Coating film with compatibility with biomaterials including block copolymers

Abstract

The purpose of the present invention is to provide a composition for forming a biocompatible coating film, said composition comprising a block copolymer having a specific structure, a coating film using the same, a cell culture substrate using the same, a method for manufacturing a cell culture substrate, and a method for producing a cell aggregate.　A composition for forming a biocompatible coating film, said composition comprising a block copolymer having unit structures represented by formulae (1) and (2) (in the formulae: R1 to R3, U1 and U2 independently represent each a hydrogen atom or an alkyl group having 1-5 carbon atoms; X1 and X2 represent an alkylene group having 1-5 carbon atoms; and n represents an integer of 1-10) and a solvent.

Description

Technical Field

[0001] The present invention relates to a composition for forming a coating film having compatibility with a biological substance containing a block copolymer, a coating film using the same, a substrate for cell culture using the same, a method for producing a substrate for cell culture, 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 medical devices such as artificial dialyzers, artificial organs, and medical instruments, and various medical application devices such as cell culture containers and substrates for cell culture.

[0003] 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 base film for cell culture and a cell culture container.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] [[ID=I43]]An object of the present invention is to provide a composition for forming a coating film having compatibility with a biological substance containing a block copolymer having a specific structure, a coating film using the same, a substrate for cell culture using the same, a method for producing a substrate for cell culture, and a method for producing cell aggregates.

[0006] The inventors have discovered that a coating film containing a block copolymer having a specific structure is particularly excellent in inhibiting protein adhesion, and also possesses the unique property of attracting cells but not adhering to them when used as a coating film for cell culture substrates, thus exhibiting excellent effects, especially in the production of cell aggregates, and have completed the present invention. [Means for solving the problem]

[0007] The present invention is as follows: [1] Equations (1) and (2): [ka] (In the formula, R 1 ~R 3 , U 1 and U 2 Each of these independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, X 1 and X 2 A coating film-forming composition having compatibility with biomaterials, comprising a block copolymer having a unit structure represented by (where each independently represents an alkylene group with 1 to 5 carbon atoms, and n1 represents an integer from 1 to 10), and a solvent. [2] The composition according to [1] above, wherein the solvent comprises water or alcohol. [3] The composition according to [1] or [2] above, wherein the compatibility with the above-mentioned biomolecules is the ability to inhibit protein adhesion. [4] The composition according to [1] or [2] above, wherein the compatibility with the above-mentioned biomaterial is for forming a base film for cell culture. [5] The composition described in [4] above, for forming a base film for cell culture, which is used to adhere cells and then detach them to obtain cell aggregates. [6] A coating film having compatibility with biomaterials, which is a coating film of a coating film forming composition having compatibility with biomaterials as described in any one of [1] to [5] above. [7] A cell culture substrate comprising, on a substrate having an ability to suppress the adhesion of a biological substance, a coating film having compatibility with the biological substance described in [6] above, at least on a part of the substrate surface. [8] A substrate having an ability to suppress the adhesion of a biological substance is a copolymer containing a repeating unit containing a group represented by the following formula (a) and a repeating unit containing a group represented by the following formula (b): [Chemical formula] (In the formula, U a1 、U a2 、U b1 、U b2 and U b3 each independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, and An - represents an anion selected from the group consisting of halide ions, inorganic acid ions, hydroxide ions, and isothiocyanate ions) is a substrate provided with a coating film on at least a part of its surface, and is the cell culture substrate according to [7] above. [9] A method for producing a cell culture substrate, comprising applying and then drying a composition for forming a coating film having compatibility with a biological substance, which comprises a block copolymer having a unit structure represented by the formulas (1) and (2): [Chemical formula] (In the formula, R 1 ~R 3 、U 1 and U 2 each independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, X 1 and X 2 each independently represents an alkylene group having 1 to 5 carbon atoms, and n1 represents an integer of 1 to 10) and a solvent, on a substrate having an ability to suppress the adhesion of a biological substance.

[10] On a substrate having an ability to suppress the adhesion of a biological substance, the formulas (1) and (2): [Chemical formula] (In the formula, R 1 ~R 3 , U 1 and U 2 Each of these independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, X 1 and X 2 A method for producing cell aggregates, comprising the steps of providing a cell culture base film on at least a portion of the substrate surface, which is made of a block copolymer having a unit structure represented by (where each independently represents an alkylene group having 1 to 5 carbon atoms, and n1 represents an integer from 1 to 10), and then seeding cells onto the base film. [Effects of the Invention]

[0008] The block copolymer of the present invention comprises hydrophobic moieties (for example, blocks having a unit structure represented by formula (1)) and hydrophilic moieties (for example, blocks having a unit structure represented by formula (2)). When formed as a coating film on a substrate, it exhibits excellent ability to inhibit protein adhesion and also possesses the unique property of attracting cells but not adhering to them. It is presumed that the hydrophobic moieties are fixed to the substrate and the hydrophilic moieties are exposed on the surface, causing the substrate surface to exhibit hydrophilicity, thereby resulting in the unique properties with respect to cells described above. [Brief explanation of the drawing]

[0009] [Figure 1] This is a schematic diagram of the flow reactor (reaction apparatus) used in synthesis examples 1-5. [Figure 2] This is the 1H-NMR chart of the polymer obtained in Synthesis Example 1. [Figure 3] This is the 1H-NMR chart of the polymer obtained in Synthesis Example 4. [Figure 4] This is a microscopic photograph of the cell adhesion observed in petri dishes coated with each of the coating film-forming compositions prepared in Test Example 4, from Preparation Example 6 to 9. [Figure 5] This is a microscopic photograph showing the formation of cell aggregates in petri dishes coated with each of the coating film-forming compositions from Preparation Examples 6 to 9 in Test Example 4. [Modes for carrying out the invention]

[0010] <Composition for forming coating films that are compatible with biomaterials> The present invention provides a coating film-forming composition compatible with biomaterials, comprising formulas (1) and (2): [ka] (In the formula, R 1 ~R 3 , U 1 and U 2 Each of these independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, X 1 and X 2 The composition for forming a coating film that is compatible with biomaterials comprises a block copolymer having a unit structure (where each independently represents an alkylene group with 1 to 5 carbon atoms, and n1 represents an integer from 1 to 10), and a solvent.

[0011] Examples of 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 , U 1 and U 2 Each of these is preferably independently selected from a hydrogen atom, a methyl group, or an ethyl group.

[0012] Examples of 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. For n1, an integer between 1 and 6 is preferred, and an integer between 2 and 4 is more preferred.

[0013] The unit structure represented by the above formula (1) is shown in the following formulas (1-1) and (2-1): [ka] It is preferable to derive from a compound represented by the formula, where R 1 ~R 3 , U 1 and U 2 , X 1 and X 2 The meaning and preferred embodiment of n1 are the same as described above.

[0014] Specific examples of monomers represented by formula (1-1) include 2-dimethylaminoethyl methacrylate, 2-diethylaminoethyl methacrylate, and 2-dipropylaminoethyl methacrylate, but it is preferable to use 2-dimethylaminoethyl methacrylate and 2-diethylaminoethyl methacrylate.

[0015] Specific examples of monomers represented by formula (2-1) include diethylene glycol monomethyl ether methacrylate and dipropylene glycol monomethyl ether methacrylate, but it is preferable to use diethylene glycol monomethyl ether methacrylate.

[0016] The block copolymer of the present invention preferably contains two types of blocks polymerized from one compound each that derives the units of formula (1) and formula (2) above, but may also contain three or more types of blocks polymerized from two or more compounds, one or both of which are derived from two or more compounds. Compounds that induce the repeating units of the third component, other than those represented by formulas (1) and (2) above, may be further polymerized.

[0017] The molar ratios of the unit structures represented by formulas (1) and (2) are preferably formula (1):(2) = 20-90:80-10, and preferably 40-70:60-30.

[0018] The molar ratio of the unit structures represented by formulas (1) and (2) to the entire block copolymer is not particularly limited as long as it does not impair compatibility with biomaterials, but for example, it may be 80 mol% or more, 90 mol% or more, or 100 mol%.

[0019] The above block copolymers can be synthesized by known methods, but may also be produced using a flow reactor equipped with a specific mixer as described in International Publication No. 2017 / 135398, for example.

[0020] The block copolymer of the present invention is a polymer composed of multiple types of polymers (blocks) that are chemically different and covalently bonded to each other. 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 organic monomer (a) as a unit structure and bonded to the organic polymer chain (A). Specific embodiments of the block copolymer used in the present invention include a polymer (block) consisting of repeating units represented by formula (1) and a polymer (block) consisting of repeating units represented by formula (2), in which both blocks are covalently bonded to each other.

[0021] The solid content of the coating film-forming composition of the present invention 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.

[0022] 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.

[0023] The number of block types present in the block copolymer can be two or more.

[0024] Furthermore, the number of blocks present in the block copolymer can be 2 or 3 or more.

[0025] 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.

[0026] 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).

[0027] 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).

[0028] The weight-average molecular weight Mw of the block copolymer used in the present invention 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 on the substrate may be poor, and if it is greater than 1,000,000, the solubility in the solvent may be poor.

[0029] The polydispersity (Mw / Mn) of the block copolymer of the present invention is preferably 1.00 to 2.00, and particularly preferably 1.00 to 1.50.

[0030] The solvent contained in the coating film-forming composition having compatibility with biomaterials of the present invention includes water, phosphate-buffered saline (PBS), alcohol, or a mixed solvent of two or more of these. Examples of alcohols include alcohols having 2 to 6 carbon atoms, such as ethanol, propanol, isopropanol, 1-butanol, 2-butanol, isobutanol, t-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 1-heptanol, 2-heptanol, 2,2-dimethyl-1-propanol (neopentyl alcohol), 2-methyl-1-propanol, 2-methyl-1-butanol, 2-methyl-2-butanol (t-amyl alcohol), 3-methyl-1-butanol, 3-methyl-3-pentanol, cyclopentanol, 1-hexanol, 2-hexanol, 3-hexanol, and 2,3-dimethyl-2-butanol. Examples include methyl-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 individually or as mixed solvents of combinations thereof. However, from the viewpoint of dissolving block copolymers, it is preferable to select from water, PBS, ethanol, and mixed solvents of two or more of these.

[0031] In the present invention, biomaterials include proteins, sugars, viruses, nucleic acids, cells, or combinations thereof.

[0032] Compatibility with biomolecules means having the ability to inhibit the adhesion of the above-mentioned biomolecules (especially proteins) and not being toxic to the above-mentioned biomolecules. Alternatively, it means that it can be used for forming a base film for cell culture, and particularly preferably for forming a base film for cell culture to obtain cell aggregates.

[0033] 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. For example, sugars include glucose, galactose, mannose, fructose, heparin, and hyaluronic acid. For example, nucleic acids include deoxyribonucleic acid (DNA) and 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).

[0034] The ability to inhibit protein adhesion is defined as the relative mass per unit area (%) compared to the case without the coating film, as measured by QCM-D using the method described in the Examples ((Mass per unit area (ng / cm²) in the Examples) 2 )) / (Mass per unit area without coating film (ng / cm 2 This means that ))) is 50% or less, preferably 30% or less, and even more preferably 20% or less; Having the ability to inhibit cell adhesion means that the relative absorbance (WST OD450nm) (%) ((absorbance of the example (WST OD450nm)) / (absorbance of the comparative example (WST OD450nm))) when measured with a fluorescence microscope using the method described in International Publication No. 2016 / 093293, compared to a sample without a coating film, is 50% or less, preferably 30% or less, and more preferably 20% or less.

[0035] Preferably, the compatibility with the above-mentioned biomolecules is defined as the ability to inhibit protein adhesion.

[0036] The compatibility with the above-mentioned biomaterials is preferably such that it is suitable for forming a base film for cell culture.

[0037] It is preferable that this material be used to form a base film for cell culture, which is used to form cell aggregates by adhering cells to it and then detaching them. A cell aggregate refers to a structure formed as a result of cell aggregation, and its shape is not limited to spherical or ring-shaped. Compared to cell aggregates produced by non-adherent culture on conventional low-adhesion cell plates, this method offers advantages such as size adjustment of the cell aggregate by specifying the adhesion area (cell aggregates of any size can be produced).

[0038] In addition to the block copolymer and solvent described above, the composition of the present invention may also contain other substances as needed, provided that they do not impair the performance of the resulting coating film. Examples of other substances include preservatives, surfactants, primers to improve adhesion to the substrate, antifungal agents, and sugars.

[0039] <Coating film with compatibility with biomaterials> The coating film compatible with biomaterials according to the present invention is a coated film of the above-mentioned coating film-forming composition compatible with biomaterials, and typically, the coating film can be formed by applying the coating film-forming composition according to the present invention to a substrate and drying it.

[0040] 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.

[0041] 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.

[0042] The drying process for the coating film according to the present invention is carried out under air or vacuum at a temperature of -200°C. The process should be carried out within a range of up to 200°C. The coating film can be formed by drying at room temperature (10°C to 35°C, e.g., 25°C), but to form the coating film more quickly, drying may be performed at, for example, 40°C to 100°C.

[0043] A more preferable drying temperature is 10°C to 180°C, and a more preferable drying temperature is 20°C to 150°C.

[0044] <Substrate for cell culture, method for manufacturing a substrate for cell culture, substrate having the ability to inhibit the adhesion of biomolecules> This is a cell culture substrate comprising a substrate that has the ability to suppress the adhesion of biomaterials, and a coating film that is compatible with biomaterials, applied to at least a portion of the substrate surface.

[0045] In this invention, the term "substrate" refers not only to a flat plate substrate, but also to a container-shaped structure having depressions such as wells or dishes (so-called cell culture vessels).

[0046] A cell culture substrate can be manufactured by applying the aforementioned coating film-forming composition, which is compatible with biomaterials, to the surface of a substrate and drying it. Here, "surface" refers to the surface that comes into contact with the contents, such as cells or cell culture medium.

[0047] The cell culture substrate of the present invention may also be a substrate having the ability to suppress the adhesion of biomaterials, on which a coating film compatible with biomaterials is provided as a plurality of spots. The shape of the spots is not particularly limited. For example, they may be approximately circular or square in shape, but approximately circular spots are preferred. The ratio of the total area of ​​spots to the surface area of ​​the substrate, the diameter of each spot, and the spacing between spots can be appropriately selected from a predetermined range depending on the type of cells and substrate used, the desired size of the cell aggregates, etc. However, the ratio of the total area of ​​spots to the surface area of ​​the substrate is preferably 30% or more, 40% or more, 50% or more, and preferably 99% or less. The diameter of each spot is preferably 50 to 5000 μm and preferably 300 to 3000 μm. The spacing between spots is preferably 30 to 1000 μm and preferably 100 to 500 μm. The cell culture substrate of the present invention allows for the formation of multiple uniformly sized cell aggregates on a single substrate (container) at once by arranging independent, micro-sized regions (spots) compatible with biomaterials at high density and preferably regularly on a substrate that has the ability to suppress the adhesion of biomaterials.

[0048] Examples of substrates (especially cell culture vessels) include petri dishes or 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, chamber slides, tubes, trays, and bottles such as roller bottles. Preferably, petri dishes or dishes, plates, and trays are used.

[0049] 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.).

[0050] 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.

[0051] 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).

[0052] The coating film-forming composition of the present invention can be applied using methods such as spin coating, dip coating, solvent casting, inkjet printing, screen printing, slit coating, or roll-to-roll, but is preferably applied using printing techniques such as inkjet printing or screen printing.

[0053] 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, such as a cell culture vessel, the coating film-forming composition is added to the container and left to stand for a predetermined time. Alternatively, if the coating film is a spot, the coating film-forming composition can be used by, for example, immersing a substrate with areas where spots are not formed in the coating film-forming composition, or adding the coating film-forming composition to a substrate (container) with areas where spots are not formed and letting it stand for a predetermined time. Addition can be done, for example, by adding 0.5 to 1 times the total volume of the container of the 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 cell culture vessels having a cell culture underlayer film on at least a portion, preferably the entire surface of, the vessel.

[0054] 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.

[0055] 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 samples to be subjected to cell culture (e.g., water, buffer, culture medium, etc., particularly preferably a culture medium (e.g., BME medium (Eagle's basal medium), DMEM medium (Dulbecc's modified Eagle's medium))) before being used as a cell culture container.

[0056] 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.

[0057] 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.

[0058] The coating film of the present invention is manufactured through the simple process described above.

[0059] 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.

[0060] The thickness of the coating film of the present invention is such that the maximum and minimum thicknesses are 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.

[0061] <Substrate with ability to inhibit the adhesion of biological substances> The substrate having the ability to inhibit the adhesion of biomaterials of the present invention may be a commercially available or other known product, but a copolymer comprising a repeating unit containing a group represented by the following formula (a) and a repeating unit containing a group represented by the following formula (b), as described in International Publication No. 2014 / 196650: [ka] (In the formula, U a1 , U a2 , U b1 , U b2 and U b3 Each of these is independently a hydrogen atom or a carbon atom. This represents alkyl groups with 1 to 5 atoms, An - These are halide ions, An anion selected from the group consisting of inorganic acid ions, hydroxide ions, and isothiocyanate ions. It is preferable to have a coating film containing ions (representing ions) on at least a portion of the substrate surface.

[0062] The above copolymer is defined by the following formulas (a1) and (b1): [ka] During the ceremony, T a , T b , U a1 , U a2 , U b1 , U b2 and U b3 Each of these independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, Q a and Q b Each of these independently represents a single bond, an ester bond, or an amide bond, and R a and R b Each of these independently represents an alkylene group having 1 to 10 carbon atoms, which may be substituted with halogen atoms, and An - (where m represents an anion selected from the group consisting of halide ions, inorganic acid ions, hydroxide ions, and isothiocyanate ions, and m represents an integer from 0 to 6.) It is preferable to include repeating units.

[0063] The alkyl groups having 1 to 5 carbon atoms mentioned above are as listed in formulas (1) and (2). The alkylene groups having 1 to 10 carbon atoms that may be substituted with halogen atoms mean alkylene groups having 1 to 10 carbon atoms, or alkylene groups having 1 to 10 carbon atoms substituted with one or more halogen atoms. Examples of alkylene groups having 1 to 10 carbon atoms include those listed in formulas (1) and (2) for alkylene groups having 1 to 5 carbon atoms, as well as hexamethylene groups, octamethylene groups, and decamethylene groups. The linear or branched alkylene groups having 1 to 10 carbon atoms substituted with one or more halogen atoms mean that one or more arbitrary hydrogen atoms of the alkylene group are replaced with halogen atoms. Examples of halogen atoms include fluorine atoms, chlorine atoms, bromine atoms, and iodine atoms. Furthermore, the above-mentioned halide ions refer to fluoride ions, chloride ions, bromide ions, or iodide ions, and the above-mentioned inorganic acid ions refer to carbonate ions, sulfate ions, phosphate ions, hydrogen phosphate ions, dihydrogen phosphate ions, nitrate ions, perchlorate ions, or borate ions. Details regarding the coating film using the above copolymer are as described in International Publication No. 2014 / 196650.

[0064] As a coating film having the ability to suppress the adhesion of other biomolecules, copolymers of ethylenically unsaturated monomers, polysaccharides, or their derivatives may be used. Examples of ethylenically unsaturated monomers include one or more ethylenically unsaturated monomers selected from the group consisting of (meth)acrylic acid and its esters; vinyl acetate; vinylpyrrolidone; ethylene; vinyl alcohol; and their hydrophilic functional derivatives. Examples of polysaccharides or their derivatives include cellulosic polymers such as hydroxyalkylcellulose (e.g., hydroxyethylcellulose or hydroxypropylcellulose), starch, dextran, and curdlan.

[0065] Hydrophilic functional derivatives refer to ethylenically unsaturated monomers that have hydrophilic functional groups or structures. Examples of hydrophilic functional groups or structures include betaine structures, amide structures, alkylene glycol residues, amino groups, and sulfinyl groups.

[0066] The betaine structure refers to a monovalent or divalent group of a compound having an amphoteric center with a quaternary ammonium-type cation structure and an acidic anionic structure, for example, a phosphorylcholine group: [ka] Examples of such structures include 2-methacryloyloxyethyl phosphorylcholine (MPC) and others.

[0067] The amide structure is represented by the following formula: [ka] [Here, R 16 , R 17 and R 18 These are, independently of each other, hydrogen atoms or organic groups (e.g., methyl group, hydroxymethyl group, or hydroxyethyl group). This refers to a group represented by . Examples of ethylenically unsaturated monomers having such a structure include (meth)acrylamide and N-(hydroxymethyl)(meth)acrylamide. Furthermore, monomers or polymers having such a structure are disclosed, for example, in Japanese Patent Application Publication No. 2010-169604.

[0068] The alkylene glycol residue refers to the alkylene oxy group (-Alk-O-) remaining after the hydroxyl groups at one or both ends of an alkylene glycol (HO-Alk-OH; where Alk is an alkylene group having 1 to 10 carbon atoms) undergo a condensation reaction with another compound, and also includes poly(alkylene oxy) groups in which alkylene oxy units are repeated. Examples of ethylenically unsaturated monomers having such a structure include 2-hydroxyethyl (meth)acrylate and methoxypolyethylene glycol (meth)acrylate. Furthermore, monomers or polymers having such a structure are disclosed, for example, in Japanese Patent Application Publication No. 2008-533489.

[0069] The amino group is represented by the formulas: -NH2, -NHR 19 or -NR 20 R 21 [Here, R 19 , R 20 and R 21The terms refer to groups that are independently organic groups (e.g., alkyl groups having 1 to 5 carbon atoms). The amino group in this invention includes quaternized or chlorinated amino groups. Examples of ethylenically unsaturated monomers having such a structure include dimethylaminoethyl (meth)acrylate, 2-(t-butylamino)ethyl (meth)acrylate, and methacryloylcholinchloride.

[0070] The sulfinyl group is represented by the following formula: [ka] [Here, R 22 This is an organic group (for example, an organic group having 1 to 10 carbon atoms, preferably an alkyl group having 1 to 10 carbon atoms and having one or more hydroxyl groups). This refers to a group represented by . Examples of polymers having such a structure include copolymers disclosed in Japanese Patent Application Publication No. 2014-48278, etc.

[0071] <Method for producing cell aggregates> The present invention provides a method for producing cell aggregates, comprising the application of formulas (1) and (2): to a substrate having the ability to suppress the adhesion of biomaterials. [ka] (In the formula, R 1 ~R 3 , U 1 and U 2 Each of these independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, X 1 and X 2 The cell culture substrate can be produced by a method comprising the steps of providing a cell culture substrate film on at least a portion of the substrate surface, which is made of a block copolymer having a unit structure represented by (where each independently represents an alkylene group having 1 to 5 carbon atoms, and n1 represents an integer from 1 to 10), and then seeding cells onto the substrate film. Cell aggregates can be produced by known methods, for example, by the method described in the following examples. [Examples]

[0072] 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.

[0073] 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.

[0074] 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 [Synthesis of block copolymers] Figure 1 shows a schematic diagram of the flow reactor (reaction apparatus) used in the synthesis examples 1 to 5 below. In Figure 1, the 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, 5 m length (synthesis examples 1, 2, 3), 2.0 mm inner diameter, 3 mm outer diameter, 4.5 m length (synthesis examples 4, 5)). The other inlet of mixer 2 was connected to a syringe pump C for supplying the second monomer liquid (Keychem-L, manufactured by YMC Co., Ltd.) with a PTFE tube (1.0 mm inner diameter, 1.6 mm outer diameter, 2 m length). The outlet of mixer 2 and the inlet of mixer 3 were connected with a PFA tube (2.0 mm inner diameter, 3 mm outer diameter, 4 m length (synthesis examples 1, 2, 3), 2.0 mm inner diameter, 3 mm outer diameter, 5 m length (synthesis examples 4, 5)). The other inlet of mixer 3 was connected to a syringe pump D for supplying the polymerization inhibitor solution (UI-22-110, manufactured by FLOM Co., Ltd.) with a PTFE tube (1.0 mm inner diameter, 1.6 mm outer diameter, 2 m length). The outlet of mixer 3 was connected with a PFA tube (2.0 mm inner diameter, 3 mm outer diameter, 0.7 m length). Furthermore, the flow paths from each pump to the outlet of mixer 3, up to 90% of their length, were immersed in a -40°C constant temperature bath to adjust the temperature.Mixer 1 used in the synthesis was a two-component mixing mixer with a double-tube structure described in International Publication No. 2017 / 135398 [using stainless steel for the joint members and cylindrical body, and as the static mixer element body, processing Noritake Co., Ltd.'s DSP-MXA3-17 (polyacetal element, 17 twisted blades, 3 mm diameter) and connecting three of them to make 51 twisted blades], Mixer 2 was also a two-component mixing mixer with a double-tube structure described in International Publication No. 2017 / 135398 [using stainless steel for the joint members and cylindrical body, and as the static mixer element body, processing Noritake Co., Ltd.'s DSP-MXA3-17 (polyacetal element, 17 twisted blades, 3 mm diameter)], and Mixer 3 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

[0075] <Synthesis Example 1: Synthesis of poly(2-dimethylaminoethyl methacrylate)-b-poly(diethylene glycol monomethyl ether methacrylate) block polymer (PDM-b-PDEGMA)> As the first monomer, a 0.5 mol / L 2-dimethylaminoethyl methacrylate THF solution was mixed with a 0.05 mol / L 1,1-diphenylhexyl lithium solution as an initiator in mixer 1 at flow rates of 10 mL / min and 1.5 mL / min, respectively, to polymerize the first monomer. Subsequently, as the second monomer, a diethylene glycol monomethyl ether methacrylate solution was mixed with mixer 2 at a flow rate of 0.92 mL / min to carry out block polymerization. Next, as a polymerization stopper, a 0.25 mol / L methanol / THF solution was mixed with mixer 3 at a flow rate of 10 mL / min to stop the polymerization. Each pump was run for 5 minutes, and the effluent was collected.

[0076] Furthermore, after the solvent was largely removed from the effluent using an evaporator, it was added dropwise to a mixture of 200 ml of n-hexane and 200 ml of diethyl ether under an ice bath. The resulting white suspension was filtered through a 0.5 μm membrane filter. Subsequently, the obtained filtrate was dissolved in 1,4-dioxane and freeze-dried to obtain 4.1 g of PDM-b-PDEGMA. The obtained polymer was analyzed by GPC and found to have Mn = 29,790 and Mw / Mn = 1.19. 1 From the 1H-NMR results, the composition ratio of 2-dimethylaminoethyl methacrylate units and diethylene glycol monomethyl ether methacrylate units was 2-dimethylaminoethyl methacrylate units:diethylene glycol monomethyl ether methacrylate units = 48:52. 1 The H-NMR chart is shown in Figure 2.

[0077] <Synthesis Example 2: Synthesis of poly(2-dimethylaminoethyl methacrylate)-b-poly(diethylene glycol monomethyl ether methacrylate) block polymer (PDM-b-PDEGMA)> PDM-b-PDEGMA was synthesized in the same manner as in Synthesis Example 1, except that diethylene glycol monomethyl ether methacrylate solution was mixed as the second monomer at 0.46 mL / min in Mixer 2 and the solution was pumped through each pump for 7 minutes, yielding 3.4 g of PDM-b-PDEGMA. The obtained polymer was analyzed by GPC and found to have Mn = 23,481 and Mw / Mn = 1.11. 1 From the 1H-NMR results, the composition ratio of 2-dimethylaminoethyl methacrylate units to diethylene glycol monomethyl ether methacrylate units was 2-dimethylaminoethyl methacrylate units:diethylene glycol monomethyl ether methacrylate units = 63:37.

[0078] <Synthesis Example 3: Synthesis of poly(2-dimethylaminoethyl methacrylate)-b-poly(diethylene glycol monomethyl ether methacrylate) block polymer (PDM-b-PDEGMA)> PDM-b-PDEGMA was synthesized in the same manner as in Synthesis Example 1, except that a solution of diethylene glycol monomethyl ether methacrylate was mixed as the second monomer at 0.19 mL / min in Mixer 2 and each pump was used for 8 minutes, yielding 4.4 g of PDM-b-PDEGMA. The obtained polymer was analyzed by GPC and found to have Mn = 15,065 and Mw / Mn = 1.17. 1 From the 1H-NMR results, the composition ratio of 2-dimethylaminoethyl methacrylate units to diethylene glycol monomethyl ether methacrylate units was 2-dimethylaminoethyl methacrylate units:diethylene glycol monomethyl ether methacrylate units = 88:12.

[0079] <Synthesis Example 4: Synthesis of poly(2-diethylaminoethyl methacrylate)-b-poly(diethylene glycol monomethyl ether methacrylate) block polymer (PDE-b-PDEGMA)> PDE-b-PDEGMA was synthesized in the same manner as in Synthesis Example 1, except that a 0.5 mol / L 2-diethylaminoethyl methacrylate THF solution was used as the first monomer and a 0.10 mol / L 1,1-diphenylhexyl lithium solution was used as the initiator, and mixed in mixer 1 at flow rates of 10 mL / min and 1.5 mL / min, respectively. A diethylene glycol monomethyl ether methacrylate solution was used as the second monomer and mixed in mixer 2 at a flow rate of 0.92 mL / min. Each pump was then used for 6 minutes to deliver the mixture, and 2.5 g of PDE-b-PDEGMA was obtained. Analysis of the obtained polymer by GPC revealed Mn = 17,555 and Mw / Mn = 1.21. 1 From the 1H-NMR results, the composition ratio of 2-diethylaminoethyl methacrylate units and diethylene glycol monomethyl ether methacrylate units was 29:71. 1 The H-NMR chart is shown in Figure 3.

[0080] <Synthesis Example 5: Synthesis of poly(2-diethylaminoethyl methacrylate)-b-poly(diethylene glycol monomethyl ether methacrylate) block polymer (PDE-b-PDEGMA)> PDE-b-PDEGMA was synthesized in the same manner as in Synthesis Example 4, except that a 0.5 mol / L 2-diethylaminoethyl methacrylate THF solution was used as the first monomer, a 0.10 mol / L 1,1-diphenylhexyl lithium solution was used as the initiator, and these were mixed in mixer 1 at flow rates of 10 mL / min and 2.0 mL / min, respectively. Diethylene glycol monomethyl ether methacrylate solution was used as the second monomer and mixed in mixer 2 at a flow rate of 1.48 mL / min, to obtain 1.6 g of PDE-b-PDEGMA. Analysis of the obtained polymer by GPC revealed Mn = 16,488 and Mw / Mn = 1.21. 1 From the 1H-NMR results, the composition ratio of 2-diethylaminoethyl methacrylate units to diethylene glycol monomethyl ether methacrylate units was 43:57.

[0081] <Comparative Synthesis Example 1: Synthesis of poly(2-dimethylaminoethyl methacrylate)-r-poly(diethylene glycol monomethyl ether methacrylate) random polymer (PDM-r-PDEGMA)> 1.0 g of 2-dimethylaminoethyl methacrylate and 1.0 g of diethylene glycol monomethyl ether methacrylate were dissolved in 18.0 g of THF and 11.1 mg of 2,2'-azodiisobutyronitrile. The mixture was then stirred for 18 hours in a water bath at 60°C under a nitrogen atmosphere. After the reaction mixture returned to room temperature, it was added dropwise to 500 mL of hexane, and the resulting white suspension was filtered through a 0.5 μm membrane filter. The resulting filtrate was then dissolved in water and freeze-dried to obtain 1.7 g of PDM-r-PDEGMA. Analysis of the obtained polymer by GPC revealed Mn = 12,317 and Mw / Mn = 2.52. 1From the 1H-NMR results, the composition ratio of 2-dimethylaminoethyl methacrylate units to diethylene glycol monomethyl ether methacrylate units was 46:54.

[0082] <Comparative Synthesis Example 2: Synthesis of Poly(2-diethylaminoethyl methacrylate)-r-poly(diethylene glycol monomethyl ether methacrylate) random polymer (PDE-r-PDEGMA)> 1.0 g of 2-diethylaminoethyl methacrylate and 1.4 g of diethylene glycol monomethyl ether methacrylate were dissolved in 21.2 g of THF and 11.8 mg of 2,2'-azodiisobutyronitrile. The mixture was then stirred for 21.5 hours under a nitrogen atmosphere in a water bath at 60°C. After the reaction mixture returned to room temperature, it was added dropwise to 300 mL of hexane, and the resulting white suspension was filtered through a 0.5 μm membrane filter. Subsequently, the filtrate was dissolved in 1,4-dioxane and freeze-dried to obtain 1.8 g of PDE-r-PDEGMA.

[0083] Analysis of the obtained polymer by GPC revealed Mn=18,107 and Mw / Mn=2.31. 1 From the 1H-NMR results, the composition ratio of 2-diethylaminoethyl methacrylate units to diethylene glycol monomethyl ether methacrylate units was 46:54.

[0084] <Comparative Synthesis Example 3: Synthesis of Poly(2-diethylaminoethyl methacrylate)-r-poly(methacrylic acid) random polymer (PDM-r-PMA)> PDM-r-PMA was obtained according to the manufacturing method described in Synthesis Example 8 of International Publication No. 2020 / 040247. The obtained polymer was analyzed by GPC and found to have Mn = 472,133 and Mw / Mn = 3.73.

[0085] <Preparation Examples 1-5, Comparative Preparation Examples 1-3: Preparation of Polymer Ethanol Solutions> The polymers from Synthesis Examples 1-5 and Comparative Synthesis Examples 1-3 were each dissolved in ethanol to a concentration of 10 mg / mL to prepare coating film-forming compositions (Preparation Examples 1-5, Comparative Preparation Examples 1-3).

[0086] <Preparation Example 6, Comparative Preparation Example 4: Preparation of Polymer Aqueous Solutions> The polymers from Synthesis Example 1 and Comparative Synthesis Example 1 were dissolved in sterile water to a concentration of 1 mg / mL to prepare coating film-forming compositions (Preparation Example 6, Comparative Preparation Example 4).

[0087] <Preparation Example 7: Preparation of Polymer Aqueous Solution> The polymer from Synthesis Example 1 was dissolved in sterile water to a concentration of 5 mg / mL to prepare a coating film-forming composition (Preparation Example 7).

[0088] <Preparation Example 8, Comparative Preparation Example 5: Preparation of Polymer Solutions> The polymers from Synthesis Example 5 and Comparative Synthesis Example 2 were dissolved in a sterile water / ethanol = 7 / 3 mixed solution to a concentration of 1 mg / mL to prepare coating film-forming compositions (Preparation Example 8, Comparative Preparation Example 5).

[0089] <Preparation Example 9: Preparation of Polymer Solution> The polymer from Synthesis Example 5 was dissolved in a sterile water / ethanol = 7 / 3 mixed solution to a concentration of 5 mg / mL to prepare a coating film-forming composition (Preparation Example 9).

[0090] <Test Example 1: Coating Film Formation Test> The coating film-forming compositions obtained in Preparation Examples 1-5 and Comparative Preparation Examples 1-3 were spin-coated onto HMDS-treated silicon wafers at 1500 rpm / 60 sec, and then dried in a 70°C oven for 24 hours as a drying step. After that, they were thoroughly washed with PBS and dried in a 70°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 in all cases using the coating film-forming composition.

[0091] [Table 1]

[0092] <Test Example 2: Protein Attachment Inhibition Test> (Fabrication of QCM sensor (PS)) A gold-deposited quartz crystal oscillator (Q-Sence, QSX301) was washed at 50mA / 3min using a soft etching apparatus (SEDE-GE, manufactured by Meiwa Forsis Co., Ltd.), and immediately afterward, it was immersed for 24 hours in a solution of 0.0772g of 2-amine ethanethiol (manufactured by Tokyo Chemical Industry Co., Ltd.) dissolved in 1000mL of ethanol. After washing the sensor surface with ethanol and allowing it to air dry, a varnish made by dissolving 1.00g of polystyrene (PS) (manufactured by Sigma-Aldrich) in 99.00g of toluene was spin-coated onto the film sensor side using a spin coater at 3500rpm / 30sec, and dried at 150℃ / 1min to produce a QCM sensor (PS).

[0093] (Fabrication of QCM sensor (SiO2)) A SiO2-deposited quartz crystal oscillator (Q-Sense, QSX303) was cleaned using a soft etching device (SEDE-GE, manufactured by Meiwa Forsis Co., Ltd.) at 50mA / 3min and then used as is.

[0094] (Preparation of surface-treated QCM sensors (PS) and surface-treated QCM sensors (SiO2)) Each coating film-forming composition obtained in Preparation Examples 1-5 and Comparative Preparation Example 1 was spin-coated onto QCM sensors (PS) at 3500 rpm / 30 sec, and then baked in a 70°C oven for 24 hours as a drying step. Subsequently, excess coating film-forming compositions were washed twice each with PBS and pure water as a cleaning step to obtain surface-treated QCM sensors (PS) (substrates No. 1-6). Similarly, each coating film-forming composition obtained in Preparation Examples 1-5 and Comparative Preparation Examples 1-2 was spin-coated onto QCM sensors (SiO2) at 3500 rpm / 30 sec, and then baked in a 100°C hot plate for 5 hours as a drying step. Subsequently, excess coating film-forming compositions were washed twice each with PBS and pure water as a cleaning step to obtain surface-treated QCM sensors (SiO2) (substrates No. 8-14). QCM sensors (PS) (substrate No. 7) and QCM sensors (SiO2) (substrate No. 15) were used as references.

[0095] (Protein adhesion experiment) Surface-treated QCM sensors (PS) (substrates No. 1-7) and surface-treated QCM sensors (SiO2) (substrates No. 8-15), prepared using the above method with a coating film-forming composition, were each mounted on a dissipative quartz crystal microbalance QCM-D (E4, Q-Sence), and PBS was flowed until a stable baseline was established where the frequency change was less than 1 Hz per hour. Next, PBS was flowed for approximately 10 minutes with the stable baseline frequency set to 0 Hz. Subsequently, a 100 μg / mL PBS solution of human serum-derived γ-globulin was flowed for approximately 30 minutes, and then PBS was flowed again for approximately 20 minutes, after which the adsorption-induced frequency shift (Δf) of the 9th overtone was read. For analysis, Q-Tools (Q-Sence) was used to measure the adsorption-induced frequency shift (Δf) as described by the Sauerbrey equation, and the adsorption-induced frequency shift (Δf) per unit area (ng / cm³). 2Table 2 below shows the amount of biomaterial attached, converted to the amount of biomaterial attached. Substrates No. 1-5 and No. 8-12, which were coated with the coating film-forming compositions of Preparation Examples 1-5 according to the present invention, showed significantly lower protein attachment compared to substrates No. 7 and 15 without a coating. Furthermore, it was shown that the coating film-forming compositions prepared in Comparative Preparation Examples 1-2 were used as coatings for substrates No. 6 and No. 13-14, which were shown to significantly suppress protein attachment.

[0096] [Table 2]

[0097] <Test Example 3: Test to inhibit the adhesion of proteins, etc., to culture media> (Fabrication of surface-treated QCM sensors (PS)) Similar to Test Example 2, after preparing the QCM sensor (PS), the coating film-forming compositions obtained in Preparation Examples 1-5 and Comparative Preparation Example 3 were spin-coated onto the QCM sensor (PS) at 3500 rpm / 30 sec, and then baked in a 70°C oven for 24 hours as a drying step. Subsequently, as a cleaning step, excess coating film-forming compositions were washed twice each with PBS and pure water to obtain surface-treated QCM sensors (PS) (substrates No. 1-5, 16).

[0098] (Experiment on the adhesion of proteins, etc., to culture medium) Surface-treated QCM sensors (PS) (substrates No. 1-5, 16) prepared using the above method with a coating film-forming composition were attached to a dissipative quartz crystal microbalance QCM-D (E4, Q-Sence), and PBS was flowed until a stable baseline was established where the frequency change was less than 1 Hz per hour. Next, PBS was flowed for approximately 10 minutes with the frequency of the stable baseline set to 0 Hz. Subsequently, BME medium (Thermo Fisher Scientific) containing 10% FBS (Sigma-Aldrich) and 1% L-glutamine-penicillin-streptomycin stabilized solution (Thermo Fisher Scientific) was flowed for approximately 30 minutes, and the adsorption-induced frequency shift (Δf) of the 9th overtone was read. For analysis, Q-Tools (Q-Sence) was used to measure the adsorption-induced frequency shift (Δf) as described by the Sauerbrey formula, and the mass per unit area (ng / cm³). 2 Table 3 below shows the amount of biomaterial attached, converted to the equivalent value. Substrates No. 1 to 5, which were coated with the coating film-forming compositions of Preparation Examples 1 to 5 according to the present invention, showed a lower amount of protein and other substances attached compared to substrate No. 16, which was coated with the coating film-forming composition of Comparative Preparation Example 3.

[0099] [Table 3]

[0100] <Test Example 4: Cell Aggregation Formation Test> (Preparation of low-adhesion cell petri dishes) A coating solution was prepared from a copolymer-containing varnish according to the manufacturing method described in Example 30 of International Publication No. 2014 / 196650. One mL of the prepared coating solution was added to each φ40 mm Azunol Petri dish (manufactured by AS ONE Corporation, #1-8549-01), and after standing at room temperature for one hour, the excess coating solution was removed and the dish was baked in a 50°C oven for 24 hours. Then, two mL of sterile water was added, and the dish was drained and washed. The same washing procedure was repeated two more times, and the dish was dried in a 50°C oven for one hour to obtain a low-cell adhesion Petri dish.

[0101] (Preparation of petri dishes for cell aggregate formation using inkjet technology) Using an inkjet device (MicroJet Co., Ltd., model number: LaboJet-600) and an inkjet head (model number: 500-SC), approximately 50 nL each of the coating film-forming compositions prepared in Preparation Examples 6-9 and Comparative Preparation Examples 4 and 5 were applied in a spot-like (circular) pattern to the low-adhesion cell petri dishes prepared above. The dishes were dried in a 70°C oven for 24 hours to prepare petri dishes for cell aggregate formation (petri dishes No. 1-6).

[0102] (Cell preparation) The cells used were mouse embryonic fibroblasts C3H10T1 / 2 (manufactured by DS Pharma Biomedical Co., Ltd.). The culture medium used for the cells was BME medium (manufactured by Thermo Fisher Scientific) containing 10% FBS (manufactured by Sigma-Aldrich) and L-glutamine-penicillin-streptomycin stabilized solution (manufactured by Thermo Fisher Scientific). The cells were cultured statically for at least 2 days in a 10 cm diameter petri dish (10 mL of medium) in 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 (manufactured by 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.

[0103] (Observation of cell adhesion) For the petri dish for forming cell aggregates prepared above, 2 mL of the cell suspension was added to each so that the concentration became 6.0×10 5 cells / cm 2 . Then, while maintaining a carbon dioxide concentration of 5%, it was left standing in a 37°C CO2 incubator for 2 hours. After standing, using an inverted microscope (manufactured by Nikon Corporation, ECLIPSE TS100-F), it was confirmed that cells had adhered to the locations where each coating agent was spotted. Then, non-adherent cells and the medium were removed with an aspirator, and only the adherent cells were left on the well by washing with PBS. After washing, 2 mL of fresh medium was added to each, and the state of the adherent cells was observed and photographed using an inverted research microscope IX73 (manufactured by Olympus Corporation). As a result, as shown in Fig. 4, it was confirmed that cells were maintained adhered to the spots for forming the coating film of Petri dishes No. 1 to 4 coated in a spot shape with the composition for forming a coating film prepared in Preparation Examples 6 to 9. On the other hand, it was confirmed that the cells adhered to the spots for forming the coating film of Petri dishes No. 5 and 6 coated in a spot shape with the composition for forming a coating film prepared in Comparative Preparation Examples 4 and 5 were removed by washing.

[0104] (Observation of cell aggregates) The petri dish in which cell adhesion was confirmed above was left standing in a 37°C CO2 incubator for an additional 2 days. After standing, the state of the cells was observed using an inverted research microscope IX73 (manufactured by Olympus Corporation). As a result, as shown in Fig. 5, it was confirmed that the cells adhered to the spots for forming the coating film of Petri dishes No. 1 to 4 coated in a spot shape with the composition for forming a coating film prepared in Preparation Examples 6 to 9 were detached from the plate and aggregated to form cell aggregates (spheroids). From this, it was suggested that the base film containing the polymer of the present invention is useful as a base film for a cell culture container. Table 4 shows the results at each stage of cell adhesion before washing, maintenance of cell adhesion after washing, and formation of cell aggregates.

[0105] [Table4] [Industrial applicability]

[0106] 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 represents a methyl group, U １ and U ２ Each of these independently represents a methyl group, an ethyl group, or a propyl group, X １ and X ２ A composition for forming a base film in cell culture, comprising a block copolymer having a unit structure represented by formula (1) and formula (2) (where each independently represents an ethylene group or a propylene group, and n1 represents an integer from 2 to 4), the molar ratio of the unit structures represented by formula (1) and formula (2) being 20 to 90:80 to 10, and the weight-average molecular weight Mw being 1,000 to 1,000,000, and a solvent.

2. The composition according to claim 1, wherein the solvent comprises water or alcohol.

3. The composition according to claim 1, for forming a base film for cell culture, which is used to obtain cell aggregates by adhering and then detaching cells.

4. A coating film for cell culture, which is a coated film of the composition according to any one of claims 1 to 3.

5. A cell culture substrate comprising a coating film according to claim 4 on at least a portion of the substrate surface, on a substrate having the ability to suppress the adhesion of biological substances.

6. A copolymer comprising a repeating unit containing a group represented by the following formula (a) and a repeating unit containing a group represented by the following formula (b), having the ability to inhibit the adhesion of biological substances: 【Chemistry 2】 (In the formula, U ａ１ , U ａ２ , U ｂ１ , U ｂ２ and U ｂ３ each independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, and An － represents an anion selected from the group consisting of halide ions, inorganic acid ions, hydroxide ions, and isothiocyanate ions), and a substrate provided with a coating film containing the same on at least a part of its surface. The substrate for cell culture according to claim 5.

7. On a substrate having the ability to suppress the adhesion of biological substances, formulas (1) and (2): 【Transformation 3】 (In the formula, R １ ~R ３ represents a methyl group, U １ and U ２ Each of these independently represents a methyl group, an ethyl group, or a propyl group, X １ and X ２ A method for producing a cell culture substrate, comprising the steps of applying a cell culture substrate composition comprising a block copolymer having a unit structure represented by formula (1) and formula (2) (where each independently represents an ethylene group or a propylene group, and n1 represents an integer from 2 to 4), the molar ratio of the unit structures represented by formula (1) and formula (2) being 20 to 90:80 to 10, and the weight-average molecular weight Mw being 1,000 to 1,000,000, and a solvent, and then drying the composition.

8. On a substrate having the ability to suppress the adhesion of biological substances, formulas (1) and (2): 【Chemistry 4】 (In the formula, R １ ~R ３ represents a methyl group, U １ and U ２ Each of these independently represents a methyl group, an ethyl group, or a propyl group, X １ and X ２ A method for producing cell aggregates, comprising the steps of providing a cell culture substrate film on at least a part of the substrate surface, comprising the steps of providing a cell culture substrate film made of a block copolymer having a unit structure represented by formula (1) and formula (2) (where each independently represents an ethylene group or a propylene group, and n1 represents an integer from 2 to 4), the molar ratio of the unit structures represented by formula (1) and formula (2) being 20 to 90:80 to 10, and the weight-average molecular weight Mw being 1,000 to 1,000,000; and then seeding cells onto the substrate film.

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