Cell culture base material for serum-free medium

Abstract

The purpose of the present invention is to provide, for the mass production of cellular aggregates: a cell culture base film capable of producing a uniform and high-quality cellular aggregate without using a serum derived from a living body; a base film-forming agent for forming the base film; and a substrate for cellular aggregate production.　The inventors of the present invention provide a cell culture base film-forming agent comprising: a polymer containing a repeating unit derived from a monomer represented by formula (I) (where Ua1, Ua2, Ra1, and Ra2 are as described in the description and the claims of the present application); a cellular adhesive material; and a solvent.

Description

Substrate material for cell culture in serum-free medium

[0001] The present invention relates to a base film forming agent for cell culture in a serum-free medium, a substrate for producing cell aggregates comprising the base film, and a method for producing cell aggregates.

[0002] Various materials have been proposed as base film forming agents for efficient cell culture. Patent Literature 1 discloses a method for producing a polymer used as a base film for cell culture and a cell culture vessel. Patent Literature 2 discloses a method for producing a cell structure. These base films for cell culture require the use of serum derived from living organisms in the process of producing (culturing) homogeneous cell aggregates to ensure uniform adhesion of cells to the base film, which has problems such as quality variations due to individual differences in proliferation ability, and safety risks such as the occurrence of allergies and viral contamination when serum derived from animals other than humans is used.

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

[0004] The present invention provides a base film for cell culture that enables the mass production of homogeneous, high-quality cell aggregates without using serum derived from a living body, a base film forming agent for forming the same, and a substrate for producing cell aggregates.

[0005] The present invention includes the following: [1] 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 [2] A base film forming agent for cell culture, comprising a polymer containing a repeating unit derived from a monomer represented by formula (II): [In the formula, R ｂrepresents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms. [3] The agent for forming a base film for cell culture according to [1] or [2], wherein the weight ratio of the polymer to the cell adhesive substance is 100:0.1 to 100:100. [4] The agent for forming a base film for cell culture according to any one of [1] to [3], wherein the cell adhesive substance comprises a glycoprotein. [5] A substrate for producing a cell aggregate, comprising, on a substrate having cell adhesion-inhibiting ability, spots of a base film for cell culture formed with the agent for forming a base film for cell culture according to any one of [1] to [4]. [6] A substrate having cell adhesion-inhibiting ability, comprising, on a substrate having cell adhesion-inhibiting ability, 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; R a２ [7] A method for producing a cell aggregate, comprising the steps of forming a base film for cell culture, the base film comprising a polymer containing a repeating unit derived from a monomer 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 [8] An agent for forming a base film for cell culture, comprising a polymer containing a repeating unit represented by the formula (IIIa), (IVa), and / or (Va) below, and a crosslinked structure, a cell adhesive substance, and a solvent. [9] The agent for forming a base film for cell culture according to [7], wherein the crosslinked structure contains a structure derived from a polyfunctional acrylate compound, a polyfunctional acrylamide compound, a polyfunctional polyester, or an isoprene compound.

[10] The agent for forming a base film for cell culture according to the formula (IIIa), (IVa), and / or (Va): [In the formula, R ｃ and Rｄ 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.

[10] The agent for forming a base film for cell culture according to [7], which comprises a structure 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

[11] A method for producing a cell aggregate, comprising the steps of forming a base film for cell culture, the base film including a polymer containing a repeating unit 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

[12] A method for producing a cell aggregate, comprising a step of seeding cells on a substrate for producing a cell aggregate, the substrate comprising a base film for cell culture, the base film comprising a polymer containing a repeating unit 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

[13] A substrate for producing cell aggregates, comprising a polymer containing a repeating unit 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 represents a linear or branched alkylene group having 1 to 5 carbon atoms], and 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] and a crosslinked structure; a cell adhesive substance; and a solvent.

[0006] The cell culture base film formed using the cell culture base film forming agent of the present invention enables uniform adhesion of cells to the base film under serum-free culture conditions derived from animals, thereby enabling the production of high-quality cell aggregates. Thus, by using the cell culture base film forming agent, mass production of homogeneous, high-quality cell aggregates for use in the field of regenerative medicine can be achieved.

[0007] 1 is a stereomicroscope photograph of the appearance of the cell aggregate production substrates produced in Examples 1 to 3 and Comparative Examples 1 and 2, which were subjected to a cell adhesion confirmation test using mouse fibroblasts in Test Example 1. 2 are stereomicroscope photographs of the appearance of the cell aggregate production substrates produced in Examples 4 and 6, which were subjected to a cell adhesion / cell aggregate formation confirmation test using mouse fibroblasts in Test Example 2, taken 2 hours and 2 days later, respectively. 3 are stereomicroscope photographs of the appearance of the cell aggregate production substrates produced in Example 5 and Comparative Example 3, which were subjected to a cell adhesion confirmation test using ADSCs in Test Example 3. 4 are stereomicroscope photographs of the appearance of the cell aggregate production substrates produced in Example 7, which were subjected to a cell adhesion confirmation test using ADSCs in Test Example 4. 1 shows stereomicroscope photographs taken 2 hours and 2 days after the appearance of the cell aggregate production substrates prepared in Examples 8 to 10, 13 to 14, 17 to 19, and 26 to 27 that were subjected to the cell adhesion / cell aggregate formation confirmation test using ADSCs in Test Example 5. 1 shows stereomicroscope photographs taken 2 hours and 1 day after the appearance of the cell aggregate production substrates prepared in Examples 20 to 25 and 28 to 31 that were subjected to the cell adhesion / cell aggregate formation confirmation test using ADSCs in Test Example 6. 1 shows stereomicroscope photographs taken 6 hours and 2 days after the appearance of the cell aggregate production substrates prepared in Examples 32 to 34 that were subjected to the cell adhesion / cell aggregate formation confirmation test using ADSCs in Test Example 7. 1 shows inverted microscope photographs taken 3 hours and 1 day after the appearance of the cell aggregate production substrates prepared in Examples 9 and 10 that were subjected to the cell adhesion confirmation test using hiPSCs in Test Example 8. 1 shows inverted microscope photographs taken two days after seeding of the cell aggregate production substrates prepared in Examples 9 and 10, which were subjected to a spheroid formation confirmation test using hiPSCs in Test Example 9. 1 shows inverted microscope photographs taken 6 hours and one day after seeding of the cell aggregate production substrates prepared in Examples 15 and 16, which were subjected to a cell adhesion confirmation test using hiPSCs in Test Example 11. 1 shows inverted microscope photographs taken two days after seeding of the cell aggregate production substrates prepared in Examples 15 and 16, which were subjected to a spheroid formation confirmation test using hiPSCs in Test Example 12.12A and 12B are stereomicroscope photographs taken 2 hours and 2 days after the appearance of the cell aggregate production substrate prepared in Comparative Example 4, which was subjected to a cell adhesion / cell aggregate formation confirmation test using mouse fibroblasts in Test Example 13. 12B are stereomicroscope photographs taken 2 hours and 2 days after the appearance of the cell aggregate production substrate prepared in Comparative Example 5, which was subjected to a cell adhesion / cell aggregate formation confirmation test using mouse fibroblasts in Test Example 14.

[0008] <Underlayer-forming agent for cell culture> The underlayer-forming agent for cell culture of the present invention has 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 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:

[0009] (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].

[0010] 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 a２Similarly, 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).

[0011] Therefore, the primer film-forming agent of the present invention is also a primer film-forming agent that contains a polymer containing a repeating unit represented by the above formula (Ia), or a polymer containing a repeating unit represented by the above formula (Ia) together with a repeating unit represented by the above formula (IIa), a cell adhesive substance, and a solvent.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] (Crosslinked Structure) 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 polymer may contain, as repeating units, 50 mol % or more, preferably 75 mol % or more, more preferably 80 mol % or more, and even more preferably 90 mol % or more of the units derived from the monomer represented by Formula (I) / Formula (II). For example, the polymer may contain units derived from the monomer represented by Formula (I) / Formula (II) and a crosslinked structure. Examples of such polymers include polymers 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.

[0018] Preferred specific examples include monomers represented by the following formulas (III) to (V).

[0019] 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.

[0020] The crosslinked structure may be a structure derived from a monomer having two or more carbon-carbon double bonds, such as a structure derived from a polyfunctional acrylate compound, a polyfunctional acrylamide compound, a polyfunctional polyester, or an isoprene compound, and is preferably a structure derived from a monomer represented by any one of the above formulas (III) to (V). The structure derived from a monomer represented by the above formula (III), (IV), or (V) is represented by the following formula (IIIa), (IVa), or (Va): [In the formula, R ｃ , R ｄ , R e and n has the same meaning as in formulas (III) to (V).

[0021] 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.

[0022] 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.

[0023] 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 %.

[0024] 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.

[0025] 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. A cell aggregate refers to a structure formed as a result of cell aggregation, and its shape is not limited to, for example, a spherical or ring shape. Furthermore, the number of cell types constituting the cell aggregate is not limited, and it may be composed of multiple cell types. Cell aggregates include various structures such as spheroids, organ primordia, and organoids. 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 aggregate 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.

[0026] (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.

[0027] 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).

[0028] 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.

[0029] 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).

[0030] 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).

[0031] 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. When the cell adhesive substance is 0.1 or more, cell adhesiveness is sufficiently exhibited, and when the cell adhesive substance is 100 or less, cell aggregation (formation of cell aggregates) after cell adhesion can be facilitated.

[0032] 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.

[0033] 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.

[0034] (Cells) In the present invention, a cell is the most basic unit constituting an animal or plant, and has as its elements a cytoplasm and various organelles inside the cell membrane. In this case, the nucleus containing DNA may or may not be contained inside the cell. For example, animal-derived cells in the present invention include germ cells such as sperm and eggs, somatic cells constituting an organism, stem cells (pluripotent stem cells, etc.), progenitor cells, cancer cells isolated from an organism, cells isolated from an organism that have acquired immortalization ability and are stably maintained outside the body (cell lines), cells isolated from an organism that have been artificially genetically modified, and cells isolated from an organism that have had their nuclei artificially exchanged. Examples of somatic cells that make up a living organism include, but are not limited to, fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, erythrocytes, platelets, macrophages, monocytes, osteocytes, bone marrow cells, pericytes, dendritic cells, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatic parenchymal cells, chondrocytes, cumulus cells, nervous system cells, glial cells, neurons, oligodendrocytes, microglia, astrocytes, cardiac cells, esophageal cells, muscle cells (e.g., smooth muscle cells or skeletal muscle cells), pancreatic beta cells, melanocytes, hematopoietic progenitor cells (e.g., CD34-positive cells derived from umbilical cord blood), and mononuclear cells. The somatic cells include cells collected from any tissue, such as skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testis, thymus, muscle, connective tissue, bone, cartilage, vascular tissue, blood (including umbilical cord blood), bone marrow, heart, cardiac muscle, eye, brain, or neural tissue. Furthermore, the somatic cells include cells induced to differentiate from stem cells or progenitor cells.

[0035] Stem cells are cells that have the ability to replicate themselves and differentiate into cells of multiple lineages. Examples include, but are not limited to, embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ stem cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, muscle stem cells, germ stem cells, intestinal stem cells, cancer stem cells, and hair follicle stem cells. Among the stem cells, pluripotent stem cells include ES cells, embryonic germ stem cells, and iPS cells. Progenitor cells are cells that are in the process of differentiating from the stem cells into specific somatic cells or germ cells. Cancer cells are cells that are derived from somatic cells and have acquired the ability to proliferate indefinitely. Cell lines are cells that have acquired the ability to proliferate indefinitely through artificial manipulation outside of the body. Among these, fibroblasts and stem cells are preferred, and among stem cells, pluripotent stem cells are more preferred.

[0036] <Substrate for Cell Aggregate Production> The substrate for cell aggregate production of the present invention comprises a substrate having cell adhesion-inhibiting properties, on which spots of a base film for cell culture are formed using the base film-forming agent for cell culture. The substrate for cell aggregate production of the present invention is produced using a substrate having cell adhesion-inhibiting properties. Prior to the formation of the spots (base film), the substrate may be subjected to a cell adhesion-inhibiting treatment. 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. The coating film-forming composition may be 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 ｂ１３each 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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).

[0041] 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.

[0042] 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.

[0043] An amino group has the formula: -NH ２ , -NHR １９ or -NR ２０ R ２１ [where R １９ , R ２０ and R ２１ are 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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).

[0048]

[0049] 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 ２ each 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.

[0050] 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).

[0051] In the water-insoluble copolymer, R １ ~R ３ are preferably each independently a hydrogen atom, a methyl group, or an ethyl group.

[0052] 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-.

[0053] 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.

[0054] 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 ２is preferably a single bond.

[0055] 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.

[0056] 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%.

[0057] 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.

[0058] 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.

[0059] Here, P represents the viscosity-average degree of polymerization, which can be determined in accordance with JIS K 6726.

[0060] 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.

[0061] In the formula, R １ , R ３ , and X １ has the same meaning as above.

[0062] 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.

[0063] In the formula, R １ ~R ３ , X １ , and X ２ has the same meaning as above.

[0064] 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)).

[0065] 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.

[0066] 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.

[0067] (Spots) The ratio of the total area of ​​the spots, preferably multiple spots (base film) provided on the substrate for producing cell aggregates of the present invention, the diameter of each spot, and the spacing between spot centers can be appropriately selected from a predetermined range 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, 50% or more, and preferably 99% or less, the diameter of each spot is 50 to 5000 μm, preferably 100 to 3000 μm, and the spacing between spot centers is preferably 100 to 6000 μm, 150 to 4000 μm, or 150 to 300 μm.

[0068] 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 cells 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 by, for example, inkjet printing, screen printing, slit coating, roll-to-roll printing, or other printing techniques, but is preferably applied by inkjet printing or screen printing.

[0069] 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 cell aggregate production.

[0070] Furthermore, the spots on the surface of the substrate obtained by this method can be used as a substrate for producing cell 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 cell 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.

[0071] 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.

[0072] 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.

[0073] (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.

[0074] 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.).

[0075] 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).

[0076] 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 may be a substrate that is flexible enough to be wound up like a conveyor belt (roll method) for mass production of cell culture aggregates, for example. Materials for the substrate used in the roll method include synthetic resins and natural polymers.

[0077] 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.

[0078] <Method for producing cell aggregates> The method for producing cell aggregates of the present invention involves applying 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; R a２ The present invention includes a step of forming a base film for cell culture, the base film comprising a polymer containing a repeating unit derived from a monomer represented by the formula [wherein ' represents a linear or branched alkylene group having 1 to 5 carbon atoms'] and a cell adhesive substance, followed by a step of seeding and culturing the cells. The cell seeding and culturing step is not particularly limited and can be carried out by a known method appropriate for the type of cell. Details of the substrate having cell adhesion-inhibiting ability, the base film forming step (production method), and the cells are as described above. The method for producing cell aggregates of the present invention is advantageous in that the cell seeding and culturing step can be carried out in the presence or absence of serum derived from a living body, and in particular, high-quality cell aggregates can be produced even when the culture medium contains low concentrations of serum derived from a living body (e.g., less than 5% by mass, particularly less than 3% by mass) or no serum is present.

[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] Synthesis Example 2: 8.00 g of 2-(dimethylamino)ethyl methacrylate (Tokyo Chemical Industry Co., Ltd.), 1.88 g of methacrylic acid (Tokyo Chemical Industry Co., Ltd.), 1.98 g of ethylene glycol dimethacrylate (Tokyo Chemical Industry Co., Ltd.), 0.12 g of dimethyl 1,1'-azobis(1-cyclohexanecarboxylate) (VE-073, Fujifilm Wako Pure Chemical Industries, Ltd.), and 43.10 g of 2-propanol were mixed and polymerized dropwise into 64.65 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 438,000 (hereinafter referred to as "Synthesis Example Polymer 2").

[0083] 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 10 mg / g to prepare a coating film-forming composition 1 having cell adhesion inhibitory properties. The resulting composition was transparent and uniform.

[0084] Preparation Example 2 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 2 having cell adhesion inhibitory properties. The resulting composition was transparent and uniform.

[0085] Preparation Example 3 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.

[0086] <Preparation Example 1> (Preparation of a substrate capable of inhibiting cell adhesion) The coating film-forming composition 1 of Preparation Example 1 was added to each well of a 24-well cell culture plate (manufactured by Corning, #351147, volume 1 mL, made of polystyrene) at 25 μL / well, and after leaving to stand at room temperature for 3 hours, it was dried in an oven at 70° C. for 24 hours to prepare a substrate 1 capable of inhibiting cell adhesion.

[0087] <Preparation Example 2> (Preparation of a substrate capable of inhibiting cell adhesion) The coating film-forming composition 1 of Preparation Example 1 was added to each well of a 24-well cell culture plate (manufactured by Corning, #351147, volume 1 mL, made of polystyrene) at 100 μL / well, and after leaving to stand at room temperature for 3 hours, it was dried in an oven at 70° C. for 24 hours to prepare a substrate 2 capable of inhibiting cell adhesion.

[0088] <Preparation Example 3> (Preparation of a substrate having the ability to inhibit cell adhesion) The coating film-forming composition 1 of Preparation Example 1 was added to each well of a Φ40 mm Petri dish (manufactured by AS ONE Corporation, #1-8549-01, volume 1 mL, made of polystyrene) at 125 μL / well, and after leaving it to stand at room temperature for 3 hours, it was dried in an oven at 70°C for 24 hours to prepare a substrate 3 having the ability to inhibit cell adhesion.

[0089] Preparation Example 4 (Preparation of a substrate capable of inhibiting cell adhesion by inkjet) 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 2 prepared in Preparation Example 2 was applied to a polystyrene substrate measuring 79 mm x 121 mm in the form of a perfect circle with a diameter of 18 mm. This was dried in an oven at 70°C for 24 hours to prepare a substrate 4 capable of inhibiting cell adhesion.

[0090] Preparation Example 5 (Preparation of a substrate capable of inhibiting cell adhesion by inkjet) 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 2 prepared in Preparation Example 2 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 5 capable of inhibiting cell adhesion.

[0091] Example 1 A primer film-forming agent was prepared by adding 99.5 g of purified water and 0.5 mL of 0.5 mg / mL vitronectin VTN-N (Gibco) to 0.0025 g of the polymer obtained in Synthesis Example 1 above and thoroughly stirring. Using an inkjet device (Microjet Corporation, model number: LaboJet-600) and an inkjet head (model number: 500-SC), an appropriate amount of the primer film-forming agent was applied to the culture surface of a cell adhesion-inhibiting culture dish (diameter: 35 mm) (Sumitomo Bakelite Co., Ltd., MS9035X), forming multiple spots. The mixture was dried in a thermostatic oven at 70°C for one day to prepare a substrate for cell aggregate production.

[0092] Example 2 A primer film-forming agent was prepared by adding 99 g of purified water and 1 mL of 0.5 mg / mL vitronectin VTN-N (Gibco) to 0.0025 g of the polymer obtained in Synthesis Example 1 above, and thoroughly stirring the mixture. The primer film-forming agent was applied using an inkjet device in the same manner as in Example 1, forming multiple spots. After drying, a substrate for producing cell aggregates was prepared.

[0093] Example 3 A primer film-forming agent was prepared by adding 99 g of purified water and 1.0 mL of 0.5 mg / mL vitronectin VTN-N (Gibco) to 0.005 g of the polymer obtained in Synthesis Example 2 above, and thoroughly stirring the mixture. The primer film-forming agent was applied using an inkjet device in the same manner as in Example 1, forming multiple spots. After drying, a substrate for producing cell aggregates was prepared.

[0094] Example 4 A primer film-forming agent was prepared by adding 99 g of purified water and 1.0 mL of 0.5 mg / mL vitronectin VTN-N (Gibco) to 0.005 g of the polymer obtained in Synthesis Example 1 above, and thoroughly stirring the mixture. The primer film-forming agent was applied using an inkjet device in the same manner as in Example 1, forming multiple spots. After drying, a substrate for producing cell aggregates was prepared.

[0095] Example 5: 0.015 g of the polymer obtained in Synthesis Example 1 above was mixed with 94 g of purified water and 6.0 mL of 0.5 mg / mL vitronectin VTN-N (Gibco) and thoroughly stirred to prepare a primer film-forming agent. Using an inkjet device (Microjet Co., Ltd., model number: LaboJet-600) and an inkjet head (model number: IJHBS-1000), an appropriate amount of primer 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), forming multiple 250 μm diameter spots. The substrate was dried for one day in a thermostatic oven at 70°C to prepare a substrate for cell aggregate production. Sterilization was performed by gamma ray irradiation at 25 kGy.

[0096] Example 6: 0.005 g of the polymer obtained in Synthesis Example 1 above was mixed with 98 g of purified water and 2.0 mL of 0.5 mg / mL vitronectin VTN-N (Gibco) and thoroughly stirred to prepare a base film-forming agent. 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 was applied to the culture surface of a cell adhesion-inhibiting culture plate (Sumitomo Bakelite Co., Ltd., PrimeSurface® Plate 24F, model number: MS-90240), forming multiple 250 μm diameter spots. The substrate was dried for one day in a thermostatic oven at 70°C to prepare a substrate for cell aggregate production. Sterilization was performed by gamma ray irradiation at 25 kGy.

[0097] Example 7: 0.005 g of the polymer obtained in Synthesis Example 1 above was mixed with 31.3 g of purified water and 2.0 mL of 0.5 mg / mL vitronectin VTN-N (Gibco) and thoroughly stirred to prepare a primer film-forming agent. Using an inkjet device (Microjet Co., Ltd., model number: LaboJet-600) and an inkjet head (model number: 200-SC), an appropriate amount of primer 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), forming multiple spots. The substrate was dried for one day in a thermostatic oven at 70°C to prepare a substrate for cell aggregate production. Sterilization was performed by gamma ray irradiation at 25 kGy.

[0098] Example 8: A primer film-forming agent was prepared by adding 0.70 g of the dilution composition obtained in Preparation Example 3, 3.69 g of sterile water, and 0.28 g of Recombinant Human Vitronectin (Peprotech) diluted to 0.5 mg / mL with sterile water, and thoroughly stirring. An inkjet device (Microjet, model number: LaboJet-600) and an inkjet head (model number: IJHBS-1000) were used to apply an appropriate amount of the primer film-forming agent to the culture surface of the cell adhesion-inhibiting substrate 4 prepared in Preparation Example 4, forming multiple 400 μm diameter spots. The substrate was dried in a thermostatic oven at 70°C for one day. The substrate was attached to a bottomless 24-well plate (CS Tech) to prepare a substrate for cell aggregate production. Sterilization was performed by irradiating with 25 kGy of gamma rays.

[0099] Example 9 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 primer film-forming agent prepared in Example 8 was applied to the culture surface of the substrate 4 having the ability to inhibit cell adhesion prepared in Preparation Example 4, forming multiple spots with a diameter of 400 μm. The substrate was dried for one day in a thermostatic dryer at 70°C. The wells were attached to a bottomless 24-well plate (CS Tech Co., Ltd.) to prepare a substrate for producing cell aggregates. Sterilization was carried out by irradiating with gamma rays at 25 kGy.

[0100] Example 10 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 primer film-forming agent prepared in Example 8 was applied to the culture surface of substrate 4 having the ability to inhibit cell adhesion, prepared in Preparation Example 4. After drying at room temperature for 5 minutes, the agent was applied again and recoated to form multiple spots with a diameter of 400 μm. The resultant was dried in a thermostatic oven at 70°C for 1 day. The resultant was attached to a bottomless 24-well plate (CS Tech Co., Ltd.) to prepare a substrate for producing cell aggregates. Sterilization was carried out by irradiating with gamma rays at 25 kGy.

[0101] Example 11 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 primer film-forming agent prepared in Example 8 was applied to the culture surface of substrate 5 having the ability to inhibit cell adhesion, prepared in Preparation Example 5, to form multiple spots with a diameter of 400 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 a substrate for producing cell aggregates. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0102] Example 12 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 primer film-forming agent prepared in Example 8 was applied to the culture surface of substrate 5 having the ability to inhibit cell adhesion, prepared in Preparation Example 5. After drying at room temperature for 5 minutes, the agent was applied again and recoated to form multiple spots with a diameter of 400 μm. The resultant was dried in a thermostatic oven at 70°C for 1 day. The resultant was attached to a bottomless 6-well plate (CS Tech Co., Ltd.) to prepare a substrate for producing cell aggregates. Sterilization was carried out by irradiating with gamma rays at 25 kGy.

[0103] Example 13: A primer film-forming agent was prepared by adding 17.9 g of sterile water and 0.60 g of Recombinant Human Vitronectin (Peprotech) diluted to 0.5 mg / mL with sterile water to 1.50 g of the dilution composition obtained in Preparation Example 3 above, and thoroughly stirring. Using an inkjet device (Microjet, model number: LaboJet-600) and an inkjet head (model number: IJHBS-1000), an appropriate amount of the primer film-forming agent was applied to the culture surface of the cell adhesion-inhibiting substrate 4 prepared in Preparation Example 4, forming multiple 400 μm diameter spots. The substrate was dried in a thermostatic oven at 70°C for one day. The substrate was attached to a bottomless 24-well plate (CS Tech) to prepare a substrate for cell aggregate production. Sterilization was performed by gamma ray irradiation at 25 kGy.

[0104] Example 14: 4.48 g of sterilized water and 0.08 g of iMatrix-511 (Matrixome Co., Ltd.) were added to 0.80 g of the dilution composition obtained in Preparation Example 3 above, and the mixture was thoroughly stirred to prepare a primer film-forming agent. Using an inkjet device (Microjet Co., Ltd., model number: LaboJet-600) and an inkjet head (model number: IJHBS-1000), an appropriate amount of primer film-forming agent was applied to the culture surface of Substrate 2 having the ability to inhibit cell adhesion, prepared in Preparation Example 2, to form multiple spots with a diameter of 400 μm. The substrate was dried in a thermostatic oven at 70°C for one day to prepare a substrate for producing cell aggregates. Sterilization was performed by irradiating with 25 kGy of gamma rays.

[0105] Example 15 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 primer film-forming agent prepared in Example 14 was applied to the culture surface of substrate 4 having the ability to inhibit cell adhesion, prepared in Preparation Example 4, to form multiple spots with a diameter of 400 μm. The substrate was dried in a thermostatic oven at 70°C for one day. The wells were attached to a bottomless 24-well plate (CS Tech Co., Ltd.) to prepare a substrate for producing cell aggregates. Sterilization was carried out by irradiating with gamma rays at 25 kGy.

[0106] Example 16 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 primer film-forming agent prepared in Example 14 was applied to the culture surface of substrate 4 having the ability to inhibit cell adhesion, prepared in Preparation Example 4. After drying at room temperature for 5 minutes, the agent was applied again and recoated to form multiple spots with a diameter of 400 μm. The resultant was dried in a thermostatic oven at 70°C for 1 day. The resultant was attached to a bottomless 24-well plate (CS Tech Co., Ltd.) to prepare a substrate for producing cell aggregates. Sterilization was carried out by irradiating with gamma rays at 25 kGy.

[0107] Example 17: 1.00 g of the dilution composition obtained in Preparation Example 3 was added with 5.59 g of sterilized water and 0.10 g of iMatrix-221 (Matrixome Co., Ltd.) and thoroughly stirred to prepare a primer film-forming agent. Using an inkjet device (Microjet Co., Ltd., model number: LaboJet-600) and an inkjet head (model number: IJHBS-1000), an appropriate amount of primer film-forming agent was applied to the culture surface of Substrate 2 having the ability to inhibit cell adhesion prepared in Preparation Example 2, forming multiple spots with a diameter of 400 μm. The substrate was dried in a thermostatic oven at 70°C for one day to prepare a substrate for producing cell aggregates. Sterilization was performed by irradiating with 25 kGy of gamma rays.

[0108] Example 18: 7.54 g of sterile water, a 0.5 mg / mL fibronectin solution purified using a spin column (Zeba Spin Desalting Columns and Plates, 7K MWCO, Thermo Fisher Scientific Co., Ltd.), and 0.30 g of human plasma-derived fibronectin (Fujifilm Wako Pure Chemical Industries, Ltd.) were added to 0.50 g of the dilution composition obtained in Preparation Example 3, and the mixture was thoroughly stirred to prepare a primer film-forming agent. An inkjet device (Microjet Corporation, model number: LaboJet-600) and an inkjet head (model number: IJHBS-1000) were used to apply an appropriate amount of the primer film-forming agent to the culture surface of a cell adhesion-inhibiting culture plate (Sumitomo Bakelite Co., Ltd., PrimeSurface® Plate 24F, model number: MS-90240) to form multiple spots with a diameter of 400 μm. A substrate for producing cell aggregates was prepared by drying for one day in a thermostatic dryer at 70° C. Sterilization was carried out by irradiating with gamma rays at 25 kGy.

[0109] Example 19: 2.81 g of the dilution composition obtained in Preparation Example 3 above was mixed with 33.35 g of sterile water and 1.28 g of 0.5 mg / mL vitronectin VTN-N (Gibco) purified using a spin column (Zeba Spin Desalting Columns and Plates, 7K MWCO, Thermo Fisher Scientific), and the mixture was thoroughly stirred to prepare a base film-forming agent. Using an inkjet device (Microjet Corporation, model number: LaboJet-600) and an inkjet head (model number: IJHBS-1000), an appropriate amount of 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), forming multiple 400 μm diameter spots. The mixture was dried in a constant-temperature dryer at 70°C for one day to prepare a substrate for cell aggregate production. Sterilization was carried out by gamma irradiation at 25 kGy.

[0110] Example 20: A primer film-forming agent was prepared by adding 13.92 g of sterile water and 0.26 g of peptide RGDS (Peptide Institute, model number: 4171-v) diluted to 0.5 mg / mL with sterile water to 2.50 g of the dilution composition obtained in Preparation Example 3 above, and thoroughly stirring. An inkjet device (Microjet, model number: LaboJet-600) and an inkjet head (model number: 200-SC) were used to apply an appropriate amount of the primer film-forming agent to the culture surface of the cell adhesion-inhibiting substrate 3 prepared in Preparation Example 3, forming multiple spots. The substrate was dried in a thermostatic oven at 70°C for one day to prepare a substrate for cell aggregate production. Sterilization was performed by irradiating with 25 kGy of gamma rays.

[0111] Example 21: A primer film-forming agent was prepared by adding 13.92 g of sterile water and 0.25 g of peptide GRGDS (Peptide Institute, model number 4189) diluted to 0.5 mg / mL with sterile water to 2.50 g of the dilution composition obtained in Preparation Example 3 above, followed by thorough stirring. An inkjet device (Microjet, model number LaboJet-600) and an inkjet head (model number 200-SC) were used to apply an appropriate amount of the primer film-forming agent to the culture surface of Substrate 3, which has the ability to inhibit cell adhesion, prepared in Preparation Example 3, to form multiple spots. The mixture was dried in a thermostatic oven at 70°C for one day to prepare a substrate for cell aggregate production. Sterilization was performed by irradiating with 25 kGy of gamma rays.

[0112] Example 22: A primer film-forming agent was prepared by adding 13.92 g of sterile water and 0.25 g of the peptide GRGDNP (Peptide Institute, model number PCI-3909-PI) diluted to 0.5 mg / mL with sterile water to 2.50 g of the dilution composition obtained in Preparation Example 3 above, and thoroughly stirring. An inkjet device (Microjet, model number LaboJet-600) and an inkjet head (model number 200-SC) were used to apply an appropriate amount of the primer film-forming agent to the culture surface of Substrate 3, which has the ability to inhibit cell adhesion, prepared in Preparation Example 3, to form multiple spots. The mixture was dried in a thermostatic oven at 70°C for one day to prepare a substrate for producing cell aggregates. Sterilization was performed by irradiating with 25 kGy of gamma rays.

[0113] Example 23 A primer film-forming agent was prepared by adding 13.92 g of sterile water and 0.25 g of peptide YIGSR-NH2 (Peptide Institute, model number: 4194-v) diluted to 0.5 mg / mL with sterile water to 2.50 g of the dilution composition obtained in Preparation Example 3 above, and thoroughly stirring the mixture. An appropriate amount of the primer film-forming agent was applied to the culture surface of Substrate 3, which has the ability to inhibit cell adhesion and was prepared in Preparation Example 3, using an inkjet device (Microjet, model number: LaboJet-600) and an inkjet head (model number: 200-SC), to form multiple spots. The substrate was dried for one day in a thermostatic oven at 70°C to prepare a substrate for cell aggregate production. Sterilization was performed by irradiating with 25 kGy of gamma rays.

[0114] Example 24: A primer film-forming agent was prepared by adding 13.92 g of sterile water and 0.25 g of peptide c[RGDfK(C)] (Peptide Institute, model number: RGD-3794-PI) diluted to 0.5 mg / mL with sterile water to 2.50 g of the dilution composition obtained in Preparation Example 3 above, and thoroughly stirring. An appropriate amount of the primer film-forming agent was applied to the culture surface of Substrate 3, which has the ability to inhibit cell adhesion, prepared in Preparation Example 3, using an inkjet device (Microjet, model number: LaboJet-600) and an inkjet head (model number: 200-SC), to form multiple spots. The substrate was dried in a thermostatic oven at 70°C for one day to prepare a substrate for cell aggregate production. Sterilization was performed by irradiating with 25 kGy of gamma rays.

[0115] Example 25: A primer film-forming agent was prepared by adding 14.12 g of sterile water and 0.05 g of peptide c[RGDfK(C)] (Peptide Institute, model number: RGD-3794-PI) diluted to 0.5 mg / mL with sterile water to 2.50 g of the dilution composition obtained in Preparation Example 3 above, and thoroughly stirring. An appropriate amount of the primer film-forming agent was applied to the culture surface of Substrate 3, which has the ability to inhibit cell adhesion and was prepared in Preparation Example 3, using an inkjet device (Microjet, model number: LaboJet-600) and an inkjet head (model number: 200-SC), to form multiple spots. The substrate was dried in a thermostatic oven at 70°C for one day to prepare a substrate for cell aggregate production. Sterilization was performed by irradiating with 25 kGy of gamma rays.

[0116] Example 26: 2.50 g of the dilution composition obtained in Preparation Example 3 was added to 30.33 g of sterile water and 0.50 g of PASAP1 (manufactured by Nanomed3D) diluted to 1.0 mg / mL with sterile water, and the mixture was thoroughly stirred to prepare a primer film-forming agent. Using an inkjet device (manufactured by Microjet Co., Ltd., model number: LaboJet-600) and an inkjet head (model number: IJHBS-1000), an appropriate amount of primer film-forming agent was applied to the culture surface of Substrate 1 with cell adhesion inhibitory ability prepared in Preparation Example 1, forming multiple spots with a diameter of 400 μm. The substrate was dried in a thermostatic oven at 70°C for one day to prepare a substrate for producing cell aggregates. Sterilization was performed by irradiating with 25 kGy of gamma rays.

[0117] Example 27: 2.50 g of the dilution composition obtained in Preparation Example 3 was added to 30.33 g of sterile water and 0.51 g of PASAP2 (manufactured by Nanomed3D) diluted to 1.0 mg / mL with sterile water, and the mixture was thoroughly stirred to prepare a primer film-forming agent. Using an inkjet device (manufactured by Microjet Co., Ltd., model number: LaboJet-600) and an inkjet head (model number: IJHBS-1000), an appropriate amount of primer film-forming agent was applied to the culture surface of the cell adhesion-inhibiting substrate 1 prepared in Preparation Example 1, forming multiple spots with a diameter of 400 μm. The substrate was dried in a thermostatic oven at 70°C for one day to prepare a substrate for producing cell aggregates. Sterilization was performed by irradiating with 25 kGy of gamma rays.

[0118] Example 28: 0.70 g of the dilution composition obtained in Preparation Example 3 was mixed with 3.45 g of sterile water, 0.23 g of ethanol, and 0.28 g of Recombinant Human Vitronectin (Peprotech) diluted to 0.5 mg / mL with sterile water, and the mixture was thoroughly stirred to prepare a primer film-forming agent. Using an inkjet device (Microjet Co., Ltd., Model No.: LaboJet-600) and an inkjet head (Model No.: 200-SC), an appropriate amount of primer film-forming agent was applied to the culture surface of a cell adhesion-inhibiting culture dish (diameter: 35 mm) (Sumitomo Bakelite Co., Ltd., MS9035X) to form multiple spots. The mixture was dried in a thermostatic oven at 70°C for one day to prepare a substrate for cell aggregate production. Sterilization was performed by gamma ray irradiation at 25 kGy.

[0119] Example 29: 0.70 g of the dilution composition obtained in Preparation Example 3 was mixed with 3.22 g of sterile water, 0.47 g of ethanol, and 0.28 g of Recombinant Human Vitronectin (Peprotech) diluted to 0.5 mg / mL with sterile water, and the mixture was thoroughly stirred to prepare a primer film-forming agent. Using an inkjet device (Microjet Co., Ltd., Model No.: LaboJet-600) and an inkjet head (Model No.: 200-SC), an appropriate amount of primer film-forming agent was applied to the culture surface of a cell adhesion-inhibiting culture dish (diameter: 35 mm) (Sumitomo Bakelite Co., Ltd., MS9035X) to form multiple spots. The mixture was dried in a thermostatic oven at 70°C for one day to prepare a substrate for cell aggregate production. Sterilization was performed by gamma ray irradiation at 25 kGy.

[0120] Example 30: 0.70 g of the dilution composition obtained in Preparation Example 3 was mixed with 3.70 g of sterile water, 0.28 g of Recombinant Human Vitronectin (Peprotech) diluted to 0.5 mg / mL with sterile water, and 0.0056 g of tannic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) diluted to 10 mg / mL with sterile water, and the mixture was thoroughly stirred to prepare a primer film-forming agent. Using an inkjet device (Microjet Corporation, Model No.: LaboJet-600) and an inkjet head (Model No.: 200-SC), an appropriate amount of primer film-forming agent was applied to the culture surface of a cell adhesion-inhibiting culture dish (diameter: 35 mm) (Sumitomo Bakelite Co., Ltd., MS9035X) to form multiple spots. The mixture was dried in a thermostatic oven at 70°C for 1 day to prepare a substrate for cell aggregate production. Sterilization was performed by gamma ray irradiation at 25 kGy.

[0121] Example 31: A primer film-forming agent was prepared by adding 4.21 g of sterile water, 0.32 g of Recombinant Human Vitronectin (Peprotech) diluted to 0.5 mg / mL with sterile water, and 0.0063 g of gallic acid hydrate (Tokyo Chemical Industry Co., Ltd.) diluted to 10 mg / mL with sterile water to 0.80 g of the dilution composition obtained in Preparation Example 3 above, and thoroughly stirring. An appropriate amount of the primer film-forming agent was applied to the culture surface of a cell adhesion-inhibiting culture dish (diameter: 35 mm) (Sumitomo Bakelite Co., Ltd., MS9035X) using an inkjet device (Microjet Co., Ltd., model number: LaboJet-600) and an inkjet head (model number: 200-SC) to form multiple spots. The substrate was dried for 1 day in a thermostatic oven at 70°C to prepare a substrate for cell aggregate production. Sterilization was performed by gamma ray irradiation at 25 kGy.

[0122] Example 32 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 primer film-forming agent prepared in Example 8 was applied to the culture surface of substrate 4 having cell adhesion inhibitory ability prepared in Preparation Example 4, so that the spot diameter was 170 μm and the spot center-to-center spacing was 250 μm. The substrate was dried for 1 day in a thermostatic oven at 70°C. The well was attached to a bottomless 24-well plate (CS Tech Co., Ltd.) to prepare a substrate for producing cell aggregates. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0123] Example 33 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 primer film-forming agent prepared in Example 8 was applied to the culture surface of substrate 4 having cell adhesion-inhibiting ability prepared in Preparation Example 4, so that the spot diameter was 400 μm and the spot center-to-center spacing was 500 μm. The substrate was dried for 1 day in a thermostatic oven at 70°C. The well was attached to a bottomless 24-well plate (CS Tech Co., Ltd.) to prepare a substrate for producing cell aggregates. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0124] Example 34 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 primer film-forming agent prepared in Example 8 was applied to the culture surface of substrate 4 having cell adhesion inhibitory ability prepared in Preparation Example 4, so as to achieve a spot diameter of 900 μm and a spot center-to-center spacing of 1000 μm. The substrate was dried in a thermostatic oven at 70°C for one day. The well was attached to a bottomless 24-well plate (CS Tech Co., Ltd.) to prepare a substrate for producing cell aggregates. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0125] Comparative Example 1 A primer film-forming agent was prepared by adding 100.0 g of pure water to 0.0025 g of the polymer obtained in Synthesis Example 1 and thoroughly stirring the mixture. The primer film-forming agent was applied using an inkjet device in the same manner as in Example 1 to form multiple spots. After drying, a substrate for producing cell aggregates was prepared.

[0126] Comparative Example 2 A primer film-forming agent was prepared by adding 100.0 g of pure water to 0.005 g of the polymer obtained in Synthesis Example 2 above and thoroughly stirring. The primer film-forming agent was applied using an inkjet device in the same manner as in Example 1 to form multiple spots. After drying, a substrate for producing cell aggregates was prepared.

[0127] Comparative Example 3: 0.005 g of the polymer obtained in Synthesis Example 1 was added to 33.3 g of pure water and thoroughly stirred to prepare a primer film-forming agent. Using an inkjet device (Microjet Corporation, model number: LaboJet-600) and an inkjet head (model number: 200-SC), an appropriate amount of primer film-forming agent was applied to the culture surface of a cell adhesion-inhibiting culture dish (diameter: 35 mm) (Sumitomo Bakelite Co., Ltd., MS9035X) to form multiple spots. The resulting mixture was dried in a thermostatic oven at 70°C for one day to prepare a substrate for cell aggregate production. Sterilization was performed by irradiating with 25 kGy of gamma rays.

[0128] Comparative Example 4 A primer film-forming agent was prepared by adding 4.8 g of pure water and 0.2 mL of 0.5 mg / mL vitronectin VTN-N (Gibco) and thoroughly stirring. The primer film-forming agent was applied using an inkjet device in the same manner as in Example 1 to form multiple spots. After drying, a substrate for producing cell aggregates was prepared.

[0129] Comparative Example 5 A primer film-forming agent was prepared by adding 2.1 g of pure water and 1.2 mL of 0.5 mg / mL vitronectin VTN-N (Gibco) and thoroughly stirring. The primer film-forming agent was applied using an inkjet device in the same manner as in Example 1 to form multiple spots. After drying, a substrate for producing cell aggregates was prepared.

[0130] <Test Example 1: Test to confirm cell adhesion with mouse fibroblasts in FBS-free media of Examples 1 to 3 and Comparative Examples 1 and 2> (Cell preparation) Mouse embryonic fibroblasts (C3H10T1 / T2 cells: manufactured by DS Pharma Biomedical Co., Ltd.) were used as the cells. For cell culture, a medium was used in which FBS (manufactured by Sigma-Aldrich) was added to the basal medium (manufactured by Gibco) to make 10% FBS (manufactured by Sigma-Aldrich) and Glutamine / Penicillin / Streptmycin (manufactured by Gibco). The cells were incubated at 37°C / CO ２ The cells were cultured in a 10 cm diameter dish (10 mL of medium) for at least two days in an incubator maintained at 5% carbon dioxide. Subsequently, the cells were washed with 3 mL of PBS solution (Fujifilm Wako Pure Chemical Industries, Ltd.), after which 3 mL of trypsin-EDTA solution (PromoCell) was added and the mixture was left to stand at room temperature for 3 minutes to detach the cells. Cells were recovered by adding 7 mL of BME medium without FBS (bovine bovine serum) or glutamine / penicillin / streptmycin. The suspension was centrifuged (Tomy Seiko Co., Ltd., Model No. LC-230, 200 × g / 3 minutes, room temperature), the supernatant was removed, and the above medium was added to prepare a cell suspension.

[0131] (Cell Adhesion Confirmation Test) 2.0 mL of cell suspension was added to the substrates for producing cell aggregates prepared in Examples 1 to 3 and Comparative Examples 1 and 2. The cell density was 1.5 × 10 for Examples 1, 2, and Comparative Example 1. ５ cells / cm 2 , 3.0 × 10 for Example 3 and Comparative Example 2 ５ cells / cm 2 After that, the medium was incubated at 37°C / CO2 with a 5% carbon dioxide concentration. ２The wells were then left to stand for 2 hours in an incubator. After standing, the non-adherent cells and medium were removed, and the wells were washed with PBS, leaving only the adherent cells on the wells. After washing, 2.0 mL of fresh medium was added, and the adherent cells were observed and photographed using a stereomicroscope SZX16 (Olympus Corporation). As a result, as shown in Figure 1 , selective cell adhesion to the base film portion on the substrates prepared in Examples 1 to 3 and Comparative Examples 1 and 2 was confirmed. In Examples 1 to 3, cell adhesion was uniform, with no gaps. In contrast, in Comparative Examples 1 and 2, cell adhesion was found to be uneven, with gaps present. From the above, it was found that the inclusion of additives that promote cell adhesion and spreading in the base film-forming agent can achieve uniform cell adhesion on the base film in serum (FBS)-free medium.

[0132] Test Example 2: Test to confirm cell adhesion and cell aggregate formation with mouse fibroblasts in FBS-free media of Examples 4 and 6 (Cell preparation) Cells were prepared in the same manner as in Test Example 1. (Cell adhesion confirmation test) A cell suspension was applied to the substrate for cell aggregate production prepared in Examples 4 and 6 at a concentration of 3.0 × 10 ５ cells / cm 2 After that, the mixture was incubated at 37°C / CO2 while maintaining a 5% carbon dioxide concentration. ２ The wells were then left to stand for 2 hours in an incubator. After standing, non-adherent cells and medium were removed, and the wells were washed with PBS, leaving only the adherent cells on the wells. After washing, 2.0 mL of fresh medium was added, and the adherent cells were observed and photographed using a stereomicroscope (Olympus Corporation) SZX16. The next day, the presence or absence of cell aggregate formation was observed and photographed. As shown in Figure 2, selective cell adhesion to the base film portion of the prepared substrate was confirmed. Furthermore, there were no gaps in the cell adhesion, and uniform adhesion occurred. After two days, the adhered cells were confirmed to have detached from the Petri dish and aggregated, forming cell aggregates (spheroids). These findings demonstrate that cells can detach and form cell aggregates after uniform cell adhesion on a base film containing additives that promote cell adhesion and spreading.

[0133] Test Example 3: Cell adhesion confirmation test in serum-free medium using human adipose tissue-derived mesenchymal stem cells of Example 5 and Comparative Example 3 (Cell preparation) Human adipose tissue-derived mesenchymal stem cells (ADSC: manufactured by Cellsource Co., Ltd.) were used. For cell culture, a low-serum medium, Mesenchymal Stem Cell Growth Medium 2 (manufactured by Takara Bio Inc.: serum concentration 2%), was used. The cells were incubated at 37°C / CO ２ The cells were cultured in a 10 cm diameter dish (10 mL of medium) for at least two days in an incubator maintained at 5% carbon dioxide. Subsequently, the cells were washed with 3 mL of PBS solution (Fujifilm Wako Pure Chemical Industries, Ltd.), after which 3 mL of trypsin-EDTA solution (PromoCell) was added and the mixture was left to stand at room temperature for 3 minutes to detach the cells. Cells were then recovered by adding 7 mL of serum-free Mesenchymal Stem Cell Growth Medium DXF medium. The suspension was centrifuged (Tomy Seiko Co., Ltd., Model No. LC-230, 200 × g / 3 minutes, room temperature), the supernatant was removed, and the above medium was added to prepare a cell suspension.

[0134] (Cell Adhesion Confirmation Test) A cell suspension was applied to the substrates for producing cell aggregates prepared in Example 5 and Comparative Example 3 at a concentration of 3.0 × 10 ５ cells / cm 2 After that, the mixture was incubated at 37°C / CO2 while maintaining a 5% carbon dioxide concentration. ２ The wells were then left to stand for 2 hours in an incubator. After standing, non-adherent cells and medium were removed, and the wells were washed with PBS, leaving only the adherent cells on the wells. After washing, 2.0 mL of fresh medium was added, and the adherent cells were observed and photographed using a stereomicroscope (Olympus Corporation) SZX16. As shown in Figure 3, selective cell adhesion to the base film portion of the substrate was confirmed in Example 5. There were no gaps in the cell adhesion, and uniform adhesion occurred. In contrast, gaps were present in the cell adhesion in Comparative Example 3, indicating uneven cell adhesion. From the above, it was found that the inclusion of additives that promote cell adhesion and spreading in the base film forming agent can achieve uniform cell adhesion on the base film in serum-free medium, even when using ADSCs.

[0135] Test Example 4: Cell Adhesion Confirmation Test in Low-Serum Medium Using Human Adipose Tissue-Derived Mesenchymal Stem Cells of Example 7 (Cell Preparation) Cells were prepared in the same manner as in Test Example 3, except that the culture medium after cell detachment was changed to Mesenchymal Stem Cell Growth Medium 2, a low-serum medium. (Cell Adhesion Confirmation Test) A cell adhesion confirmation test was performed using the cell aggregate production substrate prepared in Example 7 in the same manner as Test Example 3. As a result, as shown in Figure 4, selective cell adhesion to the base film portion on the prepared substrate was confirmed. Furthermore, there were no gaps in the cell adhesion, and uniform adhesion occurred. From the above, it was found that by including an additive that promotes cell adhesion and spreading in the base film forming agent, uniform cell adhesion can be achieved on the base film even in low-serum medium when using ADSCs.

[0136] Test Example 5: Test to confirm cell adhesion and cell aggregate formation in serum-free medium using human adipose tissue-derived mesenchymal stem cells of Examples 8 to 10, 13 to 14, 17 to 19, and 26 to 27 (Cell preparation) Cells were prepared in the same manner as in Test Example 3.

[0137] (Test for Confirming Cell Adhesion and Cell Aggregate Formation) A cell suspension was applied to the substrates for cell aggregate production prepared in Examples 8 to 10, 13 to 14, 17 to 19, and 26 to 27 at a concentration of 2.0 × 10 ５ cells / cm 2 After that, the mixture was incubated at 37°C / CO2 while maintaining a 5% carbon dioxide concentration. ２ The dish was left to stand in an incubator for 2 hours. After standing, the state of the adhered cells was observed and photographed using a stereomicroscope SZX16 (Olympus Corporation). As a result, as shown in Figure 5, selective cell adhesion to the base film portion of the prepared substrate was confirmed. Furthermore, there were no gaps in the cell adhesion, and uniform adhesion occurred. Furthermore, after 2 days, it was confirmed that the adhered cells had detached from the dish and aggregated, forming cell aggregates (spheroids). From the above, it was found that cells can detach and form cell aggregates after uniform cell adhesion on a base film containing an additive that promotes cell adhesion and spreading.

[0138] Test Example 6: Confirmation test of cell adhesion and cell aggregate formation in serum-free medium using human adipose tissue-derived mesenchymal stem cells of Examples 20 to 25 and 28 to 31 (Cell preparation) Cells were prepared in the same manner as in Test Example 3.

[0139] (Confirmation of cell adhesion, cell aggregate formation test) A cell suspension was applied to the substrates for cell aggregate production prepared in Examples 20 to 25 and 28 to 31 at a concentration of 2.0 × 10 ５ cells / cm 2 After that, the mixture was incubated at 37°C / CO2 while maintaining a 5% carbon dioxide concentration. ２ The wells were then left to stand in an incubator for 2 hours. After standing, non-adherent cells and medium were removed, and the wells were washed with PBS, leaving only the adherent cells. After washing, 2.0 mL of fresh medium was added, and the adherent cells were observed and photographed using a stereomicroscope (Olympus Corporation) SZX16. As shown in Figure 6, selective cell adhesion to the base film portion of the prepared substrate was confirmed. Furthermore, uniform adhesion occurred without gaps. After one day, the adhered cells were confirmed to have detached from the Petri dish and aggregated, forming cell aggregates (spheroids). These results demonstrate that cells can detach and form cell aggregates after uniform cell adhesion on a base film containing additives that promote cell adhesion and spreading.

[0140] Test Example 7: Confirmation test of cell adhesion and cell aggregate formation in serum-free medium using human adipose tissue-derived mesenchymal stem cells of Examples 32 to 34 (Cell preparation) Cells were prepared in the same manner as in Test Example 3.

[0141] (Cell Adhesion Confirmation Test) A cell suspension was applied to the substrates for producing cell aggregates prepared in Examples 32 to 34 at a concentration of 0.75 × 10 ５ ~3.0 x 10 ５ cells / cm 2 After that, the mixture was incubated at 37°C / CO2 while maintaining a 5% carbon dioxide concentration. ２The plates were then left in an incubator for 6 hours. After this time, the state of the adhered cells was observed and photographed using a stereomicroscope (Olympus Corporation) SZX16. After two days, the presence or absence of cell aggregate formation was also observed and photographed. As shown in Figure 7, selective cell adhesion to the base film portion of the prepared substrate was confirmed. Furthermore, there were no gaps between the cells, and uniform adhesion occurred. The cell adhesion diameter was equivalent to the coated diameter. After two days, the adhered cells were confirmed to have detached from the dish and aggregated, forming cell aggregates (spheroids). The diameter of the cell aggregates was also found to vary depending on the adhesion diameter. These results demonstrate that the base film containing additives that promote cell adhesion and spreading allows cells to detach and form cell aggregates after uniform cell adhesion. Furthermore, the cell adhesion diameter and the diameter of the cell aggregates can be controlled by the coated diameter.

[0142] Test Example 8: Cell adhesion confirmation test using human induced pluripotent stem cells of Examples 9 and 10 (Cell preparation) Human induced pluripotent stem cells (hiPSC) 1383D2 strain (obtained from the Center for iPS Cell Research and Application, Kyoto University) were used. Cells were cultured in mTeSR1 medium (manufactured by Veritas Corporation). The cells were incubated at 37°C / CO ２ In an incubator with a 5% carbon dioxide concentration, Vitronectin (VTN-N) Recombinant Human Protein, Truncated (Thermo Fisher Scientific) was added at a concentration of 0.5 μg / cm ２The cells were then cultured for at least 3 days in a 6-well plate coated with PBS (Fujifilm Wako Pure Chemical Industries, Ltd.). Subsequently, the cells were washed with 2 mL / well of PBS solution (Fujifilm Wako Pure Chemical Industries, Ltd.), and 0.5 mL of TrypLE™ Select Enzyme (1X), no phenol red (Thermo Fisher Scientific Co., Ltd.) was added. The cells were then left to stand at room temperature for 10 minutes to detach single cells. The cells were harvested using mTeSR1 medium, and the suspension was centrifuged (Tomy Seiko Co., Ltd., Model No. EIX-136, 200 × g / 3 minutes, room temperature). The supernatant was removed, and the cells were suspended in mTeSR1 medium containing 10 μM Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.). The cell suspension was then passed through a 40 μm cell strainer (Corning) to prepare a cell suspension.

[0143] (Cell Adhesion Confirmation Test) A cell suspension was applied to the substrate for producing cell aggregates prepared in Examples 9 and 10 at a concentration of 1.5 × 10 ５ cells / cm ２ After that, the mixture was incubated at 37°C / CO2 with the carbon dioxide concentration maintained at 5%. ２ The wells were then left to stand for 3 or 24 hours in an incubator. After standing, non-adherent cells and medium were removed, and the wells were washed with mTeSR1 medium, leaving only the adherent cells on the wells. After washing, 0.5 mL of fresh medium was added, and the adherent cells were observed and photographed using an inverted microscope IX73 (Olympus Corporation). As a result, hiPSC adhesion was confirmed, as shown in Figure 8. These results demonstrate that the inclusion of additives that promote cell adhesion and spreading in the base film formation agent makes it possible to achieve uniform cell adhesion on the base film, even when using hiPSCs.

[0144] Test Example 9: Test to confirm spheroid formation using human induced pluripotent stem cells of Examples 9 and 10. The cell suspension prepared in Test Example 8 was applied to the substrate for producing cell aggregates prepared in Examples 9 and 10 at a concentration of 1.5 × 10 ５ cells / cm ２ After that, the mixture was incubated at 37°C / CO2 with the carbon dioxide concentration maintained at 5%. ２The cells were then placed in an incubator. The day after seeding, the non-adherent cells and the medium were removed, and 0.5 mL of mTeSR1 medium not containing Y-27632 was added to replace the medium with a Y-27632-free medium. The cells were then placed in an incubator at 37°C / CO2, with the carbon dioxide concentration maintained at 5%. ２ The dish was left to stand in an incubator, and two days after seeding, the state of the cells was observed and photographed using an inverted microscope IX73 (Olympus Corporation). As a result, as shown in Figure 9, it was confirmed that the adhered cells had detached from the dish and aggregated, forming cell aggregates (spheroids). It was confirmed that spheroids of uniform size had been formed throughout the well.

[0145] Test Example 10: Tests for confirming cell adhesion, survival rate after spheroid formation, and undifferentiated state using human induced pluripotent stem cells of Examples 11 and 12 As in Test Example 8, human induced pluripotent stem cells (hiPSC) 1383D2 strain were used. 1.5 × 10 cell suspension was added to the substrate for cell aggregate production prepared in Examples 11 and 12. ５ cells / cm ２ After that, the mixture was incubated at 37°C / CO2 while maintaining a 5% carbon dioxide concentration. ２ The cells were then allowed to stand in an incubator. The day after seeding, the non-adherent cells and the medium were removed, and 2 mL of mTeSR1 medium not containing Y-27632 was added to replace the medium with a Y-27632-free medium. The cells were then further incubated at 37°C / CO2 with a 5% carbon dioxide concentration. ２ After incubation in an incubator, the formation of cell aggregates (spheroids) was confirmed two days after seeding, as described above. The resulting spheroids were collected in a 15 mL centrifuge tube, centrifuged at 200 × g for 3 minutes, and the supernatant was removed. The spheroids were then suspended in 0.25 mL of TrypLE™ Select Enzyme (1X), no phenol red (Thermo Fisher Scientific). Single cells were dispersed by incubating in a 37°C water bath for 5–10 minutes with pipetting. After neutralization with the addition of 750 μL of 10% FBS-containing DMEM, cells were counted using a NucleoCounter NC-200 (Chemometec). As shown in Table 1, the iPSCs that formed the resulting spheroids maintained a high viability.

[0146]

[0147] The dispersed cells were then washed with SM buffer (2% FBS / PBS), followed by the addition of PE-labeled mouse anti-human SSEA-4 antibody and AlexaFluor 647-labeled mouse anti-human TRA-1-60 antibody (BD Biosciences) and incubation at room temperature for 30 minutes. After washing twice with SM buffer, analysis was performed using a FACSLSR FortessaX-20 (BD Biosciences). As shown in Table 2, the percentage of cells co-expressing SSEA-4 and TRA-1-60 showed high expression, indicating that the spheroids created by this method maintained their undifferentiated state.

[0148]

[0149] Test Example 11: Cell adhesion confirmation test using human induced pluripotent stem cells of Examples 15 and 16. A cell suspension was applied to the substrate for producing cell aggregates prepared in Examples 15 and 16 at a concentration of 1.5 × 10 ５ cells / cm ２ After that, the mixture was incubated at 37°C / CO2 with the carbon dioxide concentration maintained at 5%. ２ After leaving the cells in the incubator for 6 hours, the state of the adhered cells was observed and photographed using an inverted microscope IX73 (Olympus Corporation). As a result, adhesion of hiPSCs was confirmed as shown in Figure 10. Subsequently, the cells were incubated at 37°C / CO2 with a 5% carbon dioxide concentration. ２ The wells were incubated in an incubator for 26 hours after seeding. After incubation, non-adherent cells and medium were removed, and the wells were washed with mTeSR1 medium, leaving only the adherent cells. After washing, 0.5 mL of fresh medium was added, and the adherent cells were observed and photographed using an inverted microscope IX73 (Olympus Corporation). As a result, hiPSC adhesion was confirmed, as shown in Figure 10. These results demonstrate that the inclusion of additives that promote cell adhesion and spreading in the base film formation agent makes it possible to achieve uniform cell adhesion on the base film, even when using hiPSCs.

[0150] Test Example 12: Test to confirm spheroid formation using human induced pluripotent stem cells of Examples 15 and 16. The cell suspension was applied to the substrate for cell aggregate production prepared in Examples 15 and 16 at a concentration of 1.5 × 10 ５ cells / cm ２ After that, the mixture was incubated at 37°C / CO2 with the carbon dioxide concentration maintained at 5%. ２ The cells were then placed in an incubator. The day after seeding, the non-adherent cells and the medium were removed, and 0.5 mL of mTeSR1 medium not containing Y-27632 was added to replace the medium with a Y-27632-free medium. The cells were then placed in an incubator at 37°C / CO2, with the carbon dioxide concentration maintained at 5%. ２ The dish was left to stand in an incubator, and two days after seeding, the state of the cells was observed and photographed using an inverted microscope IX73 (Olympus Corporation). As a result, as shown in Figure 11, it was confirmed that the adhered cells had detached from the dish and aggregated, forming cell aggregates (spheroids). It was confirmed that spheroids of uniform size had been formed throughout the well.

[0151] Test Example 13: Cell Adhesion and Cell Aggregate Formation Confirmation Test with Mouse Fibroblasts in FBS-Free Medium of Comparative Example 4 (Additives Only, No Polymer) (Cell Preparation) Cells were prepared in the same manner as in Test Example 1. (Cell Adhesion Confirmation Test) A cell adhesion and cell aggregate formation confirmation test was performed on the cell aggregate production substrate prepared in Comparative Example 4 in the same manner as in Test Example 2. As a result, as shown in Figure 12, no cell adhesion to the base film portion on the prepared substrate was confirmed. From the above, it was found that cell adhesion could not be achieved with a base film that does not contain a polymer and contains only an additive that promotes cell adhesion and spreading. This shows that the cell adhesion effect is only achieved by combining a polymer and an additive.

[0152] Test Example 14: Cell Adhesion and Cell Aggregate Formation Confirmation Test for Mouse Fibroblasts in FBS-Containing Medium of Comparative Example 5 (Polymer-Free, Additives Only) (Cell Preparation) Cells were prepared in the same manner as in Test Example 1, except that the culture medium after cell detachment was changed to BME medium containing 10% FBS (bovine serum) and 1% glutamine / penicillin / streptmycin. (Cell Adhesion Confirmation Test) A cell adhesion and cell aggregate formation confirmation test was performed on the cell aggregate production substrate prepared in Comparative Example 5 in the same manner as in Test Example 2. As a result, as shown in Figure 13, selective cell adhesion to the base film portion on the prepared substrate was confirmed. Furthermore, uniform cell adhesion occurred without gaps. Furthermore, it was confirmed that the adhered cells remained adhered to the Petri dish after two days. From the above, it was found that, although uniform cell adhesion was achieved in serum-containing medium with a base film that was polymer-free and contained only additives that promote cell adhesion and spreading, subsequent detachment did not occur, preventing the formation of cell aggregates.

[0153] The agent for forming a base film for cell culture of the present invention realizes uniform cell adhesion and subsequently enables the production of cell aggregates under culture conditions with or without animal-derived serum. Thus, by using the agent for forming a base film for cell culture, mass production of homogeneous, high-quality cell aggregates for use in the field of regenerative medicine can be achieved.

Claims

1. 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, 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 derived from a monomer represented by the formula, a cell adhesion substance, and a solvent, a basement membrane forming agent for cell culture (however, the polymer does not contain a repeating unit derived from a monomer chemically modified with a cell adhesion substance).

2. The polymer further has the formula (II): 【Chemical 2】 [wherein, R ｂ The cell culture basement membrane forming agent according to claim 1, which contains 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].

3. The cell culture substratum-forming agent according to Claim 1, wherein the weight ratio of the polymer to the cell adhesion substance is 100:0.1 to 100:

100.

4. The cell culture substratum-forming agent according to Claim 1, wherein the cell adhesion substance contains glycoprotein.

5. A substrate for producing cell aggregates, which is provided with spots of a cell culture substratum formed of the cell culture substratum-forming agent according to any one of Claims 1 to 4 on a substrate having an ability to inhibit cell adhesion.

6. The following formula (I): on a substrate having an ability to inhibit cell adhesion 【Chemical 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, 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 method for producing cell aggregates, comprising the steps of forming a basement membrane for cell culture, which comprises a polymer containing a repeating unit derived from a monomer represented by the formula and a cell adhesion substance, and then seeding cells (however, the polymer does not contain a repeating unit derived from a monomer chemically modified with a cell adhesion substance).

7. The following formula (Ia): [Chemical Formula 4] [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 comprising a repeating unit and a crosslinked structure, a cell adhesion substance, and a solvent, a basement membrane forming agent for cell culture (however, the polymer does not include a repeating unit derived from a monomer chemically modified with a cell adhesion substance).

8. The cell culture substratum-forming agent according to Claim 7, wherein the crosslinked structure contains a structure derived from a polyfunctional acrylate compound, a polyfunctional acrylamide compound, a polyfunctional polyester or an isoprene compound.

9. The crosslinked structure is the following formula (IIIa), (IVa) and / or (Va): 【Chemical Formula 5】 [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], the cell culture basement membrane forming agent according to claim 7, which contains a structure represented by the formula.

10. The following formula (Ia): on a substrate having an ability to inhibit cell adhesion 【Chemical Formula 6】 [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 comprising a repeating unit represented by and a crosslinked structure, and a cell adhesion substance, and a method for producing cell aggregates, comprising the steps of forming a basement membrane for cell culture, and then seeding cells (however, the polymer does not include a repeating unit derived from a monomer chemically modified with a cell adhesion substance).

11. The following formula (Ia): on a substrate having an ability to inhibit cell adhesion 【Chemical Formula 7】 [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 comprising a repeating unit represented by and a crosslinked structure, and a cell adhesion substance, and a method for producing cell aggregates, comprising a step of seeding cells on a substrate for producing cell aggregates provided with a basement membrane for cell culture (however, the polymer does not include a repeating unit derived from a monomer chemically modified with a cell adhesion substance).

12. The following formula (Ia): [Chemical Formula 8] [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], and a polymer containing a repeating unit represented by and a crosslinked structure, and a cell adhesion substance, and a substrate for producing cell aggregates, comprising a spot of a basement membrane for cell culture (however, the polymer does not include a repeating unit derived from a monomer chemically modified with a cell adhesion substance).

13. The following formula (Ia): 【Chemical Formula 9】 [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], and a repeating unit represented by the formula (IIa): 【Chemical Formula 10】 [wherein, R ｂ is a cell culture basement membrane forming agent comprising a polymer containing a repeating unit represented by [representing a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms], a crosslinked structure, a cell adhesion substance, and a solvent (however, the polymer does not include a repeating unit derived from a monomer chemically modified with a cell adhesion substance).

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