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

Abstract

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 to remove air bubbles during culture; and to provide a method for producing the same; as well as 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.SOLUTION: Provided is a cell culture substrate comprising: a substrate; and a layer containing a hydrophilic polymer with a thickness of 5 to 2000 nm covering at least a portion of a surface of the substrate. The hydrophilic polymer contains a phosphorylcholine group or a hydroxyl group. The cell culture substrate has the following regions (A) and (B), and an unevenness height at a boundary between each of the regions (A) and each of the regions (B) is 1 to 500 nm. Therein: the region (A) is an island-like region having cell adhesiveness and cell proliferation properties; and the region (B) is a region adjacent to region (A) and has neither cell adhesiveness nor cell proliferation properties.SELECTED DRAWING: Figure 1

Description

Technical Field

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

Background Art

[0002] Pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) are cells that have the ability to differentiate into various tissues of the body (pluripotency), and have received great attention as cell sources for regenerative medicine and drug discovery screening. In order to apply pluripotent stem cells to regenerative medicine and drug discovery screening, it is necessary to differentiate pluripotent stem cells into target cells, and in that case, it is necessary to form cell aggregates of pluripotent stem cells. In addition, although pluripotent stem cells can differentiate into various cells, it is known that the optimal size of cell aggregates differs depending on the type of cells after differentiation, and it is desirable to control the size and create cell aggregates with a uniform size.

[0003] Conventionally, as a method for forming cell aggregates, a method of spontaneously forming aggregates in pluripotent stem cells by using a substrate to which pluripotent stem cells do not adhere is known (see, for example, Patent Document 1). Although this method is excellent in the mass productivity of cell aggregates, there is a problem that cell aggregates of a uniform size cannot be obtained.

[0004] As a method for forming cell aggregates of a uniform size, a method of using a cell culture substrate having fine irregularities on the surface is known (see, for example, Patent Document 2).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] The cell culture substrate provided with fine irregularities as disclosed in Patent Document 2 has poor mass productivity and is not suitable for applications that form a large number of cell aggregates. Further, 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. Further, the inventors have found that when a culture medium is brought into contact with a cell culture substrate having fine irregularities, air bubbles are easily trapped in the fine irregularities on the surface, and there is a problem that an operation for removing air bubbles is required before starting the culture. In order to remove air bubbles, it is usually necessary to repeatedly suck and discharge the culture medium using a pipetter, which has a problem of poor workability.

[0007] An object of the present invention is to provide a cell culture substrate and a method for producing the same that can form cell aggregates, can increase the survival rate of cells in the cell aggregates, and eliminate the need for an air bubble removal operation during culture. Another object of the present invention is to provide a method for inducing differentiation of pluripotent stem cells using the cell culture substrate, which is excellent in the efficiency of inducing differentiation into three germ layer cells.

Means for Solving the Problems

[0008] The present invention includes a substrate and a layer containing a hydrophilic polymer with a layer thickness of 5 to 2000 nm covering at least a part of the surface of the substrate, wherein the hydrophilic polymer contains a phosphorylcholine group or a hydroxyl group and has the following (A) region and the following (B) region, and the uneven height at the boundary between the (A) region and the (B) region is 1 to 500 nm. The present invention relates to a cell culture substrate. (A) An island-shaped region having an area of 0.001 to 5 mm 2 with cell adhesiveness and cell proliferation ability (B) A region adjacent to the (A) region and having no cell adhesiveness or cell proliferation ability

[0009] The present invention also relates to a method for producing the above cell culture substrate, comprising: (1) 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; (2) a step of performing UV irradiation on the layer containing the hydrophilic polymer to immobilize the layer containing the hydrophilic polymer on the surface of the substrate; and (3) a step of performing plasma treatment on a part of the surface of the layer containing the immobilized hydrophilic polymer to form a region (A) in the plasma-treated part.

[0010] The present invention also relates to a method for producing the above cell culture substrate, comprising: (1') a step of using a substrate formed of a polymer containing an aromatic hydrocarbon group in a repeating unit, performing plasma treatment on the surface of the substrate, and forming the region (A) in the plasma-treated part; (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') a step of performing UV irradiation on a part of the layer containing the hydrophilic polymer to immobilize a part of the layer containing the hydrophilic polymer on the surface of the substrate; and (4') a step of washing the hydrophilic polymer with a solvent to dissolve and remove the hydrophilic polymer not immobilized on the surface from the surface of the substrate.

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

[0012] In addition, the present invention relates to a cell culture kit comprising the above 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 block copolymer.

Effects of the Invention

[0013] According to the present invention, it is possible to provide a cell culture substrate that can form cell aggregates and can increase the survival rate of cells in the cell aggregates, and can eliminate the need for bubble removal operations during culture, and a method for manufacturing 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

Figure 2

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Figure 4

Figure 5

Embodiments for Carrying Out the Invention

[0015] Hereinafter, embodiments for carrying out 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 range where the above-described effects can be obtained.

[0016] In this specification, the "cell aggregate" means a three-dimensional aggregate of cells formed by the aggregation of a plurality of cells. The shape of the three-dimensional aggregate may be an ellipsoidal shape such as a spherical shape, or may be a shape such as a hemispherical shape. These shapes may also be a shape with gaps formed by folding sheet-like cells, or may be a hollow shape.

[0017] As used herein, "temperature responsiveness" means that the degree of hydrophilicity / hydrophobicity changes with a change in temperature. Further, the boundary temperature at which the degree of hydrophilicity / hydrophobicity changes is referred to as the "response temperature".

[0018] As used herein, "biologically-derived substance" means a substance present in a living organism and a substance equivalent thereto, and a chemically synthesized substance. The substance present in a living organism may be a natural product or may be artificially synthesized by genetic recombination 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 constituting a living body, and nucleotides, nucleosides, amino acids, various sugars, which are components thereof, as well as lipids, vitamins, and hormones.

[0019] As used herein, "cell adhesiveness" indicates the ease of adhesion to a substrate or a cell culture substrate at the culture temperature, and "having cell adhesiveness" means that cells can adhere directly or via a biologically-derived substance to the substrate or the cell culture substrate at the culture temperature. Further, "not having cell adhesiveness" means that cells cannot adhere to the substrate or the cell culture substrate at the culture temperature.

[0020] As used herein, "cell proliferativity" indicates the ease of cell proliferation at the culture temperature, and "having cell proliferativity" means that cells can proliferate at the culture temperature. Further, "not having cell proliferativity" means that cells cannot proliferate at the culture temperature. "High cell proliferativity" means that more cells proliferate when compared during the same culture period.

[0021] As used herein, "trilaminar germ cells" means at least one selected from the group consisting of endodermal cells, ectodermal cells, and mesodermal cells.

[0022] The cell culture substrate according to this embodiment includes a substrate and a layer containing a hydrophilic polymer with a layer thickness of 5 to 2000 nm that covers at least a part of the surface of the substrate. Here, the hydrophilic polymer contains a phosphorylcholine group or a hydroxyl group. Further, the cell culture substrate according to this embodiment has the following region (A) and the following region (B), and the uneven height at the boundary between the region (A) and the region (B) is 1 to 500 nm. (A) An island-shaped region with an area of 0.001 to 5 mm having cell adhesiveness and cell proliferation ability 2 (B) A region adjacent to the region (A) and having no cell proliferation ability

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

[0024] The base material used for the cell culture substrate according to this embodiment is not particularly limited, but it 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, it is formed from at least one selected from the group consisting of polystyrene, polyethylene terephthalate, polycarbonate, and cycloolefin polymer. Even more preferably, it is formed from at least one selected from polystyrene, polyethylene terephthalate, and polycarbonate. Most preferably, it is formed from polystyrene or polycarbonate. Commercially available products of cycloolefin polymers include ZEONEX (manufactured by Nippon Zeon Co., Ltd.), ZEONOR (manufactured by Nippon Zeon Co., Ltd.), and ARTON (manufactured by JSR Corporation).

[0025] Since it is suitable for observing cells cultured on the cell culture substrate with a high-magnification phase-contrast microscope, the refractive index of the base material measured at the D line (wavelength 589 nm) is preferably 1.4 to 1.6, more preferably 1.45 to 1.6, and particularly preferably 1.5 to 1.55. By having the refractive index of the base material within these ranges, spherical aberration in the phase-contrast microscope observation of cells can be reduced, and a clear phase-contrast image can be obtained. Also, in order to reduce spherical aberration, the thickness of the base material 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 it is suitable for suppressing the loss of focus over the entire observation range due to the bending of the base material during microscope observation, the thickness of the base material 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, more preferably 1.45 to 1.6 and 0.05 mm or more and 0.4 mm or less, still more preferably 1.45 to 1.55 and 0.1 mm or more and 0.3 mm or less, 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 it is suitable for observing cells cultured on a cell culture substrate with a high-magnification fluorescence microscope, the fluorescence intensity (autofluorescence intensity) of the substrate at excitation wavelengths of 350 nm, 488 nm, and 647 nm (when irradiated with excitation light having these wavelengths respectively) is preferably smaller than the fluorescence intensity (autofluorescence intensity) of a polystyrene plate with a thickness of 1.2 mm excited by light having the same excitation wavelength, more preferably 80% or less of the fluorescence intensity of the polystyrene plate with a thickness of 1.2 mm, particularly preferably 50% or less of the fluorescence intensity of the polystyrene plate with a thickness of 1.2 mm, and most preferably 10% or less of the fluorescence intensity of the polystyrene plate with a thickness of 1.2 mm. Fluorescent dyes excited at excitation wavelengths of 350 nm, 488 nm, and 647 nm are frequently used in fluorescence observation of cells. By keeping the autofluorescence intensity of the substrate at these wavelengths below a certain value, a clear fluorescence 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, porous particles, a porous membrane, or a hollow fiber. Also, the shape of the substrate may generally be the shape of a container (such as a cell culture dish like a Petri dish, a flask, a plate, a bag, etc.) used for cell culture and the like. From the ease of culturing operation, the shape of the substrate is preferably a planar shape such as a plate or a film, or the shape of a porous membrane of a flat film.

[0029] The layer thickness of the layer containing the hydrophilic polymer is 5 to 2000 nm. Since the layer thickness of the layer formed by the hydrophilic polymer is 5 to 2000 nm, it is possible to adhere and grow cells only in the (A) region. Also, due to cell migration, it is easy to collect cells in the (A) region, and the cell viability of cell aggregates can be increased. When the layer thickness is less than 5 nm, cells also grow in the (B) region, so cell aggregates cannot be formed. When the layer thickness exceeds 2000 nm, cells cannot migrate, so the viability of the cells contained in the cell aggregates decreases. Here, the "layer thickness" of the layer formed by the hydrophilic polymer refers to the out-of-plane length from the interface between the substrate and the layer formed by the hydrophilic polymer to the interface on the side opposite to the substrate of the layer formed by the hydrophilic polymer (excluding the (A) region). In the range where the layer thickness exceeds 10 nm, the cross-sectional image can be measured by a transmission electron microscope using an ultra-thin section of the cell culture substrate prepared by a microtome. The distances at 10 randomly selected points are measured and averaged to calculate the value. Also, in the range where the layer thickness is 10 nm or less, it can be measured using an ellipsometer. Since it is suitable for suppressing cell adhesion in the (B) region, it is more preferably 10 nm or more, even more preferably 50 nm or more, and most preferably 100 nm or more. Also, since it is suitable for increasing the cell viability of cell aggregates by collecting cells in the (A) region due to cell migration, it 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 the hydrophilic polymer containing a phosphorylcholine group or a hydroxyl group, it is possible to make the region coated with the hydrophilic polymer a region where cells do not adhere. In addition, since such a hydrophilic polymer does not need to be completely decomposed and removed, by performing only a weak plasma treatment for a short time, the region can be made a region having cell adhesiveness and cell growth property, and it is difficult for the decomposition products of the hydrophilic polymer to mix into the cells. Except for containing a phosphorylcholine group or a hydroxyl group, the type of the hydrophilic polymer is not particularly limited, and examples of commercially available products include Lipidure(R) CM5206 (manufactured by NOF Corporation), Lipidure(R) CM2001 (manufactured by NOF Corporation), BIOSURFINE(R)-AWP (manufactured by Toyo Gosei Co., Ltd.), and the like. Further, as commercially available substrates coated with a hydrophilic polymer, PrimeSurface(R) (manufactured by Sumitomo Bakelite Co., Ltd.), EZ-BindShut(R) (manufactured by AGC Techno Glass Co., Ltd.), EZ-BindShutII(R) (manufactured by AGC Techno Glass Co., Ltd.), and the like can be preferably used.

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

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

[0034] By the hydrophilic polymer containing the compound represented by the above general formula (1), the compound represented by the above general formula (2), or the compound represented by the above general formula (3), it becomes easy to adhere and grow cells, and it is suitable for forming cell aggregate lumps with a uniform shape in the (A) region. In the above general formula (1), the above general formula (2), or the above general formula (3), R 3 R 7 , and R 10 are each preferably a hydrophobic group or a UV-reactive functional group because they are suitable for immobilizing the hydrophilic polymer on the 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, a cyclohexyl group, etc. can be preferably used. Further, examples of the UV-reactive functional group include an azide group, an acrylate group, a methacrylate group, an epoxy group, etc., and an azide group can be preferably used.

[0035] The (A) region is an island-shaped region with an area of 0.001 to 5 mm 2 having cell adhesiveness and cell growth property. By being an island-shaped region with an area of 0.001 to 5 mm 2 , it is possible to form cell aggregate lumps with a uniform particle diameter when culturing cells. Further, since it is suitable for forming cell aggregate lumps suitable for applications such as induction of differentiation of pluripotent stem cells, an area of 0.005 to 1 mm 2 is preferable, an area of 0.01 to 0.5 mm 2 is more preferable, an area of 0.015 to 0.25 mm 2 is still more preferable, and an area of 0.02 to 0.2 mm 2is most preferred.

[0036] (A) region being an island-shaped region indicates that the (A) region exists in a state independent of regions other than the (A) region. Since the (A) region is an island shape having cell adhesiveness and cell growth property, living cells concentrate and exist in the (A) region, so that cell aggregates can be produced. When the (A) region is not island-shaped, for example, in the case of a stripe structure or the like, cell aggregates 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. For example, a circle, an ellipse, a polygon, or a closed shape formed by a straight line and a curve can be mentioned. Further, since it is suitable for producing cell aggregates having a shape close to a sphere, as the shape of the island, a circle, an ellipse or a polygon is preferable, a circle, an ellipse or a rectangle is more preferable, a circle, an ellipse or a square is still more preferable, and a circle or an ellipse is most preferred.

[0037] Since it is suitable for producing cell aggregates having a uniform size and shape, it is preferable that the standard deviation / average area of the area of the (A) region is 80% or less, more preferably 50% or less, still 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, still more preferably 1.5 or less, and most preferably 1.1 or less. Here, the "aspect ratio" indicates the major axis / minor axis which is the ratio of the maximum diameter (major axis) to the minimum diameter (minor axis) of the shape.

[0039] Also, since it is suitable for the formation of cell aggregates suitable for culturing pluripotent stem cells and inducing differentiation into endodermal cells or ectodermal cells, the area of the (A) region is 0.005 to 0.2 mm 2 and the number of (A) regions is preferably 200 to 1000 per cm based on the total area of the (A) region and the (B) region. 2 The area of the (A) region is preferably 0.01 to 0.15 mm 2and the number of (A) regions is 250 to 800 per cm² based on the total area of the (A) and (B) regions 2 more preferably, the area of the (A) region is 0.02 to 0.1 mm² 2 and the number of (A) regions is 300 to 600 per cm² based on the total area of the (A) and (B) regions 2 even more preferably, the area of the (A) region is 0.03 to 0.05 mm² 2 and the number of (A) regions is 300 to 400 per cm² based on the total area of the (A) and (B) regions 2 most preferably.

[0040] Since it is suitable for the formation of cell aggregates suitable for culturing pluripotent stem cells and inducing differentiation into mesodermal cells, the area of the (A) region is 0.2 to 2 mm² 2 and the number of (A) regions is 3 to 15 per cm² based on the total area of the (A) and (B) regions 2 preferably, the area of the (A) region is 0.3 to 1.5 mm² 2 and the number of (A) regions is 4 to 12 per cm² based on the total area of the (A) and (B) regions 2 more preferably, the area of the (A) region is 0.4 to 1 mm² 2 and the number of (A) regions is 5 to 10 per cm² based on the total area of the (A) and (B) regions 2 even more preferably, the area of the (A) region is 0.5 to 0.7 mm² 2 and the number of (A) regions is 6 to 8 per cm² based on the total area of the (A) and (B) regions 2 most preferably.

[0041] Also, since it is suitable for increasing the oxygen concentration around the cells and increasing the survival rate of cell aggregates, the minimum distance between (A) regions is preferably 500 to 10000 μm, more preferably 1000 to 8000 μm, even more preferably 2000 to 5000 μm, and most preferably 3000 to 4000 μm.

[0042] The (A) region 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 the hydrophilic polymer by corona treatment, UV treatment, or plasma treatment. It is preferable to perform the modification by plasma treatment. Since the (A) region is a region where the surface of the layer containing the hydrophilic polymer is modified by plasma treatment or the like, the (A) region becomes a region having cell adhesiveness and cell growth properties. Since the (A) region can be patterned by short-time plasma treatment or the like, the mass productivity of the cell culture substrate can be increased.

[0043] In addition, when measuring XPS (X-ray photoelectron spectroscopy) for the (A) region, the ratio of the peak intensity at 287 eV to the peak intensity at 285 eV in the C1s spectrum is preferably 0.05 or more greater than the ratio of the peak intensity at 287 eV to the peak intensity at 285 eV in the C1s spectrum of the XPS measurement for the (B) region. In this case, since the difference in cell growth properties between the (A) region and the (B) region can be increased, it is possible to grow many cells in the (A) region, and it becomes easier to form uniform cell aggregates in the (A) region. Since it is more suitable for forming uniform cell aggregates, the ratio of the peak intensity at 287 eV to the peak intensity at 285 eV in the C1s spectrum of the XPS measurement for the (A) region is more preferably 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 the XPS measurement for the (B) region, still more preferably 0.1 or more greater, and most preferably 0.15 or more greater.

[0044] As a method for adjusting the ratio of the peak intensity at 287 eV to the peak intensity at 285 eV in the C1s spectrum of XPS measurement to the above-mentioned range, there is no particular limitation. However, it is preferable to perform plasma treatment, corona treatment, UV treatment, etc. only on a part of the layer containing the hydrophilic polymer formed on the substrate surface (the part that is supposed to become the (A) region). A method of performing plasma treatment or corona treatment is more preferable, and a method of performing plasma treatment is particularly preferable. As a method of performing plasma treatment, etc. only on the part that is supposed to become the (A) region, there is a method of covering the layer containing the hydrophilic polymer formed on the substrate surface with a mask made by laser processing and performing plasma treatment, etc. from above the mask. It is possible to appropriately adjust conditions such as plasma intensity and plasma irradiation time so that the difference between the ratio of the peak intensity at 287 eV to the peak intensity at 285 eV in the C1s spectrum of XPS measurement for the (A) region and the ratio of the peak intensity at 287 eV to the peak intensity at 285 eV in the C1s spectrum of XPS measurement for the (B) region falls within the above-mentioned range. In the case of plasma treatment, by using a gas containing oxygen and nitrogen as the introduced gas, it becomes easier to fall within the above-mentioned range.

[0045] (A) region may have temperature responsiveness because it is suitable for detaching the cultured cell aggregates. When the (A) region has temperature responsiveness, since cells can be cultured at a temperature close to body temperature when culturing cells on the cell culture substrate, the response temperature is preferably 50 °C or lower, more preferably 35 °C or lower. Also, since it is suitable for suppressing the detachment of cells during operations such as medium replacement during culture, the response temperature is particularly preferably 25 °C or lower. 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 higher, more preferably 10 °C or higher, and even more preferably 15 °C or higher.

[0046] When the (A) region has temperature responsiveness, for example, a layer containing a temperature-responsive polymer with a layer thickness of 1 to 100 nm may be further provided on the surface of the layer containing the 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, temperature responsiveness can be imparted 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 the hydrophilic polymer. Since it is 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 the temperature-responsive polymer is more preferably 3 to 50 nm, even more preferably 5 to 40 nm, and most preferably 10 to 35 nm. The suitable layer thickness of the temperature-responsive polymer that does not impair cell proliferation and allows cells to be detached and recovered by temperature responsiveness after culturing varies depending on the cells to be cultured and can be appropriately adjusted within the range of the 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. By the temperature-responsive polymer being such a block copolymer, the mass productivity of the cell culture substrate can be enhanced, and the incorporation of the temperature-responsive polymer into the produced cell aggregates can be suppressed. Since the constitutional unit ratio of the temperature-responsive block segment contained in the temperature-responsive polymer is suitable for rapidly detaching cell aggregates from the cell culture substrate, it is preferably 70 wt% or more, even more preferably 80 wt% or more, particularly preferably 90 wt% or more, and most preferably 92 wt% or more.

[0048] In addition, since the temperature-responsive polymer is a block copolymer having a water-insoluble block segment and a temperature-responsive block segment, it is possible to impart temperature responsiveness to the surface of the cell culture substrate by a simple method of dropping and drying a solution containing the temperature-responsive polymer on the surface of the cell culture substrate. Further, since the layer formed at this time has the layer thickness of the above-described preferred temperature-responsive polymer, even when the surface of the layer containing the hydrophilic polymer is coated with the temperature-responsive polymer, the characteristics of each of the above (A) region and (B) region are less likely to be impaired. When using the cell culture substrate of the present invention and a cell culture kit having a temperature-responsive polymer which is a block copolymer or a coating agent containing the above block copolymer, a researcher performing the culture can easily adjust the layer thickness of the temperature-responsive polymer according to the type of cells.

[0049] The coating agent may contain a solvent. Examples of the solvent that can 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, 1-butanol; acetonitrile, formamide, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, 1,4-dioxane, methyl ethyl ketone and the like. Since it is suitable for making the film thickness of the coating uniform, it is preferable to use a mixed solvent of water and alcohols. 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 other than 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, and for example, a polymer composed of only a water-insoluble block segment can be mentioned.

[0051] Examples of the 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, N-n-propylacrylamide, N-n-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-t-butylacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, and N-tetrahydrofurfurylmethacrylamide; N,N-dialkyl-substituted (meth)acrylamide derivatives such as N,N-dimethyl(meth)acrylamide, N,N-ethylmethylacrylamide, and N,N-diethylacrylamide; (meth)acrylamide derivatives having a cyclic group such as 1-(1-oxo-2-propenyl)-pyrrolidine, 1-(1-oxo-2-propenyl)-piperidine, 4-(1-oxo-2-propenyl)-morpholine, 1-(1-oxo-2-methyl-2-propenyl)-pyrrolidine, 1-(1-oxo-2-methyl-2-propenyl)-piperidine, and 4-(1-oxo-2-methyl-2-propenyl)-morpholine; vinyl ethers such as methyl vinyl ether; and proline derivatives such as N-prolinemethyl ester acrylamide. Since it is suitable for setting the response temperature to 0 to 50°C, N,N-diethylacrylamide, N-n-propylacrylamide, N-isopropylacrylamide, N-n-propylmethacrylamide, N-ethoxyethylacrylamide, N-tetrahydrofurfurylacrylamide, and N-tetrahydrofurfurylmethacrylamide are preferred, N-n-propylacrylamide and N-isopropylacrylamide are more preferred, and N-isopropylacrylamide is particularly preferred. In addition, when using a medium at room temperature during medium replacement in the culture operation, since it is suitable for setting the response temperature of the block copolymer to a temperature lower than room temperature, N-n-propylacrylamide and N-prolinemethyl ester acrylamide are preferred.

[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, n-tetradecyl methacrylate, and the like. Further, since it is suitable for firmly immobilizing the block copolymer on the substrate, those having a reactive group are preferred, and examples thereof include 4-azidophenyl acrylate, 4-azidophenyl methacrylate, 2-((4-azidobenzoyl)oxy)ethyl acrylate, 2-((4-azidobenzoyl)oxy)ethyl methacrylate, and the like. Furthermore, since it is suitable for enhancing cell proliferation, a structure having an aromatic ring is preferred, 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 repeating units that control the response temperature of the block copolymer. Examples of the repeating units that control the response temperature of the block copolymer include hydrophilic or hydrophobic components, and there is no particular limitation. For example, those having an amino group such as 2-dimethylaminoethyl acrylate, 2-dimethylaminoethyl methacrylate, 2-diethylaminoethyl acrylate, 2-diethylaminoethyl methacrylate, N-[3-(dimethylamino)propyl]acrylamide; those having a betaine such as N-(3-sulfopropyl)-N-methacryloyloxyethyl-N,N-dimethylammonium betaine, N-methacryloyloxyethyl-N,N-dimethylammonium-α-N-methylcarboxybetaine; hydroxyethyl acrylate, hydroxyethyl methacrylate, N-(2-hydroxyethyl)acrylamide, polyethylene glycol monoacrylate, polyethylene glycol monomethacrylate, polypropylene glycol monoacrylate, polypropylene glycol monomethacrylate, methoxypolyethylene glycol monoacrylate, methoxypolyethylene glycol 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, tetrahydrofurfuryl methacrylate, etc., having a polyethylene glycol group or a methoxyethyl group; those having an acrylate group such as methoxymethyl acrylate, methoxymethyl methacrylate, 2-ethoxymethyl acrylate, 2-ethoxymethyl methacrylate, 3-butoxymethyl acrylate, 3-butoxymethyl methacrylate, 3-butoxymethyl acrylamide;Examples include those having a phosphorylcholine group such as 2-methacryloyloxyethyl phosphorylcholine, 2-acryloyloxyethyl phosphorylcholine, 3-(meth)acryloyloxypropyl phosphorylcholine, 4-(meth)acryloyloxybutyl phosphorylcholine, 6-(meth)acryloyloxyhexyl phosphorylcholine, 10-(meth)acryloyloxydecyl phosphorylcholine, ω-(meth)acryloyl(poly)oxyethylene phosphorylcholine, 2-acrylamidoethyl phosphorylcholine, 3-acrylamidopropyl phosphorylcholine, 4-acrylamidobutyl phosphorylcholine, 6-acrylamidohexyl phosphorylcholine, 10-acrylamidodecyl phosphorylcholine, ω-(meth)acrylamido(poly)oxyethylene phosphorylcholine, etc.;

[0054] (B) region is adjacent to the (A) region and has no cell adhesiveness or cell proliferativeness. When the (B) region is a region adjacent to the (A) region and having no cell proliferativeness, when culturing cells, cell aggregates can be formed only in the (A) region, and a state where no cells exist around a part or all of the (A) region can be formed. Also, since it is suitable for making the size and shape of the produced cell aggregates uniform, it is preferable that the (B) region has neither cell proliferativeness nor cell adhesiveness.;

[0055] (B) region is not limited other than being adjacent to the (A) region, but since it is suitable for producing cell aggregates with uniform size and shape, it is preferable that the (B) region is adjacent to 20% or more of the length of the boundary line of the (A) region, more preferably 50% or more, still more preferably 80% or more, and most preferably the entire periphery of the (A) region is the (B) region. Also, since it is suitable for enhancing the mass productivity of the cell culture substrate, it is preferable that the (A) region is island-shaped and the (B) region is a sea-island structure in the form of a sea.;

[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, still more preferably 50% or more, and most preferably 70% or more with respect to the total area of the (A) region and the (B) region. Also, since it is suitable for providing a sufficient distance between a plurality of (A) regions and suppressing the cell aggregates of the plurality of (A) regions from fusing and becoming a non-uniform shape, the area of the (B) region is preferably 20% or more, more preferably 40% or more, still more preferably 60% or more, and most preferably 80% or more with respect to the total area of the (A) region and the (B) region.

[0057] The uneven height (the out-of-plane distance 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. By having the uneven height of 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 aggregates can be reduced. Thereby, it is possible to increase the cell viability of the cell aggregates. Also, if the uneven height is 500 nm or less, air bubbles are less likely to adhere to the uneven portions. Thereby, it becomes unnecessary to perform degassing to remove air bubbles or repeatedly discharge and aspirate the culture medium using a pipettor, and the operability is improved. By having the uneven height of 1 nm or more, live cells that have spontaneously moved (migrated) on the cell culture substrate gather in the (A) region, so it is possible to increase the cell viability of the cell aggregates. Since it is suitable for increasing the cell viability of the formed cell aggregates, the uneven height of 400 nm or less is more preferable, 100 nm or less is still more preferable, and 50 nm or less is most preferable.

[0058] The cell culture substrate may be provided with a layer containing a bio-derived substance on the surface, if necessary. The layer containing the bio-derived 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. The bio-derived substance is not particularly limited, and examples thereof include Matrigel, laminin, fibronectin, vitronectin, collagen, and the like.

[0059] These biomaterials may be natural products, may be artificially synthesized by genetic recombination technology or the like, may be fragments cleaved by restriction enzymes or the like, or may be synthetic proteins or synthetic peptides chemically synthesized from substances equivalent to these biomaterials.

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

[0061] The type of laminin is not particularly limited. 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 genetic recombination technology or the like, or may be a synthetic protein or synthetic peptide chemically synthesized from a substance equivalent to laminin. Commercially available products such as iMatrix-511 (manufactured by Nippi Inc.) can be preferably used due to their easy availability.

[0062] Vitronectin may be a natural product, may be artificially synthesized by genetic recombination technology or the like, or may be a synthetic protein or synthetic peptide chemically synthesized from a substance equivalent to vitronectin. Commercially available products such as vitronectin, human plasma-derived (manufactured by Wako Pure Chemical Industries, Ltd.), synthemax (manufactured by Corning Incorporated), Vitronectin (VTN-N) (manufactured by Thermo Fisher Scientific) can be preferably used due to their easy availability.

[0063] Fibronectin may be a natural product, may be artificially synthesized by genetic recombination technology or the like, or may be a synthetic protein or synthetic peptide obtained by chemically synthesizing a substance equivalent to fibronectin. From the perspective of easy availability, commercially available products such as fibronectin solution, derived from human plasma (manufactured by Wako Pure Chemical Industries, Ltd.), Retronectin (manufactured by Takara Bio Inc.) can be preferably used.

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

[0065] From the perspective of being able to suppress the denaturation of the biomaterial-derived substance and enhance cell proliferation, it is preferable that the biomaterial-derived substance is immobilized on the cell culture substrate by non-covalent bonds. Here, "non-covalent bond" refers to binding forces other than covalent bonds derived from intermolecular forces such as electrostatic interaction, water-insoluble interaction, hydrogen bond, π-π interaction, dipole-dipole interaction, London dispersion force, and other van der Waals interactions. The immobilization of the biomaterial-derived substance on the block copolymer may be by a single binding force or a combination of multiple ones.

[0066] The method for immobilizing the biomaterial-derived substance is not particularly limited. For example, a method of immobilizing by applying a solution of the biomaterial-derived substance to the cell culture substrate for a predetermined time, or a method of adsorbing and immobilizing the biomaterial-derived substance on the cell culture substrate by adding the biomaterial-derived substance to the culture solution when culturing cells can be preferably used.

[0067] The cell culture substrate according to this embodiment may be provided with a partition plate (for example, a plate having through holes with a cross-sectional area in the in-plane direction of 0.05 to 100 cm 2 ), etc. on the substrate, so as to provide a structure for partitioning each cell aggregate.

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

[0069] The cells cultured using the cell culture substrate according to this embodiment are not particularly limited as long as they can adhere to the surface before the stimulation by temperature drop. For example, in addition to various established cell lines such as Chinese hamster ovary-derived CHO cells, mouse connective tissue L929, human embryonic kidney-derived HEK293 cells, and human cervical cancer-derived HeLa cells, for example, epithelial cells, endothelial cells, skeletal muscle cells showing contractility, smooth muscle cells, cardiomyocytes, neuron cells, glial cells, fibroblasts, hepatocytes, non-parenchymal liver cells, and adipocytes involved in the metabolism of the living body, and as cells having differentiation ability, stem cells existing in various tissues such as mesenchymal stem cells, bone marrow cells, and Muse cells, and furthermore, pluripotent stem cells (pluripotent stem cells) having differentiation pluripotency such as ES cells and iPS cells, and cells differentiated therefrom, etc. can be mentioned. From the viewpoints of cell proliferation and detachability in the cell culture substrate according to one embodiment, stem cells or pluripotent stem cells are preferable, mesenchymal stem cells or pluripotent stem cells are more preferable, pluripotent stem cells are further preferable, and iPS cells are most preferable.

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

[0071] The cell culture substrate according to this embodiment can be manufactured, for example, by a manufacturing method including the following steps (1), (2), and (3). This manufacturing method is excellent in mass productivity. In step (3), corona treatment or UV treatment can also be performed instead of plasma treatment. Step (1): 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. Step (2): A step of irradiating the layer containing the hydrophilic polymer with UV light to immobilize the layer containing the hydrophilic polymer on the surface of the substrate by a chemical reaction. Step (3): A step of performing plasma treatment on a part of the surface of the layer containing the immobilized hydrophilic polymer to form an (A) region in the part where the plasma treatment is performed.

[0072] The cell culture substrate according to this embodiment can also be manufactured by a manufacturing method including the following steps (1'), (2'), (3'), and (4'). Step (1'): A step of using a substrate formed of a polymer containing an alicyclic hydrocarbon group or an aromatic hydrocarbon group in the repeating unit, performing plasma treatment on the surface of the substrate, and forming the above (A) region in the part where the plasma treatment is performed. Step (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. Step (3'): A step of irradiating a part of the layer containing the hydrophilic polymer with UV light to immobilize a part of the layer containing the hydrophilic polymer on the surface of the substrate. Step (4’): A step of washing the hydrophilic polymer with a solvent to dissolve the hydrophilic polymer not immobilized on the surface and removing it from the surface of the substrate.

[0073] In addition to step (1), step (2), and step (3), or step (1’), step (2’), step (3’), and step (4’), the manufacturing method according to the present embodiment may further include the following step (4) as necessary. Step (4): After step (3) or (4’), a step of laminating a plate having through-holes with an in-plane cross-sectional area of 0.05 to 100 cm 2 to the substrate on the side where the surface of the substrate coated with the layer containing the hydrophilic polymer is located.

[0074] Further, the manufacturing method according to an 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 the hydrophilic polymer that has been subjected to plasma treatment with a composition containing a temperature-responsive polymer to form a layer containing the 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 the hydrophilic polymer. By forming the layer containing the hydrophilic polymer, it is possible to achieve a state without cell adhesiveness and cell growth properties. As a method for forming the layer containing the hydrophilic polymer, there is no particular limitation, and for example, a method of forming by applying a composition containing the hydrophilic polymer to at least a part of the surface of the substrate can be mentioned. As a method for applying the composition containing the hydrophilic polymer, for example, various commonly known methods such as coating, brushing, dip coating, spin coating, bar coating, flow coating, spray coating, roll coating, air knife coating, blade coating, gravure coating, microgravure coating, and slot die coating can be used.

[0076] Figure 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 Figure 2, a layer 2 containing a hydrophilic polymer is formed on the entire surface of one side of the substrate 1.

[0077] In step (2), the layer 2 containing the hydrophilic polymer is irradiated with UV light to immobilize the layer containing the hydrophilic polymer on the surface of the substrate. By irradiating a UV-reactive hydrophilic polymer with UV light, a chemical reaction occurs between the hydrophilic polymers or between the hydrophilic polymer and the substrate, and the layer containing the hydrophilic polymer is immobilized on the surface of the substrate. By immobilizing the layer containing the hydrophilic polymer on the substrate surface, when a composition containing a temperature-responsive polymer is applied in step (5) described later, a layer containing the temperature-responsive polymer can be formed without deforming the layer containing the hydrophilic polymer. Also, by immobilizing the layer containing the hydrophilic polymer on the substrate surface, the shape of the (A) region 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), plasma treatment is performed on a part of the surface of the layer containing the immobilized hydrophilic polymer to form an (A) region in the plasma-treated part. A suitable plasma treatment method can be appropriately adjusted according to the type and layer thickness of the hydrophilic polymer used, but the plasma irradiation 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. Also, as a method for patterning the (A) region, there is no particular limitation, but examples include a method in which plasma treatment is performed on a desired part (unmasked part) by performing plasma treatment in a state of being covered with a metal mask, a silicon mask, a surface protection film, or the like.

[0079] Figure 3 is a schematic diagram (perspective view) of the cell culture substrate after step (3). In the cell culture substrate 10 shown in Figure 3, circular regions (the regions indicated by A) are arranged at equal intervals. For example, the area of each region is 0.001 to 5 mm 2This corresponds to the portion where plasma treatment was performed. The region indicated by A becomes cell - adhesive and cell - proliferative due to the surface of layer 2 containing a hydrophilic polymer being modified by plasma treatment. Also, the region other than the circular - shaped region (the region indicated by B) corresponds to the portion where plasma treatment was not performed because it was protected by, for example, a metal mask or the like. Since the region indicated by B has layer 2 whose surface contains a hydrophilic polymer, it does not have cell - adhesiveness or cell - proliferativeness.

[0080] In step (1’), a polymer substrate containing an alicyclic hydrocarbon group or an aromatic hydrocarbon group in the repeating unit is used, plasma treatment is performed on the surface of the substrate, and the above - mentioned (A) region is formed on the portion where the plasma treatment was performed. By performing plasma treatment on the surface of a polymer substrate containing an alicyclic hydrocarbon group or an aromatic hydrocarbon group, the surface of the substrate can be changed to a structure suitable for cell culture. As the conditions for plasma treatment, air, nitrogen, or oxygen is used as the introduced gas, and a gas pressure of 1 to 30 Pascals, a time of 1 second to 10 minutes is preferable, 1 second to 5 minutes is more preferable, and 1 second to 1 minute is particularly preferable. Examples of the alicyclic hydrocarbon group include cycloalkyl groups, and more specifically, cyclopropyl group, cyclobutyl group, cyclopentyl group, cyclohexyl group, cycloheptyl group, cyclooctyl group, cyclononyl group, etc. The aromatic hydrocarbon group means a group obtained by removing one or more hydrogen atoms directly bonded to the carbon atoms constituting the ring from an aromatic hydrocarbon. Examples of the aromatic hydrocarbon group include groups obtained by removing one or more hydrogen atoms directly bonded to the carbon atoms constituting the ring from benzene which is a monocyclic aromatic hydrocarbon, naphthalene which is a polycyclic aromatic hydrocarbon, etc.

[0081] Alternatively, instead of the above step (1’), a component suitable for cell culture may be coated on the surface of the substrate. As a component suitable for cell culture, a polymer material such as polycarboxystyrene can be preferably 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 the coating method, the same method as in step (1) described above can be preferably used.

[0083] In step (3’), a part of the layer containing the hydrophilic polymer is irradiated with UV light to immobilize a part of the layer containing the hydrophilic polymer on the surface of the substrate. By irradiating a part of the region of the layer with UV light, only the UV-irradiated region of the layer containing the hydrophilic polymer is immobilized on the surface of the substrate. Examples of the method of irradiating a part of the layer containing the hydrophilic polymer with UV light include a method of placing a quartz mask or the like having a region that is not UV-transmissive formed by chromium evaporation or the like in a desired pattern on the substrate and irradiating it with UV light from above.

[0084] In step (4’), the hydrophilic polymer is washed with a solvent to dissolve and remove the hydrophilic polymer that is not immobilized on the surface from the surface of the substrate. By removing the hydrophilic polymer that is not immobilized, the surface of the substrate is exposed, and region (A) can be formed. As the solvent used in step (4’), 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 azide groups, it preferably contains water and alcohol. By washing with a mixed solvent of water and alcohol, it is possible to reduce the components derived from the hydrophilic polymer remaining in region (A) and enhance the cell adhesiveness and cell growth properties of region (A). As the alcohol, a lower alcohol is preferred. For example, methanol, ethanol, 2-propanol, t-butanol, isobutanol, pentanol, and hexanol are preferred, and methanol or ethanol is more preferred. Further, 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 2A plate having a through-hole is bonded to a substrate on the surface side coated with a layer containing a hydrophilic polymer of the substrate. The cross-sectional area in the in-plane direction is 0.05 to 100 cm 2 By bonding a plate having a through-hole of 2 to a substrate, a plate having a space for containing a culture medium can be produced with high productivity. FIG. 4 is a schematic view (perspective view) of the cell culture substrate after step (4). The cell culture substrate 11 shown in FIG. 4 is, for example, the cell culture substrate 10 shown in FIG. 3, etc., to which a partition plate 20 (the cross-sectional area in the in-plane direction is 0.05 to 100 cm 2 a plate having a through-hole) is bonded.

[0086] In step (5), after step (3) or (4'), the surface of the layer containing the plasma-treated hydrophilic polymer is coated with a composition containing a temperature-responsive polymer to form a layer containing a temperature-responsive polymer. At this time, by forming the layer containing the temperature-responsive polymer with a layer thickness of 100 nm or less, the molecular chains of the temperature-responsive polymer are likely to be coated in a hydrophobic state on the surface of the (A) region formed in step (3) or (4'), and it is suitable for imparting temperature responsiveness while maintaining the function of the (A) region (having cell adhesiveness and cell growth properties). Also, by setting the layer thickness of the layer containing the temperature-responsive polymer to 1 nm or more, a cell culture substrate capable of imparting sufficient temperature responsiveness and rapidly forming cell aggregates can be manufactured, which is suitable.

[0087] As a method of coating with a composition containing a temperature-responsive polymer, a method similar to the coating method of the composition containing the aforementioned hydrophilic polymer can be preferably used.

[0088] As a method of 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, by coating using a commonly used coating method without performing patterning, the productivity of the cell culture substrate can be increased. Further, by coating the entire surface of the cell culture substrate with a composition containing a temperature-responsive polymer, temperature responsiveness is imparted to the (A) region, and the (B) region is also coated with a temperature-responsive polymer. By coating the (B) region with a temperature-responsive polymer, the cell adhesiveness of the (B) region 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 the present embodiment includes the following steps (i) to (iii). The differentiation induction method according to the present embodiment is suitably used for inducing differentiation of pluripotent stem cells into triploblastic cells. (i) A step of seeding pluripotent stem cells on the cell culture substrate according to the present invention. (ii) A step of culturing the seeded pluripotent stem cells to form hemispherical cell aggregates having a height / diameter ratio of 0.2 to 0.8. (iii) A step of inducing differentiation of the cell aggregates to form cell aggregates of triploblastic cells.

[0090] (i) The step uses the cell culture substrate according to the present invention described above, and is a step of seeding pluripotent stem cells on the cell culture substrate. "Seeding cells" means bringing a cell suspension (hereinafter referred to as "cell suspension") into contact with the cell culture substrate by applying a cell suspension on the cell culture substrate or injecting it into the cell culture substrate. By using a cell culture substrate having a cell-proliferative region, the cells can be cultured in the subsequent step (ii). If the cell culture substrate does not have a cell-proliferative region, the 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 pluripotent stem cells. The conditions effective for maintaining the undifferentiated state are not particularly limited. For example, the density of pluripotent stem cells at the start of the culture is set within the preferred range described as the cell density at the time of the following seeding, and the culture is carried out in the presence of an appropriate liquid medium. As a medium effective for maintaining the undifferentiated state of pluripotent stem cells, for example, a medium supplemented with one or more of insulin, transferrin, selenium, ascorbic acid, sodium hydrogen carbonate, basic fibroblast growth factor, transforming growth factor β (TGFβ), CCL2, activin, and 2-mercaptoethanol, which are known as factors for maintaining the undifferentiated state of pluripotent stem cells, can be preferably used. Since it is particularly suitable for maintaining the undifferentiated state of pluripotent stem cells, it is more preferable to use a medium containing insulin, transferrin, selenium, ascorbic acid, sodium hydrogen carbonate, basic fibroblast growth factor, and transforming growth factor β (TGFβ), and most preferably to use a medium supplemented with basic fibroblast growth factor.

[0092] There are no particular restrictions on the type of medium to which the basic fibroblast growth factor is added. For example, 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 Inc.), 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), ReproNaive (manufactured by REPROCELL Inc.), ReproXF (manufactured by REPROCELL Inc.), ReproFF (manufactured by REPROCELL Inc.), ReproFF2 (manufactured by REPROCELL Inc.), NutriStem (manufactured by Biological Industries), iSTEM (manufactured by Takara Bio Inc.), GS2-M (manufactured by Takara Bio Inc.), hPSC Growth Medium DXF (manufactured by PromoCell GmbH), and the like. Since it is suitable for maintaining the undifferentiated state of pluripotent stem cells, Primate ES Cell Medium (manufactured by REPROCELL Inc.), StemFit AK02N (manufactured by Ajinomoto Co., Inc.) or StemFit AK03 (manufactured by Ajinomoto Co., Inc.) is preferred, StemFit AK02N (manufactured by Ajinomoto Co., Inc.) or StemFit AK03 (manufactured by Ajinomoto Co., Inc.) is more preferred, and StemFit AK02N (manufactured by Ajinomoto Co., Inc.) is particularly preferred.

[0093] In the above step (i), there are no particular restrictions on the seeding method of pluripotent stem cells. For example, it can be carried out by injecting a cell suspension into a cell culture substrate. The cell density at the time of seeding is not particularly restricted, but in order to be able to maintain and proliferate pluripotent stem cells, 1.0×10 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 is even more preferable, and 1.2×10 3 ~1.0×10 5 cells / cm 2 is most preferable.

[0094] As the medium used in the above step (i), since it is also suitable for maintaining the survival of pluripotent stem cells, it is preferable to use a medium in which a Rho-associated kinase inhibitor is further added to the medium supplemented with the above basic fibroblast growth factor. Particularly when using human pluripotent stem cells, when the cell density of human pluripotent stem cells is low, the addition of a Rho-associated kinase inhibitor may be effective in maintaining the survival of human pluripotent stem cells. As the Rho-associated kinase inhibitor, for example, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide·2HCl·H2O (Y-27632 manufactured by Wako Pure Chemical Industries, Ltd.), 1-(5-Isoquinolinesulfonyl)homopiperazine Hydrochloride (HA1077 manufactured by Wako Pure Chemical Industries, Ltd.) can be used. The concentration of the Rho-associated kinase inhibitor added to the medium is within a range effective for maintaining the survival of human pluripotent stem cells and does not affect the undifferentiated state of human pluripotent stem cells, preferably 1 μM to 50 μM, more preferably 3 μM to 20 μM, still more preferably 5 μM to 15 μM, and most preferably 8 μM to 12 μM.

[0095] Shortly after starting the above step (i), 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 value of the diameter of the cells in the direction outside the substrate surface of the cell culture substrate. The "diameter" may be, for example, the maximum value of the diameter of the cells in the direction within the substrate surface of the cell culture substrate.

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

[0098] It is preferable to perform the first medium exchange 22 to 26 hours after starting the (ii) process. The second medium exchange is performed 48 to 72 hours after that, and then it is preferable to perform the medium exchange every 24 to 48 hours. During this period, the pluripotent stem cells proliferate and form flat cell masses called colonies. A colony is a state in which cells are two-dimensionally adhered to the cell culture substrate. The culture is continued until the size of the colony reaches about the size of the (A) region, and by further continuing the culture, three-dimensional cell aggregates are formed. The shape of the three-dimensional cell aggregates is preferably hemispherical with a height / diameter ratio of 0.2 to 0.8. The height / diameter ratio is obtained by observing a plurality of cell aggregates with a microscope, calculating the values, and averaging them. By forming hemispherical cell aggregates with a height / diameter ratio of 0.2 to 0.8, the induction of differentiation of pluripotent stem cells into triploblastic cells can be efficiently performed. Also, since it is suitable for enhancing the differentiation induction efficiency into triploblastic cells, it is more preferably 0.3 to 0.7, and still more preferably 0.4 to 0.6.

[0099] (iii) In this step, the above cell aggregates (cell aggregates of pluripotent stem cells) are induced to differentiate to form cell aggregates of triploblastic cells. Note that the (iii) step may be performed after the (ii) step or in parallel with the (ii) step. That is, immediately after the seeded pluripotent stem cells adhere to the cell culture substrate, the (ii) step and the (iii) step may be started, and differentiation induction may be performed while forming cell aggregates. Note that triploblastic cells refer to any one of endodermal cells, mesodermal cells, and ectodermal cells. In the (iii) step, the cells are cultured in a medium containing a differentiation-inducing factor.

[0100] In order to be suitable for enhancing the induction efficiency of differentiation into endoderm cells, it is preferable that the differentiation-inducing factor in step (iii) contains an endoderm-inducing factor. As the endoderm-inducing factor, in order to be suitable for enhancing the induction efficiency of differentiation into embryoid bodies, it is preferably a single or a plurality of differentiation-inducing 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] In order to be suitable for enhancing the induction efficiency of differentiation into mesoderm cells, it is preferable that the differentiation-inducing factor in step (iii) contains a mesoderm-inducing factor. As the mesoderm-inducing factor, in order to be suitable for enhancing the induction efficiency of differentiation into embryoid bodies, it is preferably a single or a plurality of differentiation-inducing factors selected from the group consisting of GSK3β inhibitor, Bone morphogenetic protein (BMP), and activin, and particularly preferably contains any one of activin A, CHIR99021 (GSK3β inhibitor), and BMP4.

[0102] In order to be suitable for enhancing the induction efficiency of differentiation into ectoderm cells, it is preferable that the differentiation-inducing factor in step (iii) contains an ectoderm-inducing factor. As the above-mentioned ectoderm-inducing factor, it is preferably contains any one of Noggin (BMP inhibitor), dorsomorphin (BMP inhibitor), SB431542 (TGF-β inhibitor), and activin inhibitor, and more preferably contains a BMP inhibitor and a TGF-β inhibitor.

[0103] The addition concentration of the differentiation-inducing factor to the medium is not particularly limited, but in order to be suitable for enhancing the induction efficiency of differentiation into the three germ layer cells, it is preferably 1000 - 500 ng / mL, more preferably 500 - 100 ng / mL, and most preferably 100 ng / mL or less.

[0104] The medium containing the differentiation-inducing factor is preferably replaced every 24 hours during the culture in order to sufficiently promote the differentiation induction. By replacing the medium within 24 hours, it is possible to prevent the shortage of the differentiation-inducing factor in the medium and uniformly differentiate all pluripotent stem cells.

[0105] The method for inducing differentiation of pluripotent stem cells according to this embodiment may further include the following step (iv). The differentiation induction method including step (iv) is preferably used for inducing differentiation of pluripotent stem cells into intestinal epithelial cells. (iv) A step of culturing 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, and expressing a marker possessed by intestinal epithelial cells.

[0106] The intestinal epithelial cells preferably have any one or a plurality of cells among intestinal cells, goblet cells, enteroendocrine cells, Paneth cells, and intestinal epithelial stem cells, and more preferably include all of intestinal cells, goblet cells, enteroendocrine cells, Paneth cells, and intestinal epithelial stem cells. Intestinal epithelial cells have specific markers. For example, as intestinal cell markers, CDX2 and VIL1; as goblet cell markers, MUC2; as enteroendocrine cell markers, CGA; as Paneth cell markers, DEFA6; and as intestinal epithelial stem cell markers, LGR5 can be mentioned.

[0107] Since the cell culture substrate according to the present invention does not have a concavo-convex structure with a high height in the out-of-substrate surface direction on the surface, it is difficult for air bubbles to adhere to the surface of the cell culture substrate when the medium is brought into contact. In the cell culture substrate according to the present invention, for example, the number of adhered air bubbles is preferably 10% or less, more preferably 5% or less, still more preferably 3% or less, and most preferably 1% or less with respect 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 survival rate because the adhered cells spontaneously aggregate as compared with the formation of cell aggregates using a conventional fine concavo-convex structure. For example, when cells with a survival rate of 50% are used, the cell survival rate of the formed cell aggregates is preferably 70% or more, more preferably 80% or more, still more preferably 85% or more, and most preferably 90% or more.

Example

[0109] Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited by the following examples. Unless otherwise specified, commercially available reagents were used.

[0110] <Measurement of the area of the (A) region> An image of the surface of the cell culture substrate was acquired using a laser microscope (manufactured by Keyence Corporation, product name VK-X200). Using the obtained image, the areas of 20 (A) regions were determined on the analysis software VK-X Viewer, and their average was taken as the area of the (A) region.

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

[0112] <Evaluation of bubble adhesion> Medium StemFit AK02N (manufactured by Ajinomoto Co., Inc.) was added to the cell culture substrate, and the presence or absence of bubble adhesion was confirmed by observing with a microscope.

[0113] <Evaluation of cell aggregates> (Culture of cell aggregates) Medium StemFit AK02N (manufactured by Ajinomoto Co., Inc.) was added to the cell culture substrate at 0.2 mL / cm 2In addition, iMatrix-511 solution (manufactured by Nippi, Inc.) was added at a concentration of 2.5 μL / mL. Using human iPS cell line 201B7 that was adjusted so that the ratio of viable cells to total cells (cell viability) became 50% by mixing dead cells, 15,000 cells (total number of live and dead cells) / cm 2 were seeded and cultured in an environment at 37°C and a CO2 concentration of 5%. Also, until 24 hours after seeding the cells, Y-27632 (manufactured by Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) was added to the culture medium. The presence or absence of cell aggregate formation was confirmed 1 day or 2 days after the start of culture.

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

[0115] (Evaluation 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 (manufactured by ThermoFisher) and 0.5 mM EDTA solution (manufactured by Invitrogen)), and then the cells were detached and collected as single cells using a cell scraper, and the 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 polymer (hydrophilic polymer and temperature-responsive polymer) coated on the substrate was determined by calculating the coating amount per unit area with the specific gravity of the polymer taken as 1 from the surface area of the substrate, the concentration of the polymer in the solution, and the volume of the solution dropped on the substrate.

[0117] (Measurement of composition of temperature-responsive polymer) The composition of the temperature-responsive polymer was determined by proton nuclear magnetic resonance spectroscopy ( 1 1H-NMR) spectrum analysis using a nuclear magnetic resonance measuring device (manufactured by JEOL Ltd., product name JNM-GSX400), or carbon nuclear magnetic resonance spectroscopy ( 13Determined by 13C-NMR spectrum analysis.

[0118] <Measurement of Molecular Weight and Molecular Weight Distribution of Temperature-Responsive Polymer> The 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 HLC-8320GPC manufactured by Tosoh Corporation. Two columns of TSKgel Super AWM-H manufactured by Tosoh Corporation were used. The column temperature was set at 40°C. The eluent was 1,1,1,3,3,3-hexafluoro-2-propanol containing 10 mM sodium trifluoroacetate or N,N-dimethylformamide containing 10 mM lithium bromide. The measurement sample was prepared at 1.0 mg / mL for measurement. A calibration curve for molecular weight was prepared using polymethyl methacrylate (manufactured by Polymer Laboratories Ltd.) with a 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 for an exposure time of 0.4 seconds, and fluorescence images were taken. The RGB values of these images were determined, 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. The same measurement was performed on a polystyrene plate with a thickness of 1.2 mm, and the relative values with respect to the values on the polystyrene plate with a thickness of 1.2 mm were shown in Table 4 as the fluorescence intensity of each substrate.

[0120] [Example 1] A water / ethanol solution containing polyvinyl alcohol having an azide group (BIOSURFINE(R)-AWP, manufactured by Toyo Gosei Co., Ltd.) as a hydrophilic polymer at a solid content concentration of 0.06 wt% was dropped in an amount of 0.8 mL onto a polystyrene (PS) dish (substrate) with a diameter of 3.5 cm, dried under reduced pressure at room temperature, and then irradiated with UV light to cure the hydrophilic polymer and form a layer of the hydrophilic polymer. A metal mask having a plurality of circular holes with a diameter of 0.2 mm was placed on the layer of the hydrophilic polymer, and plasma treatment (under a gas pressure of 20 Pa, a conductive current of 20 mA, and an irradiation time of 30 seconds) was performed from above the metal mask using a plasma irradiation device (manufactured by Vacuum Devices Co., Ltd., trade name Plasma Ion Bombarder PIB-20) to form a region ((A) region) having cell adhesiveness and cell growth properties in the plasma-treated portion. Also, a (B) region was formed in the portion masked by the metal mask.

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

[0122] Cultivation evaluation of human iPS cells was performed on this cell culture substrate, and formation of cell aggregates was confirmed 2 days after the start of cultivation. By visual judgment, the size of the formed cell aggregates was uniform. The ratio of the height / diameter of the cell aggregates was estimated to be 0.5. Also, the survival rate of the cells contained in the cell aggregates was 89%, which was a high cell survival rate. Also, no bubbles adhered 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 configuration and evaluation results of the prepared cell culture substrate are shown in Table 2. The prepared cell culture substrate had a layer thickness of the hydrophilic polymer layer of 600 nm, an area of the (A) region of 0.03 mm 2The uneven height at the boundary between the (A) region and the (B) region was 85 nm.

[0125] Cultivation evaluation of human iPS cells was performed on this cell culture substrate, and formation of cell aggregates was confirmed 2 days after the start of cultivation. By visual judgment, the sizes of the formed cell aggregates were uniform. The ratio of the height to the diameter of the cell aggregates was estimated to be 0.5. Also, the survival rate of the cells contained in the cell aggregates was 91%, indicating a high cell survival rate. Also, no bubbles adhered 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 of the plasma treatment in Example 1 was changed to 13 Pa and the plasma irradiation time was changed to 20 minutes.

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

[0128] Cultivation evaluation of human iPS cells was performed on this cell culture substrate, and formation of cell aggregates was confirmed 2 days after the start of cultivation. By visual judgment, the sizes of the formed cell aggregates were uniform. The ratio of the height to the diameter of the cell aggregates was estimated to be 0.5. Also, the survival rate of the cells contained in the cell aggregates was 78%, indicating a high cell survival rate. Also, no bubbles adhered to the cell culture substrate when the medium was added.

[0129] [Example 4] An ethanol solution containing a polymer containing a phosphorylcholine group (Lipidure-CM5210, manufactured by NOF Corporation) as a hydrophilic polymer at a solid content concentration of 0.01 wt% was dropped in an amount of 0.08 mL onto a polystyrene dish (substrate) with a diameter of 3.5 cm and dried under reduced pressure at room temperature to form a layer of the hydrophilic polymer. A metal mask having a plurality of circular holes with a diameter of 0.2 mm was placed on the layer of the hydrophilic polymer, and plasma treatment (under a gas pressure of 20 Pa, a conductive current of 20 mA, and an irradiation time of 30 seconds) was performed from above the metal mask using a plasma irradiation device (manufactured by Vacuum Device Co., Ltd., trade name Plasma Ion Bomber PIB-20) to form a region ((A) region) having cell adhesiveness and cell growth properties in the plasma-treated portion. Also, a (B) region was formed in the portion masked by the metal mask.

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

[0131] Cultivation evaluation of human iPS cells was performed on this cell culture substrate, and formation of cell aggregates was confirmed 2 days after the start of cultivation. By visual judgment, the sizes of the formed cell aggregates were uniform. The ratio of the height / diameter of the cell aggregates was estimated to be 0.5. Also, the survival rate of the cells contained in the cell aggregates was 87%, which was a high cell survival rate. Also, no bubbles adhered 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 configuration and evaluation results of the prepared cell culture substrate are shown in Table 2. The prepared cell culture substrate had a layer thickness of the hydrophilic polymer layer of 50 nm and an area of the (A) region of 0.03 mm 2 and the uneven height at the boundary between the (A) region and the (B) region was 21 nm.

[0134] The culture of human iPS cells was evaluated on this cell culture substrate, and the formation of cell aggregates was confirmed 2 days after the start of culture. By visual judgment, the sizes of the formed cell aggregates were uniform. The ratio of the height to the diameter of the cell aggregates was estimated to be 0.5. In addition, the survival rate of the cells contained in the cell aggregates was 92%, indicating a high cell survival rate. Also, no bubbles adhered 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 and evaluation results of the prepared cell culture substrate are shown in Table 2. The prepared cell culture substrate had a hydrophilic polymer layer thickness of 1500 nm and the area of the (A) region was 0.03 mm 2 , and the uneven height at the boundary between the (A) region and the (B) region was 34 nm.

[0137] The culture of human iPS cells was evaluated on this cell culture substrate, and the formation of cell aggregates was confirmed 2 days after the start of culture. By visual judgment, the sizes of the formed cell aggregates were uniform. The ratio of the height to the diameter of the cell aggregates was estimated to be 0.5. In addition, the survival rate of the cells contained in the cell aggregates was 85%, indicating a high cell survival rate. Also, no bubbles adhered 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 polyethylene terephthalate (PET) film (Lumirror, manufactured by Toray Industries, Inc.) with a film thickness of 188 μm (substrate) was used instead of a polystyrene (PS) dish with a diameter of 3.5 cm. Note that the culture was carried out by immobilizing the cut-out prepared cell culture substrate in a petri dish.

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

[0140] Cultivation evaluation of human iPS cells was performed on this cell culture substrate, and formation of cell aggregates was confirmed 2 days after the start of cultivation. By visual judgment, the sizes of the formed cell aggregates were uniform. The ratio of the height to the diameter of the cell aggregates was estimated to be 0.5. Also, the viability of the cells contained in the cell aggregates was 84%, indicating a high cell viability. Moreover, no bubbles adhered 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 a metal mask having a plurality of circular holes with a diameter of 0.2 mm.

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

[0143] Cultivation evaluation of human iPS cells was performed on this cell culture substrate, and formation of cell aggregates was confirmed 2 days after the start of cultivation. By visual judgment, the sizes of the formed cell aggregates were uniform. The ratio of the height to the diameter of the cell aggregates was estimated to be 0.5. Also, the viability of the cells contained in the cell aggregates was 89%, indicating a high cell viability. Moreover, no bubbles adhered to the cell culture substrate when the medium was added.

[0144] Using this cell culture substrate, induction of differentiation of human iPS cells into intestinal epithelial cells was carried out. 1.0 mL / dish of 1% Vitronectin (VTN-N) Recombinant Human Protein solution (manufactured by Gibco) was added to the cell culture substrate and left standing at 25 °C for 1 hour. After 1 hour, the 1% VTN solution was removed, 2.0 mL / dish of StemFit AK02N (manufactured by Ajinomoto Co., Inc.), which is an undifferentiated maintenance medium, was added, and further 3900 human iPS cell line 201B7 cells were seeded at a density of 2 per cm. The cells were then cultured in an environment at 37 °C and a CO2 concentration of 5%. Also, until 24 hours after seeding of the cells, Y-27632 (manufactured by Fujifilm Wako Pure Chemical Corporation) (concentration 10 μM) was added to the medium. By phase-contrast microscopy observation, a plurality of circular embryoid bodies adhered on the cell culture substrate were confirmed.

[0145] Twenty-four hours after seeding of the cells, 2.0 mL / dish of the following differentiation induction medium was added to initiate differentiation induction. The composition of the differentiation induction medium for induction of differentiation into intestinal epithelial cells is 85% KnockOut DMEM (manufactured by ThermoFisher), 15% KnockOut Serum Replacement XenoFree (manufactured by ThermoFisher), 0.1 mM non-essential amino acids (manufactured by Sigma-Aldrich), 2 mM Gluta Max-I Supplement (manufactured by ThermoFisher), 20 ng / mL basic fibroblast growth factor (bFGF, manufactured by PeproTech), 50 μg / mL L(+)-ascorbic acid (manufactured by Fujifilm Wako Pure Chemical Corporation), 10 ng / mL heregulin-β-1 (manufactured by Fujifilm Wako Pure Chemical Corporation), 200 ng / mL Long(R)R3IGF-I (manufactured by Sigma-Aldrich), and 1% penicillin-streptomycin solution (×100) (manufactured by Fujifilm Wako Pure Chemical Corporation). The time point when the differentiation induction medium was added was designated as "Day 0 of differentiation". Thereafter, the differentiation induction medium was replaced every 72 hours until "Day 64 of differentiation".

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

[0147] On "day 64 of differentiation", the above differentiation induction medium in the dish was removed, 2.0 mL / dish of PBS(-) was added, and the cells were washed. After washing, 1.0 mL / dish of cell detachment solution was added to the dish, and the dish was left standing for 5 minutes in an environment of 37 °C and a CO2 concentration of 5%. After leaving standing for the predetermined time, the cell detachment solution was removed, 1.0 mL / dish of differentiation induction medium was added, and the cells were dispersed. The obtained cell suspension was collected, and the cell count was measured using a Luna automatic cell counter (manufactured by Logos Biosystems).

[0148] To evaluate the induction of differentiation into intestinal epithelial cells, the housekeeping gene marker (GAPDH), intestinal cell marker (CDX2), absorptive epithelial marker (VIL1), and intestinal stem cell marker (LGR5) were adopted, and the relative expression levels of the mRNAs of the intestinal cell marker and intestinal stem cell marker were quantified. A healthy adult small intestine tissue (R1234226-50, BioChain Institute) was used as a positive control.

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

[0150]

Table 1

[0151] [Example 9] An ethanol solution containing a polymer (Lipidure-CM5210, manufactured by NOF Corporation) containing a phosphorylcholine group and a UV-reactive functional group as a hydrophilic polymer at a solid content concentration of 0.05 wt% was dropped in an amount of 0.08 mL onto a polystyrene dish (substrate) with a diameter of 3.5 cm, and dried under reduced pressure at room temperature to form a layer of the hydrophilic polymer. A metal mask having a plurality of circular holes with a diameter of 0.2 mm was placed on the layer of the hydrophilic polymer, and plasma treatment (under a gas pressure of 20 Pa, a conductive current of 20 mA, and an irradiation time of 30 seconds) was performed from above the metal mask using a plasma irradiation device (manufactured by Vacuum Devices Co., Ltd., trade name Plasma Ion Bomber PIB-20) to form a region ((A) region) having cell adhesiveness and cell growth properties in the plasma-treated portion. In addition, a (B) region was formed in the portion masked by the metal mask.

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

[0153] Cultivation evaluation of human iPS cells was performed on this cell culture substrate, and the formation of cell aggregates was confirmed 2 days after the start of cultivation. By visual judgment, the size of the formed cell aggregates was uniform. The ratio of the height / diameter of the cell aggregates was estimated to be 0.5. In addition, the survival rate of the cells contained in the cell aggregates was 86%, which was a high cell survival rate. Also, no bubbles adhered 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 the hydrophilic polymer is formed) was coated with a block copolymer (thermosensitive polymer) having a water-insoluble block segment and a thermosensitive 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 azobisisobutyronitrile 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 completion of the reaction, the reaction solvent was distilled off under reduced pressure using a rotary evaporator to concentrate the reaction solution. 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] To the test tube, 0.9 g (0.3 mmol) of the obtained n-butyl methacrylate polymer, 8.14 g (72 mmol) of N-isopropylacrylamide, and 5 mg (0.03 mmol) of azobisisobutyronitrile were added 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 completion of the reaction, 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. Further, it 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 block copolymer of N-isopropylacrylamide and n-butyl methacrylate. The synthesized block copolymer was dissolved in ethanol at 0.5 wt% and spin-coated on the surface of the cell culture substrate (the side on which the layer containing the hydrophilic polymer is formed) at 2000 rpm.

[0156] The composition unit ratio of the block copolymer was 4 wt% of n-butyl methacrylate and 96 wt% of N-isopropylacrylamide, and the number average molecular weight (Mn) of the block copolymer was 77,000. The layer 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 layer thickness of the block copolymer (temperature-responsive polymer) layer was 25 nm.

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

[0158] The culture evaluation of human iPS cells was performed on this cell culture substrate, and the formation of cell aggregates was confirmed 2 days after the start of culture. By visual judgment, the size of the formed cell aggregates was uniform. The ratio of the height / diameter of the cell aggregates was estimated to be 0.5. The cell aggregates were detached by cooling the cell culture substrate. In addition, the survival rate of the cells contained in the cell aggregates was 88%, indicating a high cell survival rate. Also, no bubbles adhered 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 the hydrophilic polymer was formed) was coated with a random copolymer composed 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 completion of the reaction, the reaction solvent was diluted with acetone, poured into 500 mL of hexane, and the precipitated solid was recovered and dried under reduced pressure. Further, it was dissolved again in acetone, poured into 500 mL of pure water, and the precipitated solid was recovered 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 on the surface of the cell culture substrate (the side on which the layer containing the hydrophilic polymer was formed) at 2000 rpm.

[0160] The compositional unit ratio of the random copolymer was 54 wt% of n-butyl methacrylate and 46 wt% of N-isopropylacrylamide, and the number average molecular weight (Mn) of the random copolymer was 158,000. The layer thickness of the random copolymer layer was determined from a calibration curve when the random copolymer was directly coated on a polystyrene dish. As a result, the layer thickness of the random copolymer layer was 25 nm. Although the random copolymer layer does not correspond to the layer made of the temperature-responsive polymer, it is described in the column of the layer made of the temperature-responsive polymer in Table 2 for convenience.

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

[0162] The culture of human iPS cells was evaluated on this cell culture substrate, and the formation of cell aggregates was confirmed 2 days after the start of culture. By visual judgment, the sizes of the formed cell aggregates were uniform. The ratio of the height / diameter of the cell aggregates was estimated to be 0.5. The cell aggregates did not detach by cooling the cell culture substrate. Also, the survival rate of the cells contained in the cell aggregates was 85%, which was a high cell survival rate. Also, no bubbles adhered to the cell culture substrate when the medium was added.

[0163] [Example 12] A polyethylene terephthalate (PET) film with a thickness of 180 μm (manufactured by Cosmo Shine, Toyobo Co., Ltd.) was subjected to plasma treatment (under a gas pressure of 20 Pa, a conductive current of 20 mA, and an irradiation time of 30 seconds). A water / ethanol solution containing polyvinyl alcohol having an azide group (BIOSURFINE(R)-AWP, manufactured by Toyo Gosei Co., Ltd.) as a hydrophilic polymer at a solid content concentration of 0.1 wt% was spin-coated on the plasma-treated surface of the above PET film at 2000 rpm. A quartz mask having a plurality of circular chromium evaporation regions with a diameter of 0.2 mm was placed on the layer of the hydrophilic polymer, and UV irradiation was performed from above the quartz mask for 10 seconds using a high-pressure mercury lamp. After washing with a mixed solvent of water / ethanol = 20 / 80 to dissolve and remove the hydrophilic polymer that was not irradiated with UV, a region ((A) region) having cell adhesiveness and cell growth properties was formed. Also, a (B) region was formed in the UV-irradiated portion. This PET film was bonded to a well plate (a plate having a through hole) with a bottom surface that was open at the bottom.

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

[0165] The culture of human iPS cells was evaluated on this cell culture substrate, and the formation of cell aggregates was confirmed 2 days after the start of culture. By visual judgment, the sizes of the formed cell aggregates were uniform. The ratio of the height to the diameter of the cell aggregates was estimated to be 0.5. In addition, the survival rate of the cells contained in the cell aggregates was 90%, indicating a high cell survival rate. Also, no bubbles adhered 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, instead of the mixed solvent of water / ethanol = 20 / 80, pure water, a mixed solvent of water / ethanol = 80 / 20, a mixed solvent of water / ethanol = 50 / 50, and ethanol were used, and cell culture substrates were prepared in the same manner as in Example 12 except for this.

[0167] The results of culturing and evaluating human iPS cells on these cell culture substrates and comparing them are shown in Table 3. It was found that by using a water / ethanol mixed solvent in the washing step, it is possible to enhance the cell adhesiveness and cell growth in the (A) region.

[0168] [Reference Example 2] As the substrate of Example 12, instead of the PET film, a PS plate with a thickness of 1.2 mm, a polycarbonate (PC) film with a thickness of 0.1 mm (Iupilon, manufactured by Mitsubishi Gas Chemical Company, Inc.), a polyethylene (PE) film, a polypropylene (PP) film, and a Permanox slide chamber (manufactured by Thermo Scientific) were used, and cell culture substrates were prepared in the same manner as in Example 12 except for this.

[0169] The culture of human iPS cells was evaluated on these cell culture substrates, and the cultured cells were observed by phase contrast at a magnification of 40 times. Also, the cells were observed by fluorescence staining and compared, and the results are shown in Table 4. It was found that when the substrate has a polymer containing an alicyclic hydrocarbon group or an aromatic hydrocarbon group as a structural unit, the cell growth in the (A) region is excellent. Also, it was found that by having a refractive index of the substrate of 1.4 to 1.6 and a thickness of 0.01 to 0.5 mm, a clear phase contrast image of the cells can be obtained. Furthermore, it was found that by having low autofluorescence of the substrate, a clear fluorescence image of the cells can 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 and evaluation results of the prepared cell culture substrate are shown in Table 2. The prepared cell culture substrate had a layer thickness of 3 nm for the hydrophilic polymer layer, an area of 0.03 mm 2 for the plasma-treated region, and the uneven height at the boundary between the plasma-treated region and the non-plasma-treated region was 25 nm.

[0172] Cultivation evaluation of human iPS cells was performed on this cell culture substrate. However, cells adhered and proliferated over the entire surface of the cell culture substrate, and cell aggregates were not 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 and evaluation results of the prepared cell culture substrate are shown in Table 2. The prepared cell culture substrate had a layer thickness of 3000 nm for the hydrophilic polymer layer, an area of 0.03 mm 2 for the plasma-treated region, and the uneven height at the boundary between the plasma-treated region and the non-plasma-treated region was 38 nm.

[0175] Cultivation evaluation of human iPS cells was performed on this cell culture substrate. However, since the cells did not adhere and proliferate, cell aggregates were not formed.

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

[0177] Cultivation evaluation of human iPS cells was performed on this cell culture substrate, and the formation of cell aggregates was confirmed 1 day after the start of cultivation. However, the survival rate of the cells contained in the cell aggregates was 65%, indicating a low cell survival rate.

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

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

[0180] The culture of human iPS cells was evaluated on this cell culture substrate. The formation of cell aggregates was confirmed 1 day after the start of the culture. However, the survival rate of the cells contained in the cell aggregates was 53%, indicating a low cell survival rate. In addition, air bubbles adhered 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 plasma treatment was not performed. The composition and evaluation results of the prepared cell culture substrate are shown in Table 2. The culture of human iPS cells was evaluated on this cell culture substrate. However, since the cells did not adhere and proliferate, cell aggregates were not 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 layer of hydrophilic polymer was not formed). The composition and evaluation results of the prepared cell culture substrate are shown in Table 2. The culture of human iPS cells was evaluated on this cell culture substrate. However, the cells adhered and proliferated over the entire surface of the cell culture substrate, and cell aggregates were not formed.

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

[0184] A... Region (A), B... Region (B), H... Concavo-convex height at the boundary between Region (A) and Region (B), 1... Substrate, 2... Layer containing hydrophilic polymer, 10, 11... Cell culture substrates, 20... Partition plate.

Claims

1. The present invention comprises a substrate and a layer containing a hydrophilic polymer and having a thickness of 5 to 2000 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) Island-like regions with cell adhesive and cell proliferation properties (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 region (A) is 0.001 to 5 mm ２ The cell culture substrate according to claim 1 or 2,

10. 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,

11. 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,

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

13. 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.

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

15. (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:

16. (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:

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

18. (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 15 or 16, 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.

19. (5) The manufacturing method according to claim 15 or 16, 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.

20. (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:

21. The method for inducing differentiation of pluripotent stem cells according to claim 20, 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;

22. 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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