Cell culture substrate and cell culture kit

Abstract

To provide a cell culture substrate that exhibits superior cell adhesion, or enables adhesion-based culture.SOLUTION: A cell culture substrate having the following regions (A) and (B): (A) a region having cell adhesiveness and cell proliferativity, and (B) a region adjacent to the region (A), having neither cell adhesiveness nor cell proliferativity, wherein the region (A) comprises a recess formed in the cell culture substrate, and the arithmetic average roughness (Ra) of the bottom surface of the recess is 0.001 μm or more.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a cell culture substrate and a cell culture kit. [Background technology]

[0002] Pluripotent stem cells, such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), have the ability to differentiate into various tissues (pluripotency), and have attracted considerable attention as a cell source for regenerative medicine and drug discovery screening. To apply pluripotent stem cells to regenerative medicine and drug discovery screening, they must be differentiated into the desired cells. This process requires the formation of pluripotent stem cell aggregates. While pluripotent stem cells can differentiate into a variety of cells, it is known that the optimal size of the cell aggregates varies depending on the type of differentiated cell. Therefore, it is desirable to control the size and create uniformly sized cell aggregates.

[0003] Several cell culture substrates for forming cell aggregates and methods for producing the same have been proposed. Patent Document 1 discloses a culture method in which cells are seeded, cultured, the medium is replaced, and recovered in a culture vessel having a plurality of depressions arranged on the bottom, and describes that the depressions consist of a bottom and an opening, and are treated to inhibit cell adhesion. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 7219303 Summary of the Invention [Problem to be solved by the invention]

[0005] In a cell suspension culture system using a cell culture substrate in which the inside of the recesses has been treated to make the cells less adhesive, such as the cell culture vessel disclosed in Patent Document 1, there is a high possibility that the cells will be sucked in when the culture medium is changed, making the operation cumbersome.

[0006] Therefore, an object of the present invention is to provide a cell culture substrate that has excellent cell adhesiveness (enabling adhesive culture). [Means for solving the problem]

[0007] The present invention provides a cell culture substrate, It has the following region (A) and the following region (B), (A) Region with cell adhesive and cell proliferation properties (B) A region adjacent to region (A) that does not have cell adhesive or cell proliferation properties. The (A) region is composed of a recess formed in a cell culture substrate, The cell culture substrate has a bottom surface of the recess having an arithmetic mean roughness (Ra) of 0.001 μm or more.

[0008] The present invention further relates to a cell culture kit comprising the cell culture substrate of the present invention provided with a partition member having a plurality of cylindrical partition walls capable of dividing the surface on which cells are cultured.

[0009] The present invention includes, for example, the following inventions. [1] A cell culture substrate, comprising: It has the following region (A) and the following region (B), (A) Region with cell adhesive and cell proliferation properties (B) a region adjacent to the region (A) and not having cell adhesiveness or cell proliferation properties the region (A) is a recess formed in the cell culture substrate, The cell culture substrate, wherein the arithmetic mean roughness (Ra) of the bottom surface of the recess is 0.001 μm or more. [2] The cell culture substrate according to [1], comprising a substrate and a layer containing a hydrophilic polymer that covers at least a portion of the surface of the substrate. [3] The cell culture substrate according to [2], wherein the entire surface of the recess is a layer containing the hydrophilic polymer. [4] The cell culture substrate according to any one of [1] to [3], wherein the Ra is 0.3 μm or more. [5] The cell culture substrate according to any one of [1] to [4], wherein the bottom surface of the well has a wrinkled surface when viewed from above. [6] A cell culture kit comprising a cell culture substrate, The cell culture substrate is a cell culture kit, in which the cell culture substrate according to any one of [1] to [5] is provided with a partition member having a plurality of cylindrical partition walls that can partition the surface on which cells are cultured. [Effects of the Invention]

[0010] According to the present invention, a cell culture substrate having excellent cell adhesiveness (enabling adhesive culture) can be provided. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram (cross-sectional view) of a cell culture substrate according to one embodiment. [Figure 2] FIG. 1 is a schematic diagram (perspective view) of a cell culture substrate according to one embodiment. [Figure 3] FIG. 3(A) shows an image of the bottom surface of region (A) in the cell culture substrate of Example 1 observed with an atomic force microscope, and FIG. 3(B) shows an image of the bottom surface of region (A) in the cell culture substrate of Example 2 observed with an atomic force microscope. [Figure 4] FIG. 4(A) shows an image of the bottom surface of region (A) in the cell culture substrate of Example 3 observed with an atomic force microscope, and FIG. 4(B) shows an image of the bottom surface of region (A) in the cell culture substrate of Example 4 observed with an atomic force microscope. [Figure 5]FIG. 5(A) shows an image of the bottom surface of region (A) in the cell culture substrate of Example 5 observed with an atomic force microscope, and FIG. 5(B) shows an image of the bottom surface of region (A) in the cell culture substrate of Example 6 observed with an atomic force microscope. [Figure 6] FIG. 6(A) shows an image of the bottom surface of region (A) in the cell culture substrate of Comparative Example 1 observed with an atomic force microscope, and FIG. 6(B) shows an image of the bottom surface of region (A) in the cell culture substrate of Comparative Example 2 observed with an atomic force microscope. [Figure 7] FIG. 7(A) shows an image of region (A) observed with a phase contrast microscope when cells were cultured using the cell culture substrate of Example 1, FIG. 7(B) shows an image of region (A) observed with a phase contrast microscope when cells were cultured using the cell culture substrate of Example 2, and FIG. 7(C) shows an image of region (A) observed with a phase contrast microscope when cells were cultured using the cell culture substrate of Example 3. [Figure 8] 8(A) and 8(B) show images of region (A) observed with a phase contrast microscope (FIG. 8(A)) or a fluorescence microscope (FIG. 8(B)) when cells were cultured using the cell culture substrate of Example 6. [Figure 9] FIG. 9(A) shows an image of region (A) observed with a phase contrast microscope when cells were cultured using the cell culture substrate of Comparative Example 1, and FIG. 9(B) shows an image of region (A) observed with a phase contrast microscope when cells were cultured using the cell culture substrate of Comparative Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0013] As used herein, the term "cell aggregate" refers to a three-dimensional aggregate of cells formed by the aggregation of multiple cells. The shape of the three-dimensional aggregate may be an ellipsoid such as a sphere, or may be a hemisphere or other shape. These shapes may have gaps formed by folding a sheet of cells, or may be hollow. An example of a cell aggregate is a spheroid.

[0014] In this specification, "temperature responsive" means that the degree of hydrophilicity / hydrophobicity changes with temperature. Furthermore, the boundary temperature at which the degree of hydrophilicity / hydrophobicity changes is referred to as the "responsive temperature."

[0015] As used herein, the term "biologically derived substance" refers to a substance present in the body of a living organism, or a chemically synthesized substance equivalent to such a substance. Substances present in the body of a living organism may be natural products, or may be artificially synthesized using recombinant DNA technology or the like. There are no particular limitations on biologically derived substances, and examples include nucleic acids, proteins, and polysaccharides, which are the basic materials that make up living organisms, and their constituent elements, such as nucleotides, nucleosides, amino acids, and various sugars, as well as lipids, vitamins, and hormones.

[0016] As used herein, "cell adhesiveness" refers to the ease with which cells adhere to a substrate or cell culture substrate at culture temperatures, "having cell adhesiveness" refers to the ability of cells to adhere to a substrate or cell culture substrate at culture temperatures directly or via a biologically derived substance, and "not having cell adhesiveness" refers to the inability of cells to adhere to a substrate or cell culture substrate at culture temperatures.

[0017] As used herein, "cell proliferation" refers to the ease with which cells proliferate at the culture temperature, "having cell proliferation" refers to the ability of cells to proliferate at the culture temperature, and "not having cell proliferation" refers to the inability of cells to proliferate at the culture temperature. "High cell proliferation" refers to the proliferation of more cells when compared over the same culture period.

[0018] The cell culture substrate according to this embodiment has the following region (A) and region (B): (A) Region with cell adhesive and cell proliferation properties (B) A region adjacent to region (A) that does not have cell adhesive or cell proliferation properties. Here, the region (A) is composed of recesses formed in the cell culture substrate, and the arithmetic mean roughness (Ra) of the bottom surface of the recesses is 0.001 μm or more.

[0019] FIG. 1 is a schematic diagram (cross-sectional view) of a cell culture substrate according to one embodiment. The cell culture substrate 10 shown in FIG. 1 has regions (A) and (B), designated by A and B in FIG. 1, formed on a substrate 1. H indicates the maximum depth of region (A). In addition, in the cell culture substrate 10 shown in FIG. 1, the surface of the recess is the substrate 1, but this is not limited thereto. For example, at least a portion or all of the surface of region (A) may be a layer containing a hydrophilic polymer, as described below.

[0020] The arithmetic mean roughness (Ra) of the bottom surface of the recesses (region (A)) of the cell culture substrate according to this embodiment may be 0.001 μm or more, and from the viewpoint of further improving cell adhesiveness, may be, for example, 0.005 μm or more, 0.01 μm or more, 0.02 μm or more, 0.03 μm or more, 0.04 μm or more, 0.05 μm or more, 0.06 μm or more, 0.08 μm or more, 0.1 μm or more, 0.2 μm or more, 0.3 μm or more, or 0.35 μm or more. The arithmetic mean roughness (Ra) of the bottom surface of the recesses (region (A)) of the cell culture substrate may be, for example, 1 μm or less.

[0021] In the cell culture substrate according to this embodiment, when the arithmetic mean roughness (Ra) of the bottom surface of the recess (region (A)) is 0.001 μm or more, for example, the surface of the bottom surface of the recess (region (A)) is formed with fine irregularities that allow cells to adhere appropriately. This increases the surface area of ​​the bottom surface of region (A), and is thought to result in a cell culture substrate with excellent cell adhesiveness (enabling adhesive culture).

[0022] The surface shape of the bottom surface of the recess (region (A)) when viewed from above is not particularly limited, but may be, for example, a wrinkled shape, a pyramidal shape, a spike shape, a moth-eye shape, or an island-in-the-sea shape, with a wrinkled shape being preferred. In this specification, "when viewed from above" refers to the case where the cell culture substrate is viewed from above along the direction of the maximum depth of region (A) (H in Figure 1). Furthermore, "wrinkled shape" refers to an uneven structure in which periodic waves are formed. Wrinkled shapes are shapes such as those shown in Figures 3 to 5.

[0023] Specifically, Ra is, for example, a laser microscope (product name: OPTELICS HYBRID + A surface roughness curve can be obtained using a laser spectroscopy (Lasertec Corporation) and calculated using the following equation (1).

[0024]

number

[0025] The recesses (areas (A)) of the cell culture substrate according to this embodiment can be formed, for example, by simply irradiating a portion of the surface of the substrate with a laser or plasma. Therefore, it is not necessary to perform other complicated steps or to prepare other components (masks, films, etc.) for such steps. Therefore, the cell culture substrate according to this embodiment can be easily produced.

[0026] The Ra of the bottom surface of the recess (region (A)) can be adjusted, for example, by appropriately setting the laser irradiation conditions (e.g., laser wavelength, output, pulse width, irradiation time, etc.) or plasma irradiation conditions (e.g., output, irradiation time, gas pressure, etc.). When the Ra of the surface of the recess serving as region (A) is within the above-mentioned range, region (A) becomes a region with excellent cell adhesiveness (capable of cell adhesion culture).

[0027] When forming recesses (regions (A)) in a cell culture substrate by irradiating with a laser, the laser used for irradiation may be a laser with a peak wavelength in the ultraviolet to infrared wavelength range. Specifically, the peak wavelength of the laser used for irradiation may be, for example, in the range of 100 nm to 1000 nm. Since this makes it easier to give the surface of the recesses a finely uneven shape (surface roughness) that allows cells to appropriately adhere, the peak wavelength of the laser used for irradiation may be in the range of 150 nm to 950 nm, 200 nm to 900 nm, 250 nm to 850 nm, 300 nm to 800 nm, 300 nm to 750 nm, 300 nm to 700 nm, or 350 nm to 550 nm.

[0028] The output power of the laser to be irradiated may be, for example, 0.1 W to 10 kW, 0.5 W to 1 kW, 1 W to 100 W, 2 W to 80 W, 3 W to 70 W, or 5 W to 50 kW.

[0029] The frequency of the irradiated laser may be, for example, 1 kHz to 2000 kHz, 10 kHz to 1000 kHz, 100 kHz to 500 kHz, 150 kHz to 400 kHz, 200 kHz to 350 kHz, or 250 kHz to 300 kHz.

[0030] The laser to be irradiated is preferably a pulsed laser. The pulse width may be, for example, 1000 picoseconds or less, preferably 1 picosecond to 100 picoseconds, more preferably 3 picoseconds to 100 picoseconds, and even more preferably 5 picoseconds to 50 picoseconds.

[0031] The laser used for irradiation may be, for example, a picosecond pulse laser, a YAG laser, a fiber laser, a green laser, or the like.

[0032] When forming recesses (region (A)) in the cell culture substrate by plasma irradiation, the current during plasma irradiation may be, for example, 10 mA to 1 A, 15 mA to 1000 mA, 20 mA to 100 mA, or 20 mA to 50 mA. The plasma irradiation time is preferably 5 seconds to 10 minutes, more preferably 10 seconds to 5 minutes, and most preferably 30 seconds to 3 minutes. The gas introduced during plasma treatment is preferably air, nitrogen, or oxygen, and more preferably oxygen. The gas pressure of the introduced gas is preferably 1 Pascal (Pa) to 50 Pascals, more preferably 1 Pascal to 30 Pascals, and even more preferably 1 Pascal to 25 Pascals. The plasma treatment may be performed, for example, by reactive ion etching (RIE). RIE is also called anisotropic etching.

[0033] The plasma to be irradiated may be, for example, a plasma device capable of localized irradiation (spot type) or a plasma device capable of other applications. Methods for forming the recessed portion (region (A)) of the cell culture substrate by irradiating with plasma include, for example, a method of irradiating the cell culture substrate with spot type plasma, or a method of irradiating the desired portion (unmasked portion) with plasma while the substrate is covered with a metal mask, a silicon mask, a surface protection film, or the like.

[0034] When forming the (A) region by laser irradiation or plasma irradiation, the Ra of the surface of the recess ((A) region) of the cell culture substrate can be increased by strengthening the treatment conditions, such as increasing the laser output or prolonging the treatment time.

[0035] The maximum depth of region (A) refers to the out-of-plane distance between the bottom surface of region (A) and the surface of region (B). The maximum depth of region (A) may be, for example, 1 nm or more and 500 nm or less. A maximum depth of region (A) of 500 nm or less can further reduce the number of dead cells trapped by the unevenness, thereby further reducing the number of dead cells mixed into the cell aggregate. This can further increase the cell viability of the cell aggregate. Furthermore, a maximum depth of region (A) of 500 nm or less further reduces the adhesion of air bubbles to the unevenness. Suppressing the adhesion of air bubbles eliminates the need for degassing or repeated dispensing and aspirating of the medium using a pipette to remove air bubbles, thereby further improving operability. A maximum depth of region (A) of 1 nm or more can more easily attract live cells that spontaneously migrate (migrate) on the cell culture substrate to region (A), thereby further increasing the cell viability of the cell aggregate. The maximum depth of region (A) is more preferably 400 nm or less, and even more preferably 350 nm or less, since this is suitable for further increasing the cell viability of the cell aggregates that are formed.

[0036] The substrate used for the cell culture substrate according to this embodiment is not particularly limited, but is preferably formed from at least one selected from the group consisting of polystyrene, polyethylene, polyethylene terephthalate, polycarbonate, cycloolefin polymer, cellulose acetate, nitrocellulose, and polyvinylidene fluoride, more preferably formed from at least one selected from the group consisting of polystyrene, polyethylene terephthalate, polycarbonate, and cycloolefin polymer, even more preferably formed from at least one selected from polystyrene, polyethylene terephthalate, and polycarbonate, and most preferably formed from polystyrene or polycarbonate.

[0037] Because the cell culture substrate is suitable for observing cells cultured on it using a high-magnification phase-contrast microscope, the refractive index of the substrate measured with D-line (wavelength 589 nm) is preferably 1.4 to 1.6, more preferably 1.45 to 1.6, and particularly preferably 1.5 to 1.55. Having the refractive index of the substrate within these ranges reduces spherical aberration during phase-contrast microscopic observation of cells, allowing for clear phase-contrast images to be obtained. Furthermore, to reduce spherical aberration, the thickness of the substrate is preferably 0.5 mm or less, more preferably 0.4 mm or less, particularly preferably 0.3 mm or less, and most preferably 0.2 mm or less. On the other hand, because the thickness of the substrate is suitable for preventing the entire observation area from being out of focus due to bending of the substrate during microscopic observation, the thickness of the substrate is preferably 0.01 mm or more, more preferably 0.05 mm or more, particularly preferably 0.1 mm or more, and most preferably 0.15 mm or more.

[0038] The thickness of the substrate is preferably 0.01 mm to 0.5 mm, more preferably 0.05 mm to 0.4 mm, even more preferably 0.1 mm to 0.3 mm, and particularly preferably 0.15 mm to 0.2 mm. When the thickness of the cell culture substrate is within this range, phase contrast images of cells become clearer.

[0039] Because the cell culture substrate is suitable for observing cells cultured on it under a high-magnification fluorescence microscope, the fluorescence intensity (autofluorescence intensity) of the substrate at excitation wavelengths of 350 nm, 488 nm, and 647 nm (when irradiated with excitation light having these wavelengths) is preferably smaller than the fluorescence intensity (autofluorescence intensity) of a 1.2 mm thick polystyrene plate excited with light having the same excitation wavelengths. It is more preferably 80% or less of the fluorescence intensity of a 1.2 mm thick polystyrene plate, particularly preferably 50% or less of the fluorescence intensity of a 1.2 mm thick polystyrene plate, and most preferably 10% or less of the fluorescence intensity of a 1.2 mm thick polystyrene plate. Fluorescent dyes excited at excitation wavelengths of 350 nm, 488 nm, and 647 nm are frequently used in fluorescent cell observation. By keeping the autofluorescence intensity of the substrate at these wavelengths below a certain value, clear fluorescent images of cells can be obtained.

[0040] The shape of the substrate is not particularly limited and may be a planar shape such as a plate or film, or may be in the shape of a fiber, porous particle, porous membrane, hollow fiber, etc. The shape of the substrate may also be the shape of a container generally used for cell culture (cell culture dish such as a Petri dish, flask, plate, bag, etc.). From the perspective of ease of culture operation, the shape of the cell culture substrate is preferably a planar shape such as a plate or film, or the shape of a flat porous membrane.

[0041] Region (A) consists of a recess formed in the cell culture substrate. Region (A) is recognized as an island-like region when the cell culture substrate is observed from the layer side where region (A) is present in a direction perpendicular to the substrate. In this case, the periphery of the island-like region corresponds to the boundary between region (A) and region (B). In this specification, the planar region defined by the boundary between region (A) and region (B) (i.e., the periphery of the island-like region) is sometimes referred to as an opening.

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

[0043] Since this is suitable for producing cell aggregates with a shape close to a sphere, the aspect ratio of the shape of the opening (the planar region defined by the boundary between region (A) and region (B)) is preferably 1 to 2, more preferably 1 to 1.5, even more preferably 1 to 1.1, and most preferably 1 to 1.05. Here, "aspect ratio" refers to the ratio of the maximum diameter (major diameter) to the minimum diameter (minor diameter) of the shape, i.e., major diameter / minor diameter.

[0044] Since this is suitable for producing cell aggregates of uniform size and shape, it is preferable that the standard deviation / average aspect ratio of the aspect ratio of the shape of the opening (the planar area defined by the boundary between area (A) and area (B)) is 80% or less, more preferably 50% or less, even more preferably 20% or less, and most preferably 5% or less.

[0045] The area of ​​the opening (the planar area defined by the boundary between the (A) and (B) areas) is, for example, 0.001 mm 2 More than 5mm 2 The area may be less than 0.005 mm 2 More than 1mm 2 The area of ​​0.01 mm or less is preferable. 2 More than 0.5mm 2 Less than 0.015mm is more preferable. 2 More than 0.25mm 2 More preferably, the area is 0.02 mm or less. 2 More than 0.2mm 2 The following are most preferred:

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

[0047] The shape of the bottom of region (A) may be the same as or different from the shape of the opening. The shape of the bottom of region (A) is not particularly limited and can be set appropriately depending on the shape of the desired cell aggregate, and examples thereof include an ellipse (including a circle), a polygon, or a closed shape formed by straight lines and curves. Furthermore, since this is suitable for producing cell aggregates with a shape close to a sphere, the shape of the opening is preferably an ellipse (including a circle) or a polygon, more preferably an ellipse (including a circle) or a rectangle, even more preferably an ellipse (including a circle) or a square, and most preferably an ellipse (including a circle).

[0048] The aspect ratio of the shape of the bottom surface of region (A) may be the same as or different from the aspect ratio of the shape of the opening. Because this is suitable for producing cell aggregates with a shape close to a sphere, the aspect ratio of the shape of the bottom surface of region (A) is preferably 1 to 2, more preferably 1 to 1.5, even more preferably 1 to 1.1, and most preferably 1 to 1.05.

[0049] Since this is suitable for producing cell aggregates of uniform size and shape, it is preferable that the standard deviation / average aspect ratio of the aspect ratio of the shape of the bottom surface of region (A) is 80% or less, more preferably 50% or less, even more preferably 20% or less, and most preferably 5% or less.

[0050] The area of ​​the bottom surface of the (A) region may be the same as or different from the area of ​​the opening. The area of ​​the bottom surface of the (A) region is, for example, 0.001 mm 2 More than 6mm 2 The area may be 0.001 mm or less. 2 More than 5mm 2 Less than 0.005mm is preferable 2 More than 1mm 2 Less than 0.01mm is more preferable. 2 More than 0.5mm 2 More preferably, the area is 0.015 mm or less. 2 More than 0.25mm 2 Even more preferably, the area is 0.02 mm or less. 2 More than 0.2mm 2 The following are most preferred:

[0051] Since this is suitable for producing cell aggregates of uniform size and shape, it is preferable that the standard deviation / average area of ​​the bottom surface of region (A) is 80% or less, more preferably 50% or less, even more preferably 20% or less, and most preferably 5% or less.

[0052] Furthermore, since this is suitable for increasing the oxygen concentration around the cells and increasing the survival rate of the cell aggregates, the minimum distance between the (A) regions is preferably 400 μm or more and 10,000 μm or less, more preferably 500 μm or more and 8,000 μm or less, even more preferably 1,000 μm or more and 5,000 μm or less, and most preferably 2,000 μm or more and 4,000 μm or less.

[0053] The region (A) may be subjected to a surface modification treatment. By performing the surface modification treatment, the cell adhesiveness of the region (A) can be improved. Examples of the surface modification treatment include plasma treatment, corona discharge treatment, and UV treatment.

[0054] The method for performing the surface modification treatment on the recessed portion that will become region (A) is not particularly limited, but examples include a method in which the surface of region (B) is covered with a silicon mask, a protective film, etc., and then the entire surface of the cell culture substrate is subjected to the surface modification treatment in this state. This allows the surface modification of only the desired portion (the uncovered surface of region (A)).

[0055] When the surface modification treatment includes a plasma treatment, the plasma treatment conditions may be, for example, as described above.

[0056] Region (A) may be temperature-responsive, as it is suitable for detaching cultured cell aggregates. When region (A) is temperature-responsive, the response temperature is preferably 50°C or lower, more preferably 35°C or lower, as this allows cells to be cultured at temperatures close to body temperature when culturing cells on the cell culture substrate. Furthermore, the response temperature is particularly preferably 25°C or lower, as this is suitable for preventing cells from detaching when performing operations such as changing the culture medium during culture. Furthermore, the response temperature is preferably 4°C or higher, more preferably 10°C or higher, and even more preferably 15°C or higher, as this allows cell aggregates to be formed by cooling operations at temperatures that do not damage the cells.

[0057] Region (B) is adjacent to region (A) and does not have cell adhesiveness or cell proliferation properties. If region (B) is adjacent to region (A) and does not have cell proliferation properties, when cells are cultured, it is possible to form cell aggregates only in region (A), and to create a state in which no cells are present around part or all of region (A). Furthermore, since this is suitable for uniformizing the size and shape of the cell aggregates produced, it is preferable that region (B) not only has no cell proliferation properties but also no cell adhesiveness.

[0058] The shape of region (B) is not limited other than being adjacent to region (A), but since this is suitable for producing cell aggregates of uniform size and shape, it is preferable that region (B) be adjacent to region (A) for at least 20% of its length, more preferably at least 50%, even more preferably at least 80%, and it is most preferable that region (A) is completely surrounded by region (B).

[0059] The area ratio of region (A) to region (B) is not particularly limited, but since this is suitable for increasing the number of cell aggregates that can be produced per unit area of ​​cell culture substrate, the area of ​​region (A) is preferably 10% or more, more preferably 30% or more, even more preferably 50% or more, and most preferably 70% or more of the total area of ​​region (A) and region (B). Furthermore, since this is suitable for providing a sufficient distance between multiple (A) regions and preventing the cell aggregates of multiple (A) regions from fusing together to form a non-uniform shape, the area of ​​region (B) is preferably 20% or more, more preferably 40% or more, even more preferably 60% or more, and most preferably 80% or more of the total area of ​​region (A) and region (B).

[0060] The cell culture substrate according to this embodiment may comprise a substrate and a layer (hydrophilic polymer layer) containing a hydrophilic polymer that covers at least a portion of the surface of the substrate. The substrate is as described above. The thickness of the hydrophilic polymer layer may be, for example, 10 nm to 500 nm. A hydrophilic polymer layer thickness of 10 nm to 500 nm facilitates cell adhesion and proliferation only in region (A), facilitates cell migration to region (A), and increases the cell viability of cell aggregates. Here, the "thickness" of the hydrophilic polymer layer refers to the out-of-plane length from the interface between the substrate and the layer containing the hydrophilic polymer to the interface of the hydrophilic polymer layer on the opposite side of the substrate (excluding region (A)). For thicknesses exceeding 10 nm, the distance can be calculated by measuring cross-sectional images of ultrathin sections of the cell culture substrate prepared using a microtome using a transmission electron microscope, measuring the distance at 10 randomly selected points, and averaging the distances. For thicknesses of 10 nm or less, the distance can be measured using an ellipsometer. The layer thickness is preferably 10 nm or more, and more preferably 50 nm or more, because it is suitable for inhibiting cell adhesion to region (B).Moreover, the layer thickness is preferably 200 nm or less, because it is suitable for increasing the cell viability of the cell aggregate by concentrating cells in region (A) through cell migration.

[0061] When the cell culture substrate according to this embodiment is provided with a hydrophilic polymer layer, as described above, at least a part or all of the surface of the recess (region (A)) in the cell culture substrate may be made of a hydrophilic polymer layer, and it is preferable that the entire surface of the recess (region (A)) in the cell culture substrate is made of a hydrophilic polymer layer.

[0062] The hydrophilic polymer contains a phosphorylcholine group or a hydroxyl group. By including a phosphorylcholine group or a hydroxyl group in the hydrophilic polymer, the area coated with the hydrophilic polymer can be made into an area to which cells do not adhere. Other than the inclusion of a phosphorylcholine group or a hydroxyl group, the type of hydrophilic polymer is not particularly limited. Examples of commercially available hydrophilic polymers include Lipidure® CM5206 (manufactured by NOF Corporation), Lipidure® CM2001 (manufactured by NOF Corporation), and BIOSURFINE®-AWP (manufactured by Toyo Gosei Co., Ltd.). Examples of commercially available substrates coated with a hydrophilic polymer include PrimeSurface® (manufactured by Sumitomo Bakelite Co., Ltd.), EZ-BindShut® (manufactured by AGC Technoglass Co., Ltd.), and EZ-BindShutII® (manufactured by AGC Technoglass Co., Ltd.).

[0063] 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).

[0064] [ka] [In general formula (1), R 1 and R 2 each independently represents a hydrogen atom or a methyl group, and R 3 represents a hydrogen atom or an arbitrary organic group, and m and n each independently represent a positive integer.

[0065] [ka] [In general formula (2), R 4 , R 5 and R 6 each independently represents a hydrogen atom or a methyl group, and R 7 represents a hydrogen atom or an arbitrary organic group, and x, y, and z each independently represent a positive integer.

[0066] [ka] [In general formula (3), R 8 R and 9 each independently represents a hydrogen atom or a methyl group, and R 10 represents a hydrogen atom or an arbitrary organic group, and a and b each independently represent a positive integer.

[0067] When the hydrophilic polymer contains a compound represented by the general formula (1), a compound represented by the general formula (2), or a compound represented by the general formula (3), cells can be easily attached and proliferated, and it is suitable for forming cell aggregates of uniform shape in the (A) region. 3 , R 7 , and R 10 are suitable for immobilizing hydrophilic polymers on substrates, and therefore are preferably hydrophobic groups or functional groups reactive to active energy rays (e.g., UV, electron beams, etc.). As the hydrophobic group, linear or cyclic alkyl groups such as methyl, ethyl, propyl, butyl, and cyclohexyl groups can be suitably used. Furthermore, as functional groups reactive to active energy rays (e.g., UV, electron beams, etc.), azide groups, acrylate groups, methacrylate groups, vinyl groups, and epoxy groups can be used, with azide groups being suitably used.

[0068] The method for forming the hydrophilic polymer layer is not particularly limited, and examples thereof include a method of forming the hydrophilic polymer layer by applying a composition containing a hydrophilic polymer to at least a portion of the surface of region (B). Examples of methods for applying the composition containing a hydrophilic polymer include various commonly known methods, such as painting, brush coating, dip coating, spin coating, bar coating, flow coating, spray coating, roll coating, air knife coating, blade coating, gravure coating, microgravure coating, and slot die coating. Another example includes a method in which at least a portion of the surface of the substrate is coated with a composition containing a hydrophilic polymer, and then a portion of the surface of the hydrophilic polymer layer is irradiated with a laser, plasma, or the like.

[0069] The hydrophilic polymer layer may be fixed to the surface of the substrate. When the hydrophilic polymer is reactive to active energy rays, the hydrophilic polymer layer may be irradiated with active energy rays to fix the hydrophilic polymer layer to the surface of the substrate. Examples of hydrophilic polymers reactive to active energy rays include the above-mentioned hydrophilic polymers having functional groups reactive to active energy rays (e.g., UV, electron beams, etc.). Examples of active energy rays include UV, electron beams, etc.

[0070] Irradiating a hydrophilic polymer that is reactive to active energy rays with active energy rays causes a chemical reaction between the hydrophilic polymers or between the hydrophilic polymer and the substrate, resulting in immobilization of a hydrophilic polymer layer on the surface of the substrate. Immobilization of the hydrophilic polymer layer on the substrate surface allows the formation of a layer containing a temperature-responsive polymer, as described below, without deforming the hydrophilic polymer layer. Furthermore, immobilization of the hydrophilic polymer layer on the substrate surface also allows the shape of region (A) formed on the cell culture substrate, as described below, to be maintained.

[0071] When region (A) is temperature-responsive, for example, the surface of the cell culture substrate (the surface including regions (A) and (B)) may further comprise a layer containing a temperature-responsive polymer and having a layer thickness of 1 nm to 100 nm. By providing a layer containing a temperature-responsive polymer with a layer thickness of 1 nm to 100 nm, temperature responsiveness can be imparted to region (A) without impairing the respective properties of regions (A) and (B) formed on the surface of the cell culture substrate. To be suitable for imparting temperature responsiveness to region (A) without impairing the respective properties of regions (A) and (B), the layer thickness of the layer containing the temperature-responsive polymer is preferably 3 nm to 50 nm, more preferably 5 nm to 40 nm, and most preferably 10 nm to 35 nm. The layer thickness of the temperature-responsive polymer suitable for imparting temperature responsiveness to region (A) without impairing cell proliferation and for enabling cells to be detached and recovered by temperature responsiveness after culture varies depending on the cells to be cultured and can be appropriately adjusted within the range of layer thickness exemplified above.

[0072] The temperature-responsive polymer is preferably a block copolymer having a water-insoluble block segment and a temperature-responsive block segment. When the temperature-responsive polymer is such a block copolymer, mass productivity of the cell culture substrate is improved and contamination of the produced cell aggregates with the temperature-responsive polymer can be suppressed. The ratio of the constituent units of the temperature-responsive block segment contained in the temperature-responsive polymer is preferably 70 wt% or more, more preferably 80 wt% or more, particularly preferably 90 wt% or more, and most preferably 92 wt% or more, because this is suitable for rapid detachment of cell aggregates from the cell culture substrate.

[0073] Furthermore, since the temperature-responsive polymer is a block copolymer having a water-insoluble block segment and a temperature-responsive block segment, temperature responsiveness can be imparted to the surface of the cell culture substrate by the simple method of dropping a solution containing the temperature-responsive polymer onto the surface of the cell culture substrate and drying it. Furthermore, since the layer formed in this manner has the preferred thickness of the temperature-responsive polymer described above, even if the entire surface of the hydrophilic polymer layer is coated with the temperature-responsive polymer, the properties of the above-mentioned regions (A) and (B) are less impaired.

[0074] Examples of the monomer unit constituting the temperature-responsive block segment include (meth)acrylamide compounds such as acrylamide and methacrylamide; N-alkyl-substituted (meth)acrylamide derivatives such as N,N-diethylacrylamide, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, Nt-butylacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, and N-tetrahydrofurfurylmethacrylamide; N,N-dialkyl-substituted (meth)acrylamide derivatives such as N,N-dimethyl(meth)acrylamide, N,N-ethylmethylacrylamide, and N,N-diethylacrylamide; 1-(1-oxo-2-propenyl)-pyrrolidine, 1-(1-oxo-2-propenyl)-pyrrolidine, and the like. Examples of the cyclic acrylamide derivatives include (meth)acrylamide derivatives having a cyclic group such as 1-(1-oxo-2-methyl-2-propenyl)-piperidine, 4-(1-oxo-2-methyl-2-propenyl)-morpholine, 1-(1-oxo-2-methyl-2-propenyl)-pyrrolidine, 1-(1-oxo-2-methyl-2-propenyl)-piperidine, and 4-(1-oxo-2-methyl-2-propenyl)-morpholine; vinyl ethers such as methyl vinyl ether; and proline derivatives such as N-proline methyl ester acrylamide. Since they are suitable for setting the response temperature to 0 to 50°C, N,N-diethylacrylamide, Nn-propylacrylamide, N-isopropylacrylamide, Nn-propylmethacrylamide, N-ethoxyethylacrylamide, N-tetrahydrofurfurylacrylamide, and N-tetrahydrofurfurylmethacrylamide are preferred, Nn-propylacrylamide and N-isopropylacrylamide are more preferred, and N-isopropylacrylamide is particularly preferred. Furthermore, when a room temperature medium is used during medium replacement in a culture operation, Nn-propylacrylamide and N-proline methyl ester acrylamide are preferred because they are suitable for setting the response temperature of the block copolymer to a temperature lower than room temperature.

[0075] Examples of monomer units constituting the water-insoluble block segment include n-butyl acrylate, n-butyl methacrylate, isobutyl acrylate, isobutyl methacrylate, t-butyl acrylate, t-butyl methacrylate, n-hexyl acrylate, n-hexyl methacrylate, n-octyl acrylate, n-octyl methacrylate, n-decyl acrylate, n-decyl methacrylate, n-dodecyl acrylate, n-dodecyl methacrylate, n-tetradecyl acrylate, and n-tetradecyl methacrylate. Furthermore, those having a reactive group are preferred because they are suitable for firmly immobilizing the block copolymer on a substrate, and examples thereof include 4-azidophenyl acrylate, 4-azidophenyl methacrylate, 2-((4-azidobenzoyl)oxy)ethyl acrylate, and 2-((4-azidobenzoyl)oxy)ethyl methacrylate. Furthermore, structures having an aromatic ring are preferred because they are suitable for enhancing cell proliferation, and examples thereof include 2-hydroxyphenyl acrylate, 2-hydroxyphenyl methacrylate, 3-hydroxyphenyl acrylate, 3-hydroxyphenyl methacrylate, 4-hydroxyphenyl acrylate, 4-hydroxyphenyl methacrylate, N-(2-hydroxyphenyl)acrylamide, N-(2-hydroxyphenyl)methacrylamide, N-(3-hydroxyphenyl)acrylamide, N-(3-hydroxyphenyl)methacrylamide, N-(4-hydroxyphenyl)acrylamide, N-(4-hydroxyphenyl)methacrylamide, and styrene.

[0076] The water-insoluble block segment may also contain a repeating unit that controls the response temperature of the block copolymer. Examples of the repeating unit that controls the response temperature of the block copolymer include hydrophilic or hydrophobic components, and are not particularly limited to those having an amino group, such as 2-dimethylaminoethyl acrylate, 2-dimethylaminoethyl methacrylate, 2-diethylaminoethyl acrylate, 2-diethylaminoethyl methacrylate, and N-[3-(dimethylamino)propyl]acrylamide; those having a betaine, such as N-(3-sulfopropyl)-N-methacryloyloxyethyl-N,N-dimethylammonium betaine and N-methacryloyloxyethyl-N,N-dimethylammonium-α-N-methylcarboxybetaine; hydroxyethyl acrylate, hydroxyethyl methacrylate, N-(2-hydroxyethyl)acrylamide, polyethylene glycol monoacrylate, polyethylene glycol monomethacrylate, polypropylene glycol monoacrylate, polypropylene glycol monomethacrylate, methoxypolyethylene glycol monoacrylate, methoxypolyethylene glycol monoacrylate, methoxypolyethylene glycol monoacrylate, methoxypolyethylene glycol monoacrylate, hydroxyethyl acrylate, hydroxyethyl methacrylate, N-(2-hydroxyethyl)acrylamide, polyethylene glycol monoacrylate, polyethylene glycol monomethacrylate, polypropylene glycol monoacrylate, polypropylene glycol monometh ... those having a polyethylene glycol group or a methoxyethyl group, such as polyethylene glycol monomethacrylate, diethylene glycol monomethyl ether acrylate, diethylene glycol monomethyl ether methacrylate, diethylene glycol monoethyl ether acrylate, diethylene glycol monoethyl ether methacrylate, 2-methoxyethyl acrylate, 2-methoxyethyl methacrylate, 2-ethoxyethyl acrylate, 2-ethoxyethyl methacrylate, 3-butoxyethyl acrylate, 3-butoxyethyl methacrylate, 3-butoxyethyl acrylamide, furfuryl acrylate, furfuryl methacrylate, tetrahydrofurfuryl acrylate, and tetrahydrofurfuryl methacrylate; those having an acrylate group, such as methoxymethyl acrylate, methoxymethyl methacrylate, 2-ethoxymethyl acrylate, 2-ethoxymethyl methacrylate, 3-butoxymethyl acrylate, 3-butoxymethyl methacrylate, and 3-butoxymethyl acrylamide;Examples of phosphorylcholine groups include 2-methacryloyloxyethyl phosphorylcholine, 2-acryloyloxyethyl phosphorylcholine, 3-(meth)acryloyloxypropyl phosphorylcholine, 4-(meth)acryloyloxybutyl phosphorylcholine, 6-(meth)acryloyloxyhexyl phosphorylcholine, 10-(meth)acryloyloxydecyl phosphorylcholine, ω-(meth)acryloyl(poly)oxyethylene phosphorylcholine, 2-acrylamidoethyl phosphorylcholine, 3-acrylamidopropyl phosphorylcholine, 4-acrylamidobutyl phosphorylcholine, 6-acrylamidohexyl phosphorylcholine, 10-acrylamidodecyl phosphorylcholine, and ω-(meth)acrylamido(poly)oxyethylene phosphorylcholine;

[0077] As a method for coating with a composition containing a temperature-responsive polymer, the same method as the above-mentioned method for applying a composition containing a hydrophilic polymer can be suitably used.

[0078] The cell culture substrate may optionally have a layer containing a biologically derived substance on its surface. The layer containing the biologically derived substance may be present on the entire surface of the cell culture substrate, or may be present only on the surface of region (A). The biologically derived substance is not particularly limited, but examples include matrigel, laminin, fibronectin, vitronectin, collagen, etc.

[0079] These biological substances may be natural products, or may be artificially synthesized using genetic engineering techniques, or may be fragments obtained by cleavage with restriction enzymes, or synthetic proteins or peptides obtained by chemically synthesizing substances equivalent to these biological substances.

[0080] As Matrigel, commercially available products such as Matrigel (manufactured by Corning Incorporated) and Geltrex (manufactured by Thermo Fisher Scientific) can be suitably used due to their ease of availability.

[0081] The type of laminin is not particularly limited, but examples that can be used include laminin 511, laminin 521, and laminin 511-E8 fragment, which have been reported to exhibit high activity against α6β1 integrin expressed on the surface of human iPS cells. Laminin may be a natural product, artificially synthesized using genetic engineering techniques, or a synthetic protein or peptide obtained by chemically synthesizing a substance equivalent to laminin. Commercially available products such as iMatrix-511 (manufactured by Nippi Corporation) are suitable for use due to their ease of availability.

[0082] Vitronectin may be a natural product, artificially synthesized using genetic recombination technology, or a synthetic protein or peptide obtained by chemically synthesizing a substance equivalent to vitronectin. Commercially available products such as human plasma-derived vitronectin (manufactured by Wako Pure Chemical Industries, Ltd.), Synthemax (manufactured by Corning Incorporated), and Vitronectin (VTN-N) (manufactured by Thermo Fisher Scientific) are suitable for use due to their availability.

[0083] Fibronectin may be a natural product, artificially synthesized by genetic recombination technology, or a synthetic protein or peptide obtained by chemically synthesizing a substance equivalent to fibronectin. In view of ease of availability, commercially available products such as fibronectin solution, human plasma-derived fibronectin (manufactured by Wako Pure Chemical Industries, Ltd.), and Retronectin (manufactured by Takara Bio Inc.) can be preferably used.

[0084] The type of collagen is not particularly limited, and examples thereof include type I collagen and type IV collagen. Collagen may be a natural product, artificially synthesized using genetic engineering techniques, or a synthetic peptide obtained by chemically synthesizing a substance equivalent to collagen. Commercially available products such as collagen I, human (manufactured by Corning Incorporated) and collagen IV, human (manufactured by Corning Incorporated) are suitable for use due to their ease of availability.

[0085] From the viewpoint of suppressing denaturation of the biological substance and enhancing cell proliferation, the biological substance is preferably immobilized on the cell culture substrate by a non-covalent bond. Here, "non-covalent bond" refers to a bonding force other than a covalent bond derived from intermolecular forces, such as electrostatic interaction, water-insoluble interaction, hydrogen bond, π-π interaction, dipole-dipole interaction, London dispersion force, or other van der Waals interaction. The biological substance may be immobilized on the block copolymer by a single bonding force or a combination of multiple bonding forces.

[0086] The method for immobilizing biological substances is not particularly limited, but suitable methods include, for example, applying a solution of the biological substance to a cell culture substrate for a predetermined period of time to immobilize the substance, and adding the biological substance to the culture medium when culturing cells to allow the biological substance to be adsorbed onto the cell culture substrate and immobilized.

[0087] The cell culture substrate according to this embodiment may be provided with a partition plate (for example, a partition plate having a cross-sectional area of ​​0.05 cm2 in the in-plane direction) on the substrate, if necessary. 2 More than 100cm 2 A structure for separating each cell aggregate may be provided by providing a partition plate (a plate having a through-hole as described below) on the substrate. FIG. 2 is a schematic diagram (perspective view) of a cell culture substrate in which a partition plate is provided on the substrate. The cell culture substrate 11 shown in FIG. 2 is, for example, a cell culture substrate 10 shown in FIG. 1 with a partition plate 20 (with a cross-sectional area in the in-plane direction of 0.05 cm2). 2 More than 100cm 2The cross-sectional area in the in-plane direction is 0.05 cm 2 More than 100cm 2 The plate having the following through-holes may be, for example, a partition member having a plurality of cylindrical partition walls that can partition the surface on the side where cells are cultured. 2 More than 100cm 2 The side walls of the following through-holes function as cylindrical partition walls.

[0088] The cell culture substrate according to this embodiment may be sterilized. There are no particular limitations on the sterilization method, but high-pressure steam sterilization, UV sterilization, gamma-ray sterilization, ethylene oxide gas sterilization, etc. can be used. From the viewpoint of suppressing denaturation of the block copolymer, high-pressure steam sterilization, UV sterilization, and ethylene oxide gas sterilization are preferred. From the viewpoint of suppressing deformation of the substrate, UV sterilization or ethylene oxide gas sterilization are more preferred. From the viewpoint of excellent mass productivity, ethylene oxide gas sterilization is preferred.

[0089] The cells to be cultured using the cell culture substrate according to this embodiment are not particularly limited as long as they can adhere to the surface before the temperature drop stimulus. Examples include various established cell lines such as Chinese hamster ovary-derived CHO cells, mouse connective tissue L929 cells, human embryonic kidney-derived HEK293 cells, and human cervical cancer-derived HeLa cells, as well as epithelial cells and endothelial cells that constitute various tissues and organs in the body, contractile skeletal muscle cells, smooth muscle cells, and cardiac muscle cells, neuronal cells, glial cells, and fibroblasts that constitute the nervous system, hepatic parenchymal cells, non-hepatic parenchymal cells, and adipocytes that are involved in the metabolism of the body, as well as differentiation-competent stem cells present in various tissues such as mesenchymal stem cells, bone marrow cells, and Muse cells, as well as pluripotent stem cells (pluripotent stem cells) such as ES cells and iPS cells, and cells induced to differentiate therefrom.

[0090] The cell culture kit according to this embodiment includes a cell culture substrate, which may be the cell culture substrate according to the present invention described above, provided with a partition member having a plurality of cylindrical partition walls that can partition the surface on which cells are cultured.

[0091] The cell culture kit according to this embodiment may include a temperature-responsive polymer or a coating agent containing a temperature-responsive polymer in addition to the cell culture substrate, which allows researchers performing culture to easily adjust the layer thickness of the temperature-responsive polymer depending on the type of cells.

[0092] The coating agent may contain a solvent. Examples of solvents that can be contained in the coating agent include water, organic solvents, and mixtures thereof. Examples of organic solvents include alcohols such as methanol, ethanol, 1-propanol, 2-propanol, and 1-butanol; acetonitrile, formamide, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, 1,4-dioxane, and methyl ethyl ketone. A mixed solvent of water and alcohols is preferably used because it is suitable for achieving a uniform coating thickness. The content of the temperature-responsive polymer, based on the total mass of the coating agent, can be 0.1 to 50 wt %, 0.2 to 10 wt %, or 0.5 to 5 wt %.

[0093] The coating agent may contain other components in addition to the temperature-responsive polymer and the solvent. Examples of other components include components for enhancing cell adhesion, such as a polymer consisting only of a water-insoluble block segment. [Example]

[0094] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0095] <Preparation of cell culture substrate> [Example 1] A 0.9 mL solution of 80 wt% ethanol containing 0.6 wt% azide-containing polyvinyl alcohol (BIOSURFINE®-AWP, Toyo Gosei Co., Ltd.) as a hydrophilic polymer was applied to a polycarbonate film (Panlite®, Teijin Limited, 0.18 mm thick) (PC substrate). The film was then spin-coated at 2000 rpm for 60 seconds using a spin coater (MS-B150, Mikasa Co., Ltd.). The film was then left under a high-pressure mercury lamp for 1 hour to cure the hydrophilic polymer via UV irradiation, forming a 75 nm thick hydrophilic polymer layer. A metal mask with 0.1 mm diameter circular holes (spots) (800 μm center-to-center spacing) was placed on the hydrophilic polymer layer. Plasma treatment was then performed on the metal mask using a plasma irradiation system (PIB-20, Vacuum Device Co., Ltd.) to form region (A). In addition, region (B) was formed in the area masked with a metal mask. The plasma treatment conditions are shown in Table 1.

[0096] [Examples 2 to 5] A cell culture substrate was prepared in the same manner as in Example 1, except that the metal mask was changed to one having a patterning diameter shown in Table 1, and the plasma treatment conditions were changed as shown in Table 1.

[0097] [Example 6] A cell culture substrate was prepared in the same manner as in Example 1, except that in the plasma treatment, the conditions for picosecond pulse laser irradiation were changed as shown in Table 1, and that the laser was irradiated from above the hydrophilic polymer layer without using a metal mask, thereby forming region (A) in the laser-irradiated area and region (B) in the non-laser-irradiated area.

[0098] [Comparative Example 1] A cell culture substrate was prepared in the same manner as in Example 1, except that the metal mask was changed to one having a patterning diameter shown in Table 1, and the plasma treatment conditions were changed as shown in Table 1.

[0099] Comparative Example 2 Except for not carrying out the plasma treatment, a cell culture substrate was produced in the same manner as in Example 1. Note that, in producing the cell culture substrate of Comparative Example 2, no metal mask was used.

[0100] <Evaluation of cell culture substrate> ((A) Evaluation of the surface shape of the area) The surface shape of region (A) in the cell culture substrates of Examples 1 to 6 and Comparative Examples 1 and 2 was evaluated by observation using an atomic force microscope (Hitachi High-Tech Corporation, product name: AFM5100N). The results are shown in Table 1 and Figures 3 to 6.

[0101] It was confirmed that fine irregularities were formed (wrinkled shape) on the bottom surfaces of region (A) in the cell culture substrates of Examples 1 to 6 (Table 1, and Figures 3(A), 3(B), 4(A), 4(B), 5(A), and 5(B) respectively). On the other hand, as shown in Table 1 and Figure 6, no fine irregularities were confirmed on the bottom surfaces of region (A) in the cell culture substrates of Comparative Examples 1 and 2 (Comparative Example 1: Table 1 and Figure 6(A); Comparative Example 2: Table 1 and Figure 6(B)).

[0102] ((A) Measurement of the average surface roughness Ra of the bottom surface of the region) (A) The average surface roughness Ra of the bottom surface of the area was measured using a laser microscope (product name: OPTELICS HYBRID + The surface roughness curve was obtained using a laser spectrometry (Lasertec Corporation) and calculated using the above formula (1). The results are shown in Table 1.

[0103] As shown in Table 1, the Ra of the cell culture substrates of Comparative Examples 1 and 2 was 0.0006 μm and 0.0003 μm, respectively. The Ra of the cell culture substrates of Examples 1 to 6 was 0.0031 μm, 0.0060 μm, 0.0047 μm, 0.0069 μm, 0.0035 μm, and 0.38 μm, respectively, all of which were 0.001 μm or greater.

[0104] (Cell culture evaluation) The cell culture substrate was attached to the bottom of a bottomless 6-well plate, which was then sterilized and used as a culture vessel. Human iPS cell line 201B7 was used, with a density of 15,000 cells / cm. 2 Cells were seeded in area (A) so that the cells were oriented in a uniform direction. They were cultured in AK02N medium (Ajinomoto Co., Inc.) (2 mL / well) at 37°C and 5% CO2. Until 24 hours after seeding, Y-27632 (Wako Pure Chemical Industries, Ltd.) (10 μM) and a cell culture protein substrate (Matrixome Co., Ltd., product name: iMatrix-511) (1.25 μg / mL) were added to the medium. The medium was changed 1 and 3 days after the initiation of culture. Five days after the initiation of culture, cell nuclei were stained with DAPI, and the cell adhesion to the cell culture substrate (whether or not the cells could be cultured) was evaluated using a fluorescence microscope (Keyence Corporation, product name: BZ-X810) and a phase-contrast microscope (Olympus Corporation, product name: IX73). The results are shown in Table 1 and Figures 7 to 9.

[0105] As shown in Table 1 and FIG. 9, in the cell culture substrates of Comparative Examples 1 and 2, cells did not adhere to region (A), and culture was not possible (Comparative Example 1: Table 1 and FIG. 9(A) ; Comparative Example 2: Table 1 and FIG. 9(B) ). On the other hand, as shown in Table 1 and FIG. 7 , in the cell culture substrates of Examples 1 to 6, cells adhered to region (A), and it was confirmed that cells could be cultured (Examples 1 to 5: Table 1 and FIGS. 7(A) to 7(C) ; Example 6: Table 1 ). FIG. 8 shows images of region (A) of the cell culture substrate of Example 6 observed with a phase contrast microscope ( FIG. 8(A) ) and a fluorescence microscope ( FIG. 8(B) ). As shown in FIG. 8 , not only was it confirmed that cells stained with DAPI were present in region (A) of the cell culture substrate of Example 6, but similar results were also obtained in region (A) of the other cell culture substrates of Examples 1 to 5.

[0106] [Table 1]

[0107] From the above results, it is considered that a cell culture substrate having an Ra of 0.001 μm or more on the bottom surface of region (A) has a rough bottom surface of region (A) and fine irregularities are formed on the surface, increasing the surface area, and therefore has excellent cell adhesion properties (enabling adhesive culture). [Explanation of symbols]

[0108] A...area (A), B...area (B), H...maximum depth of area (A), 1...substrate, 10, 11...cell culture substrate, 20...partition plate.

Claims

1. A cell culture substrate, comprising: It has the following region (A) and the following region (B), (A) Region having cell adhesive properties and cell proliferation properties (B) a region adjacent to the region (A) and not having cell adhesiveness or cell proliferation properties the region (A) is a recess formed in the cell culture substrate, The cell culture substrate, wherein the arithmetic mean roughness (Ra) of the bottom surface of the recess is 0.001 μm or more.

2. The cell culture substrate according to claim 1 , comprising a substrate and a layer containing a hydrophilic polymer that covers at least a portion of the surface of the substrate.

3. The cell culture substrate according to claim 2 , wherein the entire surface of the well is covered with a layer containing the hydrophilic polymer.

4. The cell culture substrate according to claim 1 or 2, wherein the Ra is 0.3 μm or more.

5. The cell culture substrate according to claim 1 or 2, wherein the bottom surface of the recess has a wrinkled surface when viewed from above.

6. A cell culture kit comprising a cell culture substrate, 3. A cell culture kit, comprising the cell culture substrate according to claim 1 or 2, and a partition member having a plurality of cylindrical partition walls capable of partitioning the surface on which cells are cultured.

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