Cell culture method

Abstract

The present invention addresses the problem of providing a method for enabling, in mass production of human induced pluripotent stem cell (hiPS cell)-derived cardiomyocyte aggregates, the production of homogeneous and high-quality cardiomyocyte aggregates without using a living body-derived serum.　The present invention provides a method for producing human induced pluripotent stem cell (hiPS cell)-derived cardiomyocyte aggregates, comprising: a step for forming, on a substrate having the ability to inhibit the adhesion of cells, a base membrane for cardiomyocyte culture that includes a cell adhesive substance and a polymer which contains a repeating unit derived from a monomer represented by formula (I); and a step for then seeding cardiomyocytes. (I) [In formula (1), Ua1, Ua2, Ra1, and Ra2 are as described in the specification and claims.]

Description

Cell culture method

[0001] The present invention relates to a method for producing a cardiomyocyte aggregate derived from human induced pluripotent stem cells (hiPS cells).

[0002] Heart disease is the leading cause of death worldwide, and left ventricular systolic dysfunction is a contributing factor. Transplanting sufficient numbers of ventricular muscle cells could potentially be a radical treatment for systolic heart failure (HFrEF). However, no method for efficiently producing sufficient numbers of ventricular muscle cells has been known. Meanwhile, cell aggregate (spheroid) therapy has attracted attention in the field of regenerative medicine, and the establishment of industrial technology for the stable, large-scale production of spheroids is desired. Various materials have been proposed as base film-forming agents for efficient cell culture. For example, Patent Literature 1 discloses a method for producing a polymer and a cell culture vessel used as a base film for cell culture, and Patent Literature 2 discloses a method for producing a cell structure. These cell culture base films require the use of serum derived from living organisms in the process of producing homogeneous cell aggregates (culture) to ensure uniform adhesion of cells to the base film. However, this poses problems such as quality variations due to individual differences in proliferation ability, and safety risks such as allergies and viral contamination when using serum derived from animals other than humans.

[0003] International Publication No. 2020 / 040247 Japanese Patent Application Laid-Open No. 2017-143755

[0004] The present invention aims to provide a method for mass-producing cardiomyocyte aggregates derived from human induced pluripotent stem cells (hiPS cells), which can produce homogeneous, high-quality cardiomyocyte aggregates without using serum derived from living organisms.

[0005] As a result of extensive research, the inventors discovered that by using a substrate for producing cell aggregates equipped with a base film containing a specific polymer and a cell adhesive substance, and seeding and culturing hiPS cell-derived cardiomyocytes, it is possible to produce homogeneous, high-quality cardiomyocyte aggregates without using serum derived from living organisms, and thus completed the present invention.

[0006] The present invention includes the following: [1] A substrate having cell adhesion-inhibiting properties, the substrate comprising a compound of the following formula (I): [In the formula, U ａ１ and U ａ２ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R a１ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R a２

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[0099] [ [In the formula, R ｂ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms]. [6] The method for producing a cardiomyocyte aggregate according to any one of [1] to [5], wherein the weight ratio of the polymer to the cell adhesive substance is 100:0.1 to 100:100. [7] The method for producing a cardiomyocyte aggregate according to any one of [1] to [6], wherein the cell adhesive substance contains a glycoprotein. [8] A method for producing a cardiomyocyte aggregate according to any one of [1] to [6], wherein a compound represented by the following formula (Ia): [In the formula, U ａ１ and U ａ２ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R a１ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R a２[9] A method for producing a cardiomyocyte cell aggregate derived from human induced pluripotent stem cells (hiPS cells), the method comprising the step of seeding cardiomyocytes onto a cell aggregate production substrate provided with a base film for cardiomyocyte culture, the base film comprising a polymer containing a repeating unit represented by formula (IIa): [In the formula, R ｂ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms].

[0007] The method of the present invention for producing hiPS cell-derived cardiomyocyte aggregates enables uniform adhesion of cardiomyocytes, even under culture conditions free of animal-derived serum, followed by the production of cell aggregates, thereby enabling the mass production of homogeneous, high-quality cardiomyocyte aggregates for use in the field of regenerative medicine.

[0008] 1 is a photograph of the entire well surface after 24 hours of culturing in the seeding and culturing step of Example 1, in which cardiomyocytes were seeded in each well and cultured for 48 hours. 2 is a photograph of the entire well surface after 72 hours of culturing in the seeding and culturing step of Example 1, in which cardiomyocytes were seeded in each well and cultured for 72 hours. 3 is a photograph of the entire well surface after 72 hours of culturing in the seeding and culturing step of Example 2, in which cardiomyocytes were seeded in each well of substrate 2 and cultured for 72 hours. 4 is a photograph of the entire well surface after 72 hours of culturing in the seeding and culturing step of Example 2, in which cardiomyocytes were seeded in each well of substrate 3 and cultured for 72 hours. 5 is a graph showing the results of measuring the diameter of cardiomyocytes seeded on substrate 2, cultured for 96 hours, and then recovered in the seeding and culturing step of Example 2. 6 is a graph showing the results of measuring the diameter of cardiomyocytes seeded on substrate 3, cultured for 96 hours, and then recovered in the seeding and culturing step of Example 2. In Example 3, cardiomyocytes (stock: CM1, CM2, and CM3) were seeded into each well of substrate 2, and photographs of the entire well surface were taken after culturing for 48 hours and 72 hours. In Example 3, cardiomyocytes (stock: CM1, CM2, and CM3) were seeded into each well of Elplasia plate, and photographs of the entire well surface were taken after culturing for 48 hours and 7 days. In Example 3, a graph showing the maturity of cell clusters (cardiomyocyte spheres) cultured on substrates 2 and 3 of the present invention and cell clusters (cardiomyocyte spheres) cultured on Elplasia plate in terms of the expression level of maturation markers (TNNI3 / TNNI1) analyzed by quantitative PCR (comparative Ct method). In the seeding and culturing step of Example 4, 4 x 10 ６ 10 cells were seeded into each well of the substrate 4, and after culturing for 72 hours, a photograph of the entire well was taken. ６ 4 is a photograph of the entire well of the substrate 4 after seeding 4 × 10 cardiomyocytes into each well and culturing for 72 hours. ６ 1 is a photograph of the entire well surface after seeding 3×10 cardiomyocytes into each well of the substrate 5 and culturing for 72 hours. ６4 is a photograph of the entire surface of a well after seeding 4 × 10 cardiomyocytes into each well of a substrate 5 and culturing for 72 hours. ６ 1 is a graph showing the results of measuring the diameter of cardiomyocytes seeded on a substrate 4, collected after 96 hours of culture, and then cultured in Example 4. ６ 1 is a graph showing the results of measuring the diameter of cardiomyocytes seeded on a substrate 4, collected after 96 hours of culture, and then cultured in Example 4. ６ 1 is a graph showing the results of measuring the diameter of cardiomyocytes seeded on a substrate 5, collected after 96 hours of culture, and then cultured in Example 4. ６ 10 is a graph showing the results of measuring the diameter of cardiomyocytes of the present invention, which were seeded on a substrate 5 and harvested after 96 hours of culture.

[0009] <Method of producing a cell aggregate> The method of producing a cardiomyocyte aggregate derived from hiPS cells of the present invention comprises the step of: [In the formula, U ａ１ and U ａ２ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R a１ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R a２ represents a linear or branched alkylene group having 1 to 5 carbon atoms] and a cell adhesive substance, and then seeding the cardiomyocytes.

[0010] (Underlayer formation step) The underlayer for cardiomyocyte culture according to the production method of the present invention is formed using a underlayer forming agent. The underlayer forming agent is typically a compound represented by the following formula (I): [In the formula, U ａ１ and U ａ２ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R a１ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; Ra２ represents a linear or branched alkylene group having 1 to 5 carbon atoms], a polymer containing a repeating unit derived from a monomer represented by the formula:

[0011] (Polymer) The polymer contained in the agent for forming a base film for cell culture of the present application is a polymer containing a repeating unit derived from the monomer represented by formula (I) above. The polymer contains a repeating unit derived from the monomer represented by formula (I) above together with the cationic monomer represented by formula (I) below: [In the formula, R ｂ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms].

[0012] The polymer containing a repeating unit derived from the monomer represented by the above formula (I) is represented by the following formula (Ia): [In the formula, U ａ１ , U ａ２ , R a１ and R a２ Similarly, a polymer obtained by polymerizing an anionic monomer represented by formula (II) together with a cationic monomer represented by formula (I) can be expressed as a polymer containing a repeating unit represented by formula (IIa) below together with a repeating unit represented by formula (Ia): [In the formula, R ｂ has the same meaning as formula (II).

[0013] Therefore, the production method of the present invention is also a method for producing a cardiomyocyte aggregate derived from human induced pluripotent stem cells (hiPS cells), comprising the step of seeding cardiomyocytes on a substrate having a cell adhesion-inhibiting ability, the substrate having a base film for cardiomyocyte culture comprising a polymer containing a repeating unit represented by formula (Ia) above, or a polymer containing a repeating unit represented by formula (IIa) together with a repeating unit represented by formula (Ia) above, and a cell adhesive substance.

[0014] In this specification, unless otherwise defined, examples of a "linear or branched alkyl group having 1 to 5 carbon atoms" include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, an s-butyl group, a t-butyl group, an n-pentyl group, a 1-methylbutyl group, a 2-methylbutyl group, a 3-methylbutyl group, a 1,1-dimethylpropyl group, a 1,2-dimethylpropyl group, a 2,2-dimethylpropyl group, and a 1-ethylpropyl group.

[0015] R a１ and R ｂ are preferably each independently selected from a hydrogen atom and a methyl group. ａ１ and U ａ２ are each independently preferably selected from a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, and an n-butyl group, more preferably a methyl group or an ethyl group, and most preferably a methyl group.

[0016] In this specification, unless otherwise defined, examples of a "linear or branched alkylene group having 1 to 5 carbon atoms" include a methylene group, an ethylene group, a propylene group, a trimethylene group, a tetramethylene group, a 1-methylpropylene group, a 2-methylpropylene group, a dimethylethylene group, an ethylethylene group, a pentamethylene group, a 1-methyl-tetramethylene group, a 2-methyl-tetramethylene group, a 1,1-dimethyl-trimethylene group, a 1,2-dimethyl-trimethylene group, a 2,2-dimethyl-trimethylene group, and a 1-ethyl-trimethylene group. a２ is preferably selected from an ethylene group and a propylene group.

[0017] Therefore, examples of the cationic monomer represented by formula (I) include 2-N,N-dimethylaminoethyl methacrylate and N,N-dimethylaminomethyl methacrylate, with 2-N,N-dimethylaminoethyl methacrylate being preferred. Examples of the anionic monomer represented by formula (II) include acrylic acid and methacrylic acid, with methacrylic acid being preferred.

[0018] The molar ratio of units derived from the monomer represented by formula (I) to units derived from the monomer represented by formula (II) in the polymer is 100 / 0 to 50 / 50, preferably 98 / 2 to 50 / 50, more preferably 98 / 2 to 60 / 40, and particularly preferably 98 / 2 to 70 / 30. When the molar ratio of formula (II) is 50 or less, a decrease in cell adhesive strength due to the anionic nature of the polymer can be suppressed.

[0019] (Monomer Having Two or More Carbon-Carbon Unsaturated Bonds) The polymer is not particularly limited as long as it contains units derived from the monomer represented by formula (I) and, optionally, units derived from the monomer represented by formula (II). The polymer may contain repeating units other than the units derived from the monomer represented by formula (I) / formula (II) as long as the object of the present invention is not impaired. The repeating units of the polymer may be 50 mol % or more, preferably 75 mol % or more, more preferably 80 mol % or more, and even more preferably 90 mol % or more. For example, the polymer may be a polymer obtained by polymerizing a monomer having two or more carbon-carbon unsaturated bonds together with the monomer represented by formula (I) / formula (II). The monomer having two or more carbon-carbon unsaturated bonds is specifically a monomer having two or more carbon-carbon double bonds, such as a polyfunctional acrylate compound, a polyfunctional acrylamide compound, a polyfunctional polyester, or an isoprene compound.

[0020] Preferred specific examples include monomers represented by the following formulas (III) to (V).

[0021] In the formula, R ｃ and R ｄ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R e represents a linear or branched alkylene group having 1 to 5 carbon atoms, and n represents a number from 1 to 50. Among these, the monomer represented by formula (III) is preferred.

[0022] The molar ratio of the monomers represented by formulas (III) to (V) to the entire polymer is preferably 0 to 50%, more preferably 2 to 25%. When the molar ratio of formulas (III) to (V) is 50% or less, gelation of the solid content during production due to high molecular weight caused by excessive crosslinking can be suppressed, and production can be facilitated.

[0023] R ｃ and R ｄ are preferably each independently selected from a hydrogen atom and a methyl group. ｅ is preferably selected from a methylene group, an ethylene group, and a propylene group, and is most preferably an ethylene group. n is a number from 1 to 50, preferably a number from 1 to 30, and more preferably a number from 1 to 10.

[0024] The difference between the mol % of the monomer represented by formula (II) relative to the total amount of the polymer and the mol % of the monomer represented by formula (II) relative to the total amount of monomers charged in the preparation step is 0 to 10 mol %. The polymer of the present application, produced by the production method described below, has a small difference between the monomer charge ratio and the measured value of the produced polymer, and is 0 to 10 mol %, more preferably 0 to 8 mol %.

[0025] The number average molecular weight (Mn) of the polymer is 20,000 to 1,000,000, and more preferably 50,000 to 800,000. The ratio (Mw / Mn) of the weight average molecular weight (Mw) to the number average molecular weight (Mn) of the polymer is 1.01 to 10.00, preferably 1.2 to 8.0, preferably 1.4 to 6.0, preferably 1.5 to 5.0, and preferably 1.6 to 4.5. The number average molecular weight (Mn) and number average molecular weight (Mn) can be determined, for example, by gel filtration chromatography as described in the examples.

[0026] By using the polymer of the present application as a base film for cell culture, it is possible to adhere cells and then detach them to form cell aggregates. Cell aggregates refer to structures formed as a result of cell aggregation, and are not limited to shapes such as spherical or ring-shaped. Compared to cell aggregates produced by conventional non-adhesive culture on low-adhesion plates, this method offers advantages such as the ability to adjust the size of the cell aggregates by specifying the adhesion area (cell aggregates of any size can be produced). The entire disclosures of International Publication No. 2020 / 040247 and Japanese Patent Application No. 2020-028120 are incorporated herein by reference.

[0027] (Cell Adhesive Substance) The base film forming agent for cell culture of the present invention contains a cell adhesive substance. By including a cell adhesive substance, cell adhesion, spreading, proliferation, and differentiation can be promoted. As the cell adhesive substance, known substances such as biologically derived substances such as extracellular matrix (ECM) proteins, glycoproteins, and peptides, and synthetic compounds (low molecular weight, high molecular weight) can be used, but compounds that are not biologically derived, such as synthetic compounds (low molecular weight, high molecular weight), are preferred. A low molecular weight is, for example, a compound with a weight-average molecular weight of 2,000 or less, and a high molecular weight is, for example, a compound with a weight-average molecular weight of 2,000 or more, with an upper limit of, for example, 1,000,000.

[0028] Examples of extracellular matrix (ECM) proteins include collagen (e.g., Merck's type I collagen (product numbers C9791, C7661, C1809, C2249, C2124), type II collagen (product number C9301), type IV collagen (product numbers C0543, C5533), elastin (e.g., Merck's product numbers E1625, E6527), fibronectin (e.g., Merck's product numbers F1141, F0635, F2518, F0895, F4759, F2006), laminin (e.g., Merck's product numbers L6724, L2020, L4544), laminin fragments (e.g., Matricsome's product number 892011), vitronectin (e.g., VTN-N (Gibco)), Vitronectin, Human Recombinant, Animal Free (PeproTech), Merck product numbers: V0132, V9881, V8379, 08-126, SRP3186).

[0029] The cell adhesive substance is preferably a glycoprotein, specifically selected from vitronectin, integrin, cadherin, fibronectin, laminin, tenascin, osteopontin, and bone sialoprotein, and preferably a protein having an RGD amino acid sequence.

[0030] Examples of peptides include ECM peptide (MAPTrix ​​(registered trademark) from Kollodis Bio Sciences) and RGD peptide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.: 180-01531).

[0031] Examples of synthetic compounds (polymers) include polylysine (e.g., Merck products: P4707, P4832, P7280, P9155, P6407, P6282, P7405, P5899) and polyornithine (e.g., Merck product number P4975). Examples of synthetic compounds (low molecular weights) include adhesamine (e.g., Nagase & Co.: AD-00000-0201) and synthetic cyclic RGD peptide (e.g., IRIS BIOTECH: LS-3920.0010).

[0032] The ratio (by mass) of the polymer to the cell adhesive substance in the base film-forming agent for cell culture of the present invention is not limited as long as a base film-forming agent capable of cell culture can be formed, but is preferably 100:0.1 to 100:100, and more preferably 100:10 to 100:30. When the cell adhesive substance is 0.1 or more, cell adhesiveness is sufficiently exerted, and when the cell adhesive substance is 100 or less, cell aggregation (formation of cell aggregates) after cell adhesion can be facilitated.

[0033] The agent for forming a base film for cell culture of the present invention contains a solvent. The solvent is not limited as long as it can dissolve the polymer, but is preferably an aqueous solution containing water. Examples of aqueous solutions include water, salt-containing aqueous solutions such as physiological saline or phosphate buffer solution, and mixed solvents combining water or salt-containing aqueous solutions with alcohol. Examples of alcohols include alcohols having 2 to 6 carbon atoms, such as ethanol, propanol, isopropanol, 1-butanol, 2-butanol, isobutanol, t-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 1-heptanol, 2-heptanol, 2,2-dimethyl-1-propanol (neopentyl alcohol), 2-methyl-1-propanol, 2-methyl-1-butanol, 2-methyl-2-butanol (t-amyl alcohol), 3-methyl-1-butanol, 3-methyl-3-pentanol, cyclopentanol, and 1-hexanol. Examples of suitable solvents include 2-methyl-1-pentanol, 2-methyl-2-pentanol, 2-methyl-3-pentanol, 3-methyl-1-pentanol, 3-methyl-2-pentanol, 3-methyl-3-pentanol, 4-methyl-1-pentanol, 4-methyl-2-pentanol, 4-methyl-3-pentanol, and cyclohexanol. These may be used alone or in combination. The water content in the aqueous solution is, for example, 50% to 100% by mass, 80% to 100% by mass, or 90% to 100% by mass.

[0034] In addition to the above-mentioned polymer, cell adhesive substance, and solvent, the primer film-forming agent may also contain other substances as needed within the range that does not impair the performance of the resulting primer film, such as pH adjusters, crosslinking agents, preservatives, surfactants, primers that improve adhesion to the container or substrate, antifungal agents, and sugars.

[0035] (Substrate for Producing Cell Aggregates) In one embodiment, the production method of the present invention is carried out using a substrate for producing cardiomyocyte aggregates, which comprises spots of a base film for culturing cardiomyocytes formed with the base film forming agent on a substrate having cell adhesion-inhibiting properties. The substrate for producing cardiomyocyte aggregates of the present invention is produced using a substrate having cell adhesion-inhibiting properties. The substrate may be subjected to a cell adhesion-inhibiting treatment before the formation of the spots (base film). The substrate having cell adhesion-inhibiting properties may be a commercially available cell culture dish that has been treated to reduce cell adhesion, a cell culture vessel having cell adhesion-inhibiting properties, or the like. For example, the cell culture vessel described in JP 2008-61609 A can be used, but is not limited thereto. Alternatively, the substrate may be produced by applying a known coating film-forming composition having cell adhesion-inhibiting properties. For example, the coating film-forming composition described in WO 2014 / 196650 A can be used as the coating film-forming composition. The coating film-forming composition may include a copolymer (P) containing a repeating unit containing an organic group represented by the following formula (a) and a repeating unit containing an organic group represented by the following formula (b): [In the formula, U ａ１１ , U ａ１２ , U ｂ１１ , U ｂ１２ and U ｂ１３ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; An －Preferably, the method includes a step of applying a coating film-forming composition containing a solvent and a cation bond to the surface of a container or a substrate and drying the composition. The coating film may be present on at least a portion of the substrate surface, but is preferably applied over the entire surface on which cell aggregates are produced (i.e., the surface on which the spots of the present application are present) or over the entire substrate surface. The entire disclosures of WO 2014 / 196650 and WO 2016 / 093293 are incorporated herein by reference.

[0036] Having the ability to inhibit cell adhesion means that the relative absorbance (WST O.D. 450 nm) (%) ((absorbance (WST O.D. 450 nm) of the example) / (absorbance (WST O.D. 450 nm) of the comparative example)) when compared with a case without a coating film or without a low-cell-adhesion treatment, as determined by a fluorescence microscope using the method described in the examples of WO 2016 / 093293, for example, is 50% or less, preferably 30% or less, and more preferably 20% or less.

[0037] Furthermore, a coating film having cell adhesion inhibitory properties may be prepared by copolymerizing an ethylenically unsaturated monomer, or a polysaccharide or its derivative. Examples of the ethylenically unsaturated monomer include one or more ethylenically unsaturated monomers selected from the group consisting of (meth)acrylic acid and its esters, vinyl acetate, vinylpyrrolidone, ethylene, vinyl alcohol, and their hydrophilic functional derivatives. Examples of polysaccharides or their derivatives include cellulose-based polymers such as hydroxyalkyl cellulose (e.g., hydroxyethyl cellulose or hydroxypropyl cellulose), starch, dextran, and curdlan.

[0038] The hydrophilic functional derivative refers to an ethylenically unsaturated monomer having a hydrophilic functional group or structure. Examples of the hydrophilic functional group or structure include a betaine structure, an amide structure, an alkylene glycol residue, an amino group, and a sulfinyl group.

[0039] The betaine structure means a monovalent or divalent group of a compound having an amphoteric center of a quaternary ammonium type cation structure and an acidic anion structure, and is, for example, a phosphorylcholine group: Examples of ethylenically unsaturated monomers having such a structure include 2-methacryloyloxyethyl phosphorylcholine (MPC).

[0040] The amide structure has the following formula: [where R １６ , R １７ and R １８ are each independently a hydrogen atom or an organic group (for example, a methyl group, a hydroxymethyl group, or a hydroxyethyl group). Examples of ethylenically unsaturated monomers having such a structure include (meth)acrylamide, N-(hydroxymethyl)(meth)acrylamide, and N-isopropyl(meth)acrylamide. Furthermore, monomers or polymers having such a structure are disclosed, for example, in JP-A-2010-169604.

[0041] The alkylene glycol residue refers to an alkyleneoxy group (-Alk-O-) that remains after one or both terminal hydroxyl groups of alkylene glycol (HO-Alk-OH; where Alk is an alkylene group having 1 to 10 carbon atoms) undergo a condensation reaction with another compound, and also encompasses poly(alkyleneoxy) groups in which alkyleneoxy units are repeated. Examples of ethylenically unsaturated monomers having such a structure include 2-hydroxyethyl (meth)acrylate and methoxypolyethylene glycol (meth)acrylate. Furthermore, monomers or polymers having such a structure are disclosed, for example, in JP 2008-533489 A.

[0042] An amino group has the formula: -NH ２ , -NHR １９ or -NR ２０ R ２１ [where R １９ , R ２０ and R ２１are each independently an organic group (for example, a linear or branched alkyl group having 1 to 5 carbon atoms). The amino group in the present invention includes quaternized or salified amino groups. Examples of ethylenically unsaturated monomers having such a structure include dimethylaminoethyl (meth)acrylate, 2-(t-butylamino)ethyl (meth)acrylate, and methacryloylcholine chloride.

[0043] The sulfinyl group has the following formula: [where R ２２ is an organic group (for example, an organic group having 1 to 10 carbon atoms, preferably an alkyl group having 1 to 10 carbon atoms and one or more hydroxy groups). Examples of polymers having such a structure include copolymers disclosed in JP-A-2014-48278 and the like.

[0044] Furthermore, a water-insoluble copolymer that is difficult to dissolve in phosphate buffered saline can be used as a coating film having cell adhesion inhibitory properties.

[0045] As used herein, "water-soluble" means that 1.0 g or more of the copolymer can be dissolved in 100 g of water at 25° C. "Water-insoluble" means that the copolymer does not fall under the category of "water-soluble," i.e., that the solubility in 100 g of water at 25° C. is less than 1.0 g. Therefore, a "water-insoluble copolymer" refers to a copolymer having a solubility of less than 1.0 g in 100 g of water at 25° C., and particularly refers to a copolymer having a solubility of less than 1.0 g in 100 g of phosphate buffered saline at 25° C.

[0046] The water-insoluble copolymer may be a copolymer containing a repeating unit (A) represented by the following formula (A) and a repeating unit (B) represented by the following formula (B).

[0047]

[0048] In the formula, R １ ~R ３ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; X １ and X ２each independently represents a single bond, an ester bond, an ether bond, an amide bond, or a linear or branched alkylene group having 1 to 5 carbon atoms which may be interrupted by an oxygen atom.

[0049] The water-insoluble copolymer may contain two or more types of repeating units (A) or two or more types of repeating units (B), but preferably contains one type of repeating unit (A) and one type of repeating unit (B).

[0050] In the water-insoluble copolymer, R １ ~R ３ are preferably each independently a hydrogen atom, a methyl group, or an ethyl group.

[0051] Unless otherwise defined herein, an "ester bond" means -C(=O)-O- or -O-C(=O)-, an "ether bond" means -O-, and an "amide bond" means -NHC(=O)- or -C(=O)NH-.

[0052] In this specification, unless otherwise defined, the term "a linear or branched alkylene group having 1 to 5 carbon atoms which may be interrupted by an oxygen atom" means a linear or branched alkylene group having 1 to 5 carbon atoms, or a group in which one or more carbon-carbon bonds of the linear or branched alkylene group having 1 to 5 carbon atoms are bonded via an ether bond. １ and X ２ are preferably each independently a methylene group, an ethylene group, or a propylene group.

[0053] In the water-insoluble copolymer, R １ and R ２ is a hydrogen atom, and R ３ is a methyl group, and X １ and X ２ is preferably a single bond.

[0054] The molar ratio (A:B) of the repeating units (A) to the repeating units (B) in the water-insoluble copolymer is 89:11 to 50:50. When the total number of moles of the repeating units (A) and (B) in the water-insoluble copolymer is 100, the molar ratio (A:B) of the repeating units (A) to the repeating units (B) can be expressed as (100-m):m. In this case, the range of m is 11 to 50. The lower limit of m may be 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. The upper limit of m may be 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 38, 37, 36, or 35. The range of m is, for example, 12 to 49, 12 to 48, 15 to 48, 20 to 49, 20 to 45, 22 to 49, or 22 to 45.

[0055] The total mol % of the repeating units (A) and (B) in all repeating units in the water-insoluble copolymer is not particularly limited, but is preferably 90 mol % or more, more preferably 95 mol % or more, even more preferably 99.5 mol % or more, and particularly preferably 100%.

[0056] By setting the molar ratio of the repeating unit (A) to the repeating unit (B) in the water-insoluble copolymer within a specific range, a coating film that is difficult to dissolve in phosphate-buffered saline can be obtained without crosslinking the copolymer. Therefore, the water-insoluble copolymer does not need to have a photosensitive group for crosslinking the copolymer. That is, the water-insoluble copolymer preferably does not have a photosensitive group. Examples of photosensitive groups include azide groups. As such, the water-insoluble copolymer does not need to have a photosensitive group for crosslinking the copolymer, so light irradiation to crosslink the copolymer is not required when forming a coating film. Therefore, the use of the water-insoluble copolymer can simplify the process of forming a coating film that has the ability to inhibit cell adhesion.

[0057] The viscosity-average degree of polymerization (hereinafter sometimes simply referred to as "degree of polymerization") of the water-insoluble copolymer is not particularly limited, but from the viewpoint of preferably obtaining cell adhesion inhibitory ability, it is preferably 200 to 3,000, more preferably 200 to 2,500, and particularly preferably 200 to 2,000. The viscosity-average degree of polymerization is measured after the water-insoluble copolymer has been completely saponified. The "viscosity-average degree of polymerization" of polyvinyl alcohol obtained by complete saponification is a value calculated by the following formula from the intrinsic viscosity [η] (g / dL) measured at 30°C using an Ostwald viscometer with ion-exchanged water as a solvent.

[0058] Here, P represents the viscosity-average degree of polymerization, which can be determined in accordance with JIS K 6726.

[0059] The method for producing the water-insoluble copolymer is not particularly limited, and examples thereof include a method in which a compound represented by the following formula (C) is polymerized to produce a homopolymer, and the obtained homopolymer is partially hydrolyzed by a known saponification reaction to obtain the copolymer.

[0060] In the formula, R １ , R ３ , and X １ has the same meaning as above.

[0061] Furthermore, examples of a method for producing the water-insoluble copolymer include a method of copolymerizing a compound represented by the following formula (C) with a compound represented by the following formula (D) to obtain the copolymer.

[0062] In the formula, R １ ~R ３ , X １ , and X ２ has the same meaning as above.

[0063] The water-insoluble copolymer may be a random copolymer or a block copolymer. A commercially available product may be used as the water-insoluble copolymer. A specific example of a commercially available copolymer is polyvinyl acetate (manufactured by Nippon Vinyl Acetate & Poval Co., Ltd., trade name JMR-150L (registered trademark)).

[0064] The content of the copolymer in the film-forming component in the coating film-forming composition used in the production of a substrate having cell adhesion inhibitory ability according to the present invention is not particularly limited, but is preferably 80% by mass or more, more preferably 90% by mass or more, and particularly preferably 95% by mass or more. Note that the film-forming component refers to all components of the composition excluding the solvent component.

[0065] The content of the copolymer in the coating film-forming composition used in the production of a substrate having cell adhesion-inhibiting properties according to the present invention is not particularly limited, but from the viewpoint of facilitating the formation of a coating film of a desired thickness, it is preferably 0.1 to 10% by mass, more preferably 0.3 to 8% by mass, and particularly preferably 0.5 to 5% by mass. The content of the copolymer in the coating film-forming composition may be 0.02 to 2% by mass, or 0.05 to 1% by mass.

[0066] (Spots) The ratio of the total area of ​​the spots, preferably multiple spots (base film) provided on the substrate for producing cardiomyocyte aggregates of the present invention, the diameter of each spot, and the spacing between spot centers can be appropriately selected from predetermined ranges depending on the type of cells and substrate used, the desired size of the cell aggregates, etc., but the ratio of the total area of ​​the spots to the surface area of ​​the substrate is preferably 30% or more, 40% or more, or 50% or more, and preferably 99% or less, the diameter of each spot is 50 to 5000 μm, preferably 300 to 3000 μm, more preferably 300 to 1000 μm, and most preferably 300 to 600 μm, and the spacing between spot centers is 30 to 1000 μm, preferably 50 to 800 μm, and more preferably 400 to 600 μm.

[0067] The present invention enables the simultaneous formation of multiple uniformly sized spheroids on a single substrate (container) by arranging, at high density and preferably regularly, independent micro-sized regions (spots) to which cardiomyocytes can adhere on a substrate capable of inhibiting cell adhesion. The spots can be formed by applying the primer film-forming agent. The primer film-forming agent can be applied using, for example, inkjet printing, screen printing, slit coating, roll-to-roll printing, or other printing techniques, but is preferably applied using inkjet printing or screen printing.

[0068] Other application methods include, for example, immersing a substrate, optionally with non-spotted areas protected, in the primer film-forming agent, or adding the primer film-forming agent to a substrate (container), optionally with non-spotted areas protected, and allowing it to stand for a predetermined period of time. In the case of a substrate, such as a cell culture vessel, the primer film-forming agent is added to a container, optionally with non-spotted areas protected, and allowing it to stand for a predetermined period of time. Addition can be carried out, for example, by adding the primer film-forming agent in an amount 0.5 to 1 times the total volume of the container using a syringe or the like. The time and temperature for allowing it to stand are appropriately selected depending on the material of the vessel or substrate and the type of primer film-forming agent for cell culture. For example, the time and temperature are set at 10 to 80°C for 1 minute to 24 hours, preferably 5 minutes to 3 hours. This allows the production of a substrate for producing cardiomyocyte aggregates.

[0069] Furthermore, the spots on the surface of the substrate obtained by this method can be used as a substrate for producing cardiomyocyte aggregates, either directly without a drying step or after washing with water or a medium for the sample to be subjected to cell culture (e.g., water, a buffer solution, a culture medium, etc.). That is, after the formation of the spots on the surface of the substrate, the spots can be used as a substrate for producing cardiomyocyte aggregates, either directly without a drying step or after washing with water or a medium for the sample to be subjected to cell culture (e.g., water, a buffer solution, a culture medium, etc., particularly preferably a culture medium (e.g., DMEM medium (Dulbecco's modified Eagle's medium)) within 48 hours, preferably within 24 hours, more preferably within 12 hours, more preferably within 6 hours, even more preferably within 3 hours, and even more preferably within 1 hour.

[0070] The substrate for producing cell aggregates may be subjected to a drying process. The drying process is carried out in air or under vacuum, preferably at a temperature within the range of -200°C to 200°C. The drying process removes the solvent in the base film-forming agent, thereby completely adhering it to the substrate. Spots can be formed by drying at room temperature (10°C to 35°C, preferably 20°C to 30°C, e.g., 25°C), but drying at 40°C to 80°C may be performed to form spots more quickly. Drying temperatures below -200°C require the use of an uncommon refrigerant, resulting in a lack of versatility, and drying takes a long time due to solvent sublimation, resulting in inefficiency. Drying temperatures above 200°C result in thermal decomposition of the polymer. A more preferred drying temperature is 10°C to 180°C, and even more preferred is 20°C to 150°C. The substrate for producing cell aggregates of the present application is produced through the above-described simple process.

[0071] Furthermore, in order to remove impurities remaining on the spots (undercoating films), unbonded polymers, etc., a step of washing with at least one solvent selected from water and aqueous solutions containing electrolytes may be carried out. Washing is preferably performed using running water or ultrasonic cleaning. The aqueous solution containing water and electrolytes may be heated, for example, to a temperature in the range of 40°C to 95°C. Preferred aqueous solutions containing electrolytes include PBS, saline (containing only sodium chloride), Dulbecco's phosphate-buffered saline, Tris-buffered saline, HEPES-buffered saline, and Veronal-buffered saline, with PBS being particularly preferred. After bonding, the coating film remains firmly bonded to the substrate without elution even when washed with water, PBS, alcohol, etc. The maximum and minimum film thicknesses of the spots (undercoating films) of the present application are in the range of 1 to 1,000 nm, preferably 5 to 500 nm.

[0072] (Substrate) The substrate for producing cell aggregates of the present invention can be produced by applying the base film-forming agent to the surface of a substrate and drying it. Here, "surface" refers to the surface that comes into contact with contents such as cells or cell culture medium. The shape of the substrate surface may be flat or uneven, but a flat shape is preferred.

[0073] Examples of substrate materials include glass, metals, metal-containing compounds or metalloid-containing compounds, activated carbon, and resins. Examples of metals include typical metals (aluminum group elements: Al, Ga, In; iron group elements: Fe, Co, Ni; chromium group elements: Cr, Mo, W, U; manganese group elements: Mn, Re; and precious metals: Cu, Ag, and Au). Examples of metal-containing compounds or metalloid-containing compounds include ceramics, which are sintered bodies whose basic component is a metal oxide and are hardened by heat treatment at high temperatures; semiconductors such as silicon; inorganic solid materials such as molded bodies of inorganic compounds such as metal oxides or metalloid oxides (silicon oxide, alumina, etc.), metal carbides or metalloid carbides, metal nitrides or metalloid nitrides (silicon nitride, etc.), and metal borides or metalloid borides; aluminum, nickel titanium, and stainless steel (SUS304, SUS316, SUS316L, etc.).

[0074] The resin may be a natural resin or a derivative thereof, or a synthetic resin. Preferred examples of natural resins or derivatives thereof include cellulose, cellulose triacetate (CTA), nitrocellulose (NC), and cellulose with immobilized dextran sulfate. Preferred examples of synthetic resins include polyacrylonitrile (PAN), polyimide (PI), polyester polymer alloy (PEPA), polystyrene (PS), polysulfone (PSF), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), polyvinyl alcohol (PVA), polyurethane (PU), ethylene vinyl alcohol (EVAL), polyethylene (PE), polyester, polypropylene (PP), polyvinylidene fluoride (PVDF), polyethersulfone (PES), polycarbonate (PC), cycloolefin polymer (COP), polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), ultra-high molecular weight polyethylene (UHPE), polydimethylsiloxane (PDMS), acrylonitrile-butadiene-styrene resin (ABS), and Teflon (registered trademark).

[0075] In the production of the cell aggregate production substrate of the present invention, since high-temperature treatment is not required when forming the base film, resins with low heat resistance can also be used. The substrate material may be one type or a combination of two or more types, but the cell aggregate production substrate of the present application is preferably a substrate that is flexible enough to be wound up like a conveyor belt (roll method), for example, in order to mass-produce cell culture aggregates. Materials for the substrate used in the roll method include synthetic resins and natural polymers.

[0076] The substrate of the present application may also be a substrate used in a so-called cell culture vessel, and examples thereof include Petri dishes or dishes such as Petri dishes, tissue culture dishes, and multi-dishes that are commonly used for cell culture, flasks such as cell culture flasks, spinner flasks, and multi-shelf flasks, bags such as plastic bags, Teflon (registered trademark) bags, and culture bags, plates such as microplates, microwell plates, multi-plates, and multi-well plates, chamber slides, tubes, trays, and bottles such as roller bottles.

[0077] (Seeding and Culturing Step) Following the step of forming a base film for cardiomyocyte culture, the production method of the present invention includes a step of seeding cardiomyocytes and a step of culturing the cardiomyocytes. The cardiomyocyte seeding and culturing step can be carried out by a known method suitable for cardiomyocytes. The method of producing a cardiomyocyte aggregate of the present invention is advantageous in that the cardiomyocyte seeding and culturing step can be carried out in the presence or absence of serum derived from a living body, and in particular, a high-quality cardiomyocyte aggregate can be produced even when the medium contains a low concentration of serum derived from a living body (for example, less than 5% by mass, particularly less than 3% by mass) or is absent.

[0078] (Cells) The present invention relates to a method for producing cardiomyocyte aggregates derived from human induced pluripotent stem cells (hiPS cells). The hiPS cell-derived cardiomyocytes used in the present invention refer to cardiomyocytes induced to differentiate from hiPS cells. For example, hiPS cells available from reagent suppliers or cell banks can be induced to differentiate into cardiomyocytes, followed by purification and purification. Such methods have been reported in various publications (e.g., Tohyama, et al. Cell Stem Cell 2013, Tohyama, et al. Cell Metabolism 2016, Tohyama, et al. Stem Cell Reports 2017), and hiPS cell-derived cardiomyocytes can be obtained appropriately according to these methods.

[0079] EXAMPLES The present invention will be explained in more detail below with reference to examples and comparative examples, but the present invention is not limited to the following examples.

[0080] <Method for measuring weight-average molecular weight> The weight-average molecular weights shown in the following synthesis examples are the results of gel filtration chromatography (hereinafter abbreviated as GFC). (Measurement conditions) Apparatus: HLC-8320GPC (manufactured by Tosoh Corporation) GFC column: TSKgel G 6000 + 3000 PWXL-CP Flow rate: 1.0 mL / min Eluent: salt-containing water / organic mixed solvent Column temperature: 40°C Detector: RI Injection concentration: polymer solids content 0.05 mass% Injection volume: 100 μL Calibration curve: cubic approximation curve Standard sample: polyethylene oxide (manufactured by Agilent) × 10 types

[0081] Synthesis Example 1 24.00 g of 2-(dimethylamino)ethyl methacrylate (Tokyo Chemical Industry Co., Ltd.), 1.46 g of methacrylic acid (Tokyo Chemical Industry Co., Ltd.), 5.09 g of ethylene glycol dimethacrylate (Tokyo Chemical Industry Co., Ltd.), 0.31 g of dimethyl 1,1'-azobis(1-cyclohexanecarboxylate) (VE-073, Fujifilm Wako Pure Chemical Industries, Ltd.), and 111.09 g of 2-propanol were mixed and polymerized dropwise into 166.62 g of 2-propanol at reflux temperature to synthesize a polymer. The reaction product was reprecipitated in hexane, a poor solvent, and the precipitate was collected by filtration and dried under reduced pressure. The weight-average molecular weight of this polymer measured by GFC was 228,000 (hereinafter referred to as "Synthesis Example Polymer 1").

[0082] Example 1 (Base Film Formation Step) 0.0075 g of the polymer obtained in Synthesis Example 1 above was added to 98 g of purified water and 2.0 mL of 0.5 mg / mL vitronectin VTN-N (Gibco) and thoroughly stirred to prepare base film forming agent 1. Using an inkjet device (Microjet Corporation, model number: LaboJet-600) and an inkjet head (model number: IJHBS-1000), the base film forming agent was applied to the culture surface of a cell adhesion-inhibiting culture plate (Sumitomo Bakelite Co., Ltd., PrimeSurface® Plate 24F, model number: MS-90240) at approximately 0.75 nL / spot in 13 spots (spot diameter: 500 μm, spot center-to-center spacing: 600 μm). The plate was dried in a thermostatic oven at 70°C for one day to prepare cell aggregate production substrate 1. Sterilization was performed by gamma ray irradiation at 25 kGy.

[0083] (Seeding and culture process) Cardiomyocytes induced to differentiate from human induced pluripotent stem cells (hiPS cells) were purified (see, for example, Tohyama, et al. Cell Stem Cell 2013, Tohyama, et al. Cell Metabolism 2016, Tohyama, et al. Stem Cell Reports 2017). Frozen stocks were thawed in a water bath set at 37°C and cultured at 5 x 10 cells / ml in AS301 (Ajinomoto Co., Inc.), a serum-free clinical medium. ５ The cells were suspended at a concentration of 350 μL per well. The entire cell suspension was slowly added to each well of the cell aggregate production substrate obtained in the above process, while allowing it to flow down the wall of the mold, and the cells were gently shaken by hand to distribute them evenly. Thereafter, the cells were incubated at 37°C / CO2 with a 5% carbon dioxide concentration maintained. ２ The wells were then placed in an incubator. After 24 hours, a photograph of the entire well surface was taken. The results are shown in Figure 1. 1 mL of AS301 medium was then slowly added, and the culture was continued. Photographs of the entire well surface were taken after 48 and 72 hours. The results are shown in Figures 2 and 3.

[0084] 72 hours after seeding, cardiomyospheres with a diameter of approximately 210 μm were formed on the spots.

[0085] Preparation Example 1 Polyvinyl acetate (JMR-150L (registered trademark) manufactured by Nippon Vinyl Acetate & Poval Co., Ltd. (degree of polymerization: 1480, degree of saponification: 22.7%)) was dissolved in ethanol / 1-methoxy-2-propanol (7 / 3 mass ratio) to a concentration of 3 mg / g to prepare a coating film-forming composition capable of inhibiting cell adhesion. The resulting composition was transparent and uniform.

[0086] Preparation Example 2 46.6 g of pure water was added to 0.0466 g of the polymer obtained in Synthesis Example 1 above, and the mixture was thoroughly stirred to prepare a composition for dilution.

[0087] Preparation Example 1 Preparation of a substrate capable of inhibiting cell adhesion by inkjet printing Using an inkjet device (Seiko Epson Corporation, R&D inkjet device) and an inkjet head (Seiko Epson Corporation, Precision Core head S800-A1), an appropriate amount of the coating film-forming composition prepared in Preparation Example 1 was applied to a polystyrene substrate measuring 79 mm x 121 mm in the form of a perfect circle with a diameter of 37 mm. The substrate was dried in an oven at 70°C for 24 hours to prepare a substrate capable of inhibiting cell adhesion.

[0088] Example 2 (Base Film Formation Step) 6.50 g of the dilution composition obtained in Preparation Example 2 was added to 34.20 g of sterile water and 2.61 g of Recombinant Human Vitronectin (Peprotech) diluted to 0.5 mg / mL with sterile water, and the mixture was thoroughly stirred to prepare base film forming agent 2. Using an inkjet device (Seiko Epson Corporation, R&D inkjet device) and an inkjet head (Seiko Epson Corporation, Precision Core Head S800-A1), an appropriate amount of the prepared base film forming agent 2 was applied to the culture surface of the cell adhesion-inhibiting substrate prepared in Preparation Example 1, with a spot diameter of 400 μm and a spot center-to-center spacing of 500 μm. The substrate was dried in a thermostatic oven at 70°C for one day. The substrate was attached to a bottomless 6-well plate (CS Tech Co., Ltd.) to prepare cell aggregate production substrate 2. Sterilization was performed by irradiating with 25 kGy of gamma rays.

[0089] Similarly, an appropriate amount of the prepared primer film-forming agent 2 was applied to the culture surface of the substrate with cell adhesion-inhibiting ability prepared in Preparation Example 1, with a spot diameter of 400 μm and a spot center-to-center spacing of 500 μm. After drying at room temperature for 5 minutes, the agent was applied again for recoating. The resulting mixture was dried in a thermostatic oven at 70°C for 1 day. The resulting mixture was attached to a bottomless 6-well plate (manufactured by CSTec Co., Ltd.) to prepare substrate 3 for producing cell aggregates. Sterilization was carried out by irradiating with gamma rays at 25 kGy.

[0090] (Seeding and Culturing Step) Cardiomyocytes induced to differentiate from human induced pluripotent stem cells (hiPS cells: 253G4 strain) were purified (see, for example, Tohyama, et al. Cell Stem Cell 2013, Tohyama, et al. Cell Metabolism 2016, Tohyama, et al. Stem Cell Reports 2017). Frozen stocks were thawed in a water bath set at 37°C and suspended in AS301 (Ajinomoto Co., Inc.), a serum-free clinical medium. 4 × 10 cells were then seeded into each well of the cell aggregate production substrate 2 obtained in the above step. ６ cells, and 3×10 cells were placed in each well of the cell aggregate production substrate 3. ６ The cell suspension was slowly added so that the cells were seeded, and the mixture was gently shaken by hand to distribute the cells evenly. After that, the mixture was incubated at 37°C / CO2 with a 5% carbon dioxide concentration. ２ The plates were placed in an incubator. After 72 hours, a photograph of the entire well was taken. The results for plates 2 and 3 are shown in Figures 4(A) and 4(B), respectively. Then, 1 mL of AS301 medium was slowly added, and the culture was continued. After 96 hours from seeding, a photograph of the entire well was taken. The cells were then recovered by pipetting, and their diameters were measured using a Cell3iMager duos (SCREEN Holdings Co., Ltd.). The results for plates 2 and 3 are shown in Figures 5A and 5B, respectively. As can be seen from Figure 4, 72 hours after seeding, although some cells had detached from the bottom of the plate and were floating, uniform cardiomyocyte cell clusters were observed to have formed in all wells. Furthermore, all cells recovered 96 hours after seeding had formed cardiomyocyte spheres approximately 150 μm in diameter.

[0091] Example 3 (Maturity of cells contained in cardiomyocyte spheres) A ​​stock of cardiomyocytes frozen in the same manner as above was thawed in a water bath set at 37°C, suspended in AS301 (Ajinomoto Co., Inc.), a serum-free clinical medium, and then placed in each well of the cell aggregate production substrates 2 and 3 obtained in the above step, at 4 × 10 ６ The cell suspension was slowly added so that the cells were seeded, and the mixture was gently shaken by hand to distribute the cells evenly. After that, the mixture was incubated at 37°C / CO2 with a 5% carbon dioxide concentration. ２ After leaving the plate in an incubator for 48 hours and 72 hours, the cells were collected by pipetting. For comparison, 4 × 10 cardiomyocytes were also plated in a 6-well culture plate with small dimples on the bottom (Corning Elplasia multiwell plate cat. 4440, hereinafter sometimes referred to as "Elplasia plate"). ６Cells were seeded at 1000 cells / well and cultured for 7 days, with half of the medium replaced every 2-3 days and 48 hours, as described above, and then collected by pipetting. Three different lots of frozen myocardial stocks were used in this test (referred to as CM1, CM2, and CM3). Figure 6A shows the appearance of spheres on cell aggregate production substrate 2 after 48 hours and 72 hours, and Figure 6B shows the appearance of spheres on the Elplasia plate after 48 hours and 7 days. Next, total RNA was extracted from the recovered cell masses using the ReliaPrepRNA Tissue Miniprep System (PROMEGA), and reverse-transcribed using the Superscript first strand cDNA Synthesis kit (Invitrogen) as a template. Quantitative PCR analysis was performed using primers TNNI1 (Hs00913333, Thermo Fisher), TNNI3 (Hs165957, Thermo Fisher), and GAPDH (Hs02758991, Thermo Fisher) as an endogenous control. Each sample was analyzed using the comparative Ct method, and the values ​​for each sample when the TNNI3 / TNNI1 ratio of the sample cultured for 48 hours was set to 1 are shown in Figure 6C. The TNNI3 / TNNI1 ratio was almost the same in the sample cultured for 48 hours and the sample cultured for 7 days on Elplasia plates, indicating that the cardiomyospheric spheres produced on cell aggregate production substrates 2 and 3 were as mature as the cardiomyospheric spheres produced over 7 days on Elplasia plates after a short culture period.

[0092] Example 4 (Base Film Formation Step) 1.00 g of the dilution composition obtained in Preparation Example 2 was mixed with 5.59 g of sterilized water and 0.10 g of iMatrix-221 (Matrixome Co., Ltd.) and thoroughly stirred to prepare base film forming agent 3. Using an inkjet device (Microjet Co., Ltd., model number: LaboJet-600) and an inkjet head (model number: IJHBS-1000), an appropriate amount of the base film forming agent 2 prepared above was applied to the culture surface of the cell adhesion-inhibiting substrate prepared in Preparation Example 1, with a spot diameter of 400 μm and a spot center-to-center spacing of 500 μm. The substrate was dried in a thermostatic oven at 70°C for one day. The substrate was attached to a bottomless 6-well plate (CS Tech Co., Ltd.) to prepare cell aggregate production substrate 4. Sterilization was performed by gamma ray irradiation at 25 kGy. Similarly, base film forming agent 3 was applied to prepare cell aggregate production substrate 5. Sterilization was carried out by gamma irradiation at 25 kGy.

[0093] (Seeding and Culturing Step) Cardiomyocytes induced to differentiate from human induced pluripotent stem cells (hiPS cells: 253G4 strain) were purified (see, for example, Tohyama, et al. Cell Stem Cell 2013, Tohyama, et al. Cell Metabolism 2016, Tohyama, et al. Stem Cell Reports 2017). Frozen stocks were thawed in a water bath set at 37°C and suspended in AS301 (Ajinomoto Co., Inc.), a serum-free clinical medium. 4 × 10 cells were seeded into each well of the cell aggregate production substrate 4 obtained in the above step. ６ cells and 3 x 10 ６ In order to seed the cells, 4×10 ６ cells and 3 x 10 ６ The cell suspension was slowly added so that the cells were seeded, and the mixture was gently shaken by hand to distribute the cells evenly. After that, the mixture was incubated at 37°C / CO2 with a 5% carbon dioxide concentration. ２The plates were placed in an incubator. After 72 hours, a photograph of the entire well was taken. The results for each cell seeding amount on Substrates 4 and 5 are shown in Figures 7A-D, respectively. Then, 1 mL of AS301 medium was slowly added, and the culture was continued. After 96 hours from seeding, a photograph of the entire well was taken. The cells were then recovered by pipetting, and their diameters were measured using a Cell3iMager duos (SCREEN Holdings Co., Ltd.). The results for each cell seeding amount on Substrates 4 and 5 are shown in Figures 8A-D, respectively. As is clear from Figure 7, 72 hours after seeding, although some cells had detached from the plate bottom and were floating, uniform cardiomyocyte cell clusters were observed to have formed in all wells. Furthermore, the cells recovered 96 hours after seeding all formed cardiomyocyte spheres with diameters of approximately 120-160 μm (Figure 8).

[0094] The method of the present invention for producing hiPS cell-derived cardiomyocyte aggregates enables uniform adhesion of cardiomyocytes, even under culture conditions free of animal-derived serum, followed by the production of cell aggregates, thereby enabling the mass production of homogeneous, high-quality cardiomyocyte aggregates for use in the field of regenerative medicine.

Claims

1. On a substrate having cell adhesion inhibitory ability, the following formula (I): 【Chemical 1】 [wherein, U ａ１ and U ａ２ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, and R a１ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, and R a２ represents a linear or branched alkylene group having 1 to 5 carbon atoms], a polymer containing a repeating unit derived from a monomer represented by the formula, and a method for producing a myocardial cell aggregate derived from human induced pluripotent stem cells (hiPS cells), the method comprising the steps of forming a basement membrane for myocardial cell culture, which contains the polymer and a cell adhesion substance, and then seeding myocardial cells.

2. The method for producing myocardial cell aggregates according to claim 1, comprising the step of culturing the myocardial cells in a serum-free medium.

3. The method for producing myocardial cell aggregates according to claim 1, wherein the basement membrane for culturing the myocardial cells is formed in a spot shape on the substrate.

4. The method for producing myocardial cell aggregates according to claim 3, wherein the diameter of the spot is 50 to 5000 μm.

5. The above polymer further has the formula (II): 【Chemical 2】 [wherein, R ｂ The method for producing the cardiomyocyte aggregate according to claim 1, comprising a repeating unit derived from a monomer represented by [representing a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms].

6. The method for producing myocardial cell aggregates according to any one of claims 1 to 5, wherein the weight ratio of the polymer to the cell adhesion substance is 100:0.1 to 100:

100.

7. The method for producing myocardial cell aggregates according to any one of claims 1 to 5, wherein the cell adhesion substance contains glycoprotein.

8. On a substrate having cell adhesion inhibitory ability, the following formula (Ia): 【Chemical Formula 3】 [wherein, U ａ１ and U ａ２ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, R a１ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, R a２ represents a linear or branched alkylene group having 1 to 5 carbon atoms], a polymer containing a repeating unit represented by the formula: and a cell adhesion substance, and a method for producing a cell aggregate derived from human induced pluripotent stem cells (hiPS cells), the method comprising the step of seeding cardiomyocytes on a substrate for producing a cell aggregate provided with a basement membrane for cardiomyocyte culture.

9. The above polymer further has the formula (IIa): 【Chemical Formula 4】 [wherein, R ｂ The method for producing the myocardial cell aggregate according to claim 8, which contains a repeating unit represented by [wherein R represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms].

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