real-time PCR analysis was performed on 1 μL of 5-fold diluted cDNA (corresponding to 1 / 100 of the reverse transcription product) using an oligo dT primer, a primer having the base sequence shown in Table 1 below (INTEGRATED DNA TECHNOLOGIES Co., Ltd.) and THUNDERBIRD Probe qPCR Mix (Toyobo Co., Ltd.) using a QuantStudio3 real-time PCR system (Thermo Fisher Scientific Co., Ltd.). As a result, it was confirmed that intestinal tissue-derived CDX2, VIL1, and LGR5 were expressed in cells on "differentiation day 64."

[0150] [Table 1]

[0151] [Example 9] A 0.08 mL ethanol solution containing a polymer (Lipidure-CM5210, NOF Corp.) containing phosphorylcholine groups and UV-reactive functional groups as a hydrophilic polymer at a solid concentration of 0.05 wt% was dropped onto a 3.5 cm diameter polystyrene dish (substrate) and dried under reduced pressure at room temperature to form a hydrophilic polymer layer. A metal mask with multiple circular holes with a diameter of 0.2 mm was placed on the hydrophilic polymer layer, and a plasma irradiation device (Vacuum Device Corp., product name Plasma Ion Bombarder PIB-20) was used to perform plasma treatment (20 Pa gas pressure, 20 mA conductive current, irradiation time 30 seconds) from above the metal mask to form a region (region (A)) with cell adhesiveness and cell proliferation properties in the plasma-treated area. In addition, region (B) was formed in the area masked by the metal mask.

[0152] The composition of the cell culture substrate and the evaluation results are shown in Table 2. The cell culture substrate was prepared with a hydrophilic polymer layer thickness of 50 nm and an area of ​​region (A) of 0.03 mm. 2 The height of the unevenness at the boundary between region (A) and region (B) was 33 nm.

[0153] Human iPS cells were cultured on this cell culture substrate, and the formation of cell aggregates was confirmed two days after the start of culture. Visual inspection revealed that the size of the formed cell aggregates was uniform. The height / diameter ratio of the cell aggregates was estimated to be 0.5. The survival rate of the cells contained in the cell aggregates was 86%, indicating a high cell survival rate. Furthermore, no air bubbles were attached to the cell culture substrate when the medium was added.

[0154] [Example 10] The surface of the cell culture substrate prepared in the same manner as in Example 1 (the side on which the layer containing a hydrophilic polymer was formed) was covered with a block copolymer (temperature-responsive polymer) having a water-insoluble block segment and a temperature-responsive block segment to prepare a cell culture substrate. Specifically, 0.40 g (0.1 mmol) of 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, 7.11 g (50 mmol) of n-butyl methacrylate, and 33 mg (0.2 mmol) of azobis(isobutyronitrile) were added to a test tube and dissolved in 50 mL of 1,4-dioxane. After degassing for 30 minutes by nitrogen bubbling, the mixture was reacted at 70°C for 24 hours. After the reaction was completed, the reaction solvent was distilled off under reduced pressure using a rotary evaporator, and the reaction solution was concentrated. The concentrated solution was poured into 250 mL of methanol, and the precipitated yellow oily substance was collected and dried under reduced pressure to obtain an n-butyl methacrylate polymer.

[0155] The above-obtained n-butyl methacrylate polymer (0.9 g, 0.3 mmol), N-isopropylacrylamide (8.14 g, 72 mmol), and azobisisobutyronitrile (5 mg, 0.03 mmol) were added to a test tube and dissolved in 15 mL of 1,4-dioxane. After degassing for 30 minutes by nitrogen bubbling, the mixture was reacted at 65°C for 17 hours. After the reaction was completed, the reaction solvent was diluted with acetone and poured into 500 mL of hexane, and the precipitated solid was collected and dried under reduced pressure. The mixture was also dissolved again in acetone and poured into 500 mL of pure water, and the precipitated solid was collected and dried under reduced pressure to obtain a block copolymer of N-isopropylacrylamide and n-butyl methacrylate. The synthesized block copolymer was dissolved in ethanol at 0.5 wt%, and spin-coated at 2000 rpm on the surface of the cell culture substrate (the side on which the layer containing the hydrophilic polymer was formed).

[0156] The ratio of the constituent units of the block copolymer was 4 wt% n-butyl methacrylate and 96 wt% N-isopropylacrylamide, and the number average molecular weight (Mn) of the block copolymer was 77,000. The thickness of the block copolymer (temperature-responsive polymer) layer was determined from a calibration curve when the block copolymer (temperature-responsive polymer) was directly coated on a polystyrene dish. As a result, the thickness of the block copolymer (temperature-responsive polymer) layer was 25 nm.

[0157] The composition of the cell culture substrate and the evaluation results are shown in Table 2. The cell culture substrate was prepared with a hydrophilic polymer layer thickness of 600 nm and an area of ​​region (A) of 0.03 mm. 2 The height of the unevenness at the boundary between region (A) and region (B) was 4 nm.

[0158] Human iPS cells were cultured on this cell culture substrate, and the formation of cell aggregates was confirmed two days after the start of culture. Visual inspection revealed that the size of the formed cell aggregates was uniform. The height / diameter ratio of the cell aggregates was estimated to be 0.5. The cell aggregates were detached by cooling the cell culture substrate. The survival rate of the cells contained in the cell aggregates was 88%, indicating a high cell survival rate. Furthermore, no air bubbles were attached to the cell culture substrate when the medium was added.

[0159] [Example 11] The surface of the cell culture substrate prepared in the same manner as in Example 1 (the side on which the layer containing a hydrophilic polymer was formed) was coated with a random copolymer consisting of a water-insoluble monomer unit and a temperature-responsive monomer unit to prepare a cell culture substrate. Specifically, 0.40 g (0.1 mmol) of 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, 7.11 g (50 mmol) of n-butyl methacrylate, 33 mg (0.2 mmol) of azobis(isobutyronitrile), 5.65 g (50 mmol) of N-isopropylacrylamide, and 5 mg (0.03 mmol) of azobisisobutyronitrile were added to a test tube and dissolved in 15 mL of 1,4-dioxane. After degassing for 30 minutes by nitrogen bubbling, the mixture was reacted at 70°C for 24 hours. After the reaction was completed, the reaction solvent was diluted with acetone and poured into 500 mL of hexane, and the precipitated solid was collected and dried under reduced pressure. The mixture was dissolved again in acetone and poured into 500 mL of pure water, and the precipitated solid was collected and dried under reduced pressure to obtain a random copolymer of N-isopropylacrylamide and n-butyl methacrylate. The obtained random copolymer was dissolved in ethanol at 0.5 wt% and spin-coated at 2000 rpm onto the surface of the cell culture substrate (the side on which the layer containing a hydrophilic polymer was formed).

[0160] The random copolymer had a ratio of constituent units of n-butyl methacrylate 54 wt% and N-isopropylacrylamide 46 wt%, and the number average molecular weight (Mn) of the random copolymer was 158,000. The thickness of the random copolymer layer was determined from a calibration curve obtained when the random copolymer was directly coated on a polystyrene dish. As a result, the thickness of the random copolymer layer was 25 nm. Although the random copolymer layer does not fall under the category of layers made of temperature-responsive polymers, it is listed in the column for layers made of temperature-responsive polymers in Table 2 for convenience.

[0161] The composition of the cell culture substrate and the evaluation results are shown in Table 2. The cell culture substrate was prepared with a hydrophilic polymer layer thickness of 600 nm and an area of ​​region (A) of 0.03 mm. 2 The height of the unevenness at the boundary between region (A) and region (B) was 4 nm.

[0162] Human iPS cells were cultured on this cell culture substrate, and the formation of cell aggregates was confirmed two days after the start of culture. Visual inspection revealed that the size of the formed cell aggregates was uniform. The height / diameter ratio of the cell aggregates was estimated to be 0.5. The cell aggregates did not detach when the cell culture substrate was cooled. Furthermore, the survival rate of the cells contained in the cell aggregates was 85%, indicating a high cell survival rate. Furthermore, no air bubbles were attached to the cell culture substrate when the medium was added.

[0163] [Example 12] A polyethylene terephthalate (PET) film (Cosmoshine, Toyobo Co., Ltd.) with a thickness of 180 μm was subjected to plasma treatment (20 Pa gas pressure, conductive current of 20 mA, irradiation time of 30 seconds). A water / ethanol solution containing 0.1 wt% solids of polyvinyl alcohol (BIOSURFINE®-AWP, Toyo Gosei Co., Ltd.) having an azide group as a hydrophilic polymer was spin-coated at 2000 rpm onto the plasma-treated surface of the PET film. A quartz mask having multiple circular chrome-deposited areas with a diameter of 0.2 mm was placed on the hydrophilic polymer layer, and UV irradiation was performed for 10 seconds from above the quartz mask using a high-pressure mercury lamp. The film was washed with a mixed solvent of water / ethanol = 20 / 80, and the hydrophilic polymer that was not irradiated with UV was dissolved and removed, forming a region (region (A)) with cell adhesiveness and cell proliferation properties. In addition, region (B) was formed in the UV-irradiated part. This PET film was attached to a well plate (a plate having a through hole) with a bottom that is open.

[0164] The composition of the cell culture substrate and the evaluation results are shown in Table 2. The cell culture substrate was prepared with a hydrophilic polymer layer thickness of 50 nm and an area of ​​region (A) of 0.03 mm. 2 The height of the unevenness at the boundary between region (A) and region (B) was 50 nm.

[0165] Human iPS cells were cultured on this cell culture substrate, and the formation of cell aggregates was confirmed two days after the start of culture. Visual inspection revealed that the size of the formed cell aggregates was uniform. The height / diameter ratio of the cell aggregates was estimated to be 0.5. The survival rate of the cells contained in the cell aggregates was 90%, indicating a high cell survival rate. Furthermore, no air bubbles were attached to the cell culture substrate when the medium was added.

[0166] [Reference example 1] As the solvent used in the washing step of Example 12, pure water, a mixed solvent of water / ethanol = 80 / 20, a mixed solvent of water / ethanol = 50 / 50, and ethanol were used instead of the mixed solvent of water / ethanol = 20 / 80. The rest of the procedure was the same as in Example 12 to prepare cell culture substrates.

[0167] The results of evaluating the culture of human iPS cells on these cell culture substrates and comparing them are shown in Table 3. It was found that the use of a water / ethanol mixed solvent in the washing process made it possible to increase the cell adhesiveness and cell proliferation in area (A).

[0168] [Reference example 2] As the substrates for Example 12, a 1.2 mm thick PS plate, a 0.1 mm thick polycarbonate (PC) film (Iupilon, manufactured by Mitsubishi Gas Co., Ltd.), a polyethylene (PE) film, a polypropylene (PP) film, and a Permanox slide chamber (manufactured by ThermoScientific) were used instead of a PET film, and the rest of the procedures were the same as in Example 12 to prepare cell culture substrates.

[0169] Human iPS cells were cultured on these cell culture substrates and evaluated, and the cultured cells were observed under phase contrast at a magnification of 40x. In addition, the cells were fluorescently stained and observed, and the results of the comparison are shown in Table 4. It was found that the substrate has excellent cell proliferation properties in region (A) when it contains a polymer containing an alicyclic hydrocarbon group or an aromatic hydrocarbon group as a constituent unit. It was also found that a substrate with a refractive index of 1.4 to 1.6 and a thickness of 0.01 to 0.5 mm allows clear phase contrast images of cells to be obtained. Furthermore, it was found that the low autofluorescence of the substrate allows clear fluorescent images of cells to be obtained.

[0170] [Comparative Example 1] A cell culture substrate was prepared in the same manner as in Example 4, except that the solid content concentration of the hydrophilic polymer was changed to 0.003 wt %.

[0171] The composition of the cell culture substrate and the evaluation results are shown in Table 2. The cell culture substrate had a hydrophilic polymer layer thickness of 3 nm and an area of ​​the plasma-treated region of 0.03 mm. 2 The height of the unevenness at the boundary between the plasma-treated region and the non-plasma-treated region was 25 nm.

[0172] When human iPS cells were cultured on this cell culture substrate, the cells attached and proliferated over the entire surface of the substrate, and no cell aggregates were formed.

[0173] [Comparative Example 2] A cell culture substrate was prepared in the same manner as in Example 4, except that the solid content concentration of the hydrophilic polymer was changed to 3 wt %.

[0174] The composition of the cell culture substrate and the evaluation results are shown in Table 2. The cell culture substrate has a hydrophilic polymer layer thickness of 3000 nm and an area of ​​the plasma-treated region of 0.03 mm. 2 The height of the irregularities at the boundary between the plasma-treated region and the non-plasma-treated region was 38 nm.

[0175] Human iPS cells were cultured on this cell culture substrate, but the cells did not adhere or proliferate, and no cell aggregates were formed.

[0176] [Comparative Example 3] The inner surface of a polystyrene (PS) dish with a diameter of 3.5 cm was plasma-treated (20 Pa gas pressure, 20 mA conduction current, 30 seconds exposure time) using a plasma irradiation device (Plasma Ion Bombarder PIB-20, manufactured by Vacuum Device Co., Ltd.). 0.8 mL of an ethanol solution containing 0.08 wt% solids of polyvinyl alcohol (BIOSURFINE®-AWP, manufactured by Toyo Gosei Co., Ltd.) with azide groups as a hydrophilic polymer was dropped onto the plasma-treated PS dish and dried under reduced pressure at room temperature to form a hydrophilic polymer film. A chrome mask with multiple circular black patterns with a diameter of 0.2 mm was placed on the hydrophilic polymer film, and part of the hydrophilic polymer was cured by UV irradiation. Next, the uncured hydrophilic polymer was removed by washing multiple times with pure water. The composition of the cell culture substrate prepared and the evaluation results are shown in Table 2.

[0177] Human iPS cell culture was evaluated on this cell culture substrate, and the formation of cell aggregates was confirmed one day after the start of culture. However, the survival rate of the cells in the cell aggregates was low, at 65%.

[0178] [Comparative Example 4] The inner surface of a 3.5 cm diameter polystyrene (PS) dish was plasma treated (20 Pa gas pressure, 20 mA conduction current, 30 seconds exposure time) using a plasma irradiation device (Plasma Ion Bombarder PIB-20, Vacuum Device Co., Ltd.). Multiple circular holes with a diameter of 0.2 mm were formed in a surface protection film (E-MASK, Nitto Denko Corporation) by laser processing, and this surface protection film was attached to the inner surface of the plasma-treated PS dish.

[0179] The composition of the prepared cell culture substrate and the evaluation results are shown in Table 2. The prepared cell culture substrate did not have a hydrophilic polymer layer, and the area of ​​the plasma-treated region (the region not covered with the surface protection film) was 0.03 mm 2 The height of the irregularities at the boundary between the plasma-treated region (region not covered with the surface protective film) and the region covered with the surface protective film was about 58 μm.

[0180] The culture of human iPS cells was evaluated on this cell culture substrate, and the formation of cell aggregates was confirmed one day after the start of culture, but the survival rate of the cells in the cell aggregates was low at 53%.In addition, air bubbles were found to adhere to the cell culture substrate when the medium was added.

[0181] [Comparative Example 5] A cell culture substrate was prepared in the same manner as in Example 1, except that the plasma treatment was not performed. The configuration of the prepared cell culture substrate and the evaluation results are shown in Table 2. Human iPS cell culture was evaluated on this cell culture substrate, but the cells did not adhere or proliferate, and therefore no cell aggregates were formed.

[0182] [Comparative Example 6] A cell culture substrate was prepared in the same manner as in Example 1, except that a hydrophilic polymer was not used (a hydrophilic polymer layer was not formed). The configuration of the prepared cell culture substrate and the evaluation results are shown in Table 2. Human iPS cell culture was evaluated on this cell culture substrate, and the cells adhered and proliferated over the entire surface of the cell culture substrate, with no cell aggregates being formed.

[0183] [Table 2] [Table 3] [Table 4] [Explanation of symbols]

[0184] A...region (A), B...region (B), H...height of the unevenness at the boundary between region (A) and region (B), 1...substrate, 2...layer containing hydrophilic polymer, 10, 11...cell culture substrate, 20...partition plate.

Claims

1. The present invention comprises a substrate and a layer containing a hydrophilic polymer and having a thickness of 10 to 1500 nm, the layer covering at least a part of the surface of the substrate, The hydrophilic polymer contains a phosphorylcholine group or a hydroxyl group, The present invention has the following region (A) and the following region (B): The cell culture substrate, wherein the height of the projections and recesses at the boundary between the region (A) and the region (B) is 1 to 500 nm. (A) Area having cell adhesion and cell proliferation properties of 0.001 to 5 mm ２ Island-like region of (B) a region adjacent to the region (A) that does not have cell adhesiveness or cell proliferation properties

2. The cell culture substrate according to claim 1 , wherein the hydrophilic polymer comprises a compound represented by the following general formula (1), a compound represented by the following general formula (2), or a compound represented by the following general formula (3): 【Chemistry 1】 [In general formula (1), R １ and R ２ each independently represents a hydrogen atom or a methyl group; R ３ represents a hydrogen atom or an arbitrary organic group, and m and n each independently represent a positive integer. 【Chemistry 2】 [In general formula (2), R ４ , R ５ and R ６ each independently represents a hydrogen atom or a methyl group; R ７ represents a hydrogen atom or an arbitrary organic group, and x, y, and z each independently represent a positive integer. 【Chemistry 3】 [In general formula (3), R ８ and R ９ each independently represents a hydrogen atom or a methyl group; R １０ represents a hydrogen atom or an arbitrary organic group, and a and b each independently represent a positive integer.

3. The cell culture substrate according to claim 1 or 2, wherein the hydrophilic polymer comprises a monomer unit having a UV-reactive functional group or a residue after UV reaction.

4. 3. The cell culture substrate according to claim 1, wherein a ratio of a peak intensity at 287 eV to a peak intensity at 285 eV in a C1s spectrum of an XPS measurement for the region (A) is 0.05 or more greater than a ratio of a peak intensity at 287 eV to a peak intensity at 285 eV in a C1s spectrum of an XPS measurement for the region (B).

5. a layer containing a temperature-responsive polymer and having a thickness of 1 to 100 nm is further provided on a surface of the layer containing a hydrophilic polymer, The cell culture substrate according to claim 1 or 2, wherein the temperature-responsive polymer is a block copolymer comprising a water-insoluble block segment and a temperature-responsive block segment.

6. The cell culture substrate according to claim 5 , wherein a ratio of a weight of the temperature-responsive block segment to a total weight of the water-insoluble block segment and the temperature-responsive block segment is more than 90% by weight.

7. 3. The cell culture substrate according to claim 1, wherein the refractive index of the substrate is 1.4 to 1.6, and the thickness of the substrate is 0.01 to 0.5 mm.

8. 3. The cell culture substrate according to claim 1, wherein the fluorescence intensity of the substrate excited at excitation wavelengths of 350 nm, 488 nm, and 647 nm is smaller than the fluorescence intensity of a polystyrene plate having a thickness of 1.2 mm excited at the same excitation wavelengths.

9. The area of ​​the (A) region is 0.005 to 0.2 mm ２ and The number of the (A) regions is 200 to 1000 / cm based on the total area of ​​the (A) region and the (B) region. ２ The cell culture substrate according to claim 1 or 2,

10. The area of ​​the (A) region is 0.2 to 2 mm ２ and The number of the (A) regions is 3 to 15 / cm based on the total area of ​​the (A) region and the (B) region. ２ The cell culture substrate according to claim 1 or 2,

11. 3. The cell culture substrate according to claim 1, wherein the minimum distance between the regions (A) is 500 to 10,000 μm.

12. The cell culture substrate according to claim 1 or 2, which is used for inducing differentiation of pluripotent stem cells into three germ layer cells.

13. The cell culture substrate according to claim 1 or 2, wherein the substrate is formed from polycarbonate or cycloolefin polymer.

14. (1) A step of coating at least a part of a surface of a substrate with a composition containing a UV-reactive hydrophilic polymer to form a layer containing the hydrophilic polymer; (2) irradiating the layer containing the hydrophilic polymer with UV light to fix the layer containing the hydrophilic polymer on the surface of the substrate; and (3) A step of subjecting a part of the surface of the layer containing the immobilized hydrophilic polymer to a plasma treatment to form the region (A) in the plasma-treated part. The method for producing a cell culture substrate according to claim 1 , comprising:

15. (1') using a substrate formed from a polymer containing an alicyclic hydrocarbon group or an aromatic hydrocarbon group in a repeating unit, subjecting a surface of the substrate to a plasma treatment, and forming the (A) region in the plasma-treated portion; (2') a step of coating at least a part of the surface of the substrate with a composition containing a UV-reactive hydrophilic polymer to form a layer containing the hydrophilic polymer; (3') irradiating a part of the layer containing the hydrophilic polymer with UV light to fix the part of the layer containing the hydrophilic polymer on the surface of the base material; and (4') A step of washing the hydrophilic polymer with a solvent to dissolve and remove the hydrophilic polymer that is not immobilized on the surface from the surface of the substrate. The method for producing a cell culture substrate according to claim 1 , comprising:

16. The method for producing a cell culture substrate according to claim 15, wherein the solvent used in the step (4') contains water and an alcohol.

17. (4) After the step (3) or (4'), the cross-sectional area in the in-plane direction is 0.05 to 100 cm ２ The method according to claim 14 or 15, further comprising a step of bonding a plate having a through hole to the substrate on the side of the substrate coated with the layer containing a hydrophilic polymer.

18. (5) The manufacturing method according to claim 14 or 15, further comprising a step of coating the surface of the layer containing the hydrophilic polymer that has been subjected to the plasma treatment with a composition containing a temperature-responsive polymer, after the step (3) or (4'), to form a layer containing the temperature-responsive polymer.

19. (i) seeding pluripotent stem cells onto the cell culture substrate according to claim 1 or 2; (ii) culturing the pluripotent stem cells to form hemispherical cell aggregates having a height / diameter ratio of 0.2 to 0.8; and (iii) inducing differentiation of the cell aggregate to form a cell aggregate of three germ layers. A method for inducing differentiation of pluripotent stem cells, comprising:

20. The method for inducing differentiation of pluripotent stem cells according to claim 19, further comprising the step (iv) below. (iv) culturing the cell aggregate in a medium containing at least one differentiation-inducing factor selected from the group consisting of Wnt protein, bone morphogenetic protein, insulin-like growth factor, and activin, to express a marker possessed by intestinal epithelial cells;

21. A cell culture kit comprising the cell culture substrate according to claim 1 or 2, and a block copolymer containing a water-insoluble block segment and a temperature-responsive block segment, or a coating agent containing the block copolymer.

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