Low biomolecular adhesion material made of copolymer

Abstract

The purpose of the present invention is to provide a novel coating film formation composition for the formation of a coating film having a function of inhibiting adhesion of a biological substance, a coating film using said composition, and manufacturing methods for a substrate for a cell culture, a multilayer film, and a cell aggregate using the coating film.　The present invention provides a coating film formation composition including: a polymer that includes structural units represented by formula (1) and formula (2) (in the formulas, R1-R3, X1, X2, T1, n1, n2, and n3 are as set forth in the specification and the claims) and that does not include a polysiloxane skeleton; and a solvent.

Description

Technical Field

[0001] The present invention relates to a composition for forming a coating film containing a copolymer having a specific structure, preferably a composition for forming a coating film for forming a coating film having an ability to suppress the adhesion of biological substances, a coating film using the same, a substrate for cell culture using the same, a laminated film, and a method for producing cell aggregates.

Background Art

[0002] Coating materials having the ability to suppress the adhesion of various biological substances have been proposed for suppressing the adhesion of biological substances to various medical equipment such as artificial dialyzers, artificial organs, and medical instruments, and various medical application devices such as cell culture containers and substrates for cell culture. Patent Document 1 discloses an ion complex material having the ability to control the adhesion of biological substances. Patent Document 2 discloses a method for producing a polymer used as a basement membrane for cell culture and a cell culture container.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] The present invention provides a novel composition for forming a coating film for forming a coating film having an ability to suppress the adhesion of biological substances, a coating film using the same, a substrate for cell culture using the same, a laminated film, and a method for producing cell aggregates.

Means for Solving the Problems

[0005] The present invention includes the following.

[0006] [1] Equations (1) and (2): [ka] (In the formula, R 1 ~R 3 Each of these independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, X 1 X represents a single bond, ester bond, amide bond, or alkylene group with 1 to 5 carbon atoms. 2 This represents an alkylene group with 1 to 5 carbon atoms, T 1 (P) is a copolymer (P) that contains a unit structure represented by ) and does not contain a polysiloxane skeleton. 1 A coating film-forming composition comprising ), and a solvent.

[0007] [2] The composition according to [1], wherein the solvent is selected from water, alcohol, or a combination thereof.

[0008] [3] The solvent is used as the dispersion medium, and the copolymer (P) is used as the dispersed phase. 1 A coating film-forming composition according to [1] or [2], in the form of a colloidal solution containing ).

[0009] [4] A coating film-forming composition having the ability to suppress the adhesion of biological substances, as described in any one of items [1] to [3].

[0010] [5] Equations (1) and (2): [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 represents a single bond, an ester bond, an amide bond or an alkylene group having 1 to 5 carbon atoms, X 2 represents an alkylene group having 1 to 5 carbon atoms, T 1 represents a halogen atom, an alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom, a hydroxy group, a carboxy group, a nitro group, a cyano group, a methylenedioxy group, an acetoxy group, a methylthio group, an amino group or an alkoxy group having 1 to 10 carbon atoms, n1 represents an integer of 0 to 2, n2 represents an integer of 1 to 10, and n3 represents an integer of 0 to 8), and contains a unit structure represented by, and does not contain a polysiloxane skeleton, a copolymer (P 1 ). A coating film containing

[0011] [6] The coating film according to [5], which has an ability to suppress adhesion of biomaterials.

[0012] [7] A substrate for cell culture, comprising a part of the substrate surface with the coating film according to [6].

[0013] [8] A laminated film having a layer of the coating film according to claim 5 or 6 and a layer of a basement membrane for cell culture on the upper surface thereof.

[0014] [9] The basement membrane for cell culture is represented by the following formula (I): <e [Chemical formula] [In the formula, U a1 and U a2 each independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, R a1 represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, R a2A repeating unit derived from a monomer represented by [where is an alkylene group having 1 to 5 carbon atoms], and the following formula (II): [ka] [In the formula, R b This refers to copolymers (P) containing repeating units derived from monomers represented by [a hydrogen atom or an alkyl group having 1 to 5 carbon atoms]. 2 The laminated film described in [8], including ).

[0015]

[10] A cell culture substrate comprising the multilayer film described in [8] or [9].

[0016]

[11] A method for producing cell aggregates, comprising the step of seeding cells onto a cell culture substrate as described in [7] or

[10] .

[0017]

[12] Equations (1) and (2): [ka] (In the formula, R 1 ~R 3 Each of these independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, X 1 X represents a single bond, ester bond, amide bond, or alkylene group with 1 to 5 carbon atoms. 2 This represents an alkylene group with 1 to 5 carbon atoms, T 1 (where n represents a halogen atom, an alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom, a hydroxyl group, a carboxyl group, a nitro group, a cyano group, a methylenedioxy group, an acetoxy group, a methylthio group, an amino group, or an alkoxy group having 1 to 10 carbon atoms, where n1 represents an integer from 0 to 2, n2 represents an integer from 1 to 10, and n3 represents an integer from 0 to 8) This copolymer contains a unit structure that does not contain a polysiloxane skeleton (P 1 A coating film obtained by washing a coating film containing ) with a solvent.

[0018]

[13] Equations (1) and (2): [ka] (In the formula, R 1 ~R 3 Each of these independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, X 1 X represents a single bond, ester bond, amide bond, or alkylene group with 1 to 5 carbon atoms. 2 T represents an alkylene group with 1 to 5 carbon atoms. 1 A method for producing a coating film, comprising the step of washing a coating film containing a polymer that includes a unit structure represented by ( ) and does not contain a polysiloxane skeleton, with a solvent. [Effects of the Invention]

[0019] As a result of diligent research by the inventors of this invention, they synthesized random copolymers and block copolymers containing the unit structures represented by formulas (1) and (2), and formed a coating film on a substrate. They found that these copolymers exhibited excellent ability to inhibit the adhesion of cells and proteins, thus completing the present invention. It is presumed that the hydrophobic portion of the unit structure represented by formula (1) is fixed to the substrate, and the hydrophilic portion of the unit structure represented by formula (2) is exposed on the surface, causing the substrate surface to exhibit hydrophilicity and thus possessing unique properties toward biomolecules. [Brief explanation of the drawing]

[0020] [Figure 1] This is a schematic diagram of the flow reactor (reaction apparatus) used in the synthesis example. [Figure 2](a) and (b) are 1H-NMR charts of the block copolymer obtained in Synthesis Example 1. [Figure 3] This shows the observation results of cell adhesion in Test Example 4. [Figure 4] This shows the observation results of cell adhesion in Test Example 5. [Figure 5] This shows the observation results of cell adhesion in Test Example 6. [Figure 6] This shows the observation results of cell aggregates in Test Example 6. [Figure 7] This shows the observation results of cell adhesion in Test Example 7. [Figure 8] This shows the observation results of the cell aggregates in Test Example 7. [Modes for carrying out the invention]

[0021] <Composition for forming coating films> The coating film forming composition of the present invention is Equations (1) and (2): [ka] (In the formula, R 1 ~R 3 Each of these independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, X 1 X represents a single bond, ester bond, amide bond, or alkylene group with 1 to 5 carbon atoms. 2 This represents an alkylene group with 1 to 5 carbon atoms, T 1 (where n represents a halogen atom, an alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom, a hydroxyl group, a carboxyl group, a nitro group, a cyano group, a methylenedioxy group, an acetoxy group, a methylthio group, an amino group, or an alkoxy group having 1 to 10 carbon atoms, and n1 represents an integer from 0 to 2, n2 represents an integer from 1 to 10, and n3 represents an integer from 0 to 8) copolymers containing a unit structure (P 1 ), and a solvent.

[0022] Examples of the above-mentioned "alkyl groups having 1 to 5 carbon atoms" include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, s-butyl group, t-butyl group, n-pentyl group, 1-methylbutyl group, 2-methylbutyl group, 3-methylbutyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, or 1-ethylpropyl group. 1 ~R 3 Each of these is preferably independently selected from a hydrogen atom, a methyl group, or an ethyl group.

[0023] The above "ester bond" refers to -C(=O)-O- or -OC(=O)-, and "amide bond" refers to -NHC(=O)- or -C(=O)NH-.

[0024] Examples of the above-mentioned "alkylene groups having 1 to 5 carbon atoms" include methylene group, ethylene group, propylene group, trimethylene group, tetramethylene group, 1-methylpropylene group, 2-methylpropylene group, dimethylethylene group, ethylethylene group, pentamethylene group, 1-methyltetramethylene group, 2-methyltetramethylene group, 1,1-dimethyl-trimethylene group, 1,2-dimethyl-trimethylene group, 2,2-dimethyl-trimethylene group, and 1-ethyl-trimethylene group. 1 and X 2 It is preferable that the group be selected from a methylene group, an ethylene group, and a propylene group.

[0025] Examples of the above-mentioned "halogen atoms" include fluorine atoms, chlorine atoms, bromine atoms, and iodine atoms.

[0026] The above-mentioned "alkyl groups having 1 to 5 carbon atoms that may be substituted with halogen atoms" means either the alkyl groups having 1 to 5 carbon atoms as described above, or the alkyl groups having 1 to 5 carbon atoms that are substituted with one or more halogen atoms as described above. Examples of "alkyl groups having 1 to 5 carbon atoms" are as described above. "Alkyl groups having 1 to 5 carbon atoms substituted with one or more halogen atoms" means that one or more arbitrary hydrogen atoms of the alkyl groups having 1 to 5 carbon atoms as described above are replaced with halogen atoms. Examples include fluoromethyl group, difluoromethyl group, trifluoromethyl group, chloromethyl group, dichloromethyl group, trichloromethyl group, bromomethyl group, iodomethyl group, 2,2,2-trifluoroethyl group, 2,2,2-trichloroethyl group, perfluoroethyl group, perfluorobutyl group, or perfluoropentyl group.

[0027] The above "alkoxy groups having 1 to 10 carbon atoms" include methoxy group, ethoxy group, n-propoxy group, i-propoxy group, n-butoxy group, i-butoxy group, s-butoxy group, t-butoxy group, n-pentoxy group, 1-methyl-n-butoxy group, 2-methyl-n-butoxy group, 3-methyl-n-butoxy group, 1,1-dimethyl-n-propoxy group, 1,2-dimethyl-n-propoxy group, 2,2-dimethyl-n-propoxy group, 1-ethyl-n-propoxy group, n-hexyloxy group, 1-methyl-n-pentyloxy group, 2-methyl-n-pentyloxy group, 3-methyl-n-pentyloxy group, 4 Examples include the -methyl-n-pentyloxy group, 1,1-dimethyl-n-butoxy group, 1,2-dimethyl-n-butoxy group, 1,3-dimethyl-n-butoxy group, 2,2-dimethyl-n-butoxy group, 2,3-dimethyl-n-butoxy group, 3,3-dimethyl-n-butoxy group, 1-ethyl-n-butoxy group, 2-ethyl-n-butoxy group, 1,1,2-trimethyl-n-propoxy group, 1,2,2-trimethyl-n-propoxy group, 1-ethyl-1-methyl-n-propoxy group, 1-ethyl-2-methyl-n-propoxy group, n-heptyloxy group, n-octyloxy group, and n-nonyloxy group.

[0028] The unit structure represented by the above equations (1) and (2) is given by the following equations (1-1) and (2-1): [ka] (R 1 ~R 3 , X 1 , X 2 , T 1 (n1, n2, and n3 are the same as above.) It is preferable that these compounds be derived from compounds represented by [formula]. If these compounds contain a functional group, compounds in which that functional group is appropriately protected with a known protecting group are also preferred.

[0029] In the monomer represented by formula (1-1), R 1 Preferably, represents a hydrogen atom or a methyl group, X 1 Preferably, represents a single bond, an ester bond, an amide bond, or a methylene group. Specific examples of such monomers include styrene, vinylnaphthalene, and vinylanthracene. The aryl group in the above monomer is the T 1 It may be substituted with other compounds. Among these, unsubstituted styrene is preferred.

[0030] In the monomer represented by formula (2-1), R 2 and R 3 Preferably, each independently represents a hydrogen atom or a methyl group, and X 2n2 preferably represents an ethylene group or a propylene group, and n2 represents an integer from 1 to 5. Specific examples of such monomers include ethylene glycol mono(meth)acrylate (=2-hydroxyethyl(meth)acrylate), ethylene glycol monomethyl ether(meth)acrylate, diethylene glycol mono(meth)acrylate, diethylene glycol monomethyl ether(meth)acrylate, triethylene glycol mono(meth)acrylate, triethylene glycol monomethyl ether(meth)acrylate, dipropylene glycol mono(meth)acrylate, dipropylene glycol monomethyl ether(meth)acrylate, or monomers in which their hydroxyl groups are protected as needed. However, it is preferable to use ethylene glycol monomethacrylate, diethylene glycol monomethyl ether methacrylate, or triethylene glycol monomethyl ether methacrylate. In this invention, (meth)acrylate compound means acrylate compound or methacrylate compound. For example, (meth)acrylate means acrylate or methacrylate.

[0031] The copolymer (P) of this application 1 ) may be either a random copolymer or a block copolymer. The copolymer of this application (P 1 The copolymer (P) of the present invention can be produced by polymerizing the monomers of formulas (1-1) and (2-1) in known ways. 1 Preferably, the above formulas (1) and (2) are derived from one compound each, but two or more compounds may be derived from each.

[0032] Compounds having a repeating unit of the third component, other than those represented by formulas (1) and (2) above, may be further polymerized.

[0033] The respective molar ratios of the unit structures represented by formulas (1) and (2) are, for example, formula (1): formula (2) = 1-90:99-10, preferably 1-70:99-30, more preferably 1-50:99-50, even more preferably 1-30:99-70, and particularly preferably 2-25:98-75.

[0034] The copolymer (P) of the unit structure represented by formulas (1) and (2). 1 The molar ratio to the total is not particularly limited as long as it achieves the effects of the present invention, but for example it may be 80 mol% or more, 90 mol% or more, or 100 mol%.

[0035] The copolymer (P) of this application 1 Preferably, the material does not contain a polysiloxane skeleton. A polysiloxane skeleton refers to a skeleton containing repeating siloxane unit structures: -OSiRR- (where R is hydrogen or an alkyl group, etc.).

[0036] The above block copolymer can be synthesized by known methods, but may be prepared, for example, using the mixer described in International Publication No. 2017 / 135398. The entire disclosure described in International Publication No. 2017 / 135398 is incorporated herein by reference.

[0037] The block copolymer of the present invention is a combination of multiple types of polymers (blocks) that are chemically different and covalently bonded to one another. The block copolymer used in the present invention includes an organic polymer chain (A) containing an organic monomer (a) as a unit structure, and a polymer chain (B) containing a monomer (b) different from the organic monomer (a) as a unit structure and bonded to the organic polymer chain (A).

[0038] The solid content of the coating film-forming composition of this application can be 0.01 to 50% by mass, or 0.1 to 20% by mass, or 0.1 to 10% by mass. The solid content is the proportion remaining after removing the solvent from the film-forming composition.

[0039] The proportion of block copolymer in the solid content can be 30-100% by mass, 50-100% by mass, 50-90% by mass, or 50-80% by mass. The number of blocks present in the block copolymer can be two or three or more.

[0040] By changing the polymer chain (B), for example, a neighboring structure containing monomer (c) as the unit structure can be created. It is possible to use the contacting polymer chain (C). Block polymers include combinations such as AB, ABAB, ABA, and ABC.

[0041] One method for synthesizing block copolymers involves living radical polymerization, living cationic polymerization, or living anionic polymerization, where the polymerization process consists only of an initiation reaction and a growth reaction, without side reactions that deactivate the growth ends. The growth ends can maintain their growth activity during the polymerization reaction. By preventing chain transfer, polymers of uniform length (A) can be obtained. By adding a different monomer (b) using the growth ends of this polymer (A), polymerization can proceed with this monomer (b) to form a block copolymer (AB). Homopolymer A or B is a polymerizable compound having at least one polymerizable reactive group, preferably a radically polymerizable reactive group (vinyl group or vinyl group-containing organic group).

[0042] The copolymer used in the present invention (P 1 The weight-average molecular weight Mw of the material is preferably 1,000 to 1,000,000, 5,000 to 500,000, 5,000 to 100,000, or 5,000 to 50,000. If it is less than 1,000, the coating properties to the substrate may be poor, and if it is greater than 1,000,000, the solubility in the solvent may be poor.

[0043] The copolymer (P) of this application 1The polydispersity (Mw / Mn) of ) is preferably 1.00 to 2.50, and particularly preferably 1.00 to 1.50.

[0044] <Solvent> The solvents contained in the coating film-forming composition of the present invention include water, phosphate-buffered saline (PBS), and alcohol. Examples of alcohols include alcohols having 2 to 6 carbon atoms, such as ethanol, propanol, isopropanol, 1-butanol, 2-butanol, isobutanol, t-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 1-heptanol, 2-heptanol, 2,2-dimethyl-1-propanol (neopentyl alcohol), 2-methyl-1-propanol, 2-methyl-1-butanol, 2-methyl-2-butanol (t-amyl alcohol), 3-methyl-1-butanol, 3-methyl-3-pentanol, and cyclopentyl alcohol. Examples include tanol, 1-hexanol, 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. Water, phosphate-buffered saline (PBS), and alcohols may be used as solvents, either alone or in combination thereof, but copolymers (P 1 From the viewpoint of dissolving the substance, it is preferable to select from water, alcohol, or a combination thereof, and more preferably from water, ethanol, or a combination thereof.

[0045] <Colloidal solution (sol)> The copolymer (P) of this application 1 Depending on the type of solvent, the solvent acts as a dispersion medium, and the copolymer (P 1 ) can form a colloidal solution (sol) with the dispersed phase. The particle size of the dispersed phase is, for example, in the range of 5 to 1,000 nm, preferably 5 to 500 nm, and more preferably 5 to 300 nm. The particle size is either the primary particle diameter or the secondary particle diameter. In this case, the solvent is preferably a mixed solvent of water and alcohol. A mixed solvent of water and ethanol is preferable. The water:alcohol mixing ratio (by weight) is, for example, 1-70:99-30, or 1-50:99-50.

[0046] The coating film-forming composition of the present invention is preferably a coating film-forming composition that has the ability to suppress the adhesion of biological substances.

[0047] In the present invention, biomaterials include proteins, sugars, viruses, nucleic acids, and cells or combinations thereof, or biological tissues and bodily fluids containing them. The proteins listed above include fibrinogen, bovine serum albumin (BSA), human albumin, various globulins, β-lipoproteins, various antibodies (IgG, IgA, IgM), peroxidases, various complements, various lectins, fibronectin, lysozyme, von Willebrand factor (vWF), serum γ-globulin, pepsin, ovalbumin, insulin, histones, ribonucleases, collagen, and cytochrome c. The sugars mentioned above include glucose, galactose, mannose, fructose, heparin, and hyaluronic acid. The nucleic acids mentioned above include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), The above cells include fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, erythrocytes, platelets, macrophages, monocytes, osteocytes, pericytes, dendritic cells, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatocytes, 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, mononuclear cells, embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ cells, and artificial cells. Examples include pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, muscle stem cells, germline stem cells, intestinal stem cells, cancer stem cells, hair follicle stem cells, and various cell lines (e.g., HCT116, Huh7, HEK293 (human fetal kidney cells), HeLa (human cervical cancer cell line), HepG2 (human hepatocellular carcinoma cell line), UT7 / TPO (human leukemia cell line), CHO (Chinese hamster ovary cell line), MDCK, MDBK, BHK, C-33A, HT-29, AE-1, 3D9, Ns0 / 1, Jurkat, NIH3T3, PC12, S2, Sf9, Sf21, High Five, Vero).

[0048] Furthermore, the above-mentioned antibody may also be an antibody drug. An antibody drug is a drug that utilizes antibodies, and antibodies are proteins composed of immunoglobulins. It is preferable that the above antibody drug contains at least one of the antibody and its antigen-binding fragment. It is preferable that the above antibody drug contains at least one selected from the group consisting of chimeric antibodies, human antibodies, humanized antibodies, and domain antibodies thereof.

[0049] Specific examples of the antibody drugs mentioned above include ofatumumab (trade name "Arzera®"), cetuximab (trade name "Erbitux®"), tocilizumab (trade name "Actemra®"), bevacizumab (trade name "Avastin®"), canakinumab (trade name "Ilaris®"), golimumab (trade name "Simponi®"), ustekinumab (trade name "Stelara®"), eculizumab (trade name "Soliris®"), omalizumab (trade name "Xolair®"), trastuzumab (trade name "Herceptin®"), pertuzumab (trade name "Perjeta®"), adalimumab (trade name "Humira®"), Denosumab (brand names "Prolia®" and "Xgeva®"), mogamulizumab (brand name "Poteligeo®"), rituximab (brand name "Rituxan®"), ranibizumab (brand name "Lucentis®"), infliximab (brand name "Remicade®"), aflibercept (brand name "Eylea®"), abatacept (brand name "Orencia®"), etanercept (brand name "Enbrel®"), gemtuzumab ozogamicin (brand name "Mylotarg®"), panitumumab (brand name "Vectibix®"), basiliximab (brand name "Symlect®"), certolizumab Examples include pegol (trade name "Cimzia®"), palivizumab (trade name "Synagis®"), cacilibimab / imdevimab (trade name "Ronaprive®"), and sotrovimab (trade name "Zebudi").

[0050] Among these, it is preferable that the antibody is obtained from a clone derived from a single antibody-producing cell, or that it is a monoclonal antibody which is an antibody molecule, and it is preferable that it is a monoclonal antibody consisting of an anti-human CD20 human antibody, and it is preferable that it contains rituximab or abatacept. Furthermore, the antibody drug may contain one type of antibody, or it may contain two or more types of antibodies. In other words, the antibody drug of the present invention may contain both an antibody and an antigen-binding fragment, or it may contain two or more types of antibodies, or it may contain two or more types of antigen-binding fragments.

[0051] Having protein adhesion inhibitory ability means that, in IgG antibody HRP measurement performed by the method described in the examples, the relative average absorbance (%) ((average absorbance in the examples / (average absorbance without coating)) compared to no coating film is 50% or less, preferably 30% or less, and more preferably 20% or less; Having the ability to inhibit cell adhesion means that, under microscopic observation, no cell adhesion or spread is observed on the target, and cell aggregates (spheroids) are formed.

[0052] The above-mentioned biomolecules are preferably cells and proteins. If the above-mentioned biomolecule is a cell, the copolymer (P) of this application 1 ) is preferably the aforementioned block copolymer.

[0053] <Coating film> The coating film of the present invention is based on formulas (1) and (2): [ka] (In the formula, R 1 ~R 3 Each of these independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, X 1 X represents a single bond, ester bond, amide bond, or alkylene group with 1 to 5 carbon atoms. 2 This represents an alkylene group with 1 to 5 carbon atoms, T 1A copolymer (P) containing a unit structure represented by a halogen atom, an alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom, a hydroxyl group, a carboxyl group, a nitro group, a cyano group, a methylenedioxy group, an acetoxy group, a methylthio group, an amino group, or an alkoxy group having 1 to 10 carbon atoms, where n1 represents an integer from 0 to 2, n2 represents an integer from 1 to 10, and n3 represents an integer from 0 to 8. 1 It is a coating film containing ).

[0054] The coating film of the present invention is a coated film of the above-mentioned coating film-forming composition. The coating film-forming composition according to the present invention is applied to a substrate and dried to form a coating film.

[0055] As the substrate for forming the coating film of the present invention, the same substrate as that described below for cell culture substrates can be used.

[0056] To form the coating film of the present invention, the above-mentioned coating film-forming composition is applied to at least a portion of the surface of a substrate. There are no particular restrictions on the application method, and conventional application methods such as spin coating, dip coating, and solvent casting can be used.

[0057] The drying process for the coating film according to the present invention is carried out under air or vacuum at a temperature in the range of -200°C to 200°C. The coating film can be formed by drying at room temperature (10°C to 35°C, for example, 25°C), but to form the coating film more quickly, it may be dried at a temperature of, for example, 40°C to 100°C. A more preferable drying temperature is 10°C to 180°C, and a more preferable drying temperature is 20°C to 150°C.

[0058] The coating film of this application is preferably a coating film that has the ability to suppress the adhesion of biological substances. The biological substances are preferably proteins and cells. If the above-mentioned biological substance is a cell, the copolymer (P) contained in the coating film of this application 1) is preferably the aforementioned block copolymer.

[0059] The coating film of the present invention may be a coating film obtained by further cleaning after forming a coating film by the above process. The above cleaning may be carried out by known methods, but running water cleaning or ultrasonic cleaning is preferable. Examples of cleaning solvents include water and aqueous solutions containing electrolytes. The cleaning solvent is usually used at room temperature (e.g., 10-35°C), but may also be heated to a range of 40-95°C. Preferred aqueous solutions containing electrolytes include PBS, physiological saline (containing only sodium chloride), Dulbecco phosphate-buffered saline, Tris-buffered saline, HEPES-buffered saline, and Veronal-buffered saline, with PBS being particularly preferred. After deposition, the coating film remains firmly attached to the substrate without eluting even when washed with water, PBS, and alcohol. In particular, the copolymer (P) contained in the coating film is important. 1 When the material is a block copolymer, washing with water or phosphate-buffered saline (PBS) results in minimal change in film thickness before and after washing, meaning that the coating film is less likely to dissolve into the solvent.

[0060] <Substrates, cell culture substrates, methods for manufacturing cell culture substrates> In this invention, the term "substrate" refers not only to a flat plate substrate having a surface, but also to containers, devices, etc., having any structure. Examples of such substrates include instruments for collecting or delivering the above-mentioned biological substances (e.g., blood glucose meters, injection needles, catheters, etc.), containers for storing the above-mentioned biological substances (e.g., bags, bottles, vials, etc., specifically blood bags, storage containers for antibody drugs, etc.), instruments for separating, isolating, or analyzing the above-mentioned biological substances (e.g., microscope peripheral instruments such as carriers and cover slips, microfluidic devices including flow cytometers such as cell sorters, cuvettes, cell culture substrates, cell spheroid arrays, cell separation columns, microchannel chips, microwell array chips, assay chips, biochips, magnetic beads, measuring cells for fully automated analyzers, etc.), bioprocessing instruments (e.g., reaction vessels, transfer tubes, transfer pipes, purification instruments, cell culture plates, etc.), prosthetic materials (e.g., implants, bone fixation materials, sutures, adhesion prevention membranes, artificial blood vessels, etc.), as well as drug delivery media such as vesicles, microparticles, and nanoparticles, materials for diagnostic equipment such as gastroscopes, and medical application materials such as microfibers, nanofibers, and magnetic particles. By applying the aforementioned coating film-forming composition to the surface of a substrate and drying it, a substrate with the ability to suppress the adhesion of biological substances can be manufactured. Here, "surface" refers to the surface that comes into contact with biological substances.

[0061] The cell culture substrate of the present invention is a cell culture substrate having the coating film on at least a portion of the substrate surface. Preferably, the coating film is formed over the entire surface on which cell culture is performed. In particular, examples of cell culture substrates include dishes (Petri dishes) such as Petri dishes, tissue culture dishes, and multi-dishes commonly used for cell culture, flasks such as cell culture flasks and spinner flasks, bags such as plastic bags, Teflon® bags, and culture bags, plates such as microplates, microwell plates, multi-plates, and multi-well plates, bottles such as chamber slides, tubes, trays, and roller bottles, culture vessels having internal stirring blades for agitating cell suspensions, large culture tanks, and bioreactors. Preferably, dishes, plates, and trays are used.

[0062] Furthermore, the substrate material can be, for example, glass, metal, metal-containing compound or metalloid-containing compound, activated carbon, or resin. Examples of metals include typical metals (alkali metals: Li, Na, K, Rb, Cs; alkaline earth metals: Ca, Sr, Ba, Ra), magnesium group elements: Be, Mg, Zn, Cd, Hg; aluminum group elements: Al, Ga, In; rare earth elements: Y, La, Ce, Pr, Nd, Sm, Eu; tin group elements: Ti, Zr, Sn, Hf, Pb, Th; iron group elements: Fe, Co, Ni; iron group elements: V, Nb, Ta; chromium group elements: Cr, Mo, W, U; manganese group elements: Mn, Re; precious metals: Cu, Ag, Au; platinum group elements: Ru, Rh, Pd, Os, Ir, Pt, etc. Examples of metal-containing compounds or metalloid-containing compounds include ceramics, which are sintered bodies whose basic component is a metal oxide and are hardened by heat treatment at high temperatures; semiconductors such as silicon; inorganic solid materials such as molded bodies of inorganic compounds including metal oxides or metalloid oxides (silicon oxide, alumina, etc.), metal carbides or metalloid carbides, metal nitrides or metalloid nitrides (silicon nitride, etc.), metal borides or metalloid borides; and aluminum, nickel titanium, and stainless steel (SUS304, SUS316, SUS316L, etc.).

[0063] The resin may be a natural resin or its derivative, or a synthetic resin. Preferred natural resins or their derivatives include cellulose, cellulose triacetate (CTA), nitrocellulose (NC), cellulose immobilized with dextran sulfate, etc. Preferred synthetic resins include polyacrylonitrile (PAN), polyester polymer alloy (PEPA), polystyrene (PS), polysulfone (PSF), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), polyvinyl alcohol (PVA), polyurethane (PU), ethylene vinyl alcohol (EVAL), polyethylene (PE), polyester, polypropylene (PP), polyvinylidene fluoride (PVDF), polyethersulfone (PES), polycarbonate (PC), polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), ultra-high molecular weight polyethylene (UHPE), polydimethylsiloxane (PDMS), acrylonitrile-butadiene-styrene resin (ABS), or Teflon®. In the production of the cell culture substrate of the present invention, since high-temperature treatment is not required when coating the substrate surface with the coating film-forming composition so that it is present on at least a portion of the substrate surface, resins with low heat resistance can also be used.

[0064] The substrate material may be one type or a combination of two or more types. Among these materials, it is preferable that the substrate be glass, silicon, silicon oxide, polystyrene (PS), polypropylene (PP), polyethersulfone (PES), polyethylene terephthalate (PET), polycarbonate (PC), polyvinyl chloride (PVC), Teflon (registered trademark), cycloolefin polymer (COP), polydimethylsiloxane (PDMS), or stainless steel (SUS304, SUS316, SUS316L, etc.) alone or a combination selected from these, and it is particularly preferable that the substrate be glass, polystyrene (PS), polypropylene (PP), stainless steel (SUS304, SUS316, SUS316L, etc.), or polydimethylsiloxane (PDMS).

[0065] The coating film-forming composition of this application can be applied using methods such as spin coating, inkjet printing, screen printing, slit coating, or roll-to-roll, but is preferably applied using printing techniques such as inkjet printing or screen printing.

[0066] Other coating methods include, for example, immersing the container in the coating film-forming composition, adding the coating film-forming composition to the container and letting it stand for a predetermined time, or applying the coating film-forming composition to the surface of the container or substrate. In the case of a container, specifically a cell culture vessel, the coating film-forming composition is added to the container and left to stand for a predetermined time. Addition can be performed, for example, by adding 0.5 to 1 times the total volume of the container's coating film-forming composition using a syringe or the like. The standing time is performed by appropriately selecting the time and temperature depending on the material of the container or substrate and the type of cell culture substrate-forming agent, but for example, it is performed from 1 minute to 24 hours, preferably from 5 minutes to 3 hours, at 10 to 80°C. This makes it possible to manufacture a cell culture vessel on at least a part, preferably the entire surface of the container.

[0067] Furthermore, the coating film on the surface of a container or substrate obtained by this method can be used as a cell culture container either 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, buffer solution, culture medium, etc.), following a step of bringing the coating film into contact with at least a portion of the surface of the container or substrate, preferably by adding a coating film-forming composition and letting it stand for a predetermined time.

[0068] In other words, after the step of bringing the container or substrate into contact with at least a portion of its surface, preferably a coating film-forming composition is added, and after a predetermined period of standing, the container can be used as is without a drying step within 48 hours, preferably within 24 hours, more preferably within 12 hours, more preferably within 6 hours, more preferably within 3 hours, and more preferably within 1 hour, or after washing with water or a medium for the sample to be subjected to cell culture (e.g., water, buffer solution, culture medium, etc., particularly preferably a culture medium (e.g., DMEM medium (Dulbecc's modified Eagle medium))) before being used as a cell culture container.

[0069] The container may be subjected to a drying process. The drying process is carried out under air or vacuum, preferably at a temperature in the range of -200°C to 200°C. The drying process removes the solvent from the above-mentioned undercoat forming agent, causing it to completely adhere to the substrate. The coating film can be formed by drying at room temperature (10°C to 35°C, preferably 20°C to 30°C, for example 25°C), but to form the base film more quickly, it may be dried at, for example, 40°C to 100°C. A more preferable drying temperature is 10°C to 180°C, and a more preferable drying temperature is 20°C to 150°C. The coating film of this invention is manufactured through the simple process described above.

[0070] Furthermore, in order to remove impurities, unreacted monomers, etc. remaining in the coating film, a washing step may be performed with at least one solvent selected from aqueous solutions containing water and electrolytes. Washing is preferably done by running water or ultrasonic cleaning. The aqueous solution containing water and electrolytes may be heated to a temperature range of, for example, 40°C to 95°C. The aqueous solution containing electrolytes is preferably PBS, physiological saline (containing only sodium chloride), Dulbecco's phosphate-buffered physiological saline, Tris-buffered physiological saline, HEPES-buffered physiological saline, and Veronal-buffered physiological saline, with PBS being particularly preferred. After setting, the coating film remains firmly attached to the substrate without eluting even when washed with water, PBS, alcohol, etc.

[0071] The thickness of the coating film of this application is in the range of 1 to 1000 nm, preferably in the ranges of 5 to 500 nm, 10 to 300 nm, 10 to 200 nm, 10 to 100 nm, and 10 to 50 nm.

[0072] <Laminated film> The laminated film of the present invention is a laminated film having a layer of the coating film and a layer of a cell culture substrate film on its upper surface. The cell culture substrate is the following formula (I) described in International Publication No. 2020 / 040247: [ka] [In the formula, U a1 and U a2 Each of these independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, and R a1 R represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. a2 A repeating unit derived from a monomer represented by [where is an alkylene group having 1 to 5 carbon atoms], and the following formula (II): [ka] [In the formula, R b This refers to copolymers (P) containing repeating units derived from monomers represented by [a hydrogen atom or an alkyl group having 1 to 5 carbon atoms]. 2 The film may be a multilayer film containing the above. The examples of "alkyl groups having 1 to 5 carbon atoms" and "alkylene groups having 1 to 5 carbon atoms" are the same as those given in the explanation of formulas (1) and (2).

[0073] The cell culture substrate of the present invention may include the cell culture substrate described in International Publication No. 2020 / 040247. The entire disclosure of International Publication No. 2020 / 040247 is incorporated herein by reference. The range of the maximum and minimum thickness of the cell culture substrate is, for example, in the range of 1 to 1000 nm, preferably in the range of 5 to 500 nm. A cell culture substrate may be provided with the aforementioned multilayer film. The description of the substrate is as described above.

[0074] <Method for producing cell aggregates> The present invention's method for producing cell aggregates includes the step of seeding cells onto the cell culture substrate. Cell aggregates can be produced by known methods, including static culture, in which cells are cultured after seeding, and agitated culture (including shaking culture), in which the cell suspension is agitated using a stirring blade after seeding. In agitated culture, agitation may be stopped for a predetermined time before, during, and after agitation, allowing the cell suspension to settle, and the agitation and resting may be repeated. For example, they can be produced by the method described in the examples below. There are no limitations on the type of cells, and the aforementioned cells are just specific examples. [Examples]

[0075] The present invention will be described more specifically below with reference to examples and comparative examples, but the present invention is not limited to the following examples.

[0076] The weight-average molecular weight (Mw) of polymer (A) shown in the synthesis example below was measured by gel permeation chromatography (GPC). The measurement conditions are as follows. GPC column: Plgel 3μm MIXED-E (manufactured by Agilent Technologies) Column temperature: 40°C Solvent: Tetrahydrofuran (THF) Flow rate: 1.00mL / min Detector: RI detector Standard sample: Polystyrene

[0077] [Synthesis of block copolymers] Figure 1 shows schematic diagrams of the flow reactors used in synthesis examples 1-4 and 6 below. In Figure 1, arrows indicate the direction of liquid flow. A plunger pump A (UI-22-110, manufactured by FLOM Co., Ltd.) was used to deliver the first monomer liquid, and a PTFE tube (1.0 mm inner diameter, 1.6 mm outer diameter, 2 m length) was used to connect plunger pump A to mixer 1. A syringe pump B (Keychem-L, manufactured by YMC Co., Ltd.) was used to deliver the initiator solution, and a PTFE tube (1.0 mm inner diameter, 1.6 mm outer diameter, 2 m length) was used to connect syringe pump B to mixer 1. The outlet of mixer 1 and the inlet of mixer 2 were connected by a PFA tube (2.0 mm inner diameter, 3 mm outer diameter, 1 m length). The other inlet of mixer 2 was connected to syringe pump C (YMC Co., Ltd., Keychem-L) for delivering reaction regulator solution using PTFE tubing (inner diameter 1.0 mm, outer diameter 1.6 mm, length 2 m). The outlet of mixer 2 and the inlet of mixer 3 were connected with PFA tubing (inner diameter 2.0 mm, outer diameter 3 mm, length 1 m). The other inlet of mixer 3 was connected to syringe pump D (YMC Co., Ltd., Keychem-L) for delivering the second monomer liquid using PTFE tubing (inner diameter 1.0 mm, outer diameter 1.6 mm, length 2 m). The outlet of mixer 3 and the inlet of mixer 4 were connected with PFA tubing (inner diameter 2.0 mm, outer diameter 3 mm, length 5 m). The other inlet of mixer 4 was connected to a polymerization inhibitor solution delivery plunger pump E (manufactured by From Co., Ltd., UI-22-110) using a PTFE tube (inner diameter 1.0 mm, outer diameter 1.6 mm, length 2 m). A PFA tube (inner diameter 2.0 mm, outer diameter 3 mm, length 0.7 m) was connected to the outlet of mixer 4. The flow paths from pumps A, B, and C to 90% of the length of the tube connected to the outlet of mixer 2 were immersed in a water bath, and the flow paths from pumps D and E to 90% of the length of the tube connected to the outlet of mixer 4 were immersed in a -40°C constant temperature bath to adjust the temperature.Mixer 1 used in the synthesis was a Comet X-01 (stainless steel) manufactured by Techno Applications Co., Ltd. Mixers 2 and 3 were two-component mixing mixers with a double-tube structure as described in International Publication No. 2017 / 135398 [using stainless steel for the joint members and cylindrical body, and a modified DSP-MXA3-17 (polyacetal element, 17.3 mm diameter with twisted blades) manufactured by Noritake Co., Ltd. as the static mixer element] Mixer 4 was a general, simple double-tube mixer. The connection method for each mixer was as follows: Mixer 1 was connected to the inlet of the first monomer solution tube and to the inlet of the inner tube with the initiator solution tube. Mixer 2 was connected to the inlet of A polymerization inhibitor solution was connected to the inlet of mixer 4, and a tube connected to the outlet of mixer 3 was connected to the inlet of the inner tube.

[0078] <Synthesis Example 1: Synthesis of Polystyrene-b-Poly(Diethylene Glycol Monomethyl Ether Methacrylate) Block Copolymer (PS-b-PDEGMA)> As the first monomer, a 0.25 mol / L styrene THF solution (containing 210 mg of lithium chloride as an additive salt) and as an initiator, a 0.05 mol / L n-butyllithium toluene / hexane solution were mixed in mixer 1 at flow rates of 10 mL / min and 2.0 mL / min, respectively, to polymerize the first monomer. Subsequently, 1,1-diphenylethylene (DPE) was mixed in mixer 2 at a flow rate of 19 μL / min to adjust the reactivity with the second monomer. As the second monomer, a diethylene glycol monomethyl ether methacrylate solution was mixed in mixer 3 at a flow rate of 1.85 mL / min to undergo block polymerization. Subsequently, a 0.25 mol / L methanol / THF solution was mixed in mixer 4 at a flow rate of 10 mL / min to stop the polymerization. Each pump was run for 3 minutes, and the effluent was collected. Furthermore, after the solvent was largely removed from the effluent using an evaporator, it was added dropwise to a mixture of 300 ml of diethyl ether, and a precipitate was obtained by removing the supernatant from the resulting white suspension. Subsequently, the obtained precipitate was vacuum-dried to obtain 1.60 g of PS-b-PDEGMA. Analysis of the obtained copolymer by GPC revealed Mn = 41,258 and Mw / Mn = 1.15. 1 From the 1H-NMR results, the composition ratio of polystyrene units to diethylene glycol monomethyl ether methacrylate units was 24:76. 1 The H-NMR chart is shown in Figure 2.

[0079] <Synthesis Example 2: Synthesis of Polystyrene-b-Poly(Diethylene Glycol Monomethyl Ether Methacrylate) Block Copolymer (PS-b-PDEGMA)> The copolymer was synthesized in the same manner as in Synthesis Example 1, except that the initiator solution concentration was 0.09 mol / L, DPE was mixed in Mixer 2 at 35 μL / min, and diethylene glycol monomethyl ether methacrylate solution was mixed as the second monomer in Mixer 3 at 2.77 mL / min, yielding 3.29 g of PS-b-PDEGMA. The obtained copolymer was analyzed by GPC and found to have Mn = 24,751 and Mw / Mn = 1.39. 1 From the 1H-NMR results, the composition ratio of polystyrene units to diethylene glycol monomethyl ether methacrylate units was 11:89.

[0080] <Synthesis Example 3: Synthesis of Polystyrene-b-Poly(Diethylene Glycol Monomethyl Ether Methacrylate) Block Copolymer (PS-b-PDEGMA)> The first monomer solution was prepared at a concentration of 0.20 mol / L (containing 395 mg of lithium chloride as an added salt), the initiator solution at a concentration of 0.09 mol / L, and DPE was mixed in mixer 2 at 35 μL / min. Diethylene glycol monomethyl ether methacrylate solution was added as the second monomer in mixer 3 at 4.06 mL / min, and each pump was run for 1 minute. The synthesis was carried out in the same manner as in Synthesis Example 1, yielding 1.50 g of PS-b-PDEGMA. Analysis of the obtained copolymer by GPC revealed Mn = 21,721 and Mw / Mn = 1.50. 1 From the 1H-NMR results, the composition ratio of polystyrene units to diethylene glycol monomethyl ether methacrylate units was 8:92.

[0081] <Synthesis Example 4: Synthesis of Polystyrene-b-Poly(Diethylene Glycol Monomethyl Ether Methacrylate) Block Copolymer (PS-b-PDEGMA)> The polymer was synthesized in the same manner as in Synthesis Example 1, except that the solution concentration of the first monomer was 0.10 mol / L (containing 356 mg of lithium chloride as an added salt), the initiator solution concentration was 0.09 mol / L, DPE was mixed in mixer 2 at 35 μL / min, and diethylene glycol monomethyl ether methacrylate solution was mixed in mixer 3 at 4.06 mL / min as the second monomer, yielding 4.70 g of PS-b-PDEGMA. The obtained polymer was analyzed by GPC and found to have Mn = 32,175 and Mw / Mn = 1.28. 1 From the 1H-NMR results, the composition ratio of polystyrene units to diethylene glycol monomethyl ether methacrylate units was 3:97.

[0082] <Synthesis Example 5: Synthesis of Polystyrene-r-Poly(Diethylene Glycol Monomethyl Ether Methacrylate) Random Copolymer (PS-r-PDEGMA)> 55.3 mg of styrene and 2.0 g of diethylene glycol monomethyl ether methacrylate were dissolved in 18.50 g of toluene and 11.1 mg of 2,2'-azodiisobutyronitrile. The mixture was then stirred for 24 hours under a nitrogen atmosphere in a water bath at 75°C. After the reaction mixture returned to room temperature, the solvent was mostly removed using an evaporator. The mixture was then added dropwise to a 300 mL mixture of diethyl ether, and the supernatant was removed from the resulting white suspension to obtain a precipitate. The precipitate was then vacuum-dried to obtain 0.64 g of PS-b-PDEGMA. Analysis of the copolymer by GPC revealed Mn = 10,361 and Mw / Mn = 2.26. 1 From the 1H-NMR results, the composition ratio of polystyrene units to diethylene glycol monomethyl ether methacrylate units was 7:93.

[0083] <Synthesis Example 6: Synthesis of Polystyrene-b-Poly(Hydroxyethyl Methacrylate) Block Copolymer (PS-b-PHEMA)> The first monomer solution was prepared at a concentration of 0.10 mol / L (containing 117 mg of lithium chloride as an added salt), the initiator solution at a concentration of 0.05 mol / L, DPE was mixed in mixer 2 at 17 μL / min, and 2-(trimethylsiloxy)ethyl methacrylate solution was mixed in mixer 3 at 1.94 mL / min as the second monomer. The synthesis was carried out in the same manner as in Synthesis Example 1, yielding 1.25 g of PS-b-PHEMA precursor. Analysis of the obtained polymer by GPC revealed Mn = 28,877 and Mw / Mn = 1.67. Subsequently, the obtained precursor polymer was suspended in 500 mL of diethyl ether, 500 μL of 35% hydrochloric acid was added, and the mixture was heated overnight at room temperature. This suspension was filtered through a 1.0 μm membrane filter to obtain 1.08 g of PS-b-PHEMA. 1¹H-NMR results confirmed that the peak originating from the trimethylsilyl protecting group had completely disappeared. Furthermore, the composition ratio of polystyrene units to polyhydroxyethyl methacrylate units was 6.5:93.5.

[0084] <Preparation Examples 1-5: Preparation of Polymer Ethanol / Aqueous Solution> The polymers from Synthesis Examples 1 to 5 were each dissolved in ethanol / water (95 / 5 weight percent concentration) to a concentration of 10 mg / g to prepare coating film-forming compositions (Preparation Examples 1 to 5).

[0085] <Preparation Example 6: Preparation of Polymer Ethanol Solution> The polymer from Synthesis Example 6 was dissolved in ethanol to a concentration of 10 mg / g to prepare a coating film-forming composition (Preparation Example 6).

[0086] <Comparative Preparation Example 1: Preparation of Polymer Aqueous Solution> Polyethylene glycol 20000 (manufactured by Junsei Chemical Co., Ltd., weight-average molecular weight 15,500-253,000) was dissolved in pure water to a concentration of 10 mg / g to prepare a coating film-forming composition (comparative preparation example 1).

[0087] <Reference example 1> A coating film-forming composition (Reference Example 1) was prepared by adding 49.75g of ethanol to 0.25g of Lipidure-CM5206 (manufactured by NOF Corporation) and stirring thoroughly.

[0088] <Test Example 1: Coating Film Formation Test> The coating film-forming compositions obtained in Preparation Examples 1-6 and Comparative Preparation Example 1 were spin-coated onto HMDS-treated silicon wafers at 1500 rpm / 60 seconds, and then dried in a 50°C oven for 24 hours as a drying step. After that, they were thoroughly washed with PBS and dried in a 50°C oven for 1 hour to obtain a coating film on the HMDS-treated silicon wafer. The film thickness of the coating film on the HMDS-treated silicon wafer, measured using a spectroscopic ellipsometer, is shown in Table 1 below. A coating film was formed when any of the coating film-forming compositions obtained in Preparation Examples 1-6 were used. When the coating film-forming composition obtained in Comparative Preparation Example 1 was used, it repelled on the substrate and did not form a uniform film.

[0089] [Table 1]

[0090] <Test Example 2: Colloidal Particle Size Measurement Test> When the coating film-forming compositions obtained in Preparation Examples 1, 2, and 4 were placed in a glass container and illuminated from the side with an LED light, the Tyndall effect was observed, confirming that they were colloidal dispersions rather than true solutions. Next, the average particle size of the coating film-forming compositions obtained in Preparation Examples 1, 2, and 4 was measured using dynamic light scattering (DLS) with a Zetasizer Nano ZS (manufactured by Malvern Panalytical).

[0091] [Table 2]

[0092] <Test Example 3: Protein Attachment Inhibition Test> (Fabrication of coated plates) The coating film-forming compositions prepared in Preparation Examples 1-6 were added to separate wells of a 96-well cell culture plate (Corning, #9017, 0.36 mL, polystyrene) at a concentration of 20 μL / well for 5 wells each. After the solvent evaporated at room temperature, the wells were dried in an oven at 50°C for 24 hours. Subsequently, each coated well was washed three times with 100 μL of pure water, dried in an oven at 50°C for 1 hour, and then used for testing. As a positive control, the coating film-forming composition prepared in Reference Example 1 was used to coat the wells of a 96-well cell culture plate (Corning, #9017, 0.36 mL, polystyrene) using the same method as above. As a negative control, wells of an uncoated 96-well cell culture plate (Corning, #9017, 0.36 mL, polystyrene) were used.

[0093] (Preparation of diluted IgG-HRP solution) Goat anti-mouse IgG antibody HRP conjugate (Southern Biotechnology Associates) was diluted with PBS to a concentration of 1 mg / g to prepare an IgG-HRP dilution.

[0094] (Protein adhesion experiment) For each well of the plate prepared as described above, as well as for the positive and negative controls, 100 μL / well of IgG-HRP diluent was added, and the plates were allowed to stand at room temperature for 30 minutes. After 30 minutes, the IgG-HRP diluent was drained, and each well was washed three times with 200 μL of PBS. 100 μL / well of TMB solution (SureBlue, sera care) was added, and after 1 minute, 100 μL / well of TMB STOP solution (sera care) was added. Using a microplate reader (infinite M200PRO, TECAN), the absorbance at 450 nm and 650 nm was measured. The average absorbance of 5 wells for each coating film-forming composition was calculated by subtracting the absorbance at 650 nm from the absorbance at 450 nm. The average absorbance in the negative control well was set as 100% protein adsorption, and the protein adsorption rates of the wells coated with each coating film-forming composition were calculated and are shown in Table 3. In all wells coated with any of the coating film-forming compositions, the amount of protein adhesion was lower compared to the uncoated wells (negative control).

[0095] [Table 3]

[0096] <Test Example 4: Cell adhesion inhibition test using mouse fibroblasts> (Fabrication of coated plates) The coating film-forming compositions prepared in Preparation Examples 1-6 were added to separate wells of a 96-well cell culture plate (Corning, #351172, 0.37 mL, polystyrene) at a concentration of 200 μL / well. After standing at room temperature for 1 hour, excess varnish was removed. The plates were then dried in an oven at 50°C for 24 hours. Subsequently, each coated well was washed three times with 250 μL of pure water, dried in an oven at 50°C for 1 hour, and then used for testing. As a positive control, the coating film-forming composition prepared in Reference Example 1 was used to coat the wells of a 96-well cell culture plate (Corning, #351172, 0.37 mL, polystyrene) using the same method as described above. As a negative control, an uncoated 96-well cell culture plate (Corning, #351172, 0.37 mL, polystyrene) was used.

[0097] (Cell preparation) The cells used were mouse embryonic fibroblasts C3H10T1 / 2 (DS Pharma Biomedical). The culture medium used for the cells was BME medium (Thermo Fisher Scientific) containing 10% FBS (Sigma-Aldrich) and L-glutamine-penicillin-streptomycin stabilized solution (Thermo Fisher Scientific). The cells were cultured statically for at least 2 days in a 10 cm diameter petri dish (10 mL of medium) at a 37°C / CO2 incubator while maintaining a 5% carbon dioxide concentration. Subsequently, the cells were washed with 5 mL of PBS, and then 1 mL of 0.25 w / v% trypsin-1 mmol / L EDTA solution (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to detach the cells, and each cell was suspended in 10 mL of the above medium. After centrifuging the suspension (using a Tommy Seiko Co., Ltd. LC-200, 1000 rpm / 3 min, at room temperature), the supernatant was removed, and the above-mentioned culture medium was added to prepare the cell suspension.

[0098] (Cell adhesion experiment) For each well of the plate prepared above, as well as for the positive control and negative control, add 1 × 10⁶ cells of the cell suspension to each well. 4150 μL was added to each cell / well to achieve a cell / well ratio. The cells were then left to stand in a CO2 incubator at 37°C for 3 days while maintaining a 5% carbon dioxide concentration.

[0099] (Observation of cell attachment) After 3 days of culture, cell adhesion to each well of the plates prepared as described above, as well as to the positive and negative controls, was compared based on observations using an inverted microscope (Nikon Corporation, ECLIPSE TS100-F). The results are shown in Table 4. As shown in Table 4, in plates treated with the coating film-forming compositions prepared in Preparation Examples 1-6 and Reference Example 1 (positive control), cell adhesion and spread were suppressed, and the formation of cell aggregates (spheroids) was observed in the wells. On the other hand, in the uncoated plate (negative control), although slight spheroid formation was observed, most cells adhered to and spread on the bottom surface of the plate. As a representative example of cell observation, micrographs of each well of the plate coated with the coating film-forming composition of Preparation Example 2 and the positive control (Reference Example 1), and the negative control are shown in Figure 3.

[0100] [Table 4]

[0101] <Test Example 5: Cell adhesion inhibition test using human adipose tissue-derived mesenchymal stem cells> (Fabrication of coated plates) Using the coating film-forming compositions prepared in Preparation Examples 3, 6, and Reference Example 1, a coating plate was prepared in the same manner as in Test Example 4.

[0102] (Cell preparation) Human adipose tissue-derived mesenchymal stem cells (ADSCs) (CellSource, Inc.) were used. The culture medium used for the cells was Mesenchymal Stem Cell Growth Medium 2 (PromoCell), a low-serum medium containing L-glutamine-penicillin-streptomycin stabilized solution (Thermo Fisher Scientific). The cells were cultured statically for at least 2 days in a 10 cm diameter petri dish (10 mL of medium) at a 37°C / CO2 incubator while maintaining a 5% carbon dioxide concentration. Subsequently, the cells were washed with 5 mL of PBS, and then 1 mL of TrypLE Select Enzyme (Thermo Fisher Scientific) was added to detach the cells. Each cell was then suspended in 10 mL of the above medium. The suspensions were centrifuged (Tommy Seikou Co., Ltd., model LC-200, 1000 rpm / 3 min, room temperature), the supernatant was removed, and the above medium was added to prepare the cell suspension.

[0103] (Cell adhesion experiment) For each well of the plate prepared above, as well as for the positive control and negative control, add 1 × 10⁶ cells of the cell suspension to each well. 4 150 μL was added to each cell / well to achieve a cell / well ratio. The cells were then left to stand in a CO2 incubator at 37°C for 3 days while maintaining a 5% carbon dioxide concentration.

[0104] (Observation of cell attachment) After 3 days of culture, cell adhesion to each well of the plates prepared as described above, as well as to the positive and negative controls, was compared based on observations using an inverted microscope (Olympus CKX31). The results are shown in Table 4. As shown in Table 4, in plates treated with the coating film-forming compositions prepared in Preparation Examples 3, 6, and Reference Example 1 (positive control), cell adhesion and spread were suppressed, and the formation of cell aggregates (spheroids) was observed in the wells. On the other hand, in the uncoated plate (negative control), although slight spheroid formation was observed, most cells adhered to and spread on the bottom surface of the plate. Micrographs of each well of the plates coated with the coating film-forming compositions of Preparation Examples 3, 6, and the positive control (Reference Example 1), as well as the negative control, are shown in Figure 4.

[0105] [Table 5]

[0106] <Test Example 6: Cell Aggregation Formation Test Using Mouse Fibroblasts> (Preparation of low-adhesion cell plates) The coating film-forming composition prepared in Preparation Example 3 was applied to a 24-well cell culture plate (Corning, #351147, culture area 2 cm²). 2 The varnish was added to separate wells (made of polystyrene) at a concentration of 0.5 mL / well, allowed to stand at room temperature for 1 hour, and then the excess varnish was removed. The plates were dried in an oven at 50°C for 24 hours to obtain low-adhesion cell plates.

[0107] (Preparation of a base layer forming agent for cell culture) A polymer for use as a cell culture substrate was obtained according to the manufacturing method described in Synthesis Example 8 of International Publication No. 2020 / 040247. The obtained polymer was dissolved in water to concentrations of 2000, 1000, 500, and 150 μg / mL to prepare cell culture substrates.

[0108] (Preparation of plates for cell aggregate formation using inkjet technology) Using an inkjet device (MicroJet Co., Ltd., model number: LaboJet-600) and an inkjet head (model number: IJHBS-1000), an appropriate amount of the cell culture substrate forming agent prepared above was applied to the low-adhesion cell petri dish prepared above. The plates were dried in a 70°C oven for 24 hours to prepare cell aggregate formation plates (wells No. 1-4).

[0109] (Cell preparation) Cell preparation was carried out using the same method as in Test Example 4.

[0110] (Observation of cell adhesion) Add the cell suspension to the petri dish prepared above for cell aggregate formation. 5 cells / cm 2 0.8 mL of each was added to achieve the desired result. The mixture was then left to stand for 2 hours in a 37°C CO2 incubator while maintaining a 5% carbon dioxide concentration. After standing, the cell adhesion to the coated areas of each coating agent was observed using an inverted microscope (Olympus CKX31). As a result, as shown in Figure 5, it was confirmed that cells, which were dispersed throughout the entire surface immediately after seeding, did not adhere to or spread to the areas in wells No. 1 to 4 where the coating film-forming composition prepared in Preparation Example 3 was exposed, but selectively adhered to the areas where the cell culture base film-forming agent was applied to the upper layer.

[0111] (Observation of cell aggregates) The petri dishes in which cell adhesion was confirmed as described above were left to stand for another day in a 37°C CO2 incubator. After standing, the cells were observed using an inverted microscope (Olympus CKX31). As a result, as shown in Figure 6, it was confirmed that the cells that had adhered to the areas in wells No. 1 to 4 coated with the cell culture substrate were detached from the plate and aggregated, forming cell aggregates (spheroids). Even at this time, cells did not adhere to or spread in the areas where the coating film-forming composition prepared in Preparation Example 3 was exposed. From this, it was suggested that the substrate film containing the polymer of the present invention is useful as a low-cell adhesion film for cell culture vessels. The results at each stage of cell adhesion maintenance 2 hours after seeding and cell aggregate formation are shown in Table 6.

[0112] [Table 6]

[0113] <Test Example 7: Cell Aggregation Formation Test Using Mouse Fibroblasts> (Preparation of low-adhesion cell plates) The coating film-forming composition prepared in Preparation Example 3 was spin-coated onto a PS sheet at 1500 rpm / 60 seconds, and then dried in a 50°C oven for 24 hours as a drying step. After that, it was adhered to a 24-well type open-bottom plate to obtain a low-cell adhesion plate.

[0114] (Preparation of a base layer forming agent for cell culture) A polymer to be used as a base film-forming agent for cell culture was obtained using the same method as in Test Example 5. The obtained polymer was dissolved in water to concentrations of 2000, 1000, and 500 μg / mL to prepare base film-forming agents for cell culture.

[0115] (Preparation of plates for cell aggregate formation using inkjet technology) Using an inkjet device (manufactured by Microjet Co., Ltd., model number: LaboJet-600) and an inkjet head (model number: 500-S-C), an appropriate amount of the cell culture basement membrane forming agent prepared above was applied to the cell low-adhesion petri dish prepared above. It was dried in an oven at 70 °C for 24 hours to prepare a plate for forming cell aggregates (well Nos. 5 to 7).

[0116] (Preparation of cells) Cells were prepared in the same manner as in Test Example 4.

[0117] (Observation of cell adhesion) For the petri dish for forming cell aggregates prepared above, 0.8 mL of the cell suspension was added to each so that it became 3.0×10 5 cells / cm 2 Thereafter, it was left standing in a CO2 incubator at 37 °C for 2 hours while maintaining a 5% carbon dioxide concentration. After standing, using an inverted microscope (manufactured by Olympus, CKX31), the state of cell adhesion to the coated portions of each coating agent was confirmed. Thereafter, the floating cells and the medium were removed with an aspirator and washed with PBS to leave only the adherent cells on the well. After washing, 0.8 mL of fresh medium was added to each, and the state of the adherent cells was observed using an inverted microscope (manufactured by Olympus, CKX31). As a result, as shown in Fig. 7, the cells that were dispersed over the entire surface immediately after seeding selectively adhered to the portions where the coating film forming composition prepared in Preparation Example 3 was exposed in well Nos. 5 to 7, and did not adhere and spread to the portions where the cell culture basement membrane forming agent was applied to the upper layer. It was confirmed that cell adhesion was maintained even after medium replacement.

[0118] (Observation of cell aggregates) The petri dishes in which cell adhesion was confirmed as described above were left to stand for another day in a 37°C CO2 incubator. After standing, the cells were observed using an inverted microscope (Olympus CKX31). As a result, as shown in Figure 8, it was confirmed that the cells that had adhered to the areas in wells No. 5-7 coated with the cell culture base film forming agent had detached from the plate and aggregated, forming cell aggregates (spheroids). Even at this time, cells did not adhere to or spread in the areas where the coating film forming composition prepared in Preparation Example 3 was exposed. From this, it was suggested that the base film containing the polymer of the present invention is useful as a low-cell adhesion film for cell culture vessels. The results at each stage—cell adhesion before washing, maintenance of cell adhesion after washing, and formation of cell aggregates—are shown in Table 7.

[0119] [Table 7] [Industrial applicability]

[0120] According to the present invention, a coating film-forming composition having compatibility with biomaterials, a coating film using the same, and a cell culture substrate using the same can be provided.

Claims

1. Equations (1) and (2): 【Chemistry 1】 (In the formula, R １ ~R ３ Each of these independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, X １ X represents a single bond, ester bond, amide bond, or alkylene group having 1 to 5 carbon atoms. ２ This represents an alkylene group with 1 to 5 carbon atoms, T １ (where n1 represents a halogen atom, an alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom, a hydroxyl group, a carboxyl group, a nitro group, a cyano group, a methylenedioxy group, an acetoxy group, a methylthio group, an amino group, or an alkoxy group having 1 to 10 carbon atoms, where n1 represents an integer from 0 to 2, n2 represents an integer from 1 to 10, and n3 represents an integer from 0 to 8) is a copolymer (P) that contains a unit structure and does not contain a polysiloxane skeleton. １ ), and a solvent, A coating film-forming composition wherein the molar ratio of the unit structure represented by formula (1) to the unit structure represented by formula (2) is 1 to 30:99 to 70.

2. The composition according to claim 1, wherein the solvent is selected from water, alcohol, or a combination thereof.

3. The solvent is used as a dispersion medium, and the copolymer (P) is used as a dispersed phase. １ The coating film-forming composition according to claim 1 or 2, in the form of a colloidal solution containing ).

4. The coating film forming composition according to any one of claims 1 to 3, wherein the unit structure represented by formula (1) is derived from styrene.

5. The coating film forming composition according to any one of claims 1 to 4, wherein the unit structure represented by formula (2) is derived from ethylene glycol monomethacrylate, diethylene glycol monomethyl ether methacrylate, or triethylene glycol monomethyl ether methacrylate.

6. A coating film-forming composition having the ability to suppress the adhesion of biological substances, according to any one of claims 1 to 5.

7. Equations (1) and (2): 【Chemistry 2】 (wherein, R １ ~R ３ each independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, X １ represents a single bond, an ester bond, an amide bond or an alkylene group having 1 to 5 carbon atoms, X ２ represents an alkylene group having 1 to 5 carbon atoms, T １ represents a halogen atom, an alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom, a hydroxy group, a carboxy group, a nitro group, a cyano group, a methylenedioxy group, an acetoxy group, a methylthio group, an amino group or an alkoxy group having 1 to 10 carbon atoms, n1 represents an integer of 0 to 2, n2 represents an integer of 1 to 10, n3 represents an integer of 0 to 8), and contains a copolymer (P １ ) which does not contain a polysiloxane skeleton, Herein, the coating film is characterized in that the molar ratio of the unit structure represented by formula (1) to the unit structure represented by formula (2) is 1 to 30:99 to 70.

8. The coating film according to claim 7, wherein the unit structure represented by formula (1) is derived from styrene.

9. The coating film according to claim 7 or 8, wherein the unit structure represented by formula (2) is derived from ethylene glycol monomethacrylate, diethylene glycol monomethyl ether methacrylate, or triethylene glycol monomethyl ether methacrylate.

10. A coating film according to any one of claims 7 to 9, which has the ability to suppress the adhesion of biological substances.

11. A cell culture substrate comprising a coating film according to any one of claims 7 to 10 on a portion of the substrate surface.

12. A laminated film having a layer of a coating film according to any one of claims 7 to 10 and a layer of a cell culture underlayer film on its upper surface.

13. The cell culture substrate is given by the following formula (I): 【Transformation 3】 [In the formula, U ａ１ and U ａ２ Each of these independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, R a１ R represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. a２ A repeating unit derived from a monomer represented by [where is an alkylene group having 1 to 5 carbon atoms], and the following formula (II): 【Chemistry 4】 [In the formula, R ｂ This is a copolymer (P) containing repeating units derived from monomers represented by [a hydrogen atom or an alkyl group having 1 to 5 carbon atoms]. ２ The laminated film according to claim 12, comprising )

14. A cell culture substrate comprising the multilayer film according to claim 12 or 13.

15. A method for producing cell aggregates, comprising the step of seeding cells onto a cell culture substrate according to claim 11 or 14.

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