Cell culture substrate, method for producing cell culture substrate, and method for producing spheroids

Abstract

To provide a cell culture substrate capable of efficiently forming spheroids of cells, capable of forming a uniform size and optional shape of spheroids with high survival rate of cells, a production method of a cell culture substrate, and a spheroid production method excellent in cell survival rate of cell in the spheroid using the cell culture substrate.SOLUTION: There is provided a cell culture substrate comprising a substrate and a stimulation responsive polymer which covers the substrate, the stimulation responsive polymer is a block copolymer comprising a water insoluble block segment and the stimulation responsive block segment, and the cell culture substrate comprises following two regions (A) and (B), in which the region (A) is an island-shaped region whose area is 0.001-5 mmand which has cell growth property and stimulation responsiveness, and the region (B) is a region which is adjacent to the region (A) and has no cell growth property.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a cell culture substrate that enables efficient spheroid formation and can form spheroids of uniform size and arbitrary shape, a method for producing the cell culture substrate, and a method for producing spheroids using the cell culture substrate. [Background technology]

[0002] Pluripotent stem cells, such as embryonic cells (ES cells) and artificial cells (iPS cells), are cells that possess the ability to differentiate into various tissues in the body (pluripotency), and are attracting significant attention as a cell source for regenerative medicine and drug discovery screening. To apply pluripotent stem cells to regenerative medicine and drug discovery screening, it is necessary to differentiate them into the target cells, which requires the formation of spheroids (embryoid bodies) of the pluripotent stem cells. Furthermore, although pluripotent stem cells can differentiate into various cell types, it is known that the optimal spheroid size differs depending on the type of cell after differentiation, and it is desirable to control the size and create spheroids of uniform size.

[0003] Conventionally, a method for forming spheroids is known in which pluripotent stem cells spontaneously form aggregates by using a substrate to which they do not adhere (see, for example, Patent Document 1). Although this method is excellent for mass production of spheroids, it has the problem that it is not possible to obtain spheroids of uniform size.

[0004] A known method for forming spheroids of uniform size is to use a cell culture substrate with fine irregularities on its surface (see, for example, Patent Document 2). However, such cell culture substrates with fine irregularities have poor mass production capabilities and are unsuitable for applications involving the formation of large quantities of spheroids. Furthermore, the spheroids that can be formed are limited to a spherical shape, and it is not possible to form spheroids of any other shape. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 8-140673 [Patent Document 2] Japanese Patent Publication No. 2015-073520 [Overview of the project] [Problems that the invention aims to solve]

[0006] The object of the present invention is to provide a cell culture substrate that enables efficient spheroid formation of cells and can form spheroids of uniform size and arbitrary shape, a method for producing the cell culture substrate, and a method for producing spheroids using the cell culture substrate. [Means for solving the problem]

[0007] In light of the above points, the inventors conducted extensive research and, as a result, discovered that the above problems can be solved by a substrate having a region with specific stimulus responsiveness, thus completing the present invention.

[0008] In other words, the present invention consists of the following [1] to

[12] . [1] A cell culture substrate comprising a base material and a stimulus-responsive polymer coated on the base material, wherein the stimulus-responsive polymer is a block copolymer having a water-insoluble block segment and a stimulus-responsive block segment, and the cell culture substrate is characterized in that it has the following two regions (A) and (B). (A) Possesses cell proliferation and stimulus responsiveness, with an area of ​​0.001-5 mm² 2 An island-like region. (B) A region adjacent to the region (A) above that does not have cell proliferation properties. [2] The cell culture substrate according to [1], characterized in that the (A) region consists of the following two regions (A1) and (A2), and the (B) region consists of the following two regions (B1) and (B2). (A1) Cell proliferation zone (A2) Stimulation-responsive region (B1) Substrate adhesion region (B2) Stimulation-responsive region [3] The cell culture substrate according to [1] or [2], wherein the (A) region is formed by dispersing temperature-responsive regions having a diameter of 10 to 500 nm in a cell-proliferative region, or by dispersing cell-proliferative regions having a diameter of 10 to 500 nm in a temperature-responsive region having no cell proliferation property. [4] A cell culture substrate having a substrate having two regions, a cell-proliferative region and a region having no cell proliferation property, and a layer containing a stimulation-responsive polymer formed on the substrate, wherein the ratio of the average roughness of the layer to the layer thickness is 0.5 or more and 1 or less, according to any one of [1] to [3] above. [5] The cell culture substrate according to [4] above, wherein the stimulation-responsive polymer is a block copolymer having a water-insoluble block segment and a stimulation-responsive block segment exceeding 90 wt%. [6] The cell culture substrate according to any one of [1] to [5] above, wherein the (A) region is a plasma-treated region. [7] The cell culture substrate according to any one of [1] to [6] above, which is for forming spheroids of pluripotent stem cells. [8] A method for manufacturing the cell culture substrate according to any one of [1] to [7] above, characterized by having the following steps (1) and (2). (1) A step of forming island-shaped regions having a cell proliferation property and an area of 0.001 to 5 mm on the surface of a substrate having no cell proliferation property. 2 (2) A step of forming a layer of a stimulation-responsive substance on the surface of the substrate. [9] In the step (1), a region having cell proliferation property is formed on the surface of the substrate having no cell proliferation property by any one of plasma treatment, UV treatment, corona treatment, or a combination of a plurality of these, according to the method for manufacturing the cell culture substrate according to [8] above.

[10] A method for producing a cell culture substrate according to [8] or [9], characterized in that the substrate used in step (1) has cell adhesion properties but does not have cell proliferation properties.

[11] The above (1) step is performed on an area of ​​0.001 to 10 mm 2 A method for producing a cell culture substrate according to [9] or

[10] , characterized by comprising the step of attaching a surface protective film having holes to a substrate.

[12] A method for producing spheroids, characterized by following the steps (i) to (iii) below. (i) A step of seeding cells onto a cell culture substrate according to any of the above [1] to [8]. (ii) A step of culturing the seeded cells and forming colonies of cells attached to the cell culture substrate. (iii) A step of detaching at least a portion of the colony from the cell culture substrate by applying an external stimulus to form a cell spheroid. [Effects of the Invention]

[0009] The present invention provides a cell culture substrate that enables efficient spheroid formation of cells, has a high cell viability rate, and can form spheroids of uniform size and arbitrary shape, as well as a method for producing the cell culture substrate and a method for producing spheroids with excellent cell viability inside the spheroids using the cell culture substrate. [Brief explanation of the drawing]

[0010] [Figure 1] A schematic diagram (perspective view) of the cell culture substrate of the present invention. [Figure 2] Schematic diagram (cross-sectional view) of a cell culture substrate having a layer of stimulus-responsive polymer. [Figure 3] A schematic diagram showing the state of cells after step (i) of the spheroid production method of the present invention. [Figure 4] A schematic diagram showing the state of cells after step (ii) of the spheroid manufacturing method of the present invention. [Figure 5] A schematic diagram showing the state of cells after step (iii) of the spheroid production method of the present invention. [Figure 6] Phase-contrast microscope image showing the spheroid from Example 1. [Figure 7] Phase-contrast microscope image showing colonies from Example 2. [Figure 8] Phase-contrast microscope image showing the spheroid from Example 2. [Figure 9] Phase-contrast microscope image showing the spheroid from Example 4. [Figure 10] Phase-contrast microscope image showing the spheroid from Example 5. [Figure 11] Atomic force microscope image of the cell culture substrate from Example 7. [Figure 12] Atomic force microscope image of the cell culture substrate from Example 8. [Figure 13] Phase-contrast microscope image showing the spheroid from Example 8. [Modes for carrying out the invention]

[0011] The following describes in detail embodiments for carrying out the present invention (hereinafter simply referred to as "these embodiments"). These embodiments are illustrative for explaining the present invention and are not intended to limit the present invention to the following content. The present invention can be appropriately modified and implemented within the scope of its spirit.

[0012] In this specification, "spheroid" refers to a three-dimensional aggregate of cells formed by the aggregation of multiple cells, excluding cell sheets formed when cells are adhered to a substrate. A spherical shape is preferred, but a spherical shape with gaps formed by the folding of sheet-like cells or a hollow shape is also acceptable. Furthermore, in this specification, "colony" refers to a cell sheet-like group of cells formed when cells are adhered to a substrate.

[0013] Furthermore, in this specification, "stimulus responsiveness" refers to a change in structure or degree of hydrophilicity / hydrophobicity in response to external stimuli. Here, "external stimuli" in this specification refers to mechanical stimuli such as ultrasound, vibration, and convection; electromagnetic stimuli such as light, electricity, and magnetism; and thermodynamic stimuli such as heating and cooling, but excludes biological reactions such as enzymatic reactions.

[0014] Furthermore, in this specification, "temperature responsiveness" refers to a change in the degree of hydrophilicity / hydrophobicity due to temperature changes. In addition, the boundary temperature at which the degree of hydrophilicity / hydrophobicity changes is referred to as the "response temperature."

[0015] Furthermore, in this specification, "bio-derived substance" refers to a substance present in the body of an organism, which may be a natural product, an artificially synthesized substance using genetic engineering technology, or a chemically synthesized substance based on the aforementioned bio-derived substance. There are no particular limitations on bio-derived substances, but examples include nucleic acids, proteins, and polysaccharides, which are the basic building blocks of living organisms, as well as their constituent elements such as nucleotides, nucleosides, amino acids, various sugars, lipids, vitamins, and hormones.

[0016] Furthermore, in this specification, "cell adhesion" refers to the ease of adhesion to a cell culture substrate at culture temperature, and "having cell adhesion" indicates that cells can adhere to the substrate or cell culture substrate directly or via a bio-derived substance at culture temperature. Conversely, "not having cell adhesion" indicates that cells cannot adhere to the substrate or cell culture substrate at culture temperature.

[0017] Furthermore, in this specification, "cell proliferation" refers to the ease with which cells proliferate at the culture temperature, and "having cell proliferation" means that cells adhere directly to the substrate or cell culture substrate, either directly or via a bio-derived substance, at the culture temperature and are capable of further proliferation. "Not having cell proliferation" means that cells cannot adhere to the substrate or cell culture substrate at the culture temperature, or that they adhere but cannot proliferate. Moreover, "high cell proliferation" means that a larger number of cells proliferate when compared over the same culture period.

[0018] A cell culture substrate according to one aspect of the present invention has the following two regions, (A) and (B). (A) Possesses cell proliferation and stimulus responsiveness, with an area of ​​0.001-5 mm² 2 An island-like region. (B) A region adjacent to the region (A) above that does not have cell proliferation properties.

[0019] The aforementioned region (A) is cell-proliferative and stimulus-responsive. Because it is cell-proliferative and stimulus-responsive, the cell culture substrate of the present invention can produce spheroids by culturing cells to form colonies and detaching the colonies with external stimuli. If it lacks cell-proliferative or stimulus-responsive properties, it is impossible to culture cells to form colonies or detach colonies with external stimuli, and therefore spheroids cannot be produced.

[0020] The stimulus responsiveness is not particularly limited as long as the colony can be detached by an external stimulus, but examples include temperature responsiveness, photoresponsiveness, pH responsiveness, magnetic responsiveness, electric field responsiveness, and mechanical stimulus responsiveness. It is preferable that the stimulus be any one of temperature responsiveness, photoresponsiveness, pH responsiveness, or mechanical stimulus responsiveness, or a combination of these, more preferably any one of temperature responsiveness, photoresponsiveness, or mechanical stimulus responsiveness, or a combination of these, particularly preferably any one of temperature responsiveness, mechanical stimulus responsiveness, or a combination of these, and most preferably temperature responsiveness.

[0021] (A) region is also an island-shaped region with an area of 0.001 to 5 mm 2 The island-shaped region with an area of 0.001 to 5 mm 2 Since it is an island-shaped region, when culturing cells, colonies with an optimal size and shape for producing spheroids can be formed. Also, it is possible to control the shape of the colonies according to the shape of the island-shaped region, enabling the production of spheroids other than spherical ones. If the area is less than 0.001 mm 2 or exceeds 5 mm 2 when culturing cells, colonies of an optimal size cannot be formed, and the shape of the spheroid is distorted or the spheroid cannot be produced. Also, since it is suitable for forming spheroids suitable for applications such as induction of differentiation of pluripotent stem cells, an area of 0.005 to 1 mm 2 is preferred, an area of 0.01 to 0.5 mm 2 is more preferred, an area of 0.015 to 0.25 mm 2 is particularly preferred, and an area of 0.02 to 0.2 mm 2 is most preferred.

[0022] Also, since it is suitable for producing spheroids with a uniform size and shape, it is preferred that the standard deviation / mean area of the area of the (A) region is 80% or less, more preferably 50% or less, particularly preferably 20% or less, and most preferably 5% or less.

[0023] In the present invention, "island-like" refers to an independent region arranged on a plane, similar to the island structures found in sea-island structures (for example, the part indicated by symbol A in Figure 1). (A) Because the region is island-like, the cell culture substrate of the present invention can be used to culture cells to form colonies, and spheroids can be produced by detaching the colonies with external stimuli. If the region is not island-like, for example, in the case of a stripe structure, the colonies are prone to breaking during detachment, and spheroids of uniform size and shape cannot be produced. There are no particular limitations on the shape of the island-like region, and it can be appropriately set according to the desired shape of the spheroid, but examples include circles, ellipses, polygons, and closed shapes consisting of irregular straight or curved lines. Furthermore, since it is suitable for producing spheroids with a shape close to a sphere, circles or ellipses and polygons are preferred as island-like shapes, circles or ellipses and rectangles are more preferred, circles or ellipses and squares are particularly preferred, and circles or ellipses are most preferred.

[0024] Furthermore, in the present invention, since it is suitable for manufacturing spheroids with a shape close to a sphere, the aspect ratio of the island shape is preferably 5 or less, more preferably 2 or less, particularly preferably 1.5 or less, and most preferably 1.1 or less. Here, in the present invention, "aspect ratio" refers to the ratio of the maximum diameter (major axis) to the minimum diameter (minor axis) of the shape, i.e., major axis / minor axis.

[0025] Region (B) is adjacent to region (A) and does not have cell proliferation properties. Because it is adjacent to region (A) and does not have cell proliferation properties, when cells are cultured, it is possible to create a state in which colonies form only in region (A) and no colonies are present around region (A), and spheroids can be produced by detaching the colonies with external stimuli. If region (B) is not adjacent to region (A) or has cell proliferation properties, when cells are cultured, colonies will also be present around region (A), so when colonies in region (A) are detached with external stimuli, spheroids cannot be produced because the colonies in region (A) are fixed by the surrounding colonies, or spheroids of uniform size and shape cannot be produced because the surrounding colonies adhere to the colonies in region (A). Furthermore, it is preferable that region (B) does not have cell proliferation properties as well as cell adhesion properties, as this is suitable for uniformizing the size and shape of the produced spheroids.

[0026] The shape of region (B) is not limited to being adjacent to region (A), but it is preferable that region (B) is adjacent to region (A) for a length of 20% or more of the boundary line between region (A) and region (A), more preferably 50% or more, particularly preferably 80% or more, and most preferably that region (B) is entirely surrounding region (A). Furthermore, it is preferable that region (A) is island-like and region (B) is sea-island-like in order to improve the mass productivity of the cell culture substrate (for example, the part indicated by the symbol B in Figure 1). Regions other than region (B) adjacent to region (A) include regions that have cell proliferation properties but do not respond to stimuli.

[0027] The (B) region is also preferably responsive to stimuli. If the (B) region is responsive to stimuli, similar to the (A) region, it is not necessary to pattern the (A) and (B) regions with a stimuli-responsive substance during the production of the cell culture substrate of the present invention. Instead, the entire surface of the cell culture substrate can be uniformly coated with the stimuli-responsive substance, thus increasing the mass productivity of the cell culture substrate. Furthermore, coating the (B) region with a stimuli-responsive substance reduces the cell adhesion of the (B) region, making it easier to produce colonies with a uniform shape and spheroids of a uniform size and shape, which is preferable.

[0028] While there are no particular limitations on the area ratio of region (A) to region (B), it is preferable that the area of ​​region (A) be 10% or more of the total substrate, more preferably 30% or more, particularly preferably 50% or more, and most preferably 70% or more, as this is suitable for increasing the amount of spheroids that can be produced per unit area of ​​the culture substrate. Furthermore, it is preferable that the area of ​​region (B) be 20% or more of the total substrate, more preferably 40% or more, particularly preferably 60% or more, and most preferably 80% or more, as this is suitable for providing sufficient distance between multiple regions (A) and suppressing the fusion of colonies in multiple regions (A) to form an uneven shape.

[0029] In the present invention, it is preferable that the stimulus responsiveness is due to a layer containing a stimulus-responsive polymer, as this is suitable for increasing the mass productivity of the cell culture substrate. Here, when "substrate" is used in the present invention, it refers to the base substrate of the article of the present invention (for example, the part indicated by reference numeral 1 in Figure 2). Also, when "cell culture substrate" is used, it refers to the entire article for spheroid formation (for example, the part indicated by reference numeral 10 in Figure 2).

[0030] There are no particular limitations on the type of stimulus-responsive polymer, but block copolymers coated on a substrate, copolymers immobilized on a substrate via reactive groups such as azide groups, and polymers immobilized on a substrate by coating a monomer onto the substrate and performing electron beam polymerization or radical polymerization on the substrate are preferably used.

[0031] As the stimulus-responsive polymer, a temperature-responsive polymer is preferred because it is suitable for detaching colonies with a weak stimulus and producing spheroids without damaging the cells. Furthermore, since cells can be cultured at a temperature close to body temperature when culturing cells on a cell culture substrate, a response temperature of 50°C or lower is preferred, more preferably 35°C or lower, and particularly preferably 25°C or lower, and most preferably 15°C or lower, as it is suitable for suppressing cell detachment when performing operations such as changing the culture medium during cultivation. Moreover, since spheroids can be formed by cooling at a temperature that does not damage the cells, a response temperature of 0°C or higher is preferred, more preferably 5°C or higher, particularly preferably 10°C or higher, and most preferably 15°C or higher.

[0032] There are no particular limitations on the type of temperature-responsive polymer, but examples of monomer units include (meth)acrylamide compounds such as acrylamide and methacrylamide; N,N-diethylacrylamide, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, Nt-butylacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylamide N-alkyl-substituted (meth)acrylamide derivatives such as crillamide 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)-piperidine, 4-(1-oxo-2-propenyl)-morpholine, 1-(1-oxo-2-methyl-2-propenyl)-pyrrolidine, 1-(1-oxo-2-methyl-2-propenyl)- Examples of suitable materials include (meth)acrylamide derivatives having cyclic groups such as 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 these are suitable for setting the response temperature to 0-50°C, N,N-diethylacrylamide, Nn-propylacrylamide, N-isopropylacrylamide, Nn-propylmethacrylamide, N-ethoxyethylacrylamide, N-tetrahydrofurfurylacrylamide, and N-tetrahydrofurfurylmethacrylamide are preferred, with Nn-propylacrylamide and N-isopropylacrylamide being more preferred, and N-isopropylacrylamide being particularly preferred. Furthermore, when using a medium at room temperature during culture medium exchange, Nn-propylacrylamide and N-proline methyl ester acrylamide are preferred as they are suitable for setting the response temperature of the block copolymer to a temperature lower than room temperature.

[0033] Furthermore, the temperature-responsive polymer may also be a copolymer. For example, a copolymer consisting of at least two monomer units selected from the monomer units mentioned above can be used. In particular, a copolymer of N-isopropylacrylamide and Nt-butylacrylamide is preferred because the response temperature can be controlled in the range of 5 to 30°C by varying the Nt-butylacrylamide content in the range of 5 to 60 mol%. Moreover, a copolymer of the monomer units constituting the temperature-responsive segment and other monomer units is also acceptable. For example, a copolymer with hydrophilic monomer units can shift the response temperature to the higher temperature side, and a copolymer with hydrophobic monomer units can shift the response temperature to the lower temperature side. The copolymer arrangement can be random, alternating, or block.

[0034] In the present invention, the ratio of temperature-responsive constituent units 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, as it is suitable for rapidly performing the process of forming spheroids from colonies.

[0035] The stimulus-responsive polymer is preferably a block copolymer having a stimulus-responsive block segment and a water-insoluble block segment, or a block copolymer having a stimulus-responsive block segment and a reactive segment. The use of the stimulus-responsive polymer as the block copolymer enhances the mass productivity of the cell culture substrate and suppresses contamination of the spheroids with the stimulus-responsive polymer. The stimulus-responsive block segment is not particularly limited and can be a polymer consisting of the aforementioned temperature-responsive monomer units. Furthermore, the inclusion of a water-insoluble block segment or a reactive segment in the block copolymer is preferable because it suppresses contamination of the block copolymer into the culture medium, allowing cells to be cultured without contamination, thus suppressing contamination of the spheroids with the stimulus-responsive polymer. Here, in this specification, "water-insoluble" of a block segment means that at least a portion of the homopolymer consisting only of the monomer units constituting the block segment is insoluble in water. "Reactive segment" means that the block segment has a reactive group and can be immobilized on the substrate by external stimuli such as heat, pH changes, or light irradiation.

[0036] 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, n-tetradecyl methacrylate, and the like. Furthermore, since it is suitable for firmly immobilizing the block copolymer on a substrate, those having reactive groups are preferred, for example, 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. Examples 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.

[0037] In the present invention, examples of monomer units constituting the reactive block segment include 4-azidophenyl acrylate, 4-azidophenyl methacrylate, 2-((4-azidobenzoyl)oxy)ethyl acrylate, and 2-((4-azidobenzoyl)oxy)ethyl methacrylate.

[0038] In the present invention, the water-insoluble block segment or the reactive block segment may also include repeating units that control the response temperature of the block copolymer. Examples of repeating units that control the response temperature of the block copolymer include hydrophilic or hydrophobic components, and are not particularly limited, but include 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-methacroyloxyethyl-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 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, tetrahydrofurfuryl methacrylate, etc., having polyethylene glycol groups and methoxyethyl groups; acrylates having acrylate groups such as methoxymethyl acrylate, methoxymethyl methacrylate, 2-ethoxymethyl acrylate, 2-ethoxymethyl methacrylate, 3-butoxymethyl acrylate, 3-butoxymethyl methacrylate, 3-butoxymethyl acrylamide;Examples of phosphorylcholine compounds include those having a phosphorylcholine group, such as 2-methacryloyloxyethyl phosphorylcholine, 2-acryloyloxyethyl phosphorylcholine, 3-(meth)acryloyloxypropyl phosphorylcholine, 4-(meth)acryloyloxybutyl phosphorylcholine, 6-(meth)acryloyloxyhexyl phosphorylcholine, 10-(meth)acryloyloxydecyl phosphorylcholine, ω-(meth)acryloyl(poly)oxyethylene phosphorylcholine, 2-acrylamidoethyl phosphorylcholine, 3-acrylamidopropyl phosphorylcholine, 4-acrylamidobutyl phosphorylcholine, 6-acrylamidohexyl phosphorylcholine, 10-acrylamidodecyl phosphorylcholine, and ω-(meth)acrylamido(poly)oxyethylene phosphorylcholine.

[0039] The water-insoluble block segment or reactive block segment may be a copolymer, and the copolymer may be arranged in a random, alternating, or block configuration.

[0040] The ratio of constituent units of the water-insoluble block segment or reactive block segment is preferably 1 wt% or more, more preferably 2 wt% or more, particularly preferably 5 wt% or more, and most preferably 10 wt% or more, from the viewpoint of suppressing the contamination of cells by the block copolymer into the culture medium and also from the viewpoint of maintaining an undifferentiated state in subculturing.

[0041] There are no particular restrictions on the molecular weight of the stimulus-responsive polymer in the present invention, but it is preferable that the number average molecular weight be 1000 to 1,000,000, more preferably 2000 to 500,000, particularly preferably 50000 to 300,000, and most preferably 10,000 to 200,000, as this is suitable for increasing the strength of the stimulus-responsive polymer.

[0042] In the present invention, since it is suitable for suppressing the contamination of spheroids with stimulus-responsive polymers, it is preferable that the component with a number average molecular weight of 5000 or less contained in the stimulus-responsive polymer is 50% or less, more preferably 30% or less, particularly preferably 10% or less, and most preferably 5% or less. Furthermore, it is preferable that the component with a number average molecular weight of 10000 or less is 50% or less, more preferably 30% or less, particularly preferably 10% or less, and most preferably 5% or less. Moreover, it is preferable that the component with a number average molecular weight of 30000 or less is 50% or less, more preferably 30% or less, particularly preferably 10% or less, and most preferably 5% or less. The content of components with a specific molecular weight or less contained in the block copolymer can be measured by gel permeation chromatography.

[0043] In the present invention, since it is suitable for suppressing the incorporation of the stimulus-responsive polymer into the spheroid, the amount of stimulus-responsive polymer eluted into water is preferably 50% or less, more preferably 30% or less, particularly preferably 10% or less, and most preferably 5% or less. The amount of stimulus-responsive polymer eluted into water is the percentage of weight loss of the stimulus-responsive polymer before and after immersing a substrate on which a layer of stimulus-responsive polymer has been formed in water at 25°C for 1 hour. Here, there are no particular restrictions on the method for measuring the weight loss of the stimulus-responsive polymer, and it can be measured by a commonly used method, for example, the FT-IR method, the average film thickness measurement method, etc. can be used.

[0044] The stimulus-responsive polymer in the present invention may optionally contain chain transfer agents, polymerization initiators, polymerization inhibitors, etc. There are no particular restrictions on the chain transfer agent, and commonly used ones can be suitably used, but examples include dithiobenzoate, trithiocarbonate, 4-cyano-4-[(dodecylsulfonylthiocarbonyl)sulfonyl]pentanoic acid, 2-cyanopropan-2-yl N-methyl-N-(pyridine-4-yl)carbamodithioate, and methylmethyl(4-pyridinyl)carbamodithioate 2-propionate. There are no particular restrictions on the polymerization initiator, and commonly used ones can be suitably used, but examples include azobisisobutyronitrile, 1,1'-azobis(cyclohexanecarbonile), di-tert-butyl peroxide, tert-butyl hydroperoxide, hydrogen peroxide, potassium peroxodisulfate, benzoyl peroxide, triethylborane, and diethylzinc. Furthermore, there are no particular restrictions on the polymerization inhibitor, and commonly used ones can be suitably used, but examples include hydroquinone, p-methoxyphenol, triphenylfeldazyl, 2,2,6,6-tetramethylpiperidinyl-1-oxyl, and 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxyl.

[0045] The method for synthesizing the stimulus-responsive polymer in the present invention is not particularly limited, but the living radical polymerization technology described in "Radical Polymerization Handbook," pp. 161-225 (2010), published by NTS Corporation, can be used.

[0046] In the present invention, the thickness of the stimuli-responsive polymer layer is preferably 1000 nm or less, more preferably 200 nm or less, particularly preferably 100 nm or less, and most preferably 50 nm or less, as it is suitable for enhancing cell proliferation. Furthermore, the thickness of the stimuli-responsive polymer layer is preferably 5 nm or more, more preferably 20 nm or more, particularly preferably 30 nm or more, and most preferably 35 nm or more, as it is suitable for rapidly performing the process of forming spheroids from colonies. Here, in the present invention, the "thickness" of the stimuli-responsive polymer layer refers to the out-of-plane length from the interface between the substrate and the stimuli-responsive polymer layer to the peak portion of the surface structure of the stimuli-responsive polymer layer. The thickness of the layer made of stimulus-responsive polymer can be calculated by immersing a cell culture substrate in water at culture temperature for 24 hours or more, rapidly drying it by blowing air onto it, etc., and for samples with a layer thickness exceeding 10 nm, measuring the cross-sectional image using a transmission electron microscope with ultrathin sections of the cell culture substrate prepared with a microtome, measuring the distance at 10 randomly selected points, and averaging the result. For layers with a thickness of 10 nm or less, it can be measured using an ellipsometer.

[0047] In the present invention, it is preferable that the layer made of the stimulus-responsive polymer has an average surface roughness / thickness ratio of 0.3 or higher, as it is suitable for enhancing cell proliferation and rapidly forming spheroids from colonies. By having an average surface roughness / thickness ratio of 0.3 or higher, it is possible to create areas where cells adhere strongly and areas where they do not, thereby enhancing cell proliferation and rapidly forming spheroids from colonies. It is even more preferable that the average surface roughness / thickness ratio of the layer made of the stimulus-responsive polymer be 0.5 or higher, particularly preferable that be 0.7 or higher, and most preferable that be 0.9 or higher, as it is suitable for enhancing cell proliferation and rapidly forming spheroids from colonies. In this invention, the "average roughness" of the surface of the layer made of stimulus-responsive polymer refers to the average height of the contour curve determined in accordance with JIS B0601:2013. This can be calculated by measuring atomic force microscope images of a representative surface of the cell culture substrate and determining the average height of 10 randomly selected roughness curves, with 1 μm as the reference length. Similar to the measurement of the layer thickness described above, the measurement is performed on a sample in which the cell culture substrate has been immersed in water at the culture temperature for 24 hours or more and rapidly dried by blowing air on it.

[0048] In a cell culture substrate according to one aspect of the present invention, it is preferable that the (A) region consists of the following two regions (A1) and (A2), and the (B) region consists of the following two regions (B1) and (B2).

[0049] (A1) Cell proliferation zone (A2) Stimuli-responsive area (B1) Base material adhesion area (B2) Stimuli-responsive area Here, the "substrate adhesion region" refers to the region where the components coated on the cell culture substrate adhere to the substrate. Since the stimulus-responsive region has the effect of reducing cell proliferation, the fact that region (A) consists of two regions, (A1) and (A2), allows for the suitability of spheroid formation and detachment from the cell culture substrate in response to stimuli, which are difficult to promote by adhering to and proliferating pluripotent stem cells on the cell culture substrate. Furthermore, the presence of (B1) in region (B) is suitable for firmly fixing the components coated on the cell culture substrate to the substrate. Moreover, the presence of (B2) in region (B) suppresses the proliferation of pluripotent stem cells in region (B) and is suitable for forming uniform spheroids. Regions (A1) and (A2), and (B1) and (B2) can be confirmed by measuring atomic force microscope images or transmission electron microscope images of the surface of the cell culture substrate and determining the main components present in each region. For example, when a copolymer consisting of cell-proliferating monomer units and stimulus-responsive monomer units is applied, the monomer units present in each region can be determined from the phase image in an atomic force microscope image or the contrast in a transmission electron microscope image.

[0050] The regions (A1) and (A2), (B1) and (B2) can be suitably formed by coating with a block copolymer consisting of a combination of monomer units with significantly different degrees of hydrophilicity / hydrophobicity. However, the composition of the copolymer and the amount of coating that can form the regions (A1) and (A2), (B1) and (B2) vary depending on the type of block copolymer. When the block copolymer is a diblock of butyl methacrylate and N-isopropylacrylamide, a diblock in which one component exceeds 90% is preferred, more preferably N-isopropylacrylamide exceeds 90%, particularly preferably N-isopropylacrylamide exceeds 92%, and most preferably N-isopropylacrylamide exceeds 94%. Furthermore, a film thickness of 1 to 200 nm is preferred, more preferably 5 to 100 nm, particularly preferably 10 to 50 nm, and most preferably 15 to 35 nm.

[0051] In the present invention, it is preferable that the (A) region consists of a cell proliferation region in which a temperature-responsive region with a diameter of 10 to 500 nm is dispersed, or a temperature-responsive region that does not have cell proliferation properties in which a cell proliferation region with a diameter of 10 to 500 nm is dispersed. By having the (A) region consist of the dispersed region with a diameter of 10 to 500 nm, spheroid formation of pluripotent stem cells and detachment from the cell culture substrate in response to stimulation can be suitably performed.

[0052] In the present invention, it is preferable that the (A) region is a plasma-treated region in order to improve the mass production of spheroids and to form uniform spheroids. By having the (A) region as a plasma-treated region, a large number of cells can be concentrated only in the (A) region, making it easier to form a large quantity of uniform spheroids.

[0053] In the present invention, in order to improve the mass production of spheroids and to further facilitate the formation of uniform spheroids, it is preferable that the difference in zeta potential between region (A) and region (B) be 5 mV or more, more preferably 10 mV or more, particularly preferably 20 mV or more, and most preferably 40 mV or more.

[0054] In the present invention, it is preferable that there are no septa around region (A) that cells cannot cross, as this is suitable for increasing the mass production of spheroids. The absence of septa around region (A) that cells cannot cross allows cells to move freely, and many cells can gather in region (A), thereby increasing the mass production of spheroids. Furthermore, it is even more preferable that there are no septa with a height of 1 μm or more, particularly preferable that there are no septa with a height of 0.1 μm or more, and most preferable that there are no septa with a height of 0.05 μm or more, as this is suitable for increasing the mass production of spheroids.

[0055] In the present invention, the cell culture substrate may contain a bio-derived substance on its surface as needed. The bio-derived substance is not particularly limited, but examples include Matrigel, laminin, fibronectin, vitronectin, collagen, and the like.

[0056] These bio-derived substances may be natural products, artificially synthesized using genetic engineering techniques, or fragments obtained by cleaving with restriction enzymes, or synthetic proteins or peptides based on these bio-derived substances.

[0057] In the present invention, as the Matrigel, commercially available products such as Matrigel (manufactured by Corning Incorporated) or Geltrex (manufactured by Thermo Fisher Scientific) can be suitably used due to their availability.

[0058] The type of laminin is not particularly limited, but for example, laminin 511, laminin 521, or laminin 511-E8 fragment, which have been reported to show high activity against α6β1 integrin expressed on the surface of human iPS cells, can be used. The laminin may be a natural product, artificially synthesized by genetic engineering or the like, or a synthetic protein or synthetic peptide based on the laminin. Due to its availability, iMatrix-511 (manufactured by Nippi Corporation) can be preferably used as a commercially available product.

[0059] The aforementioned vitronectin may be a natural product, artificially synthesized using genetic engineering technology, or a synthetic protein or peptide based on the vitronectin. Due to their availability, commercially available products such as vitronectin derived from human plasma (manufactured by Wako Pure Chemical Industries, Ltd.), synthemax (manufactured by Corning Incorporated), and Vitronectin (VTN-N) (manufactured by Thermo Fisher Scientific) can be suitably used.

[0060] The fibronectin may be a natural product, artificially synthesized using genetic engineering technology, or a synthetic protein or peptide based on the fibronectin. Due to their 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 suitably used.

[0061] The type of collagen is not particularly limited, but for example, type I collagen or type IV collagen can be used. The collagen may be a natural product, artificially synthesized using genetic engineering technology, or a synthetic peptide based on the collagen. Due to their availability, commercially available products such as Collagen I, Human (manufactured by Corning Incorporated) and Collagen IV, Human (manufactured by Corning Incorporated) can be suitably used.

[0062] In the present invention, it is preferable that bio-derived substances are immobilized on the cell culture substrate by non-covalent bonds rather than covalent bonds, in order to suppress the denaturation of bio-derived substances and enhance cell proliferation. Here, in the present invention, "non-covalent bonds" refer to bonding forces other than covalent bonds that originate from intermolecular forces, such as electrostatic interactions, water-insoluble interactions, hydrogen bonds, π-π interactions, dipole-dipole interactions, London dispersion forces, and other van der Waals interactions. The immobilization of bio-derived substances on block copolymers may be by a single bonding force or a combination of multiple bonding forces.

[0063] In the present invention, the method for immobilizing bio-derived substances is not particularly limited, but for example, a method of immobilizing by applying a solution of bio-derived substances to a cell culture substrate for a predetermined time, or a method of immobilizing by adsorbing bio-derived substances onto a cell culture substrate by adding bio-derived substances to the culture medium when culturing cells, can be suitably used.

[0064] In the present invention, the material of the substrate is not particularly limited, but in addition to materials such as glass and polystyrene that are normally used in cell culture, materials that can generally be given shape, such as polymer compounds such as polycarbonate, polyethylene terephthalate, polyvinylidene fluoride, polytetrafluoroethylene, polyethylene, polypropylene, and polymethyl methacrylate, as well as ceramics and metals, can be used. For ease of culture operations, it is preferable that the material of the substrate contains at least one of glass, polystyrene, polycarbonate, polyethylene terephthalate, polyethylene, and polypropylene, and it is even more preferable that it contains at least one of glass, polystyrene, polycarbonate, polyethylene terephthalate, and polyethylene. Polystyrene, polycarbonate, polyethylene terephthalate, and polyethylene are particularly preferred because they are suitable for increasing flexibility. Furthermore, polystyrene, polycarbonate, and polyethylene terephthalate are most preferred because they are suitable for giving cell proliferation properties by patterning by hydrophilization treatment described later.

[0065] There are no particular restrictions on the shape of the substrate; it may be a flat shape such as a plate or film, or it may be a fiber, porous particle, porous membrane, or hollow fiber. It may also be a container commonly used for cell culture (such as a Petri dish, flask, or plate). For ease of culture operations, a flat shape such as a plate or film, or a flat porous membrane, is preferred. Furthermore, a structure for separating each spheroid may be provided on the substrate, such as by adding a partition plate, as needed.

[0066] In the present invention, it is preferable that the substrate is a porous substrate, and that the pore diameter of the porous substrate is smaller than that of a cell, as this is suitable for rapidly performing the process of forming spheroids from colonies, and furthermore, even when producing large spheroids, it is possible to distribute nutrients evenly throughout the interior of the colony. Also, as it is suitable for rapidly performing the process of forming spheroids from colonies, the pore diameter is preferably 0.01 to 8 μm, more preferably 0.01 to 3 μm, particularly preferably 0.01 to 1 μm, and most preferably 0.1 to 1 μm. Here, in the present invention, the "pore diameter" of the porous substrate refers to the average value of the diameter of the pores in the porous substrate along the in-plane direction of the porous substrate, and can be calculated by measuring the diameter of 20 or more pores in a laser microscope image, scanning electron microscope image, or transmission electron microscope image of the porous substrate and determining the average value.

[0067] The porous substrate is also suitable for rapidly performing the process of forming spheroids from colonies, and therefore preferably has a porosity of 0.01 to 60%, more preferably 0.01 to 20%, particularly preferably 0.01 to 4%, and most preferably 0.01 to 1.5%. Here, in the present invention, the "porosity" of the porous substrate is the value obtained by dividing the total area of ​​the pores by the substrate area for one main surface of the porous substrate, and indicates the extent of voids on the substrate surface as an area ratio. This can be measured by observing a square region with sides at least 200 times the pore diameter of the porous substrate in a laser microscope image, scanning electron microscope image, or transmission electron microscope image of the porous substrate.

[0068] The cell culture substrate of the present invention may be sterilized. There are no particular limitations on the sterilization method, but autoclaving, UV sterilization, gamma ray sterilization, ethylene oxide gas sterilization, etc., can be used. Autoclaving, UV sterilization, and ethylene oxide gas sterilization are preferred because they are suitable for suppressing the denaturation of the block copolymer, UV sterilization or ethylene oxide gas sterilization are more preferred because they are suitable for suppressing deformation of the substrate, and ethylene oxide gas sterilization is particularly preferred because it is excellent in terms of mass productivity.

[0069] The cells cultured using the cell culture substrate of the present invention are not particularly limited as long as they can adhere to the surface before stimulation by temperature reduction. For example, in addition to various cell lines such as Chinese hamster ovary-derived CHO cells, mouse connective tissue L929, human fetal kidney-derived HEK293 cells, and human cervical cancer-derived HeLa cells, other cells that can be used include, for example, epithelial cells and endothelial cells that make up various tissues and organs in the body, contractile skeletal muscle cells, smooth muscle cells, cardiomyocytes, neurons, glial cells, fibroblasts that make up the nervous system, hepatocytes, non-parenchymal cells and adipocytes that are involved in the metabolism of the body, and as cells with differentiation potential, stem cells that exist in various tissues such as mesenchymal stem cells, bone marrow cells, and Muse cells, as well as pluripotent stem cells (pluripotent stem cells) that have differentiation potential such as ES cells and iPS cells, and cells differentiated from them. From the viewpoint of cell proliferation and detachability in the culture substrate of the present invention, stem cells or pluripotent stem cells are preferred, mesenchymal stem cells or pluripotent stem cells are more preferred, pluripotent stem cells are particularly preferred, and iPS cells are most preferred.

[0070] The present invention also relates to a method for producing the cell culture substrate that is excellent in terms of mass productivity. The method is characterized by comprising the following steps (1) and (2). (1) On the surface of a substrate that does not have cell proliferation properties, a cell proliferation property with an area of ​​0.001 to 5 mm² 2 The process of forming island-like regions. (2) A step of forming a layer of stimulus-responsive substance on the surface of the substrate.

[0071] The following describes in detail steps (1) and (2) of the method for producing the cell culture substrate of the present invention.

[0072] In the method for producing a cell culture substrate of the present invention, step (1) uses a substrate that does not have cell proliferation properties, and has cell proliferation properties with an area of ​​0.001 to 5 mm². 2 Island-like regions are formed on the surface of the substrate. By using a substrate that does not have cell proliferation properties and forming cell proliferation-promoting regions by patterning, cell proliferation-promoting regions of any shape and size can be efficiently produced. As a method for forming cell proliferation-promoting regions, hydrophilization treatments such as plasma treatment, UV treatment, and corona treatment, or coating the substrate with a substance that has cell proliferation properties are preferred, as these are suitable for increasing the mass productivity of cell culture substrates. A method of imparting cell proliferation properties to a part of the substrate by hydrophilization treatments such as plasma treatment, UV treatment, and corona treatment is even more preferred, and plasma treatment is particularly preferred among these.

[0073] The patterning method is not particularly limited, but examples include a method of hydrophilizing the substrate to a desired shape by performing a hydrophilization treatment such as plasma treatment, UV treatment, or corona treatment while the substrate is covered with a metal mask, a silicon mask, or a surface protective film, or a method of coating the substrate with a cell proliferation-promoting substance in a desired shape using photolithography or inkjet. To improve the mass production of cell culture substrates, it is preferable to perform a hydrophilization treatment such as plasma treatment, UV treatment, or corona treatment while a surface protective film with holes formed by laser processing is attached to the substrate.

[0074] Furthermore, as a method of patterning other than the above-mentioned step (1), a method of forming a region that does not exhibit cell proliferation properties on the surface of a substrate that exhibits cell proliferation properties is also available. Examples of this method include using a substrate that exhibits cell proliferation properties, or imparting cell proliferation properties to the entire surface of a substrate that does not exhibit cell proliferation properties, and then coating the substrate with a substance that does not exhibit cell proliferation properties in a desired shape using photolithography or inkjet technology.

[0075] In the method for producing a cell culture substrate of the present invention, step (2) involves forming a layer of a stimulus-responsive substance on the surface of the substrate. By forming a layer of the stimulus-responsive substance on the surface of the substrate with a thickness of 100 nm or less, even when step (2) is performed after step (1), the molecular chains of the stimulus-responsive substance can be easily coated on the cell proliferation surface formed in the patterning step in a sparse state, which is suitable for imparting stimulus responsiveness while maintaining the effect of patterning. Furthermore, by setting the layer thickness to 1 nm or more, it is possible to produce a cell culture substrate that can be given sufficient stimulus responsiveness and that allows the spheroid formation step to be performed rapidly, which is preferable.

[0076] As a method for coating the substrate with the stimulus-responsive substance, a method of coating the substrate with the stimulus-responsive substance by chemical bonding to form a layer, or a method of coating the substrate with the stimulus-responsive substance by physical interaction, can be used alone or in combination. Specifically, as a method of chemical bonding, ultraviolet irradiation, electron beam irradiation, gamma ray irradiation, plasma treatment, corona treatment, etc., can be used. Furthermore, if the stimulus-responsive polymer and the substrate have appropriate reactive functional groups, commonly used organic reactions such as radical reactions, anionic reactions, and cationic reactions can be utilized. As a method of physical interaction, a matrix with good compatibility with the stimulus-responsive polymer and good coating properties can be used as a medium, and various commonly known methods such as coating, 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 can be used.

[0077] As a method for coating the cell culture substrate with the stimulus-responsive substance, it is also preferable to coat the entire surface of the cell culture substrate with the stimulus-responsive substance. By coating the cell culture substrate with the stimulus-responsive substance using a commonly used coating method without patterning, the mass production of the cell culture substrate can be increased. Furthermore, by coating the entire surface of the cell culture substrate with the stimulus-responsive substance, stimulus responsiveness is imparted to region (A), and region (B) is also coated with the stimulus-responsive substance. By coating region (B) with the stimulus-responsive substance, the cell adhesion of region (B) can be reduced. For this reason, when using a substrate that has cell adhesion but not cell proliferation properties, region (B) will have cell adhesion in the patterning step alone, but by coating region (B) with the stimulus-responsive substance, region (B) can be made into a region that does not have cell adhesion, making it easier to form spheroids of uniform size and shape, which is preferable. At this time, the difference in the amount of coating between region (A) and region (B) is 1 μg / cm². 2 Preferably, it is 0.8 μg / cm³. 2 The following is even more preferable: 0.6 μg / cm³ 2 The following is particularly preferred: 0.4 μg / cm³ 2 The following is most preferable. Furthermore, the coverage amount for regions (A) and (B) should be 0.1 to 10 μg / cm². 2 Preferably, 0.5 to 7 μg / cm³ 2 More preferably, 1-5 μg / cm³ 2 This is particularly preferred, at 2-4 μg / cm³ 2 Most preferable.

[0078] In the method for producing cell culture substrates of the present invention, the order in which steps (1) and (2) are performed does not matter. It is preferable to perform step (2) after step (1) because it is suitable for minimizing the effects of step (1), such as scratching the layer of surface irritation-responsive substance.

[0079] The present invention also relates to a method for producing spheroids using the cell culture substrate. The method is characterized by producing spheroids through the following steps (i) to (iii). (i) A step of seeding cells onto the cell culture substrate. (ii) A step of culturing the seeded cells and forming colonies of cells attached to the cell culture substrate. (iii) A step of detaching the colony from the cell culture substrate by applying an external stimulus to form a cell spheroid.

[0080] The following describes in detail the steps (i) to (iii) in the spheroid manufacturing method of the present invention.

[0081] Step (i) in the method for producing spheroids of the present invention is a step of seeding cells onto the cell culture substrate using the cell culture substrate. In the present invention, "seeding cells" means bringing the cell suspension (hereinafter referred to as "cell suspension") into contact with the cell culture substrate by coating the cell culture substrate with a culture medium in which cells are dispersed (hereinafter referred to as "cell suspension") or by injecting it into the cell culture substrate. By using a cell culture substrate having a cell proliferation region, cells can be cultured in step (ii) described later. If the cell culture substrate does not have a cell proliferation region, cells cannot be cultured in step (ii). Furthermore, if the cell proliferation region also has a stimulus-responsive region, colonies can be detached from the cell culture substrate by external stimulation in step (iii) described later, and spheroids can be formed. If the cell culture substrate does not have stimulus responsiveness, spheroids cannot be formed by external stimulation in step (iii).

[0082] In steps (i) to (iii) above, the culture is carried out under conditions effective in maintaining the undifferentiated state of the cells. There are no particular limitations on the conditions effective in maintaining the undifferentiated state, but examples include setting the cell density at the start of culture to the preferred range described below as the cell density at seeding, and carrying out the culture in the presence of an appropriate liquid medium. As a medium effective in maintaining the undifferentiated state of the cells, for example, a medium containing one or more of the following known factors for maintaining the undifferentiated state of cells can be suitably used. It is even more preferable to use a medium containing insulin, transferrin, selenium, ascorbic acid, sodium bicarbonate, basic fibroblast growth factor, and transforming growth factor β (TGFβ) because it is particularly suitable for maintaining the undifferentiated state of the cells, and it is most preferable to use a medium containing basic fibroblast growth factor.

[0083] There are no particular restrictions on the type of culture medium to which the basic fibroblast growth factor is added, but commercially available examples include DMEM (manufactured by Sigma-Aldrich Co. LLC), Ham's F12 (manufactured by Sigma-Aldrich Co. LLC), D-MEM / Ham's F12 (manufactured by Sigma-Aldrich Co. LLC), Primate ES Cell Medium (manufactured by REPROCELL Co., Ltd.), StemFit AK02N (manufactured by Ajinomoto Co., Inc.), StemFit AK03 (manufactured by Ajinomoto Co., Inc.), mTeSR1 (manufactured by STEMCELL TECHNOLOGIES), and TeSR-E8 (manufactured by STEMCELL TECHNOLOGIES). Examples include TECHNOLOGIES, ReproNaive (REPROCELL, Inc.), ReproXF (REPROCELL, Inc.), ReproFF (REPROCELL, Inc.), ReproFF2 (REPROCELL, Inc.), NutriStem (Biological Industries, Inc.), iSTEM (Takara Bio Inc.), GS2-M (Takara Bio Inc.), hPSC Growth Medium DXF (PromoCell, Inc.), etc. Because they are suitable for maintaining the undifferentiated state of cells, Primate ES Cell Medium (manufactured by REPROCELL Inc.), StemFit AK02N (manufactured by Ajinomoto Co., Inc.), or StemFit AK03 (manufactured by Ajinomoto Co., Inc.) are preferred, StemFit AK02N (manufactured by Ajinomoto Co., Inc.) or StemFit AK03 (manufactured by Ajinomoto Co., Inc.) are more preferred, and StemFit AK02N (manufactured by Ajinomoto Co., Inc.) is particularly preferred.

[0084] In step (i) above, there are no particular restrictions on the method of seeding the cells, but for example, it can be done by injecting a cell suspension into a cell culture substrate. There are no particular restrictions on the cell density at the time of seeding, but 1.0 × 10 is recommended so that the cells can be maintained and proliferated. 2 ~1.0×10 6 cells / cm 2 Preferably, 5.0 × 10 2 ~5.0×10 5 cells / cm 2 More preferably, 1.0 × 103 ~2.0×10 5 cells / cm 2 This is particularly preferred, 1.2 × 10 3 ~1.0×10 5 cells / cm 2 Most preferable.

[0085] As the culture medium used in step (i) above, it is preferable to use a medium to which a Rho-binding kinase inhibitor has been further added to the culture medium to which the basic fibroblast growth factor has been added, as this is suitable for maintaining cell survival. In particular when using human cells, the addition of a Rho-binding kinase inhibitor may be effective in maintaining the survival of human cells when the cell density of human cells is low. Examples of Rho-binding kinase inhibitors that can be used include (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide·2HCl·H2O (Y-27632, manufactured by Wako Pure Chemical Industries, Ltd.) and 1-(5-Isoquinolinesulfonyl)homopiperazine Hydrochloride (HA1077, manufactured by Wako Pure Chemical Industries, Ltd.). The concentration of the Rho-binding kinase inhibitor added to the culture medium is within a range that is effective in maintaining the survival of human cells and does not affect the undifferentiated state of human cells, preferably 1 μM to 50 μM, more preferably 3 μM to 20 μM, even more preferably 5 μM to 15 μM, and most preferably 8 μM to 12 μM.

[0086] Shortly after step (i) above is started, the cells begin to adhere to the cell culture substrate.

[0087] In step (ii) of the method for producing spheroids of the present invention, the seeded cells are cultured. The culture temperature is preferably 30 to 42°C, more preferably 32 to 40°C, particularly preferably 36 to 38°C, and most preferably 37°C, as it is suitable for maintaining the cell's proliferative ability, physiological activity, and function.

[0088] It is preferable to perform the first culture medium change 22 to 26 hours after starting step (ii). A second culture medium change is performed 48 to 72 hours later, and thereafter, it is preferable to change the culture medium every 24 to 48 hours. During this time, the cells proliferate and form flat cell clusters called colonies. The colonies are attached to the cell culture substrate. Culture is continued until the size of the colonies is about the size of area (A), and then the process proceeds to step (iii).

[0089] In step (iii) of the method for producing spheroids according to the present invention, an external stimulus is applied to the cell culture substrate in which cells are cultured, and at least a portion of the colony is detached from the cell culture substrate. At this time, by gradually detaching from the periphery of the colony, the colony folds up and becomes a round mass, making it easy to form a spheroid with a uniform shape. If the entire surface of a single colony is detached from the substrate, a floating spheroid can be obtained. Alternatively, by detaching only the outer periphery of the colony from the substrate and not detaching the center of the colony, a spheroid adhered to the substrate can be formed. When cooling is used as the external stimulus, in order to detach the cells in a short time and reduce damage caused by cooling, the cooling temperature is preferably 0 to 30°C, more preferably 3 to 25°C, and even more preferably 5 to 20°C. In addition, in order to reduce damage to the cells, the cooling time is preferably 120 minutes or less, more preferably 60 minutes or less, particularly preferably 30 minutes or less, and most preferably 15 minutes or less.

[0090] There are no particular restrictions on the method of cooling the cell culture substrate in step (iii) above, but for example, a method of cooling the cell culture substrate by placing it in a refrigerator, a method of cooling the cell culture substrate by placing it on a cool plate, or a method of replacing it with cooled culture medium or buffer and letting it stand for a predetermined time can be used.

[0091] Furthermore, step (iii) may include a step of generating convection in the liquid containing the cultured cells in order to detach the cells in a short time and reduce damage from external stimuli. There are no particular limitations on the method of generating convection, but examples include methods of mechanically generating forced convection in the liquid by pipetting the culture medium or by using a pump or agitator.

[0092] The spheroids produced by the spheroid production method of the present invention are suitable for uniformly treating all cells in culture after spheroid formation, such as during differentiation induction. Therefore, it is preferable that the variability of the spheroid particle size (standard deviation of particle size / average particle size) is 20% or less, more preferably 15% or less, particularly preferably 10% or less, and most preferably 5% or less. [Examples]

[0093] The present invention will be described in detail below with reference to embodiments for carrying it out. However, these are merely examples to illustrate the present invention and are not intended to limit the present invention to the following. Furthermore, the present invention can be implemented with appropriate modifications within the scope of its essence. Unless otherwise specified, commercially available reagents were used. <Composition of polymer> Proton nuclear magnetic resonance spectroscopy using a nuclear magnetic resonance measuring instrument (manufactured by JEOL Ltd., product name JNM-GSX400) 1 (H-NMR) spectral analysis, or carbon nuclear magnetic resonance spectroscopy using a nuclear magnetic resonance spectrometer (Bruker, product name AVANCEIIIHD500) 13 The result was obtained from 13C-NMR spectral analysis. <Molecular weight and molecular weight distribution of polymers> Weight-average molecular weight (Mw), number-average molecular weight (Mn), and molecular weight distribution (Mw / Mn) were measured by gel permeation chromatography (GPC). A Tosoh Corporation HLC-8320GPC was used for the GPC apparatus, with two Tosoh Corporation TSKgel Super AWM-H columns. The column temperature was set to 40°C, and the eluent was either 1,1,1,3,3,3-hexafluoro-2-isopropanol containing 10 mM sodium trifluoroacetate or N,N-dimethylformamide containing 10 mM lithium bromide. Samples were prepared at 1.0 mg / mL for measurement. A molecular weight calibration curve was created using polymethyl methacrylate (Polymer Laboratories Ltd.) with a known molecular weight. <Polymer layer thickness> The thickness of the polymer layer coating the substrate was measured using an AFM device (SPM-9600, Shimadzu Corporation). A BL-AC40TS-C2 cantilever was used, and the polymer layer thickness was measured by scratching the surface with tweezers and measuring the depth of the scratches. [Example 1] 0.40 g (0.1 mmol) of 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, 7.11 g (50 mmol) of n-butyl methacrylate, and 33 mg (0.2 mmol) of azobis(isobutyronitrile) were added to a test tube and dissolved in 50 mL of 1,4-dioxane. After degassing by nitrogen bubbling for 30 minutes, the mixture was reacted at 70°C for 24 hours. After the reaction was complete, the reaction solvent was removed by vacuum distillation using a rotary evaporator, and the reaction solution was concentrated. The concentrate was poured into 250 mL of methanol, and the precipitated yellow oily substance was collected and dried under vacuum to obtain the n-butyl methacrylate polymer.

[0094] In a test tube, 0.9 g (0.3 mmol) of the 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 by nitrogen bubbling for 30 minutes, the mixture was reacted at 65°C for 17 hours. After the reaction was complete, the reaction solvent was diluted with acetone, poured into 500 mL of hexane, and the precipitated solid was collected and dried under reduced pressure. The mixture was then dissolved again in acetone, poured into 500 mL of pure water, and the precipitated solid was collected and dried under reduced pressure to obtain a diblock copolymer of N-isopropylacrylamide and n-butyl methacrylate.

[0095] A 10 cm diameter dish (made by AS ONE, material: polystyrene) that does not support cell proliferation was covered with a metal mask having multiple circular holes with a diameter of 0.3 mm. Plasma treatment (under a gas pressure of 20 Pa, conductive current of 20 mA, irradiation time of 10 seconds) was performed on the metal mask using a plasma irradiation device (made by Vacuum Device Co., Ltd., product name: Plasma Ion Bombardier PIB-20) to create a pattern that forms a region suitable for cell proliferation. The block copolymer was dissolved in ethanol to prepare a 0.6 wt% solution, and this solution was spin-coated onto the surface of the patterned substrate at 2000 rpm for 60 seconds. After drying at room temperature for 1 hour, a substrate coated with the block copolymer was prepared.

[0096] The constituent unit ratio of the block copolymer was 4 wt% n-butyl methacrylate and 96 wt% N-isopropylacrylamide, with a molecular weight of Mn 77,000. The thickness of the block copolymer coating on the polystyrene dish was 25 nm.

[0097] The substrate coated with the aforementioned patterning and block copolymer was then coated with 0.2 mL / cm³ of culture medium StemFitAK02N (manufactured by Ajinomoto Co., Inc.). 2 In addition, 1300 human iPS cells 201B7 strain were added per cm³. 2iMatrix-511 solution (manufactured by Nippi Corporation) was added at a concentration of 2.5 μL / mL. The cells were cultured at 37°C with a CO2 concentration of 5%. In addition, Y-27632 (manufactured by Wako Pure Chemical Industries, Ltd.) (concentration 10 μM) was added to the culture medium until 24 hours after cell seeding.

[0098] At 24, 96, and 144 hours after cell seeding, the cells were observed using a phase-contrast microscope. In all cases, cell adhesion and proliferation were confirmed along the patterned shape, and the culture medium was replaced with fresh medium. After the medium change 144 hours after cell seeding, the substrate was cooled to room temperature for 20 minutes and observed again with a phase-contrast microscope. As the substrate cooled, the colonies detached from the surrounding area, and the formation of spheroids on the substrate was observed. Furthermore, by tapping the sides of the substrate by hand, the spheroids detached from the substrate, and uniformly sized, spherical spheroids could be collected. [Example 2] A metal mask with multiple 0.5 mm x 1 mm rectangular holes was used as the metal mask in Example 1, and the cell culture substrate was prepared in the same manner as in Example 1.

[0099] Cell culture was performed in the same manner as in Example 1. At 24, 96, and 144 hours after cell seeding, the cells were observed using a phase-contrast microscope. In all cases, cell adhesion and proliferation were confirmed along the patterned shape, and the culture medium was replaced with fresh medium. After the medium change 144 hours after cell seeding, the substrate was cooled at room temperature for 20 minutes, and the sides of the substrate were tapped by hand. Uniformly sized, elongated, roll-shaped spheroids were successfully collected. [Example 3] A metal mask with multiple circular holes with a diameter of 0.5 mm was used as the metal mask in Example 1, and the cell culture substrate was prepared in the same manner as in Example 1.

[0100] Cell culture was performed in the same manner as in Example 1. At 24, 96, and 144 hours after cell seeding, the cells were observed using a phase-contrast microscope. In all cases, cell adhesion and proliferation were confirmed along the patterned shape, and the culture medium was replaced with fresh medium. After the medium change 144 hours after cell seeding, the substrate was cooled at room temperature for 20 minutes and then cultured again at 37°C for 1 hour. As the cells cooled, the colonies detached from the surrounding area, forming spheroids, and spheroids adhered to the substrate were obtained. [Example 4] In Example 1, a metal mask with multiple circular holes with a diameter of 0.2 mm was used, and instead of the Plasma Ion Bombardier PIB-20, a VUV Aligner manufactured by Ushio Inc. was used. The cell culture substrate was prepared in the same manner as in Example 1. Cell culture was performed in the same manner as in Example 1. At 24, 96, and 144 hours after cell seeding, the cells were observed using a phase-contrast microscope. In all cases, cell adhesion and proliferation were confirmed along the patterned shape, and the culture medium was replaced with fresh medium. After the medium change 144 hours after cell seeding, the substrate was cooled at room temperature for 20 minutes, and the sides of the substrate were tapped by hand. Spherical spheroids of uniform size were recovered. The particle size of the recovered spheroids was measured, and the particle size variability (standard deviation of particle size / average particle size) was calculated to be 6.6%, indicating low variability. [Example 5] In Example 1, a metal mask with multiple circular holes with a diameter of 0.5 mm was used, and instead of the Plasma Ion Bombardier PIB-20, a VUV Aligner manufactured by Ushio Inc. was used. The cell culture substrate was prepared in the same manner as in Example 1. Cell culture was performed in the same manner as in Example 1. At 24, 96, and 144 hours after cell seeding, the cells were observed using a phase-contrast microscope. In all cases, cell adhesion and proliferation were confirmed along the patterned shape, and the culture medium was replaced with fresh medium. After the medium change 144 hours after cell seeding, the substrate was cooled to room temperature for 20 minutes, the cells were detached by tapping the sides of the substrate by hand, and then cultured for another day. Spherical spheroids of uniform size were successfully collected. The particle size of the collected spheroids was measured, and the particle size variability (standard deviation of particle size / average particle size) was calculated to be 14.2%, indicating low variability. [Example 6] Instead of the metal mask used in Example 1, a surface protection film (E-MASK manufactured by Nitto Denko) with multiple circular holes of 0.2 mm in diameter formed by laser processing was used. This surface protection film was attached to the substrate and subjected to plasma treatment, after which the surface protection film was peeled off. Subsequently, a block copolymer coating was applied in the same manner as in Example 1 to prepare a cell culture substrate.

[0101] Cell culture was performed in the same manner as in Example 1. At 24, 96, and 144 hours after cell seeding, the cells were observed using a phase-contrast microscope. In all cases, cell adhesion and proliferation were confirmed along the patterned shape, and the culture medium was replaced with fresh medium. After the medium change 144 hours after cell seeding, the substrate was cooled at room temperature for 20 minutes, and the sides of the substrate were tapped by hand. Spherical spheroids of uniform size were recovered. The particle size of the recovered spheroids was measured, and the particle size variability (standard deviation of particle size / average particle size) was found to be 4.2%, indicating low variability. [Example 7] A metal mask with multiple circular holes with a diameter of 0.2 mm was used as the metal mask in Example 1, and the cell culture substrate was prepared in the same manner as in Example 1.

[0102] Atomic force microscopy images of the prepared cell culture substrate were measured, and region (A) consisted of a region (A2) with a diameter of approximately 90 nm and a high concentration of N-isopropylacrylamide polymer, as well as other regions. Similarly, region (B) also consisted of a region (B2) with a diameter of approximately 90 nm and a high concentration of N-isopropylacrylamide polymer, as well as other regions.

[0103] Cell culture was performed in the same manner as in Example 1. At 24, 96, and 144 hours after cell seeding, the cells were observed using a phase-contrast microscope. In all cases, cell adhesion and proliferation were confirmed along the patterned shape, and the culture medium was replaced with fresh medium. After the medium change 144 hours after cell seeding, the substrate was cooled at room temperature for 20 minutes, and the sides of the substrate were tapped by hand. Spherical spheroids of uniform size were recovered. The particle size of the recovered spheroids was measured, and the particle size variability (standard deviation of particle size / average particle size) was found to be 4.5%, indicating low variability. [Example 8] In Example 1, a metal mask having multiple circular holes with a diameter of 0.2 mm was used, and the block copolymer was synthesized and coated using the method described below.

[0104] 0.40 g (0.1 mmol) of 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, 7.11 g (50 mmol) of n-butyl methacrylate, and 33 mg (0.2 mmol) of azobis(isobutyronitrile) were added to a test tube and dissolved in 50 mL of 1,4-dioxane. After degassing by nitrogen bubbling for 30 minutes, the mixture was reacted at 70°C for 24 hours. After the reaction was complete, the reaction solvent was removed by vacuum distillation using a rotary evaporator, and the reaction solution was concentrated. The concentrate was poured into 250 mL of methanol, and the precipitated yellow oily substance was collected and dried under vacuum to obtain the n-butyl methacrylate polymer.

[0105] In a test tube, 0.9 g (0.3 mmol) of the n-butyl methacrylate polymer, 0.26 g (2.4 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 by nitrogen bubbling for 30 minutes, the mixture was reacted at 65°C for 17 hours. After the reaction was complete, the reaction solvent was diluted with acetone, poured into 500 mL of hexane, and the precipitated solid was collected and dried under reduced pressure. The mixture was then dissolved again in acetone, poured into 500 mL of pure water, and the precipitated solid was collected and dried under reduced pressure to obtain a copolymer of N-isopropylacrylamide and n-butyl methacrylate.

[0106] A 10 cm diameter dish (made by AS ONE, material: polystyrene) that does not support cell proliferation was covered with a metal mask having multiple circular holes with a diameter of 0.2 mm. Plasma treatment (under a gas pressure of 20 Pa, conductive current of 20 mA, irradiation time of 10 seconds) was performed on the metal mask using a plasma irradiation device (manufactured by Vacuum Devices Co., Ltd., product name: Plasma Ion Bombardier PIB-20) to create a pattern that forms a region suitable for cell proliferation. The block copolymer was dissolved in ethanol to prepare a 1 wt% solution, and this solution was spin-coated onto the surface of the patterned substrate at 2000 rpm for 60 seconds. After immersion in pure water for 1 hour, the substrate was dried at room temperature for 24 hours to produce a substrate coated with the block copolymer.

[0107] The constituent unit ratio of the block copolymer was 50 wt% n-butyl methacrylate and 50 wt% N-isopropylacrylamide, with a molecular weight of Mn 0.6 million. The thickness of the block copolymer coating on the polystyrene dish was 50 nm.

[0108] Atomic force microscopy images of the prepared cell culture substrate were measured, and it was confirmed that regions (A) and (B) were homogeneous.

[0109] Cell culture was performed in the same manner as in Example 1. At 24, 96, and 144 hours after cell seeding, the cells were observed using a phase-contrast microscope. In all cases, cell adhesion and proliferation were confirmed along the patterned shape, and the culture medium was replaced with fresh medium. After the medium change 144 hours after cell seeding, the substrate was cooled to room temperature for 20 minutes. The sides of the substrate were tapped by hand, but the cells did not detach. Therefore, pipetting was performed 20 times to detach the cells. Uniformly sized, elongated, roll-shaped spheroids were recovered. The particle size of the recovered spheroids was measured, and the particle size variability (standard deviation of particle size / average particle size) was calculated to be 14.6%. Although the variability was small, it was larger than that in Example 7. [Comparative Example 1] In Example 1, the cell culture substrate was prepared in the same manner as in Example 1, but without plasma treatment. Because there was no cell proliferation region, the iPS cells died 24 hours after seeding, and spheroids could not be formed. [Comparative Example 2] In Example 1, the cell culture substrate was prepared in the same manner as in Example 1, except that the block copolymer was not coated. Because there were no stimulus-responsive regions, the colonies did not detach from the substrate, and spheroids could not be recovered. In addition, the shape of the colonies was non-uniform. [Comparative Example 3] In Example 1, no patterning was performed, and the entire substrate was plasma-treated. The cell culture substrate was otherwise prepared in the same manner as in Example 1. The size and shape of the colonies were non-uniform. The particle size of the recovered spheroids was measured, and the particle size variability (standard deviation of particle size / average particle size) was found to be 36.5%, indicating a large variability. [Comparative Example 4] In Example 1, a metal mask with numerous circular holes with a diameter of 4 mm was used as the metal mask, and the cell culture substrate was prepared in the same manner as in Example 1. The size and shape of the colonies were non-uniform. The particle size of the recovered spheroids was measured, and the particle size variability (standard deviation of particle size / average particle size) was calculated to be 31.1%, indicating a large variability. [Comparative Example 5] Using a commercially available cell culture vessel for spheroid formation (manufactured by AGC Techno Glass Co., Ltd., product name EZSPHERE) with an uneven surface and a cell-non-adherent surface, cell seeding was performed in the same manner as in Example 1, and spheroids were formed by suspension culture. Although the formed spheroids were generally spherical, several aggregates formed by the aggregation of multiple spheroids were observed. The particle size of the collected spheroids was measured, and the particle size variability (standard deviation of particle size / average particle size) was calculated to be 22.3%, indicating a large variability. [Reference example] The zeta potentials of regions (A) and (B) in Example 1 were measured by the following method. The entire surface of a 10 cm diameter dish that does not allow for cell proliferation was plasma-treated using a plasma irradiation device (Shinku Device Co., Ltd., product name Plasma Ion Bombardier PIB-20) (under a gas pressure of 20 Pa, conductive current of 20 mA, irradiation time of 10 seconds) to plasma-treat the entire substrate surface. Block copolymers were then coated onto this plasma-treated substrate and an untreated substrate in the same manner as in Example 1 to prepare a sample in which the entire substrate surface was region (A) of Example 1 and a sample in which the entire substrate surface was region (B) of Example 1. The zeta potentials of these substrates were measured (using SurPASS manufactured by Anton Paar Co., Ltd.), and the zeta potential of region (B) was 8 mV higher than that of region (A).

[0110] [Table 1] [Explanation of Symbols]

[0111] A (A) area B (B) area 1 Base material 2. Stimulus-responsive polymers 10 Cell culture substrate 20 cells 21 Colonies 22 Spheroids

Claims

1. A cell culture substrate comprising a base material and a stimulus-responsive polymer coated on the base material, wherein the stimulus-responsive polymer is a block copolymer having water-insoluble block segments and stimulus-responsive block segments, and the cell culture substrate has the following two regions (A) and (B): The substrate has a cell-proliferating region and a region that does not promote cell proliferation, and a layer containing the stimulus-responsive polymer is formed on these cell-proliferating and non-cell-proliferating regions. The thickness of the aforementioned layer is 25 nm or more and 50 nm or less. A cell culture substrate characterized in that the ratio of the average roughness of the layer to the thickness of the layer is 0.3 or more and 1 or less. (A) Possesses cell proliferation and stimulus responsiveness, with an area of ​​0.001 to 5 mm² ２ An island-like region. (B) A region adjacent to the region (A) that does not have cell proliferation properties.

2. The cell culture substrate according to claim 1, characterized in that the (A) region consists of the following two regions (A1) and (A2), and the (B) region consists of the following two regions (B1) and (B2). (A1) Cell proliferation zone (A2) Stimulus responsive area (B1) Base material adhesion area (B2) Stimulus responsive area

3. The cell culture substrate according to claim 1 or 2, characterized in that the (A) region is composed of a cell proliferation region in which a temperature-responsive region with a diameter of 10 to 500 nm is dispersed, or a temperature-responsive region that does not have cell proliferation properties in which a cell proliferation region with a diameter of 10 to 500 nm is dispersed.

4. A cell culture substrate according to any one of claims 1 to 3, characterized in that the ratio of the average roughness of the layer to the thickness of the layer is 0.5 or more and 1 or less.

5. The cell culture substrate according to claim 4, characterized in that the stimulus-responsive polymer is a block copolymer having water-insoluble block segments and stimulus-responsive block segments exceeding 90 wt%.

6. The cell culture substrate according to any one of claims 1 to 5, characterized in that the (A) region is a plasma treatment region.

7. A cell culture substrate according to any one of claims 1 to 6, characterized in that it is for the formation of spheroids of pluripotent stem cells.

8. A method for producing a cell culture substrate according to any one of 1 to 7, characterized by having the following steps (1) and (2). (1) On the surface of a substrate that does not have cell proliferation properties, a material that has cell proliferation properties and an area of ​​0.001 to 5 mm ２ The process of forming island-like regions. (2) A step of forming a layer of stimulus-responsive substance on the surface of the substrate.

9. The method for producing a cell culture substrate according to claim 8, characterized in that, in step (1) above, a region having cell proliferation properties is formed on the surface of a substrate that does not have cell proliferation properties by plasma treatment, UV treatment, corona treatment, or a combination thereof.

10. A method for producing a cell culture substrate according to claim 8 or 9, characterized in that the substrate used in step (1) above has cell adhesion properties but does not have cell proliferation properties.

11. The above step (1) is performed on an area of ​​0.001 to 10 mm ２ A method for producing a cell culture substrate according to claim 9 or 10, characterized by comprising the step of attaching a surface protective film having holes to a substrate.

12. A method for producing spheroids, characterized by following the steps (i) to (iii) below. (i) A step of seeding cells onto a cell culture substrate according to any one of claims 1 to 7. (ii) A step of culturing the seeded cells and forming colonies of cells attached to the cell culture substrate. (iii) A step of detaching at least a portion of the colony from the cell culture substrate by applying an external stimulus to form a cell spheroid.

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