Structure for cells, method for producing structure for cells, and method for cell culture

Abstract

To provide a structure for cells and the like that allows preservation with suppressed reduction in cell viability and facilitates simple cell culturing.SOLUTION: A structure for cells includes a substrate and multiple spots arranged at intervals on the surface of the substrate, each of the multiple spots having a polymer-containing cell culture base membrane, with cultured cells disposed on the cell culture base membrane, the cultured cells being in a frozen state.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] The present invention relates to a cell structure, a method for producing a cell structure, and a cell culture method. [Background technology]

[0002] Various materials have been proposed as underlayer-forming agents for forming underlayers for efficient cell culture. Patent Document 1 discloses a method for producing a polymer used as a base membrane for cell culture and a cell culture vessel, while Patent Document 2 discloses a method for producing a cell structure. In the process of producing (cultivating) homogenous cell aggregates, these cell culture base membranes require the use of serum derived from living organisms to ensure uniform adhesion of cells to the base membrane. This poses 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 using serum derived from animals other than humans.

[0003] Therefore, the present applicant has proposed a base film for cell culture that enables the mass production of homogeneous, high-quality cell aggregates without using serum derived from living organisms, a base film forming agent for forming the same, and a substrate for producing cell aggregates (Patent Document 3). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2020 / 040247 Brochure [Patent Document 2] JP 2017-143755 A [Patent Document 3] International Publication No. 2023 / 282253 Brochure Summary of the Invention [Problem to be solved by the invention]

[0005] Cryopreservation of cells is utilized for the purpose of preventing cell deterioration and bacterial contamination during cell culture, long-term stable cell supply, cell transportation, etc. However, when cell structures are cryopreserved, the viability of the cells decreases.

[0006] The present invention aims to provide a cell structure that can store cells while suppressing a decrease in cell viability and that can easily culture cells, a method for producing the cell structure, and a cell culture method using the cell structure. [Means for solving the problem]

[0007] The present inventors have found that the above-mentioned problems can be solved and have completed the present invention.

[0008] That is, the present invention includes the following aspects. [1] A substrate; a plurality of spaced apart spots on a surface of the substrate; A cell structure comprising: Each of the plurality of spots has a cell culture substratum containing a polymer; The cell culture base film has cells for culture thereon, The cell structure, wherein the cells for culture are in a frozen state. [2] The cell structure described in [1], further comprising a cell adhesive substance disposed on the cell culture base membrane. [3] further comprising a cryopreservative; The cell structure according to [1] or [2], wherein the cells for culture are covered with the cryopreservation agent. [4] A cell structure according to any one of [1] to [3], which is a cell culture vessel. [5] The cell structure according to any one of [1] to [4], wherein the polymer is a polymer (A) containing a repeating unit represented by the following formula (Ia): [ka] (In formula (Ia), 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. [6] The cell structure described in [5], wherein the polymer (A) includes a crosslinked structure. [7] The cell structure according to [6], wherein the polymer (A) contains at least one of a structure represented by the following formula (IIIa), a structure represented by the following formula (IVa), and a structure represented by the following formula (Va). [ka] (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. [8] The surface of the substrate has a coating film having a cell adhesion inhibitory ability, The cell structure described in any one of [1] to [7], wherein the spots are arranged on the coating film. [9] A step of applying droplets of a cell culture base film forming agent containing a polymer and a solvent to a substrate to form a cell culture base film in the area to be the spot; Seeding cells for culture on the cell culture base film; Freezing the cells for culture; A method for producing a cell structure comprising the steps of:

[10] A method for producing a cell structure according to [9], comprising the step of contacting the cells for culture with a cryopreservation agent prior to the freezing step.

[11] The method for producing a cell structure described in [9] or

[10] , wherein the application of the droplets of the cell culture base film forming agent to the substrate is carried out by an inkjet method.

[12] A method for producing a cell structure described in any of [9] to

[11] , further comprising a step of applying droplets containing a cell adhesive substance to the cell culture base membrane prior to the seeding step, thereby disposing the cell adhesive substance on the cell culture base membrane.

[13] The method for producing a cell structure described in

[12] , wherein the application of droplets containing the cell adhesive substance to the cell culture base membrane is carried out by an inkjet method.

[14] The method for producing a cell structure described in any one of [9] to

[13] , wherein the polymer is a polymer containing a repeating unit represented by the following formula (Ia): [ka] (In formula (Ia), 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.

[15] In the cell structure to be produced, the surface of the substrate has a coating film having a cell adhesion inhibitory ability, and the plurality of spots are arranged on the coating film; The method for producing a cell structure described in any of [9] to

[14] , further comprising a step of applying a coating agent containing a polymer capable of inhibiting cell adhesion onto the substrate to form the coating film.

[16] A method for producing a cell structure described in any one of [9] to

[15] , wherein the cell structure to be produced is a cell culture vessel.

[17] A step of thawing the frozen cells for culture in the cell structure according to any one of [1] to [8]; Culturing the cells for culture; A cell culture method comprising:

[18] The cell culture method according to

[17] , for producing a cell structure. Effect of the Invention

[0009] According to the present invention, it is possible to provide a cell structure that can store cells while suppressing a decrease in cell viability and can easily culture cells, a method for manufacturing the cell structure, and a cell culture method using the cell structure. [Brief description of the drawings]

[0010] [Figure 1A] FIG. 1A is a perspective view of an example of a cell structure before cells are placed thereon. [Figure 1B] FIG. 1B is a top view of an example of a cell structure before cells are placed thereon. [Figure 2A] FIG. 2A is a perspective view of another example of a cell structure before cells are placed thereon. [Figure 2B] FIG. 2B is a top view of another example of a cell structure before cells are arranged thereon. [Figure 3A] FIG. 3A is a perspective view of another example of a cell structure before cells are placed therein. [Figure 3B] FIG. 3B is a top view of another example of a cell structure before cells are placed thereon. [Figure 4A] FIG. 4A shows the results of observation 3 hours after inoculation in the test of Example 1. [Figure 4B] FIG. 4B shows the results of observation after replacement with a freezing preservation solution in the test of Example 1. [Figure 4C] FIG. 4C shows the results of observation after thawing two weeks after freezing and addition of a medium in the test of Example 1. [Figure 4D] FIG. 4D shows the observation results after one day of culture following freezing and thawing in the test of Example 1. [Figure 5A] FIG. 5A shows the results of observation 3 hours after inoculation in the test of Example 2. [Figure 5B]FIG. 5B shows the results of observation after two days of culture following freeze-thawing in the test of Example 2. [Figure 6] FIG. 6 shows the results of observation of spheroid formation after 3 days of culture in the test of Comparative Example 1. [Figure 7A] FIG. 7A shows the results of fluorescence observation in Example 2. [Figure 7B] FIG. 7B shows the bright field observation results of Example 2. [Figure 8A] FIG. 8A shows the results of fluorescence observation in Comparative Example 1. [Figure 8B] FIG. 8B shows the bright field observation results of Comparative Example 1. [Figure 9] FIG. 9 shows the observation results of the process of cell aggregate formation in Test Example 4. [Figure 10] FIG. 10 shows size distribution data of spheroids in Test Example 4. [Figure 11] FIG. 11 shows the observation results of the process of cell aggregate formation in Test Example 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] (Structure for cells) The cell structure of the present invention comprises a substrate and a plurality of spots. The plurality of spots are spaced apart on the surface of the substrate. The spot, in one embodiment, comprises a cell culture substrate membrane. In another embodiment, the spot has a cell culture base membrane and a cell adhesive substance disposed on the cell culture base membrane. Here, the "surface of the substrate" refers to the surface that comes into contact with contents such as cell culture medium.

[0012] Furthermore, in one embodiment, the cell structure has cells for culture on the cell culture base membrane. Furthermore, in another embodiment, the cell structure has cells for culture adhered to a cell adhesive substance disposed on a cell culture base membrane. In the cell structure, the cells for culture are in a frozen state.

[0013] The present inventors have found that cryopreservation of cell structures reduces the survival rate of the cells, and as a result of extensive research, have found that cryopreservation of culture cells placed on a cell culture base membrane prior to culturing to prepare a cell structure prevents a decrease in the survival rate of the cells, and that culturing the culture cells on the cell culture base membrane after thawing allows the cell structure to be easily prepared.

[0014] <Cells for culture> The term "cells for culture" refers to cells that are cultured to prepare cell structures.

[0015] A cell is the most basic unit constituting an animal or a 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 (cell lines) isolated from an organism that have acquired immortalization ability and are stably maintained outside the body, cells isolated from an organism that have been artificially genetically modified, cells isolated from an organism that have been artificially replaced with a nucleus, etc. Examples of somatic cells that constitute a living body include, but are not limited to, fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, red blood cells, platelets, macrophages, monocytes, bone cells, 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, neural tissue, hair, etc. Furthermore, the somatic cells include cells induced to differentiate from stem cells or progenitor cells.

[0016] Stem cells are cells that have the ability to replicate themselves and differentiate into cells of multiple lineages, and examples of such cells 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, hair follicle stem cells, etc. Among the above stem cells, pluripotent stem cells include ES cells, embryonic germ stem cells, and iPS cells. A precursor cell is a cell that is at an intermediate stage in the differentiation of the stem cell into a specific somatic cell or germ cell. Cancer cells are cells that derive from somatic cells and have acquired the ability to proliferate indefinitely. A cell line is a type of cell that has acquired the ability to proliferate indefinitely through artificial manipulation outside of a living organism. Among these, fibroblasts, stem cells, and among stem cells, pluripotent stem cells are more preferable.

[0017] In the cell structure, the cells for culture are in a frozen state.

[0018] The method for freezing the cells for culture is not particularly limited, and examples thereof include the freezing methods described in the method for producing a cell structure described below.

[0019] The temperature at which the cell structure having cells for culture is stored is not particularly limited, and is, for example, -250°C to -5°C, preferably -200°C to -20°C, and more preferably -200°C to -80°C.

[0020] In the cell for culture in the cell structure, a plurality of cells may be aggregated, and in this case, the aggregate of the plurality of cells may be in a two-dimensional structure state (sheet-like) in which the cells are adhered to each other. However, in order for the cryoprotectant described later to be distributed to each cell, the cells do not have to be adhered to each other. If the aggregate of the plurality of cells is in a three-dimensional state (cell mass) such as a spheroid or organoid, the cells are damaged by ice crystals inside and outside the cells during freezing or after thawing, and the survival rate decreases.

[0021] <Cryopreservation agent> The cell structure preferably comprises a cryopreservation agent. In the cell structure, the cells for culture are preferably covered with a cryopreservation agent. By covering the cells for culture with a cryopreservation agent in the cell structure, damage to the cells caused by ice crystals inside and outside the cells during freezing or thawing can be prevented, and a decrease in the survival rate of the cells can be further prevented.

[0022] The cryopreservation agent is not particularly limited, and any cryopreservation solution used for cryopreservation of cells can be used. The cryopreservation agent contained in the cell structure is, for example, a cryopreservation solution in a frozen state. A cryopreservation solution is a solution for reducing damage to cells caused by cryopreservation. There are no particular limitations on the cryopreservation solution, so long as it is suitable for cryopreservation of cells. The cryopreservation solution may contain culture solution components contained in the culture solution, cryoprotectants, and the like. The culture solution components, such as sugars, amino acids, vitamins, inorganic salts, trace metals, and additives, may be any that satisfy the explanation in the above (culture solution) section. In addition, it is preferable that the cryopreservation solution has a coagulation start temperature in the range of -15°C or higher and -5°C or lower.

[0023] Cryoprotectants are substances used to reduce damage to cells caused by freezing and thawing during cryopreservation. Examples of cryoprotectants include cell-non-permeable cryoprotectants and cell-permeable cryoprotectants. Specific examples of cell-impermeable cryoprotectants include, for example, albumin, sucrose, trehalose, dextran, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polylysine, and the like. Specific examples of cell-permeable cryoprotectants include, for example, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, propanediol, and the like. These cryoprotectants may be used alone or in combination of two or more. These cryoprotectants may be appropriately selected from known ones depending on the type of cells, the composition of the cryopreservation solution, etc.

[0024] Examples of commercially available cryopreservation solutions that do not contain DMSO include Stem Cell Banker (registered trademark) DMSO-free GMP grade (Nihon Zenyaku Kogyo Co., Ltd.), Bambanker (registered trademark) DMSO-free (Nihon Genetics Co., Ltd.), CryoScarless (registered trademark) DMSO-free (BioVerde), Stem Cell Keep (BioVerde), CryoNovo (registered trademark) X12 (Cosmo Bio Co., Ltd.), CryoNovo (registered trademark) P24 (Cosmo Bio Co., Ltd.), DMSO-free cell cryopreservation solution for cryopreservation of human ES / iPS cells (ReproCell Co., Ltd.), CellReservoirOne (Nacalai Tesque, Inc.), TheliKeep (registered trademark: BioVerde Co., Ltd.), Cellvation (registered trademark: Protide Pharmaceuticals Co., Ltd.), ReproCryoRM (ReproCell Co., Ltd.), and SOFOROCryo (SARAYA Co., Ltd.). In addition, commercially available cryopreservation solutions containing DMSO include, for example, Cellbanker 1 (Nihon Zenyaku Kogyo Co., Ltd.), Stem Cellbanker (registered trademark) (Nihon Zenyaku Kogyo Co., Ltd.), Bambanker (registered trademark) hRM (Nihon Genetics Co., Ltd.), and Bambanker (registered trademark) (Nihon Genetics Co., Ltd.).

[0025] <Spot> The spots have a cell culture base membrane. A spot is a location on a cell structure where cells are anchored or cultured (eg, structured).

[0026] The number of spots spaced apart on the surface of the substrate is not particularly limited. For example, the number of spots may be 1 cm. 2 The number of particles per unit is preferably 50 to 5,000, and more preferably 100 to 2,000. The distance between adjacent spots is not particularly limited, and is preferably 100 μm to 6,000 μm, more preferably 150 μm to 4,000 μm, and particularly preferably 150 μm to 3,000 μm, where the distance refers to the length between the centers of the two spots. The size of the spot surface is not particularly limited, and for example, the circle equivalent diameter is preferably 25 μm to 5,000 μm, and more preferably 50 μm to 3,000 μm. The equivalent circle diameter is the diameter of a perfect circle that corresponds to the area of ​​the target surface. If the surface of the spot is a perfect circle, the equivalent circle diameter refers to the diameter of the perfect circle, and if the surface of the spot is other than a perfect circle, the equivalent circle diameter refers to the diameter of a perfect circle that has the same area as the area of ​​the target surface.

[0027] The area ratio of the spots on the substrate is not particularly limited, but is preferably 30% or more, 40% or more, 50% or more, and is preferably 99% or less.

[0028] The spot preferably comprises a cell adhesive substance. In this case, the cell adhesive substance is disposed on a cell culture base membrane. In this case, the area occupied by the cell culture base membrane is the spot. Therefore, the surface of the cell culture base membrane corresponds to the spot. The surface size of the cell adhesive material placed on the cell culture base membrane is not particularly limited as long as it is less than the surface size of the cell culture base membrane, but is preferably 1 / 80 to 1 / 3, and more preferably 1 / 50 to 1 / 4, of the area of ​​the cell culture base membrane.

[0029] <<Cell adhesive substances>> The cell adhesive substance is present, for example, at one spot.

[0030] Cell adhesive substances promote cell adhesion, spreading, proliferation and differentiation. As the cell adhesive substance, known substances such as biological substances such as extracellular matrix (ECM) proteins, glycoproteins, peptides, and synthetic compounds (low molecular weight, high molecular weight) can be used, but compounds not of biological origin, 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.

[0031] 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., Matrixome'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).

[0032] The cell adhesive substance is preferably a glycoprotein, specifically selected from vitronectin, integrin, cadherin, fibronectin, laminin, tenascin, osteopontin, and bone sialoprotein, and is preferably a protein having an RGD sequence as an amino acid sequence.

[0033] Examples of peptides include ECM peptides (MAPTrix ​​(registered trademark) from Kollodis Bio Sciences) and RGD peptides (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.: 180-01531).

[0034] 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 weight) include adhesamine (e.g., Nagase & Co.: AD-00000-0201) and synthetic cyclic RGD peptide (e.g., IRIS BIOTECH: LS-3920.0010).

[0035] <<Cell culture base membrane>> The cell culture substrata contain a polymer, which is a material different from the cell adhesive material and is typically a synthetic polymer. The cell culture base membrane may further contain a cell adhesive substance.

[0036] The thickness of the cell culture base film is not particularly limited, but is, for example, in the range of 1 to 1000 nm, and preferably in the range of 5 to 500 nm.

[0037] The polymer is not particularly limited as long as it is a polymer that can be used for a base membrane for cell culture. From the viewpoint of optimally obtaining the effects of the present invention, however, a polymer (A) containing a repeating unit represented by the following formula (Ia) is preferred. [ka] (In formula (Ia), 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.

[0038] The polymer (A) preferably further contains a repeating unit represented by the following formula (IIa). [ka] (In formula (IIb), R b represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms.

[0039] 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.

[0040] R a1 and R b are preferably each independently selected from a hydrogen atom and a methyl group.

[0041] U a1 and U a2 are each preferably independently selected from a hydrogen atom, a methyl group, an ethyl group, a n-propyl group, an isopropyl group, and a n-butyl group, more preferably a methyl group or an ethyl group, and most preferably a methyl group.

[0042] In this specification, unless otherwise defined, examples of the "straight-chain 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.

[0043] Examples of the monomer (cationic monomer) that provides the repeating unit represented by formula (Ia) to the polymer (A) include 2-N,N-dimethylaminoethyl methacrylate, N,N-dimethylaminomethyl methacrylate, etc., and 2-N,N-dimethylaminoethyl methacrylate (2-(dimethylamino)ethyl methacrylate) is preferred.

[0044] Examples of the monomer (anionic monomer) that gives the repeating unit represented by formula (IIa) to the polymer (A) include acrylic acid and methacrylic acid, with methacrylic acid being preferred.

[0045] The molar ratio of the repeating unit (Ia) represented by formula (Ia) to the repeating unit (IIa) represented by formula (IIa) in the polymer (A) [(Ia) / (IIa)] is, for example, 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 [(Ia) / (IIa)] is 1 or more, a decrease in cell adhesive strength due to the anionic nature of the polymer can be suppressed.

[0046] The polymer (A) may have other repeating units. In the polymer (A), the total of the repeating units represented by formula (Ia) and the repeating units represented by formula (IIa) relative to all repeating units is, for example, 50 mol % or more, preferably 75 mol % or more, more preferably 80 mol % or more, and even more preferably 90 mol % or more.

[0047] From the viewpoint of suitably obtaining the effects of the present invention, it is preferable that the polymer (A) further contains a crosslinked structure. The crosslinked structure can be obtained by using a monomer having two or more carbon-carbon unsaturated bonds when synthesizing the polymer (A). Specifically, the monomer having two or more carbon-carbon unsaturated bonds is 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.

[0048] The polymer (A) contains, for example, a structure derived from at least one of a polyfunctional acrylate compound, a polyfunctional acrylamide compound, a polyfunctional polyester, and an isoprene compound. The polymer (A) preferably contains at least one of a structure represented by the following formula (IIIa), a structure represented by the following formula (IVa), and a structure represented by the following formula (Va). Among these, the structure represented by the formula (IIIa) is preferred. [ka] (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, n represents a number from 1 to 50.)

[0049] R c and R d are preferably each independently selected from a hydrogen atom and a methyl group. R e is preferably selected from a methylene group, an ethylene group, and a propylene group, and more preferably an ethylene group. Although n is a number from 1 to 50, n is preferably a number from 1 to 30, and more preferably a number from 1 to 10.

[0050] The molar ratio of the structures represented by formulae (IIIa) to (Va) to the entire polymer (A) is preferably from 0 mol % to 50 mol %, and more preferably from 2 mol % to 25 mol %. When the molar ratio of the structures represented by formulae (IIIa) to (Va) is 50% or less of the entire polymer (A), gelation of the solid content during production due to high molecular weight caused by excessive crosslinking can be suppressed, making production easier.

[0051] The synthesis method of the polymer (A) is not particularly limited, and examples thereof include known radical polymerization methods. In addition, the method described in International Publication No. 2020 / 040247 may be used.

[0052] The number average molecular weight (Mn) of the polymer is, for example, 20,000 to 1,000,000, and preferably 50,000 to 800,000. The number average molecular weight (Mw) of the polymer is, for example, 50,000 to 2,000,000, and preferably 10,000 to 1,200,000. The ratio (Mw / Mn) of the weight average molecular weight (Mw) to the number average molecular weight (Mn) of the polymer is, for example, 1.01 to 10.00, preferably 1.2 to 8.0, more preferably 1.4 to 6.0, further more preferably 1.5 to 5.0, and particularly preferably 1.6 to 4.5. The number average molecular weight (Mn) and the weight average molecular weight (Mw) can be determined, for example, by gel filtration chromatography.

[0053] <Substrate> The material, shape, size and structure of the substrate are not particularly limited. The surface of the substrate may be flat or uneven, but is preferably flat.

[0054] Examples of the material of the substrate include glass, metal, a metal-containing compound or a metalloid-containing compound, activated carbon, and resin. 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, Au, etc.). Examples of the metal-containing compound or metalloid-containing compound include ceramics, which are sintered bodies whose basic component is a metal oxide and are sintered 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.).

[0055] The resin may be either a natural resin or a derivative thereof, or a synthetic resin. Examples of the natural resin or a derivative thereof include cellulose, cellulose triacetate (CTA), nitrocellulose (NC), and cellulose immobilized with dextran sulfate. Examples of the synthetic resin include polyacrylonitrile (PAN), polyimide (PI), polyester polymer alloy (PEPA), polystyrene (PS), polysulfone (PSF), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), polyvinyl alcohol (PVA), and vinyl acetate-modified polyethylene. (EVA), polyurethane (PU), ethylene-vinyl alcohol copolymer (EVOH), 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), or Teflon (registered trademark) are preferably used.

[0056] The substrate may be in the form of a plate or a film, and the thickness of the substrate is not particularly limited. The substrate may be, for example, a film having a thickness of 1 mm or less.

[0057] The substrate may be a substrate used in a so-called cell culture vessel, and examples of such substrates include dishes or dishes, such as Petri dishes, tissue culture dishes, and multi-dishes, 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, that are generally used for cell culture.

[0058] <<Coating film>> The surface of the substrate may have a coating film having a cell adhesion suppressing ability, in which case the spots are arranged on the coating film. The coating film can be obtained, for example, by applying a coating agent containing a polymer capable of inhibiting cell adhesion onto a substrate. The shape of the coating film on the surface of the substrate is not particularly limited, so long as the spots are arranged thereon; one film may uniformly cover the entire surface of the substrate, one film may be formed within the plane of the surface of the substrate, or multiple films may be scattered on the surface of the substrate.

[0059] Here, having the ability to inhibit cell adhesion means that the relative absorbance (WST OD450nm) (%) ((absorbance (WST OD450nm) of the example) / (absorbance (WST OD450nm) of the comparative example)) when compared to a case without a coating film or without a low cell adsorption treatment, measured using a fluorescence microscope performed, for example, by the method described in the examples of WO 2016 / 093293, is 50% or less, preferably 30% or less, and more preferably 20% or less.

[0060] The coating film having cell adhesion suppression ability is not particularly limited, but examples thereof include the following coating films.

[0061] <<<Coating film according to the first embodiment>>> The coating film of the first embodiment includes a copolymer. The copolymer of the first embodiment contains 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). The copolymer is, for example, a copolymer described in WO2014 / 196652. The contents of WO2014 / 196652 are incorporated herein by reference to the same extent as if fully set forth herein. [ka]

[0062] (In the formula, U a1 , U a2 , U b1 , U b2 and U b3 each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; and An - represents an anion selected from the group consisting of a halide ion, an inorganic acid ion, a hydroxide ion, and an isothiocyanate ion.

[0063] The copolymer is not particularly limited as long as it is a copolymer containing a repeating unit containing an organic group represented by the above formula (a) and a repeating unit containing an organic group represented by the above formula (b). The copolymer is preferably one obtained by radical polymerization of a monomer containing an organic group represented by the above formula (a) and a monomer containing an organic group represented by the above formula (b), but one obtained by polycondensation or polyaddition reaction can also be used. Examples of copolymers include vinyl polymerization polymers reacted with olefins, polyamides, polyesters, polycarbonates, polyurethanes, etc., among which vinyl polymerization polymers reacted with olefins or (meth)acrylic polymers polymerized with (meth)acrylate compounds are particularly desirable. In the present invention, the (meth)acrylate compound means both an acrylate compound and a methacrylate compound. For example, (meth)acrylic acid means acrylic acid and methacrylic acid.

[0064] Preferably, the monomers containing the organic groups represented by the above formulae (a) and (b) are monomers represented by the following formulae (A) and (B), respectively. [ka] (In the formula, T a , T b , U a1 , U a2 , U b1 , U b2 and U b3 each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; Q a and Q beach independently represents a single bond, an ester bond, or an amide bond; R a and R b each independently represents a linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted with a halogen atom; An - represents an anion selected from the group consisting of a halide ion, an inorganic acid ion, a hydroxide ion, and an isothiocyanate ion, and m represents an integer of 0 to 6.

[0065] Therefore, the repeating units derived from the monomers represented by formulae (A) and (B) are represented by the following formulae (a1) and (b1), respectively. [ka] (In the formula, T a , T b , U a1 , U a2 , U b1 , U b2 and U b3 , Q a and Q b , R a and R b , An - and m has the same meaning as above.

[0066] In the present invention, an "ester bond" means -C(=O)-O- or -OC(=O)-, and an "amide bond" means -NHC(=O)- or -C(=O)NH-.

[0067] In the present invention, the term "linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted with one or more halogen atoms" means a linear or branched alkylene group having 1 to 10 carbon atoms, or a linear or branched alkylene group having 1 to 10 carbon atoms which is substituted with one or more halogen atoms. Here, the "straight-chain or branched alkylene group having 1 to 10 carbon atoms" refers to a divalent organic group in which one hydrogen atom has been further removed from the above-mentioned alkyl group, and examples thereof 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, a 1-ethyl-trimethylene group, a hexamethylene group, an octamethylene group, and a decamethylene group. Among these, ethylene, propylene, octamethylene and decamethylene are preferred, and linear or branched alkylene groups having 1 to 5 carbon atoms, such as ethylene, propylene, trimethylene and tetramethylene, are more preferred, with ethylene or propylene being particularly preferred. "Linear or branched alkylene groups having 1 to 10 carbon atoms substituted with one or more halogen atoms" means such alkylene groups in which any one or more hydrogen atoms have been replaced with halogen atoms, and particularly preferred is ethylene or propylene group in which some or all of the hydrogen atoms have been replaced with halogen atoms.

[0068] In the present invention, the "halogen atom" includes a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. In the present invention, the term "halide ion" refers to an anion of a halogen atom, and examples of such an anion include a fluoride ion, a chloride ion, a bromide ion, and an iodide ion, with a chloride ion being preferred. In the present invention, the term "inorganic acid ion" means a carbonate ion, a sulfate ion, a phosphate ion, a hydrogen phosphate ion, a dihydrogen phosphate ion, a nitrate ion, a perchlorate ion, or a borate ion. The above An -Preferred as the cation are halide ions, sulfate ions, phosphate ions, hydroxide ions and isothiocyanate ions, and particularly preferred are halide ions.

[0069] In formulas (A) and (B), T a and T b are preferably each independently a hydrogen atom, a methyl group, or an ethyl group, and more preferably each independently a hydrogen atom or a methyl group.

[0070] In the formula (a), the formula (b), the formula (A) and the formula (B), U a1 , U a2 , U b1 , U b2 and U b3 is preferably each independently a hydrogen atom, a methyl group or an ethyl group. a1 and U a2 is more preferably a hydrogen atom. b1 , U b2 (and U b3 ) is more preferably a methyl group or an ethyl group, and particularly preferably a methyl group.

[0071] In formulas (A) and (B), Q a and Q b are preferably each independently an ester bond (-C(=O)-O- or -OC(=O)-) or an amide bond (-NHC(=O)- or -C(=O)NH-), more preferably each independently -C(=O)-O- or -C(=O)NH-, and particularly preferably -C(=O)-O-.

[0072] In formulas (A) and (B), R a and R bare preferably each independently a linear or branched alkylene group having 1 to 3 carbon atoms which may be substituted with a halogen atom, more preferably each independently an ethylene group or a propylene group, or an ethylene group or a propylene group substituted with one chlorine atom, and particularly preferably an ethylene group or a propylene group.

[0073] In formulae (A) and (B), m preferably represents an integer of 0 to 3, more preferably an integer of 1 or 2, and particularly preferably 1.

[0074] Specific examples of the above formula (A) include vinylphosphonic acid, acid phosphooxyethyl (meth)acrylate, 3-chloro-2-acid phosphooxypropyl (meth)acrylate, acid phosphooxypropyl (meth)acrylate, acid phosphooxymethyl (meth)acrylate, acid phosphooxypolyoxyethylene glycol mono(meth)acrylate, and acid phosphooxypolyoxypropylene glycol mono(meth)acrylate. Of these, vinyl phosphonic acid and acid phosphooxyethyl methacrylate (=2-(methacryloyloxy)ethyl phosphate) are preferably used.

[0075] The structural formulas of vinylphosphonic acid, acid phosphooxyethyl methacrylate (=2-(methacryloyloxy)ethyl phosphate), and acid phosphooxypolyoxyethylene glycol monomethacrylate are represented by the following formulas (A-1) to (A-3), respectively. [ka]

[0076] In the synthesis, in addition to these compounds, a (meth)acrylate compound having two functional groups, such as those represented by general formula (C) or (D) described later, may be used in combination.

[0077] Specific examples of the above formula (B) include dimethylaminoethyl (meth)acrylate, diethylaminoethyl (meth)acrylate, dimethylaminopropyl (meth)acrylate, 2-(t-butylamino)ethyl (meth)acrylate, methacryloylcholine chloride, and the like. Among these, dimethylaminoethyl (meth)acrylate, methacryloylcholine chloride, and 2-(t-butylamino)ethyl (meth)acrylate are preferably used.

[0078] The structural formulas of dimethylaminoethyl acrylate (= 2-(dimethylamino)ethyl acrylate), dimethylaminoethyl methacrylate (= 2-(dimethylamino)ethyl methacrylate), methacryloylcholine chloride and 2-(t-butylamino)ethyl methacrylate (= 2-(t-butylamino)ethyl methacrylate) are represented by the following formulas (B-1) to (B-4), respectively.

[0079] [ka]

[0080] The proportion of the repeating unit containing an organic group represented by formula (a) (or the repeating unit represented by formula (a1)) in the copolymer is 20 mol % to 80 mol %, preferably 30 mol % to 70 mol %, and more preferably 40 mol % to 60 mol %. The copolymer may contain two or more kinds of repeating units containing an organic group represented by formula (a) (or the repeating units represented by formula (a1)).

[0081] The proportion of the repeating unit containing an organic group represented by formula (b) (or the repeating unit represented by formula (b1)) in the above copolymer may be the entire remainder after subtracting the proportion of the above formula (a) (or formula (a1)) from the total copolymer, or may be the remainder after subtracting the total proportion of the above formula (a) (or formula (a1)) and the third component described below. The copolymer may contain two or more kinds of repeating units containing an organic group represented by formula (b) (or repeating units represented by formula (b1)).

[0082] Furthermore, the copolymer may be copolymerized with any third component. For example, a (meth)acrylate compound having two or more functional groups may be copolymerized as the third component, and a part of the polymer may be partially three-dimensionally crosslinked. Examples of such a third component include bifunctional monomers represented by the following formula (C) or (D). [ka]

[0083] (In the formula, T c , T d and U d each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R c and R d each independently represents a linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted with a halogen atom. That is, the copolymer preferably contains a crosslinked structure derived from such a bifunctional monomer.

[0084] In formulas (C) and (D), T c and T d are preferably each independently a hydrogen atom, a methyl group, or an ethyl group, and more preferably each independently a hydrogen atom or a methyl group.

[0085] In formulas (C) and (D), U d is preferably a hydrogen atom, a methyl group or an ethyl group, and more preferably a hydrogen atom.

[0086] In formulas (C) and (D), R c and R dare preferably each independently a linear or branched alkylene group having 1 to 3 carbon atoms which may be substituted with a halogen atom, more preferably each independently an ethylene group or a propylene group, or an ethylene group or a propylene group substituted with one chlorine atom, and particularly preferably an ethylene group or a propylene group.

[0087] The bifunctional monomer represented by formula (C) preferably includes ethylene glycol di(meth)acrylate, triethylene glycol di(meth)acrylate, propylene glycol di(meth)acrylate, etc. The bifunctional monomer represented by formula (D) preferably includes bis(methacryloyloxymethyl) phosphate, bis[2-(methacryloyloxy)ethyl] phosphate, bis[2-(methacryloyloxy)propyl] phosphate, etc.

[0088] The optional third component may be a trifunctional monomer, such as phosphinylidineditris(oxy-2,1-ethanediyl) triacrylate.

[0089] Among these, ethylene glycol di(meth)acrylate represented by the following formula (C-1) and bis[2-(methacryloyloxy)ethyl]phosphate represented by the following formula (D-1) are particularly preferred.

[0090] [ka]

[0091] The copolymer may contain one or more of these third components. Among the above, the bifunctional monomer represented by formula (D) is preferred, and the bifunctional monomer represented by formula (D-1) is particularly preferred. The ratio of the third component (for example, a crosslinked structure derived from a bifunctional monomer represented by the above formula (C) or (D)) in the above copolymer is 0 mol % to 50 mol %.

[0092] The method for producing the copolymer is not particularly limited, and examples thereof include the copolymer production method described in WO2014 / 196652 pamphlet.

[0093] <<<Coating film according to the second embodiment>>> The coating film may be a coating film obtained from a composition for forming a coating film described in WO2021 / 167037. The contents of WO2021 / 167037 are incorporated herein by reference to the same extent as if fully set forth herein. Hereinafter, the coating film-forming composition described in WO2021 / 167037 will be described. In this specification, it is referred to as a coating film-forming composition of the second aspect.

[0094] The composition for forming a coating film according to the second embodiment has the following formula (1): [ka] (In the formula, R 1 represents a hydrogen atom or a methyl group, and R 2 represents an alkylene group having 1 to 6 carbon atoms, and n represents an integer of 1 to 30. A compound represented by the following formula (2): [ka] (In the formula, R 3 represents a hydrogen atom or a methyl group, and R 4 represents a monovalent organic group having cationic properties. and a compound represented by the following formula (3): [ka] (In the formula, R 5 each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms; R 6represents a linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted with a halogen atom, and m represents an integer of 1 to 30. The present invention includes a monomer mixture containing a compound represented by formula (3), wherein the ratio of the compound represented by formula (3) to the total mass of the monomer mixture is 2 to 40 mass %, and a solvent.

[0095] In the compound of formula (1) which is a monomer component of the copolymer according to the second embodiment, R 1 represents a hydrogen atom or a methyl group, and R 2 represents an alkylene group having 1 to 6 carbon atoms, preferably a linear or branched alkylene group having 1 to 6 carbon atoms, more preferably a linear or branched alkylene group having 2 to 5 carbon atoms, particularly preferably an ethylene group or a propylene group; n represents an integer of 1 to 30, preferably an integer of 1 to 20, more preferably an integer of 2 to 10, and particularly preferably an integer of 3 to 6.

[0096] Specific examples of the compound of the above formula (1) include acid phosphooxyethyl (meth)acrylate, 3-chloro-2-acid phosphooxypropyl (meth)acrylate, acid phosphooxypropyl (meth)acrylate, acid phosphooxymethyl (meth)acrylate, acid phosphooxypolyoxyethylene glycol mono(meth)acrylate, and acid phosphooxypolyoxypropylene glycol mono(meth)acrylate, and among these, acid phosphooxyethyl methacrylate (=2-(methacryloyloxy)ethyl phosphate), acid phosphooxypolyoxyethylene glycol monomethacrylate, and acid phosphooxypolyoxypropylene glycol monomethacrylate are preferably used.

[0097] The structural formulas of acid phosphooxyethyl methacrylate (=2-(methacryloyloxy)ethyl phosphate), acid phosphooxy polyoxyethylene glycol monomethacrylate, and acid phosphooxy polyoxypropylene glycol monomethacrylate are represented by the following formulas (1-1) to (1-3), respectively.

[0098] [ka]

[0099] In the compound of formula (2) which is a monomer component of the copolymer according to the second embodiment, R 3 represents a hydrogen atom or a methyl group, and R 4 represents a monovalent organic group having cationic properties, and is typically a monovalent group having a primary amine, secondary amine, tertiary amine or quaternary ammonium structure. Examples of the monovalent group having a primary amine, secondary amine, tertiary amine or quaternary ammonium structure include those represented by the formula: -R 4a -NH2, -R 4a -NHR, -R 4a -NRR', -R 4a -N + RR'R'' [where R 4a is an alkylene group having 1 to 6 carbon atoms which may be interrupted by an ester bond, an amide bond, an ether bond or a phosphodiester bond, and R, R' and R'' are each independently a straight-chain or branched alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 10 carbon atoms, or an aralkyl group having 7 to 16 carbon atoms. In the second embodiment, the monovalent group having a primary amine, secondary amine or tertiary amine structure may be quaternized or salified, and similarly, the monovalent group having a quaternary ammonium structure may be salified.

[0100] Specific examples of the compound of the above formula (2) include dimethylaminoethyl (meth)acrylate, diethylaminoethyl (meth)acrylate, dimethylaminopropyl (meth)acrylate, 2-(t-butylamino)ethyl (meth)acrylate, (meth)acryloylcholine chloride, and 2-(meth)acryloyloxyethyl phosphorylcholine (MPC).

[0101] The structural formulas of 2-(dimethylamino)ethyl methacrylate, methacryloylcholine chloride, and 2-methacryloyloxyethyl phosphorylcholine (MPC) are represented by the following formulas (2-1) to (2-3), respectively.

[0102] [ka]

[0103] The compound of formula (3), which is a monomer component of the copolymer according to the second embodiment, is a bifunctional monomer having crosslinking properties. 5 each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms, preferably a hydrogen atom or a methyl group; R 6 represents a linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted with a halogen atom, preferably a linear or branched alkylene group having 1 to 6 carbon atoms which may be substituted with a halogen atom, more preferably a linear or branched alkylene group having 1 to 6 carbon atoms which may be substituted with a chlorine atom, particularly preferably an ethylene group, propylene group or trimethylene group, and m represents an integer of 1 to 30, preferably an integer of 1 to 20, more preferably an integer of 1 to 10, even more preferably an integer of 1 to 6, and particularly preferably an integer of 1 to 4.

[0104] Specific examples of the compound of the above formula (3) include poly(ethylene glycol) di(meth)acrylate, poly(trimethylene glycol) di(meth)acrylate, poly(propylene glycol) di(meth)acrylate, and the like.

[0105] The structural formulas of poly(ethylene glycol) dimethacrylate, poly(trimethylene glycol) dimethacrylate, and poly(propylene glycol) dimethacrylate are represented by the following formulas (3-1) to (3-3), respectively.

[0106] [ka]

[0107] The copolymer according to the second aspect is obtained by polymerizing a monomer mixture containing the compounds of the above formulae (1), (2) and (3) as monomer components. Such a monomer mixture may contain an ethylenically unsaturated monomer, or a polysaccharide or a derivative thereof as an optional fourth component, as long as the adhesion inhibitory ability of the biological material is not impaired. 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 hydrophilic functional derivatives thereof. Examples of polysaccharides or derivatives thereof include cellulose-based polymers such as hydroxyalkyl cellulose (e.g., hydroxyethyl cellulose or hydroxypropyl cellulose), starch, dextran and curdlan. The copolymer according to the second aspect is preferably obtained by polymerizing a monomer mixture containing only the compounds of the above formulae (1), (2) and (3) as monomer components.

[0108] The copolymer according to the second aspect is obtained by polymerizing a monomer mixture containing the compounds of the above formulas (1), (2) and (3) as monomer components, and characterized in that the ratio of the compound of the formula (3) to the total mass of the monomer components contained in the monomer mixture is 2 to 40 mass%. By containing the compound of the formula (3) as a bifunctional monomer in the range of 2 to 40 mass%, preferably in the range of 3 to 35 mass%, more preferably in the range of 5 to 30 mass%, relative to the total mass of the monomer components contained in the monomer mixture, the coating film forming composition of the second aspect containing the copolymer obtained from the monomer mixture can obtain excellent adhesion suppression ability of biological materials. The monomer mixture may contain two or more compounds of formula (3).

[0109] In the monomer mixture, the ratio of the compound of formula (1) to the total mass of the monomer components is 10 to 95 mass%, preferably 10 to 80 mass%, and more preferably 45 to 75 mass%. The monomer mixture may contain two or more types of compounds of formula (1).

[0110] In the monomer mixture, the ratio of the compound of formula (2) to the total mass of the monomer components is 3 to 90 mass%, preferably 10 to 70 mass%, and more preferably 15 to 35 mass%. The monomer mixture may contain two or more types of compounds of formula (2).

[0111] The copolymer according to the second aspect can be synthesized by a method such as radical polymerization, anionic polymerization, or cationic polymerization, which is a general method for synthesizing (meth)acrylic polymers. The form of the copolymer can be various methods such as solution polymerization, suspension polymerization, emulsion polymerization, and bulk polymerization. In one embodiment, the copolymer can be prepared by a production method including a step of reacting (polymerizing) a monomer mixture containing the compounds of the above formulas (1), (2), and (3) in a solvent at a total concentration of the monomer components of 0.01 to 20% by mass.

[0112] The solvent in the polymerization reaction may be water, a phosphate buffer solution, an alcohol such as ethanol, or a mixed solvent of these, but preferably contains water or ethanol. It is more preferable that the solvent contains 10% by mass or more and 100% by mass or less of water or ethanol. It is more preferable that the solvent contains 50% by mass or more and 100% by mass or less of water or ethanol. It is more preferable that the solvent contains 80% by mass or more and 100% by mass or less of water or ethanol. It is more preferable that the solvent contains 90% by mass or more and 100% by mass or less of water or ethanol. The total of water and ethanol is preferably 100% by mass.

[0113] In order to efficiently advance the polymerization reaction, it is desirable to use a polymerization initiator. Examples of the polymerization initiator include a "thermal radical polymerization initiator" or a "photoradical polymerization initiator." Examples of the "thermal radical polymerization initiator" include 2,2'-azobis(isobutyronitrile), 2,2'-azobis(2-methylbutyronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile) (FUJIFILM Wako Pure Chemical Industries, Ltd. product name: V-65, 10-hour half-life temperature: 51°C), 4,4'-azobis(4-cyanovaleric acid), 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile), 1,1'-azobis(cyclohexane-1-carbonitrile), 1-[(1-cyano 2,2'-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (FUJIFILM Wako Pure Chemical Industries, Ltd.; VA-044, 10-hour half-life temperature; 44°C), 2,2'-Azobis[2-(2-imidazolin-2-yl)propane] (FUJIFILM Wako Pure Chemical Industries, Ltd.; VA-061, 10-hour half-life temperature; 61°C), 2,2'-Azobis(2-methylpropionamidine)dihydrochloride, 2,2'-Azo(2-methyl-N-(2-hydroxyethyl) Propionamide (FUJIFILM Wako Pure Chemical Industries, Ltd. product name: VA-086, 10-hour half-life temperature: 86°C), benzoyl peroxide (BPO), 2,2'-azobis(N-(2-carboxyethyl)-2-methylpropionamidine) n-hydrate (FUJIFILM Wako Pure Chemical Industries, Ltd. product name: VA-057, 10-hour half-life temperature: 57°C), 4,4'-azobis(4-cyanopentanoic acid) (FUJIFILM Wako Pure Chemical Industries, Ltd. product name: VA-501), 2,2'-azobis[2-(2-imidazoline 1,1'-azobis(1-cyclohexanecarboxylate)(FUJIFILM Wako Pure Chemical Industries, Ltd. product name: VE-073), and the like.

[0114] Examples of the "photoradical polymerization initiator" include acetophenone, chloroacetophenone, hydroxyacetophenone, 2-aminoacetophenone, dialkylaminoacetophenone, 2,2-diethoxyacetophenone, 2,2-dimethoxy-2'-phenylacetophenone (BASF product name: Irgacure 651), 2-hydroxy-2-methyl-1-phenylpropanone (BASF product name: Irgacure 1173), 1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxymethylpropanone (BASF product name: Irgacure 1173), and the like. Acetophenones such as 2-hydroxy-1-{4-[4-(2-hydroxy-2-methylpropionyl)benzyl]phenyl}-2-methyl-1-propan-1-one (BASF product name: Irgacure 127), 2-methyl-1-[4-(methylthio)phenyl]-2-morpholinopropan-1-one (BASF product name: Irgacure 907), and 2-benzyl-2-(dimethylamino)-4-morpholinobutyrophenone (BASF product name: Irgacure 369) Benzoins such as benzoin, benzoin methyl ether, benzoin ethyl ether, benzoin isopropyl ether, benzoin isobutyl ether, and 1-hydroxycyclohexyl phenyl ketone (BASF product name: Irgacure 184); benzophenone, benzoylbenzoic acid, methyl benzoylbenzoate, methyl-o-benzoylbenzoate, 4-phenylbenzophenone, hydroxybenzophenone, hydroxypropylbenzophenone, acrylic benzophenone, 4,4'-bis(dimethylamino)benzoate, benzophenones such as 4,4'-dichlorobenzophenone, 4,4'-dichlorobenzophenone, etc.; thioxanthones such as thioxanthone, 2-chlorothioxanthone, 2-methylthioxanthone, diethylthioxanthone, dimethylthioxanthone, etc.; acylphosphine oxides such as 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (BASF product name: Irgacure TPO), bis(2,4,6-trimethylbenzoyl)-phenylphosphine oxide (BASF product name: Irgacure 819), etc.;Examples of the oxyphenylacetic acid, 2-[2-oxo-2-phenylacetoxyethoxy]ethyl ester and oxyphenylacetic acid, 2-(2-hydroxyethoxy)ethyl ester mixture (BASF product name: Irgacure 754), methyl benzoylformate (BASF product name: Irgacure MBF), α-acyloxime ester, benzyl-(o-ethoxycarbonyl)-α-monoxime, glyoxyester, 2-ethylanthraquinone, camphorquinone, tetramethylthiuram sulfide, azobisisobutyronitrile, benzoyl peroxide, dialkyl peroxide, tert-butyl peroxypivalate, etc.;

[0115] In consideration of solubility in water, ion balance and interaction with the monomer, it is preferable to select from 2,2'-azo(2-methyl-N-(2-hydroxyethyl)propionamide, 2,2'-azobis(N-(2-carboxyethyl)-2-methylpropionamidine) n-hydrate, 4,4'-azobis(4-cyanopentanoic acid), 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, 2,2'-azobis[2-(2-imidazolin-2-yl)propane]disulfate dihydrate, 2,2'-azobis[2-(2-imidazolin-2-yl)propane], 2,2'-azobis(2-amidinopropane)dihydrochloride and peroxodisulfuric acid. Considering the solubility in organic solvents, ion balance, and interaction with monomers, it is desirable to use 2,2'-azobis(2,4-dimethylvaleronitrile) or 2,2'-azobis(isobutyronitrile).

[0116] The amount of the polymerization initiator added is 0.05% by mass to 10% by mass based on the total mass of the monomer components used in the polymerization.

[0117] The reaction conditions are that the reaction vessel is heated to 50 to 200°C in an oil bath or the like, and stirred for 1 to 48 hours, more preferably at 80 to 150°C for 5 to 30 hours, to allow the polymerization reaction to proceed and obtain the copolymer of the second embodiment. The reaction atmosphere is preferably a nitrogen atmosphere.

[0118] As for the reaction procedure, all the reactants may be placed in a reaction solvent at room temperature and then heated to the above temperature to polymerize, or the whole or part of the mixture of reactants may be added dropwise in small portions to a pre-heated solvent.

[0119] The weight average molecular weight of the copolymer according to the second embodiment may be several thousand to several million, preferably 5,000 to 5,000,000, more preferably 10,000 to 2,000,000. The copolymer may be a random copolymer, a block copolymer, or a graft copolymer, and is preferably a random copolymer. Furthermore, the copolymer produced in this manner is considered to be a three-dimensional polymer since it contains a bifunctional monomer, and is in a dissolved or dispersed state in a solution containing water or alcohol.

[0120] The coating film-forming composition according to the second embodiment may be prepared by isolating and purifying the copolymer thus obtained, and then diluting the copolymer with a desired solvent to a predetermined concentration. Furthermore, the composition for forming a coating film according to the second embodiment may be prepared from a reaction solution (that is, a copolymer-containing varnish) obtained after the polymerization reaction.

[0121] Examples of the solvent contained in the coating film forming composition of the second embodiment include water, phosphate buffered saline (PBS), and alcohol. Examples of the alcohol 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, 1-hexanol, 2-hexanol, 3-hexanol, 2,3-dimethyl-1-propanol, 2-methyl-1-butanol, 2-methyl-2-butanol (=t-amyl alcohol), 2-methyl-1-butanol, 2-methyl-3-pent ... Examples of suitable solvents include ethyl-2-butanol, 3,3-dimethyl-1-butanol, 3,3-dimethyl-2-butanol, 2-ethyl-1-butanol, 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 solvents may be used alone or in combination, but from the viewpoint of dissolving the copolymer, it is preferable to select from water, PBS, ethanol and propanol.

[0122] The concentration of solids in the composition for forming a coating film according to the second embodiment is preferably 0.01 to 50% by mass in order to form a uniform coating film. The concentration of the copolymer in the composition for forming a coating film is preferably 0.01 to 5% by mass, more preferably 0.01 to 4% by mass, and particularly preferably 0.01 to 3% by mass. If the concentration of the copolymer is 0.01% by mass or less, the concentration of the copolymer in the composition for forming a coating film obtained is too low to form a coating film with a sufficient thickness, and if it is 5% by mass or more, the storage stability of the composition for forming a coating film is deteriorated, and precipitation or gelation of the dissolved matter may occur.

[0123] Furthermore, in addition to the copolymer and the solvent, the composition for forming a coating film of the second embodiment may contain other substances as necessary within a range that does not impair the performance of the resulting coating film. Examples of other substances include preservatives, surfactants, primers that improve adhesion to the substrate, antifungal agents, and sugars.

[0124] In order to adjust the ion balance of the copolymer in the composition for forming a coating film according to the second embodiment, the process for obtaining the coating film of the second embodiment may further include a step of adjusting the pH of the composition for forming a coating film in advance. The pH adjustment may be performed, for example, by adding a pH adjuster to a composition containing the copolymer and a solvent, and adjusting the pH of the composition to 2 to 13.5, preferably 2 to 8.5, more preferably 3 to 8, or preferably 8.5 to 13.5, more preferably 10 to 13.5. The type and amount of the pH adjuster that can be used are appropriately selected depending on the concentration of the copolymer, the abundance ratio of its anion and cation, and the like. Examples of pH adjusters include organic amines such as ammonia, diethanolamine, pyridine, N-methyl-D-glucamine, and tris(hydroxymethyl)aminomethane; alkali metal hydroxides such as potassium hydroxide and sodium hydroxide; alkali metal halides such as potassium chloride and sodium chloride; inorganic acids such as sulfuric acid, phosphoric acid, hydrochloric acid, and carbonic acid, or alkali metal salts thereof; quaternary ammonium cations such as choline, or mixtures thereof (e.g., buffer solutions such as phosphate buffered saline).Among these, ammonia, diethanolamine, sodium hydroxide, choline, N-methyl-D-glucamine, and tris(hydroxymethyl)aminomethane are preferred, and ammonia, diethanolamine, sodium hydroxide, and choline are particularly preferred.

[0125] <<<Coating film according to the third embodiment>>> The coating film may be a coating film obtained from a composition for forming a coating film described in WO2022 / 259998. The contents of WO2022 / 259998 are incorporated herein by reference to the same extent as if expressly set forth in their entirety. Hereinafter, the coating film-forming composition described in WO2022 / 259998 will be described. In this specification, it is referred to as a coating film-forming composition of the third aspect.

[0126] The coating film-forming composition of the third embodiment is used to inhibit adhesion of biological materials. The coating film-forming composition of the third embodiment contains at least a copolymer, and further contains other components such as a solvent, if necessary. The coating film-forming composition of the third embodiment can form a coating film that is difficult to dissolve in phosphate buffered saline. However, the use of the coating film-forming composition of the third embodiment is not particularly limited as long as it is used to inhibit adhesion of biological materials, and is not limited to the formation of a coating film that comes into contact with phosphate buffered saline.

[0127] The copolymer according to the third embodiment is water-insoluble. Here, "water-soluble" means that it can dissolve at least 1.0 g in 100 g of water at 25° C. "Water-insoluble" means that it does not fall under the category of "water soluble", that is, the solubility in 100 g of water at 25° C. is less than 1.0 g. The copolymer according to the third embodiment has a repeating unit (A) represented by the following formula (A) and a repeating unit (B) represented by the following formula (B). The molar ratio (A:B) of the repeating unit (A) to the repeating unit (B) in the copolymer according to the third embodiment is from 89:11 to 50:50. [ka] (In the formula, R 1 ~R 3 each independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms; X 1 and X2 each independently represents a single bond, an ester bond, an ether bond, an amide bond, or an alkylene group having 1 to 5 carbon atoms which may be interrupted by an oxygen atom.

[0128] The copolymer according to the third embodiment may have two or more kinds of repeating units (A). The copolymer according to the third embodiment may have two or more kinds of repeating units (B). The copolymer according to the third embodiment preferably has one type of repeating unit (A) and one type of repeating unit (B).

[0129] Examples of the 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. R 1 ~R 3 are each preferably independently a hydrogen atom, a methyl group, or an ethyl group.

[0130] The above "ester bond" means -C(=O)-O- or -OC(=O)-, "ether bond" means -O-, and "amide bond" means -NHC(=O)- or -C(=O)NH-.

[0131] The alkylene group having 1 to 5 carbon atoms may be interrupted by an oxygen atom. Examples of the 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. 1and X 2 is preferably a methylene group, an ethylene group, or a propylene group. The phrase "optionally interrupted by an oxygen atom" means that one or more carbon-carbon bonds in the alkylene group having 1 to 5 carbon atoms are bonded via an ether bond.

[0132] The copolymer according to the third aspect comprises R 1 and R 2 is a hydrogen atom, and R 3 is a methyl group, and X 1 and X 2 A copolymer in which is a single bond is preferred.

[0133] The molar ratio (A:B) of the repeating unit (A) to the repeating unit (B) is 89:11 to 50:50. In the copolymer according to the third aspect, when the total number of moles of the repeating unit (A) and the repeating unit (B) is 100, the molar ratio (A:B) of the repeating unit (A) to the repeating unit (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.

[0134] The total mole percentage of the repeating unit (A) and the repeating unit (B) in all repeating units in the copolymer related to the third aspect is not particularly limited, but is preferably 90 mole% or more, more preferably 95 mole% or more, even more preferably 99.5 mole% or more, and particularly preferably 100%.

[0135] In the third embodiment, the molar ratio of the repeating unit (A) to the repeating unit (B) in the copolymer is set to a specific range in order to obtain a coating film that is difficult to dissolve in phosphate buffered saline. Therefore, in the third embodiment, a coating film that is difficult to dissolve in phosphate buffered saline is obtained without crosslinking the copolymer. Therefore, the copolymer does not need to have a photosensitive group for crosslinking the copolymer. That is, it is preferable that the copolymer does not have a photosensitive group. An example of the photosensitive group is an azide group. In the third embodiment, the copolymer does not need to have a photosensitive group for crosslinking the copolymer. Therefore, when forming a coating film, it is not necessary to perform light irradiation for crosslinking the copolymer. Therefore, the process for forming the coating film can be simplified.

[0136] The viscosity average degree of polymerization of the copolymer according to the third aspect (hereinafter, simply referred to as "degree of polymerization") is not particularly limited, but from the viewpoint of favorably obtaining the effects of the third aspect, 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 in a completely saponified state of the copolymer. The "viscosity average degree of polymerization" of polyvinyl alcohol obtained by complete saponification is a value calculated from the intrinsic viscosity [η] (g / dL) measured at 30°C using an Ostwald viscometer with ion-exchanged water as a solvent, using the following formula. log(P) = 1.613 × log([η] × 10 4 / 8.29) Here, P represents the viscosity average degree of polymerization. The viscosity average degree of polymerization can be determined in accordance with JIS K 6726.

[0137] The method for producing the copolymer according to the third embodiment is not particularly limited, but may be, for example, 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. [ka] (In the formula, R 1 , R 3 , and X 1 is the same as above.)

[0138] In addition, an example of a method for producing the copolymer related to the third aspect is a method of copolymerizing a compound represented by the following formula (C) and a compound represented by the following formula (D) to obtain the copolymer. [ka] (In the formula, R 1 ~R 3 , X 1 , and X 2 is the same as above.)

[0139] The copolymer according to the third embodiment may be a random copolymer or a block copolymer. As the copolymer according to the third embodiment, a commercially available product may be used, specifically, polyvinyl acetate (manufactured by Nippon Acetic Acid Vinyl Poval, trade name JMR-10L (registered trademark)).

[0140] The content of the copolymer in the film-forming component in the coating film-forming composition of the third embodiment 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. The film-forming component refers to all the components of the composition excluding the solvent component.

[0141] The content of the copolymer in the composition for forming a coating film of the third embodiment is not particularly limited, but from the viewpoint of easily forming a coating film of a desired thickness, it is preferably 0.1 to 10 mass%, more preferably 0.3 to 8 mass%, and particularly preferably 0.5 to 5 mass%. The content of the copolymer in the composition for forming a coating film may be 0.02 to 2 mass%, or 0.05 to 1 mass%.

[0142] Examples of the solvent according to the third embodiment include water, phosphate buffered saline (PBS), alcohol, and water-soluble organic solvents (excluding alcohol).

[0143] The alcohol may be one having 2 to 6 carbon atoms. Examples of alcohols include 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, 1-heptanol, Examples of the cyclohexanol include cyclohexanol, 2-hexanol, 3-hexanol, 2,3-dimethyl-2-butanol, 3,3-dimethyl-1-butanol, 3,3-dimethyl-2-butanol, 2-ethyl-1-butanol, 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 can be used alone or in combination of two or more.

[0144] The water-soluble organic solvent refers to an organic solvent that can be mixed with water and alcohol in any ratio and does not separate after mixing. Examples of the water-soluble organic solvent include ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, methyl cellosolve acetate, ethyl cellosolve acetate, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, propylene glycol, propylene glycol monomethyl ether, propylene glycol monoethyl ether, propylene glycol monomethyl ether acetate, and propylene glycol propyl ether acetate. These can be used alone or in combination of two or more.

[0145] In the composition for forming a coating film of the third embodiment, water, phosphate buffered saline (PBS), alcohol or a water-soluble organic solvent may be used alone as the solvent. In the composition for forming a coating film of the third embodiment, two or more of water, phosphate buffered saline (PBS), alcohol, and a water-soluble organic solvent may be used in combination as a solvent. From the viewpoint of the solubility of the copolymer, the solvent is preferably selected from water, alcohol, a water-soluble organic solvent, and a combination of two or more thereof, and more preferably selected from water, ethanol, a water-soluble organic solvent, and a combination of two or more thereof.

[0146] The following combinations of solvents are preferred: ·Water and alcohol Water, alcohol and water-soluble organic solvents Alcohols and water-soluble organic solvents

[0147] As the combination of solvents, the following combinations are more preferable. Water and ethanol Water, ethanol and propylene glycol monomethyl ether Ethanol and propylene glycol monomethyl ether

[0148] In the composition for forming a coating film of the third embodiment, the mass ratio of water:alcohol is, for example, 1:99 to 70:30, or 1:99 to 50:50. In the composition for forming a coating film of the third embodiment, the mass ratio of water:alcohol:water-soluble organic solvent (A:B:C) is, for example, 5-30:65-92:1-30 (wherein A+B+C is 100). In the composition for forming a coating film of the third embodiment, the mixing ratio (mass ratio) of alcohol:water-soluble organic solvent is, for example, 30:70 to 97:3.

[0149] The content of the solvent in the composition for forming a coating film of the third embodiment is not particularly limited, but from the viewpoint of easily forming a coating film of a desired thickness, it is preferably 90% by mass or more, more preferably 92% by mass or more, and particularly preferably 95% by mass or more.

[0150] The coating film-forming composition of the third embodiment can also contain other components as necessary. Examples of other ingredients include pH adjusters, preservatives, surfactants, anti-mold agents, and sugars.

[0151] An example of a cell structure before cells are arranged therein will be described with reference to the drawings. A cell structure 100 before cells are arranged thereon, shown in FIGS. 1A and 1B, has a main body 1 and a cell culture base membrane 3 which is a spot. The main body 1 has an outer peripheral wall 1A, an inner bottom surface 1B, and an opening 1C. The opening 1C is an opening formed by the outer peripheral wall 1A, and the inner bottom surface 1B exists below the opening 1C. A plurality of cell culture base membranes 3 are arranged at intervals on the inner bottom surface 1B.

[0152] The cell structure 100 shown in FIGS. 2A and 2B before cells are arranged thereon has a main body 1, a cell adhesive substance 2, and a cell culture base membrane 3 which is a spot. The main body 1 has an outer peripheral wall 1A, an inner bottom surface 1B, and an opening 1C. The opening 1C is an opening formed by the outer peripheral wall 1A, and the inner bottom surface 1B exists below the opening 1C. A plurality of cell culture base membranes 3 are arranged at intervals on the inner bottom surface 1B. A cell adhesive substance 2 is disposed on a cell culture base membrane 3 .

[0153] The cell structure 100 shown in FIGS. 3A and 3B before cells are arranged thereon comprises a main body 1, a cell adhesive substance 2, a cell culture base film 3 which is a spot, and a coating film 4. The main body 1 has an outer peripheral wall 1A, an inner bottom surface 1B, and an opening 1C. The opening 1C is an opening formed by the outer peripheral wall 1A, and the inner bottom surface 1B exists below the opening 1C. The surface of the inner bottom surface 1B has a coating film 4 having a cell adhesion suppressing ability. A plurality of cell culture base films 3 are arranged on the coating film 4 at intervals. A cell adhesive substance 2 is disposed on a cell culture base membrane 3 .

[0154] In one embodiment of the cell structure, a plurality of cell adhesive substances are arranged at intervals on the substrate, so that cells or cell structures can be retained at a predetermined interval. In this embodiment, cells can be attached on the cell adhesive substance and cultured for a long period of time. Depending on the type of cell, the attached cells can grow and produce a cell structure (e.g., a hemispherical spheroid).

[0155] In another embodiment of the cell structure, a plurality of cell culture base membranes are arranged at intervals on a substrate. In this embodiment, a cell structure (e.g., a spheroid) can be produced on the cell culture base membrane. Then, by aligning the shape and size of the cell culture base membrane of each spot, a cell structure of uniform size can be produced. Furthermore, since the cell culture base membrane has a cell adhesive substance, the produced cell structure is less likely to detach from the cell culture base membrane compared to a case where the cell adhesive substance is not present on the cell culture base membrane. Therefore, it is possible to prevent the size of the cell structures from becoming non-uniform due to the fusion of the detached cell structures, and the number of cell structures from fusion.

[0156] The cell structure is preferably a cell culture vessel.

[0157] (Method of manufacturing cell structures) The method for producing a cell structure of the present invention comprises the following steps (I) to (III). Step (I): A step of applying droplets of a cell culture base film forming agent to a substrate to form a cell culture base film in the areas to be spots. Step (II): Seeding cells for culture on the cell culture base film Step (III): Freezing the cells for culture

[0158] The method for producing a cell structure may further include at least one of the following steps (IV) to (VI). Step (IV): A step of applying a coating agent containing a polymer capable of inhibiting cell adhesion onto a substrate to form a coating film. Step (V): A step of applying droplets containing a cell adhesive substance to the cell culture base film to arrange the cell adhesive substance on the cell culture base film. Step (VI): Contacting the cells for culture with a cryopreservation agent The step (IV) is carried out before the step (I). Step (V) is carried out after step (I) and before step (II). Step (VI) is carried out after step (II) and before step (III).

[0159] The method for producing a cell structure of the present invention is, for example, a method for producing a cell structure of the present invention.

[0160] <Process (I)> Step (I) is a step of applying droplets of a cell culture base film forming agent to a substrate to form a cell culture base film in areas that will become spots.

[0161] Examples of the substrate include the substrates mentioned in the description of the cell structure of the present invention.

[0162] The cell culture base film forming agent (hereinafter sometimes referred to as "base film forming agent") contains a polymer and a solvent, and may further contain other components. Examples of the polymer include the polymers mentioned in the description of the cell structure of the present invention. The solvent is not particularly limited, but is preferably an aqueous solution containing water. The aqueous solution may be water, a salt-containing aqueous solution such as physiological saline or a phosphate buffer solution, or a mixed solvent of water or a salt-containing aqueous solution with an alcohol. The alcohol may be an alcohol 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, 1-hexanol, etc. Examples of suitable solvents include cyclohexanol, 2-hexanol, 3-hexanol, 2,3-dimethyl-2-butanol, 3,3-dimethyl-1-butanol, 3,3-dimethyl-2-butanol, 2-ethyl-1-butanol, 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 solvents may be used alone or in combination. The water content in the aqueous solution is, for example, 50% by mass to 100% by mass, 80% by mass to 100% by mass, or 90% by mass to 100% by mass. In addition to the above-mentioned polymer, cell adhesive substance, and solvent, the undercoat film forming agent may also contain other substances as necessary within the range that does not impair the performance of the obtained undercoat film. Examples of other substances include pH adjusters, crosslinking agents, preservatives, surfactants, primers that improve adhesion to containers or substrates, antifungal agents, and sugars.

[0163] The method for applying droplets of the undercoat film-forming agent to the substrate is not particularly limited, and may be an ink-jet method, a screen printing method, a slit coating method, a roll-to-roll method, or the like. Preferably, the method is performed by a printing technique such as an ink-jet method or screen printing. As another application method, for example, a method of immersing a substrate with a portion that is not to be a spot protected in the above-mentioned primer film forming agent, a method of adding a primer film forming agent to a substrate (container) with a portion that is not to be a spot protected and leaving it to stand for a predetermined time, etc. are used. In one embodiment, when the cell structure is a cell culture container, the primer film forming agent is added to a container with a portion that is not to be a spot protected and left to stand for a predetermined time. The addition can be performed, for example, by adding the primer film forming agent in an amount of 0.5 to 1 times the total volume of the container using a syringe or the like. The time and temperature for leaving it to stand are appropriately selected depending on the material of the container or substrate and the type of primer film forming agent for cell culture, and it is performed, for example, for 1 minute to 24 hours, preferably 5 minutes to 3 hours, at 10 to 80°C.

[0164] Furthermore, after forming a cell culture base film on the surface of a substrate by such a method, the subsequent steps may be carried out directly without a drying step, or after washing with water or the medium of the sample to be subjected to cell culture (e.g., water, a buffer solution, a culture medium, etc.).

[0165] <Process (II)> Step (II) is a step of seeding cells for culture on the cell culture base membrane. An example of the step of seeding cells is adding a medium in which cells are dispersed to a substrate on which a cell culture base film has been formed. For example, the following total number of cells are dispersed in medium and the medium is added to the cell construct: The lower limit of the total number of cells is, for example, equal to or greater than the number of spots (n) present in the cell structure. The medium is adjusted according to the type of cells to be cultured. One type of cell may be used, or two or more types of cells may be used.

[0166] The cell concentration in the medium in which the cells are dispersed is not particularly limited.

[0167] The medium can be appropriately selected depending on the type of cells used, etc. For example, when the purpose is to culture mammalian cells, a medium generally used for culturing mammalian cells can be used as the medium. Examples of media for mammalian cells include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12 medium, McCoy's 5A medium, Eagle's Minimum Essential Medium (EMEM), alpha Modified Eagle's Minimum Essential Medium (αMEM), Minimum Essential Medium, RPMI1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, IPL41 medium, Fischer's medium, StemPro34 (Invitrogen), X-VIVO 10 (Cambrex), X-VIVO 15 (Cambrex), HPGM (Cambrex), StemSpan Examples of such media include H3000 (manufactured by Stem Cell Technology), StemSpanSFEM (manufactured by Stem Cell Technology), StemlineII (manufactured by Sigma-Aldrich), QBSF-60 (manufactured by Quality Biologicals), StemProhESCSFM (manufactured by Invitrogen), mTeSR1 or 2 medium (manufactured by Stem Cell Technology), Sf-900II (manufactured by Invitrogen), Opti-Pro (manufactured by Invitrogen), and HuMedia-KG2 (manufactured by Kuraray Industries, Inc.).

[0168] Those skilled in the art may freely add sodium, potassium, calcium, magnesium, phosphorus, chlorine, various amino acids, various vitamins, antibiotics, serum, fatty acids, sugars, etc. to the above-mentioned medium according to the purpose. When culturing mammalian cells, those skilled in the art may also add one or more combinations of other chemical components or biological components according to the purpose.

[0169] Components that can be added to culture media for mammalian cells include fetal bovine serum, human serum, horse serum, insulin, transferrin, lactoferrin, cholesterol, ethanolamine, sodium selenite, monothioglycerol, 2-mercaptoethanol, bovine serum albumin, sodium pyruvate, polyethylene glycol, various vitamins, various amino acids, agar, agarose, collagen, methylcellulose, various cytokines, various hormones, various growth factors, various extracellular matrices, and various cell adhesion molecules.

[0170] Examples of cytokines that can be added to the medium include interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-14 (IL-14), interleukin-15 (IL-15), interleukin-16 (IL-16), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-19 (IL-19), interleukin-20 (IL-20), interleukin-21 (IL-21), interleukin-22 (IL-22), interleukin-23 (IL-23), interleukin-24 (IL-24), interleukin-25 (IL-25), interleukin-26 (IL-26), interleukin-27 (IL-27), interleukin-28 (IL-28), interleukin-29 (IL-29), interleukin-30 (IL-29), interleukin-31 (IL-29), interleukin-32 (IL-29), interleukin-33 (IL-29), interleukin-34 (IL-29), interleukin-35 (IL-29), interleukin-36 (IL-29), interleukin-37 (IL-29), interleukin-38 (IL-29), interleukin-39 (IL-39), interleukin-40 (IL- These include, but are not limited to, interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interferon-α (IFN-α), interferon-β (IFN-β), interferon-γ (IFN-γ), granulocyte colony-stimulating factor (G-CSF), monocyte colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), flk2 / flt3 ligand (FL), leukemia cell inhibitory factor (LIF), oncostatin M (OM), erythropoietin (EPO), and thrombopoietin (TPO).

[0171] Hormones that may be added to the medium include melatonin, serotonin, thyroxine, triiodothyronine, epinephrine, norepinephrine, dopamine, anti-Mullerian hormone, adiponectin, adrenocorticotropic hormone, angiotensinogen and angiotensin, antidiuretic hormone, atrial natriuretic peptide, calcitonin, cholecystokinin, corticotropin-releasing hormone, erythropoietin, follicle-stimulating hormone, gastrin, ghrelin, glucagon, gonadotropin-releasing hormone, growth hormone-releasing hormone, human chorionic gonadotropin, human placental lactogen, growth hormone, inhibin, insulin, insulin-like growth factor, leptin, luteinizing hormone, melanocyte-stimulating hormone, and the like. Examples of hormone-releasing hormones include, but are not limited to, oxytocin, parathyroid hormone, prolactin, secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone, thyrotropin-releasing hormone, cortisol, aldosterone, testosterone, dehydroepiandrosterone, androstenedione, dihydrotestosterone, estradiol, estrone, estriol, progesterone, calcitriol, calcidiol, prostaglandins, leukotrienes, prostacyclin, thromboxane, prolactin-releasing hormone, lipotropin, brain natriuretic peptide, neuropeptide Y, histamine, endothelin, pancreatic polypeptide, renin, and enkephalins.

[0172] Growth factors that can be added to the medium include transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), macrophage inflammatory protein-1α (MIP-1α), epidermal growth factor (EGF), fibroblast growth factor-1, 2, 3, 4, 5, 6, 7, 8, or 9 (FGF-1, 2, 3, 4, 5, 6, 7, 8, 9), nerve growth factor (NGF), hepatocyte growth factor (HGF), leukemia inhibitory factor (LIF), protease inhibitory factor (PI), and NF-α. These include, but are not limited to, pleiotrophin I, protease nexin II, platelet-derived growth factor (PDGF), cholinergic differentiation factor (CDF), chemokine, Notch ligand (Delta1, etc.), Wnt protein, angiopoietin-like protein 2, 3, 5 or 7 (Angpt2, 3, 5, 7), insulin-like growth factor (IGF), insulin-like growth factor binding protein (IGFBP), and pleiotrophin.

[0173] In addition, it is also possible to add cytokines or growth factors whose amino acid sequences have been artificially altered using recombinant gene technology, such as IL-6 / soluble IL-6 receptor complex or Hyper IL-6 (a fusion protein of IL-6 and soluble IL-6 receptor).

[0174] Examples of various extracellular matrices and various cell adhesion molecules include collagen I to XIX, fibronectin, laminin-1 to 12, nitrogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, various elastins, various proteoglycans, various cadherins, desmocollins, desmogleins, various integrins, E-selectin, P-selectin, L-selectin, the immunoglobulin superfamily, Matrigel, poly-D-lysine, poly-L-lysine, chitin, chitosan, Sepharose, hyaluronic acid, alginate gel, various hydrogels, and cleaved fragments of these.

[0175] Examples of antibiotics that can be added to the medium include sulfa preparations, penicillin, phenethicillin, methicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, ampicillin, penicillin, amoxicillin, cyclacillin, carbenicillin, ticarcillin, piperacillin, azlocillin, mexocillin, mecillinam, anginocillin, cephalosporin and its derivatives, oxolinic acid, amifloxacin, temafloxacin, nalidixic acid, piromidic acid, ciprofloxacin, cinoxacin, norfloxacin, perfloxacin, rozaxacin, ofloxacin, enoxacin, pipemi. These include penicillin, sulbactam, clavulinic acid, β-bromopenicillanic acid, β-chloropenicillanic acid, 6-acetylmethylene-penicillanic acid, cefoxazole, sultampicillin, adinocillin and the formaldehyde fodolate ester of sulbactam, tazobactam, aztreonam, sulfazetine, isosulfazetine, nocardicin, methyl phenylacetamidophosphonate, chlortetracycline, oxytetracycline, tetracycline, demeclocycline, doxycycline, methacycline, and minocycline.

[0176] As mentioned above, serum and / or serum substitutes may be added to the medium. The concentration of serum to be added to the medium may be appropriately set depending on the type of cells, culture conditions, culture purpose, etc., but in one embodiment, the concentration of serum (and / or serum substitute) in the medium can be 15% by weight or less, 10% by weight or less, 9% by weight or less, 8% by weight or less, 7% by weight or less, 6% by weight or less, 5% by weight or less, 4% by weight or less, 3% by weight or less, 2% by weight or less, 1% by weight or less, 0.9% by weight or less, 0.8% by weight or less, 0.7% by weight or less, 0.6% by weight or less, 0.5% by weight or less, 0.4% by weight or less, or 0.3% by weight or less. The serum concentration may be kept constant during the culture period, or may be increased or decreased as necessary when changing the medium, etc.

[0177] <Process (III)> Step (III) is a step of freezing the cells for culture. The freezing method is not particularly limited, and for example, conventional means used in technical fields such as medicine, pharmaceuticals, quasi-drugs, cosmetics, food, and veterinary medicine, as well as basic technical fields such as regenerative medicine and bioengineering, can be used. Examples of freezing methods include slow freezing using a program freezer and simple freezing using a deep freezer. In the slow freezing method using a program freezer, the sample is cooled at a preset rate of, for example, 1 to 3°C / min, i.e., the temperature can be lowered at a rate of 1 to 3°C / min. On the other hand, in the simple freezing method, the sample can be placed in a deep freezer and slowly or rapidly frozen. Freezing can be carried out, for example, by placing the substrate having the cells for culture in a cooling device (eg, a programmable freezer, a deep freezer) and leaving it to stand.

[0178] The storage temperature after freezing is not particularly limited and is, for example, -250°C to -5°C, preferably -200°C to -20°C, and more preferably -200°C to -80°C.

[0179] Before carrying out step (III), it is preferable to remove the medium (culture solution) used in step (II) from the substrate having the cells for culture. The method of removal is not particularly limited, and for example, a method of removing using a pipette is exemplified. The amount of removal is not particularly limited, and may be the entire amount, an amount close to the entire amount, or a part, but is preferably the entire amount.

[0180] <Process (IV)> Step (IV) is a step of applying a coating agent containing a polymer capable of inhibiting cell adhesion onto a substrate to form a coating film. The coating agent is not particularly limited as long as it contains a polymer capable of inhibiting cell adhesion, and for example, contains a polymer capable of inhibiting cell adhesion and a solvent. Examples of the coating film and coating agent include the coating films and coating agents mentioned in the description of the cell structure of the present invention. The method for applying the coating agent onto the substrate is not particularly limited, and may be, for example, an inkjet method, a screen printing method, a slit coating method, or a roll-to-roll method. Preferably, the coating agent is applied by a printing technique such as an inkjet method or screen printing.

[0181] <Process (V)> Step (V) is a step of providing droplets containing a cell adhesive substance onto the cell culture base membrane, thereby disposing the cell adhesive substance on the cell culture base membrane.

[0182] Examples of the cell adhesive substance include the cell adhesive substances mentioned in the explanation of the cell structure of the present invention.

[0183] The droplets containing the cell adhesive substance are formed, for example, from a liquid containing the cell adhesive substance. The liquid containing the cell adhesive substance contains, for example, the cell adhesive substance and a solvent. Examples of the solvent include the solvents mentioned in the description of the cell culture base film forming agent.

[0184] The method for applying droplets containing the cell adhesive substance is not particularly limited, and may be an inkjet method, a screen printing method, a slit coating method, a roll-to-roll method, or the like. Preferably, the method is performed using a printing technique such as an inkjet method or screen printing.

[0185] <Process (VI)> Step (VI) is a step of contacting the cells for culture with a cryopreservation agent. Step (VI) can be carried out, for example, by adding a cryopreservation solution, which is a liquid cryopreservation agent, to the substrate having the cells for culture, and immersing the cells for culture in the cryopreservation solution.

[0186] Examples of cryopreservation solutions that are liquid cryopreservation agents include the cryopreservation agents mentioned in the description of the cell structure of the present invention.

[0187] Step (III), which is carried out after step (VI), is preferably carried out while the cells for culture are immersed in a cryopreservation solution.

[0188] (Cell culture method) The cell culture method of the present invention includes the following steps (1) and (2). Step (1): A step of thawing the frozen culture cells in the cell structure of the present invention. Step (2): Culturing cells for culture

[0189] The cell culture method may further include the following steps (A) and (B). Step (A): After step (1), a step of removing the cryopreservation agent (freezing solution) from the substrate having the cells for culture. Step (B): A step of washing the substrate having the cells for culture Step (B) is preferably carried out after step (A) and before step (2).

[0190] The cell structure is preferably a cell culture vessel.

[0191] <Process (1)> Step (1) is a step of thawing the frozen cells for culture in the cell structure of the present invention.

[0192] As a thawing method, a conventional method used in technical fields such as medicine, pharmaceuticals, quasi-drugs, cosmetics, food, and veterinary medicine, and in basic technical fields such as regenerative medicine and bioengineering can be used. Methods for thawing include, for example, using a water bath, an incubator, a hot plate, or immersion in a melting liquid at a temperature higher than the freezing temperature. Thawing may also be carried out by leaving the cell structure of the present invention at room temperature.

[0193] <Process (2)> Step (2) is a step of culturing cells for culture. The method for culturing the cells for culture is not particularly limited, and examples include a method in which a medium (culture solution) is added to a cell structure containing thawed cells for culture, and then the cell structure is allowed to stand under certain culture conditions.

[0194] The medium is not particularly limited, and examples thereof include the media mentioned in the explanation of step (II).

[0195] Culturing is carried out, for example, by placing the cell construct in an incubator and leaving it to stand at 37° C. under a constant CO 2 concentration (for example, 5%).

[0196] The culture period is not particularly limited, and examples include within 28 days, within 21 days, within 14 days, within 7 days, within 5 days, and within 3 days.

[0197] <Process (A)> Step (A) is a step of removing the cryopreservation agent (cryopreservation solution) from the substrate having the cells for culture after thawing. The method of removal is not particularly limited, and for example, a method of removing using a pipette is exemplified. The amount of removal is not particularly limited, and may be the entire amount, an amount close to the entire amount, or a part, but is preferably the entire amount.

[0198] <Process (B)> Step (B) is a step of washing the substrate having the cells for culture after thawing. The washing liquid used for washing is not particularly limited, and examples thereof include culture medium, PBS, etc. Examples of the culture medium include the culture medium described in the explanation of step (II). The washing method is not particularly limited, and examples thereof include adding a washing liquid to the substrate having the cells for culture and then removing the washing liquid. The removal method is not particularly limited, and examples thereof include a method of removing the cells using a pipette.

[0199] One example of the cell culture method of the present invention is a method for producing a cell structure. The method for producing a cell structure includes the following step (2-1) as step (2), and may further include the following steps (3) to (4). Step (2-1): A step of culturing cells to form a cell structure Step (3): Replacing the medium Step (4): Maturating the cell structure

[0200] The method for producing a cell structure may further include the following steps (5) and (6). Step (5): Suspending the cell structure in a medium Step (6): Recovering the cell structure

[0201] A cell structure is a cell aggregate formed by self-assembly of cells into a three-dimensional aggregate. The cell structure is also called a cell aggregate, a spheroid, a cell cluster, etc. The shape of the cell structure is not particularly limited, and examples include a sphere and a ring shape.

[0202] <Process (2-1)> The step (2-1) is a step of culturing cells to form a cell structure. In the step (2-1), for example, cells are cultured in a cell structure for 12 hours or more to form a cell structure. When a medium is added to the cell structure, the cells dispersed in the medium remain in the spots and are cultured in each spot. It is preferable that a certain number of cells remain in each spot, and it is preferable that one cell structure is formed in each spot. In order to culture cell structures at a high density, it is preferable that cell structures are formed in all spots, and therefore it is preferable that at least one cell is present in each spot.

[0203] <Process (3)> Step (3) is a step of replacing the medium. In changing the medium, a certain amount of the medium in the cell structure is aspirated, and then the same amount of fresh medium is poured in. It is preferable that the medium is changed at least once during cell culture. The certain amount may be, for example, 20% to 80% by mass, or 20% to 50% by mass of the medium. Examples of the medium include the media exemplified in step (II).

[0204] <Process (4)> Step (4) is a step of maturing the cell structure. In step (4), steps (2-1) to (2-3) are repeated multiple times to mature the cell structure. "Maturation" refers to, for example, enlarging the cell structure to a desired size, forming an organoid structure, or inducing differentiation into a target tissue. When inducing differentiation, it is preferable that after forming a cell structure on the spot, the medium is replaced with a differentiation-inducing medium to induce differentiation.

[0205] <Process (5)> Step (5) is a step of suspending the cell structure in a medium. In step (5), for example, after the cell structures are grown to a desired size, the medium is stirred to suspend the cell structures cultured in each spot in the medium. For example, this is carried out by stirring the medium. Specifically, the medium can be stirred by (i) shaking the cell structures to stir the medium, (ii) aspirating and discharging the medium (pipetting operation) to stir the medium, (iii) installing a stirring blade to stir the medium, or (iv) inserting a stir bar into the cell structures to stir the medium. Two or more of these may be combined.

[0206] <Process (6)> Step (6) is a step of recovering the cell structure. In step (6), for example, the medium in which the cell structures of the cell structure are suspended is sucked up with an aspirator, and the cell structures suspended in the medium are collected. EXAMPLES

[0207] The present invention will be described in more detail below based on Synthesis Examples, Examples, Test Examples, etc., but the present invention is not limited to these.

[0208] <Method of measuring molecular weight> The weight average molecular weights shown in the following synthesis examples are the results obtained by gel filtration chromatography (hereinafter abbreviated as GFC). (Measurement conditions) Equipment: HLC-8320GPC (Tosoh Corporation) GFC column: TSKgel G 6000 + 3000 PWXL-CP ·Flow rate: 1.0mL / min Eluent: Salt-containing water / organic mixed solvent Column temperature: 40℃ Detector: RI Injection concentration: polymer solids 0.05% by mass ·Injection volume: 100μL Calibration curve: Cubic approximation curve Standard sample: Polyethylene oxide (Agilent) x 10 types

[0209] <Synthesis Example 1> 24.00g of 2-(dimethylamino)ethyl methacrylate (Tokyo Chemical Industry Co., Ltd.), 1.46g of methacrylic acid (Tokyo Chemical Industry Co., Ltd.), 5.09g of ethylene glycol dimethacrylate (Tokyo Chemical Industry Co., Ltd.), 0.31g of dimethyl 1,1'-azobis(1-cyclohexanecarboxylate) (VE-073, Fujifilm Wako Pure Chemical Industries, Ltd.), and 111.09g of 2-propanol were mixed and polymerized dropwise into 166.62 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 by GFC was 228,000.

[0210] <Preparation Example 1> Polyvinyl acetate (JMR-150L (registered trademark) manufactured by Nippon Vinyl Acetate & Poval Co., Ltd. (degree of polymerization 1480, degree of saponification 22.7%)) was dissolved in ethanol / 1-methoxy-2-propanol (7 / 3 mass ratio) to a concentration of 3.0 mg / g to prepare a coating film-forming composition 1 having cell adhesion inhibitory properties. The obtained composition was transparent and uniform.

[0211] <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 (mass ratio 85 / 15) to a concentration of 20 mg / g to prepare a coating film-forming composition 2 having cell adhesion inhibitory properties. The obtained composition was transparent and uniform.

[0212] <Preparation Example 3> 49.95 g of sterilized water was added to 0.05 g of the polymer obtained in Synthesis Example 1 and thoroughly stirred. 24.38 g of sterilized water was added to 4.30 g of the obtained solution and thoroughly stirred to prepare base film forming agent 1.

[0213] <Preparation Example 4> 49.95 g of sterile water was added to 0.05 g of the polymer obtained in Synthesis Example 1 above, and the mixture was thoroughly stirred. 41.97 g of pure water and 0.60 g of GRGDS (manufactured by Peptide Institute, Inc.) diluted to 2.5 mg / mL with sterile water were added to 7.50 g of the obtained solution, and the mixture was thoroughly stirred to prepare base film forming agent 2.

[0214] <Preparation Example 5> 16.41 g of sterile water was added to 0.016 g of cell adhesive peptide PASAP2 (manufactured by Nanomed3D) and thoroughly stirred. 17.40 g of sterile water and 0.60 g of V-02 (manufactured by Nisshinbo Chemical Co., Ltd.) diluted to 5.0 mg / mL with sterile water were added to 6.0 g of the obtained solution and thoroughly stirred to prepare temporary film forming agent 1.

[0215] <Preparation Example 6> 49.5 g of sterilized water was added to 0.05 g of the polymer obtained in Synthesis Example 1 and thoroughly stirred. 49.50 g of sterilized water was added to 0.50 g of the obtained solution and thoroughly stirred to prepare base film forming agent 3.

[0216] <Production Example 1> (Inkjet printing to create a substrate capable of inhibiting cell adhesion) Using an inkjet device (Seiko Epson Corporation, inkjet device for R&D) and an inkjet head (Seiko Epson Corporation, Precision Core head S800-A1), an appropriate amount of the composition 1 for forming a coating film prepared in Preparation Example 1 was applied to a polystyrene substrate having a size of 79 mm x 121 mm in a perfect circle with a diameter of 18 mm. The polystyrene substrate after application was dried in an oven at 70°C for 24 hours and attached to a bottomless 24-well plate (CS Tech Co., Ltd.) to prepare a substrate 1 having cell adhesion inhibitory properties.

[0217] <Production Example 2> (Preparation of a substrate capable of inhibiting cell adhesion using a film applicator) Using a film applicator 100-3 (manufactured by Allgood Co., Ltd.) and a bar (wet film thickness 10 μm, manufactured by Allgood Co., Ltd.), an appropriate amount of the coating film-forming composition 2 prepared in Preparation Example 2 was applied to a polystyrene substrate having a size of 79 mm × 121 mm. The polystyrene substrate after application was dried in an oven at 70°C for 24 hours and attached to a bottomless 24-well plate (manufactured by CSTec Co., Ltd.) to prepare a substrate 2 capable of inhibiting cell adhesion.

[0218] <Preparation Example 3> Using an inkjet device (Seiko Epson Corporation, inkjet device for R&D) and an inkjet head (Seiko Epson Corporation, Precision Core head S800-A1), an appropriate amount of the base film forming agent 1 prepared in Preparation Example 3 was applied to the culture surface of the substrate 1 having cell adhesion inhibitory ability prepared in Preparation Example 1, forming a large number of spots with a diameter of 400 μm. The substrate with a large number of spots formed thereon was dried at room temperature, and then dried in a thermostatic dryer at 70° C. for one day, and attached to a bottomless 24-well plate (CS Tech Co., Ltd.) to prepare a substrate 1 for producing cell aggregates. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0219] <Preparation Example 4> Using an inkjet device (Seiko Epson Corporation, inkjet device for R&D) and an inkjet head (Seiko Epson Corporation, Precision Core Head S800-A1), an appropriate amount of the base film forming agent 2 prepared in Preparation Example 4 was applied to the culture surface of the substrate 2 having cell adhesion suppression ability prepared in Preparation Example 2, and many spots with a diameter of 900 μm were formed. After drying at room temperature, an appropriate amount of the temporary fixing film forming agent 1 prepared in Preparation Example 5 was applied to the center of the created spots with a diameter of 900 μm, and many spots with a diameter of 100 to 150 μm were formed. The substrate with many spots formed was dried for one day in a thermostatic dryer at 70° C. and attached to a bottomless 24-well plate (C.S.T.E. Co., Ltd.) to prepare a substrate 2 for producing cell aggregates. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0220] <Preparation Example 5> Using an inkjet device (Seiko Epson Corporation, inkjet device for R&D) and an inkjet head (Seiko Epson Corporation, Precision Core head S800-A1), an appropriate amount of the base film forming agent 2 prepared in Preparation Example 4 was applied to the culture surface of the substrate 2 having cell adhesion inhibitory ability prepared in Preparation Example 2, forming a large number of spots with a diameter of 400 μm. The substrate with a large number of spots formed was dried in a thermostatic dryer at 70° C. for one day and attached to a bottomless 6-well plate (CS Tech Co., Ltd.) to prepare a substrate 3 for producing cell aggregates. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0221] <Preparation Example 6> Using an inkjet device (Seiko Epson Corporation, inkjet device for R&D) and an inkjet head (Seiko Epson Corporation, Precision Core head S800-A1), an appropriate amount of the base film forming agent 3 prepared in Preparation Example 6 was applied to the culture surface of the substrate 2 having cell adhesion inhibitory ability prepared in Preparation Example 2, forming a large number of spots with a diameter of 400 μm. The substrate with a large number of spots formed thereon was dried at room temperature, and then dried in a thermostatic dryer at 70° C. for one day, and attached to a bottomless 24-well plate (CS Tech Co., Ltd.) to prepare a substrate 4 for producing cell aggregates. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0222] <Example 1: Freezing test using human adipose tissue-derived mesenchymal stem cells in a cell-adherent state in serum medium> (Cell preparation) The cells used were human adipose tissue-derived mesenchymal stem cells (ADSCs: Cellsource, Inc.). A low-serum medium, Mesenchymal Stem Cell Growth Medium GM2 (Takara Bio, Inc.), was used for cell culture. The cells were statically cultured for more than 2 days in a 10 cm diameter petri dish (10 mL of medium) at 37°C / CO2 incubator with 5% carbon dioxide concentration. The cells were then 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 cells were allowed to stand at room temperature for 3 minutes to detach. 7 mL of the above low-serum medium was added to recover the cells. The suspension was centrifuged (Tomy Seiko Co., Ltd., model number LC-230, 200 × g / 3 minutes, room temperature), the supernatant was removed, and the above low-serum medium was added to detach the cells at a concentration of 4.0 × 10 5 A cell suspension of 100 cells / mL was prepared.

[0223] (cell seeding, freezing, thawing) 1 mL of the cell suspension prepared above was added to the cell aggregate production substrate 1 prepared in Preparation Example 3. Then, the plate was left to stand in a 37°C / CO2 incubator with a carbon dioxide concentration of 5%. After 3 hours, the entire medium was aspirated, and 300 μL of cryopreservation solution was added. Cellbanker1 (manufactured by Nippon Zenyaku Kogyo Co., Ltd.) and STEM CELLBANKER (manufactured by Nippon Zenyaku Kogyo Co., Ltd.) were used as cryopreservation solutions. The plate was then frozen at -85°C for 14 days in a deep freezer (LAB8 (manufactured by Kanou Reiki Co., Ltd.)). After 14 days, the plate was removed and thawed at room temperature. Then, 1 mL of serum medium warmed to 37°C was added, and the entire amount was aspirated. 1 mL of serum medium was added, and the plate was left to stand in a 37°C / CO2 incubator.

[0224] <Example 2: Freezing test using human adipose tissue-derived mesenchymal stem cells in a cell-adherent state in serum-free medium> (Cell preparation) The same method was used to prepare 4.0 × 10 cells, except that the serum-free medium Mesenchymal Stem Cell Growth Medium XF (Takara Bio Inc.) was used instead of the low-serum medium used in the preparation of cells in Example 1 after cell detachment. 5 A cell suspension of 100 cells / mL was prepared.

[0225] (cell seeding, freezing, thawing) 1 mL of the cell suspension prepared above was added to the cell aggregate production substrate 2 prepared in Preparation Example 4. Then, the plate was left to stand in a 37°C / CO2 incubator with the carbon dioxide concentration maintained at 5%. After 3 hours, the entire medium was aspirated, and 300 μL of cryopreservation solution was added. Cellbanker1 was used as the cryopreservation solution. Then, the plate was frozen at -85°C in a deep freezer (LAB8 (Kano Refrigeration Co., Ltd.)) for 7 days. After 7 days, the plate was removed and thawed at room temperature. Then, 1 mL of serum-free medium warmed to 37°C was added, and the entire amount was aspirated. 1 mL of serum-free medium was added, and the plate was left to stand in a 37°C / CO2 incubator.

[0226] <Example 3: Freezing test using human adipose tissue-derived mesenchymal stem cells in a cell-adherent state in serum-free medium, and culture test in the presence of medium additives after thawing> (Cell preparation) In the same manner as in Example 2, 7.5 × 10 5 A cell suspension of 100 cells / mL was prepared.

[0227] (cell seeding, freezing, thawing) The cell suspension prepared above was added to the cell aggregate production substrate 3 prepared in Preparation Example 5 in 4 mL. Then, it was left to stand in a 37 ° C / CO2 incubator with 5% carbon dioxide concentration. After 3 hours, the entire medium was aspirated, and 1000 L of cryopreservation liquid was added. Cellbanker 1 was used as the cryopreservation liquid. Then, the plate was frozen at -85 ° C in a deep freezer (LAB8 (Kano Refrigeration Co., Ltd.)) for 14 days. After 14 days, it was removed and thawed at room temperature. Then, 1 mL of serum-free medium warmed to 37 ° C was added, and the entire amount was aspirated. 1 mL of serum-free medium was added and left to stand in a 37 ° C / CO2 incubator. After 4 days, 3 mL of medium was aspirated and removed, and 3 mL of serum-free medium prepared so that FCeM advance-CR (Nissan Chemical Co., Ltd.) was added to a concentration of 0.04 wt%. Thereafter, the plate was left to stand in a 37° C. / CO 2 incubator for one week, while carrying out Test Example 4 described below.

[0228] <Example 4: Cryopreservation test on CAT using HCT116 cells> (Cell preparation) Human colon cancer cell line (HCT116: Sumitomo Pharma Co., Ltd.) was used for cell culture. McCoy's 5A (Modified) Medium (Thermo Fisher Scientific) supplemented with 10v / v% fetal bovine serum (McCoy's 5A+10%FBS) was used for cell culture. The cells were cultured in a 10cm diameter petri dish (10mL of medium) for more than 2 days at 37℃ / CO2 incubator with 5% carbon dioxide concentration. The cells were then washed with 10mL of PBS solution (Fujifilm Wako Pure Chemical Co., Ltd.), and 1mL of 0.25w / v% trypsin-1mmol / L EDTA·4Na solution (containing phenol red) (Fujifilm Wako Pure Chemical Co., Ltd.) was added and the cells were detached by leaving the cells for 3 minutes at 37℃ / CO2 incubator with 5% carbon dioxide concentration. The cells were collected by adding 4 mL of the above McCoy's 5A + 10% FBS. The suspension was centrifuged (Tomy Seiko Co., Ltd., Model No. LC-200, 200 × g / 3 minutes, room temperature), the supernatant was removed, and the above McCoy's 5A + 10% FBS was added to prepare a suspension.

[0229] (cell seeding, freezing, thawing) The cell suspension was added to the cell aggregate production substrate 4 prepared in Preparation Example 6 at a rate of 4.2 × 10 5 cells / cm 2 Then, 1.0 mL was added so that the concentration of carbon dioxide in the wells was 5%. After one day, the entire medium was aspirated, and 300 μL of Cellbanker1 (manufactured by Nippon Zenyaku Kogyo Co., Ltd.) was added per well as a freezing medium. The plate was then frozen at -80°C for 12 days in a deep freezer (manufactured by Nippon Freezer Co., Ltd., model number CLN-31UW). After 12 days, the plate was removed and thawed using a plate heater (manufactured by Innovex Science Co., Ltd.) heated to 40°C. Then, 1 mL of the above McCoy's 5A+10% FBS warmed to 37°C was added, and the entire medium was aspirated. Furthermore, 1 mL of the above McCoy's 5A+10% FBS was added, and the plate was left to stand in a 37°C / CO2 incubator, and the entire medium was replaced every day. After thawing, the cells were left to stand in a 37° C. / CO 2 incubator for 3 days, during which Test Example 5 described below was carried out.

[0230] <Comparative Example 1: Freezing test of spheroids in serum-free medium> (Cell preparation) In the same manner as in Example 2, 4.0 × 10 5 A cell suspension of 100 cells / mL was prepared.

[0231] (cell seeding, spheroid freezing, thawing) 1 mL of the cell suspension prepared above was added to the cell aggregate production substrate 2 prepared in Preparation Example 4. Then, the plate was left to stand in a 37°C / CO2 incubator with the carbon dioxide concentration maintained at 5%. After 3 days, it was confirmed that spheroids had been formed. The entire amount of medium was aspirated, and 300 μL of cryopreservation solution was added. Cellbanker1 was used as the cryopreservation solution. The plate was then frozen at -85°C for 7 days in a deep freezer (LAB8 (Kano Refrigeration Co., Ltd.)). After 7 days, the plate was removed and thawed at room temperature. Then, 1 mL of PBS warmed to 37°C was added, and the entire amount was aspirated. 500 μL of PBS was added.

[0232] <Test Example 1: Cell Observation> The state of the cells was observed and photographed at each step using a stereomicroscope SZX16 (Olympus Corporation). The results are shown in Figures 4A to 4D, 5A to 5B, and 6. FIG. 4A shows the results of observation 3 hours after inoculation in the test of Example 1. FIG. 4B shows the results of observation after replacement with a freezing preservation solution in the test of Example 1. FIG. 4C shows the results of observation after thawing two weeks after freezing and addition of a medium in the test of Example 1. FIG. 4D shows the observation results after one day of culture following freezing and thawing in the test of Example 1. FIG. 5A shows the results of observation 3 hours after inoculation in the test of Example 2. FIG. 5B shows the results of observation after two days of culture following freeze-thawing in the test of Example 2. FIG. 6 shows the results of observation of spheroid formation after 3 days of culture in the test of Comparative Example 1.

[0233] In Example 1, selective cell adhesion to the base film portion on the prepared substrate was confirmed 3 hours after seeding. Furthermore, no change was observed in the state of cell adhesion even after replacement with a cryopreservation solution. Furthermore, even after thawing two weeks after freezing and adding a medium, cell adhesion was maintained and no change was observed. Furthermore, after culturing for one day, it was confirmed that the adhered cells peeled off from the dish and aggregated, forming cell aggregates (spheroids) of uniform size. From the above, it was shown that cell aggregates can be formed after thawing even if a freezing process is performed after cells are adhered to the base film.

[0234] In Example 2, selective adhesion of cells to the base film portion on the prepared substrate was confirmed 3 hours after seeding. Furthermore, it was confirmed that when the substrate was thawed after freezing and cultured for 2 days after adding medium, the adhered cells peeled off from the dish and aggregated to form cell aggregates (spheroids) of uniform size. From the above, it was demonstrated that cell aggregates can be formed after thawing even under culture conditions of serum-free medium and when a freezing process is performed after cells are adhered to the base film.

[0235] In Comparative Example 1, it was confirmed that uniform-sized cell aggregates (spheroids) were formed after 3 days of culture (before freezing). No difference was observed in the rate of spheroid formation between Example 2 and Comparative Example 1.

[0236] <Test Example 2: Survival Rate Evaluation> The spheroids formed in Examples 1 and 2 and Comparative Example 1 were all collected in a centrifuge tube by pipetting. After aspirating the supernatant, 500 μL of PBS was added and aspirated. 500 μL of Accumax (manufactured by Nacalai Tesque) was added and left to stand for 20 minutes in a 37°C / CO2 incubator. After pipetting, 50 μL was dispensed into a 96-well plate and 2.5 μL of Solution 18 (manufactured by Chemometec) was added. The cells were supported on NC Slide A2 (manufactured by Chemometec) and the survival rate was measured using a Nucleocounter NC-250 (manufactured by Chemometec). At the same time, the number of live cells was measured using a Nucleocounter NC-250, and the cell recovery rate was calculated by dividing the number of live cells by the number of cells seeded. The results are shown below. Table 1 shows the quantitative results of cell viability in the test of Example 1. Table 2 shows the quantitative results of cell viability and cell recovery rate in Example 2 and Comparative Example 1.

[0237] [Table 1]

[0238] [Table 2]

[0239] It was shown in Example 1 that a high survival rate of 93% or more was achieved by using the cryopreservation solutions Cellbanker 1 and STEMCELLBANKER. This shows that the survival rate of cells can be maintained high even when the cells are attached to the cell culture base membrane and frozen in contact with the cryopreservation solution for two weeks.

[0240] Moreover, in Example 2, it was shown that the survival rate was high at 98% by using the freezing preservation solution Cellbanker 1. On the other hand, when the cells were frozen in a spheroid state in Comparative Example 1, the survival rate dropped to 87%. It is considered that in the spheroid state, the freezing preservation solution was less likely to penetrate into the inside of the spheroid, and cell damage occurred due to ice crystal formation.

[0241] The recovery rate was calculated from the number of cells detected relative to the number of cells seeded. The recovery rate was improved by about 20% in Example 2 compared to Comparative Example 1. It is believed that the recovery rate decreased due to the lysis of single dead cells during the spheroid disintegration treatment using Accumax. Since more cells were recovered in Example 2, it is believed that the cell survival rate was higher than in Comparative Example 1. These results suggest that cryopreservation of cells while attached to a cell culture base membrane allows for a higher survival rate of cells compared to cryopreservation after spheroid formation.

[0242] <Test Example 3: Cell viability staining of spheroids> The spheroids formed in Example 2 and Comparative Example 1 were all collected in a centrifuge tube by pipetting. After aspirating the supernatant, 500 μL of PBS was added and aspirated. 500 μL of Cellstain Propidium Iodide solution (manufactured by Dojindo Laboratories) adjusted to a concentration of 8 μM was added. The solution was dispensed into a 24-well plate (manufactured by Nunc) and left to stand for 15 minutes in a 37°C / CO2 incubator to stain dead cells. Fluorescence observation and bright field observation were performed using an all-in-one microscope BZ-9000 (manufactured by Keyence Corporation). The exposure time during fluorescence observation was 1 / 12 sec. The results are shown in the figure. FIG. 7A shows the results of fluorescence observation in Example 2. FIG. 7B shows the bright field observation results of Example 2. FIG. 8A shows the results of fluorescence observation in Comparative Example 1. FIG. 8B shows the bright field observation results of Comparative Example 1.

[0243] In Example 2, almost no fluorescently labeled dead cells were observed. On the other hand, in Comparative Example 1, dead cells were distributed throughout the spheroids. It was also found that the intensity was higher and the number of dead cells was greater compared to Example 2. From the above, it was demonstrated that cells can be cryopreserved with a higher survival rate by cryopreserving them while attached to the base membrane for cell culture, compared to cryopreserving them after forming spheroids.

[0244] <Test Example 4: Cell Observation> Using a cell observation device Cell3imager (manufactured by Screen Holdings), the state of the cells cultured in Example 3 was observed and photographed 0, 1, 3, and 7 days after the addition of the FCeM-containing medium. The results are shown in Figure 9. Furthermore, the Cell3imager was used to obtain size distribution data of the spheroids. The results 7 days after the addition of the FCeM-containing medium are shown in Figure 10. The vertical axis of the graph in Figure 10 represents the number of detected spheroids of each size shown on the horizontal axis.

[0245] In Example 3, after thawing from a frozen state, spheroids could be cultured for a total of 11 days (7 days after adding FCeM-containing medium) by culturing in culture medium supplemented with FCeM advance-CR. Furthermore, when the size distribution of the spheroids was confirmed, it was shown that spheroids of relatively uniform size were formed, with a peak top at around 150 μm in diameter. From the above, it was shown that when frozen and stored while attached to a cell culture base membrane, long-term culture of approximately 11 days (7 days after adding FCeM-containing medium) is possible while maintaining a uniform spheroid size by combining with culture medium supplemented with FCeM advance-CR after thawing.

[0246] <Test Example 5: Cell Observation> For Example 4, photographs of the entire well surface were taken over time using a cell observation device, Cell3imager duos (manufactured by SCREEN Corporation). The results are shown in FIG. Photographs taken with a Cell3imager duos (SCREEN Corporation) confirmed selective cell adhesion to the base film portion of the substrate on the day of seeding. When the substrate was thawed after freezing and culture was continued after adding medium, it was confirmed that the attached cells proliferated on the base film of the substrate and formed cell aggregates (spheroids). From the above, it was demonstrated that even cells used for drug discovery, such as cancer cells, can be attached to a base film, undergo a freezing process, and then form cell aggregates after thawing.

[0247] According to the present invention, cells can be preserved with a higher survival rate than by freezing spheroids by adhering the cells to the substrate for forming cell aggregates. By applying the present invention to various cells, the plate can be used as a plate for forming cell aggregates for use in drug discovery screening and animal alternative testing. Furthermore, since delivery of cells in an adhering state becomes easier, it is possible to develop applications such as adhering necessary cells in regenerative medicine, freezing and storing them, transporting them when necessary to form cell aggregates, and using them for transplantation. [Explanation of symbols]

[0248] 1 Main unit 1A outer wall 1B Inner bottom surface 1C aperture 2 Cell adhesive substance 3 Cell culture base membrane 4. Coating film 100 cell structures

Claims

1. A substrate; a plurality of spaced apart spots on a surface of the substrate; A cell structure comprising: Each of the plurality of spots has a cell culture substratum containing a polymer; The cell culture base film has cells for culture thereon, The cell structure, wherein the cells for culture are in a frozen state.

2. The cell structure according to claim 1 , further comprising a cell adhesive substance disposed on the cell culture base membrane.

3. Further comprising a cryopreservative, The cell structure according to claim 1 , wherein the cells for culture are covered with the cryopreservation agent.

4. The cell structure according to claim 1 , which is a cell culture vessel.

5. The cell structure according to claim 1 , wherein the polymer is a polymer (A) containing a repeating unit represented by the following formula (Ia): 【Chemistry 1】 (In formula (Ia), 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.

6. The cell structure according to claim 5 , wherein the polymer (A) includes a crosslinked structure.

7. The cell structure according to claim 6, wherein the polymer (A) comprises at least one of a structure represented by the following formula (IIIa), a structure represented by the following formula (IVa), and a structure represented by the following formula (Va). 【Chemistry 2】 (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.

8. the surface of the substrate has a coating film having a cell adhesion inhibitory ability; The cell structure according to claim 1 , wherein the spots are disposed on the coating film.

9. A step of applying droplets of a cell culture base film forming agent containing a polymer and a solvent to a substrate to form a cell culture base film in an area to be a spot; Seeding cells for culture on the cell culture base film; Freezing the cells for culture; A method for producing a cell structure comprising the steps of:

10. The method for producing a cell structure according to claim 9 , further comprising a step of contacting the cells for culture with a cryopreservation agent prior to the freezing step.

11. The method for producing a cell structure according to claim 9 , wherein the droplets of the cell culture base film forming agent are applied to the substrate by an inkjet method.

12. The method for producing a cell structure described in claim 9, further comprising a step of applying a droplet containing a cell adhesive substance to the cell culture base membrane prior to the seeding step, thereby disposing the cell adhesive substance on the cell culture base membrane.

13. The method for producing a cell structure according to claim 12, wherein the droplets containing the cell adhesive substance are applied to the cell culture base membrane by an inkjet method.

14. The method for producing a cell structure according to claim 9 , wherein the polymer is a polymer containing a repeating unit represented by the following formula (Ia): 【Chemistry 3】 (In formula (Ia), 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.

15. In the cell structure to be produced, the surface of the substrate has a coating film having a cell adhesion inhibitory ability, and the plurality of spots are arranged on the coating film, The method for producing a cell structure described in claim 9, further comprising the step of applying a coating agent containing a polymer capable of inhibiting cell adhesion onto the substrate to form the coating film.

16. The method for producing a cell structure according to claim 9, wherein the cell structure to be produced is a cell culture vessel.

17. A step of thawing the frozen cells for culture in the cell structure according to any one of claims 1 to 8; Culturing the cells for culture; A cell culture method comprising:

18. The cell culture method according to claim 17, which produces a cell structure.

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