WO2022202652A1

WO2022202652A1 - Coating agent for cell culture, cell culture container and method for producing same, and cell culture method

Info

Publication number: WO2022202652A1
Application number: PCT/JP2022/012561
Authority: WO
Inventors: 慧蟹江; 竜司加藤; 雅善百木; 優樹石嶋
Original assignee: 国立大学法人東海国立大学機構; 荒川化学工業株式会社
Priority date: 2021-03-23
Filing date: 2022-03-18
Publication date: 2022-09-29
Also published as: JPWO2022202652A1

Abstract

The present invention relates to: a coating agent for cell culture, said coating agent comprising an aqueous polyurethane resin and one or two or more compounds selected from the group consisting of compounds having a terpene skeleton; a method for producing a cell culture container (10), comprising coating a culture surface (11a) using the coating agent for cell culture; a cell culture container (10) in which a culture surface (11a) has been coated by a coating agent comprising an aqueous polyurethane resin and one or two or more compounds selected from the group consisting of compounds having a terpene skeleton; and a cell culture method of culturing cells using the cell culture container (10).

Description

CELL CULTURE COATING AGENT, CELL CULTURE CONTAINER AND METHOD FOR MANUFACTURING THE SAME, AND CELL CULTURE METHOD

TECHNICAL FIELD The present invention relates to a coating agent for cell culture, a cell culture vessel and its manufacturing method, and a cell culture method.
This application claims priority based on Japanese Patent Application No. 2021-048054 filed in Japan on March 23, 2021, the content of which is incorporated herein.

Conventionally, cell culture vessels made of plastic such as polystyrene have been used as substrates for culturing cells. In order to effectively perform cell culture, the cell culture vessel may be plasma-treated on the culture surface, or the culture surface may be coated with a polymeric material that enhances cell adhesion strength.

Polymer materials are required to have low cytotoxicity, excellent cell adhesiveness and coating stability.
Known polymer materials include biopolymer materials such as collagen, fibronectin, and laminin, and synthetic polymer materials such as poly-L-lysine, poly-D-lysine, and poly-L-ornithine.
For example, Patent Document 1 discloses a cell culture vessel having a culture surface coated with poly-L-lysine.

JP-A-5-227944

Although poly-L-lysine is widely used as a coating agent for cell culture, further cost reduction, low cytotoxicity, excellent cell adhesion and coating stability are required.
An object of the present invention is to provide a low-cost, low-cytotoxicity, cell-adhesive and coating-stable coating agent for cell culture, a cell culture vessel and its production method, and a cell culture method.

The present invention has the following aspects.
[1] A coating agent for cell culture, containing one or more compounds selected from the group consisting of an aqueous polyurethane resin and a compound having a terpene skeleton.
[2] The coating agent for cell culture according to [1] above, wherein the compound having a terpene skeleton contains one or more compounds selected from the group consisting of terpene resins and rosin resins.
[3] The coating agent for cell culture according to [1] or [2] above, wherein the rosin resin comprises an α,β-unsaturated carboxylic acid-modified rosin.
[4] A method for producing a cell culture vessel, comprising coating a culture surface with the cell culture coating agent according to any one of [1] to [3].
[5] A cell culture vessel having a culture surface coated with a coating agent, wherein the coating agent contains one or more compounds selected from the group consisting of an aqueous polyurethane resin and a compound having a terpene skeleton. cell culture vessel.
[6] A cell culture method, wherein cells are cultured using the cell culture vessel of [5] above.

According to the present invention, it is possible to provide a cell culture coating agent with low cytotoxicity, cell adhesion, and excellent coating stability, a cell culture vessel and its manufacturing method, and a cell culture method.

It is a perspective view showing an example of a cell culture container concerning the present invention. FIG. 2 is a cross-sectional view taken along line XX in FIG. 1; 1 is a graph showing fluorescence intensity values in Examples 1-1 to 1-7. 2 is a graph showing fluorescence intensity values in Examples 2-1 to 2-7 and Comparative Examples 2-1 and 2-2. 2 is a graph showing fluorescence intensity values in Examples 2-8 to 2-14 and Comparative Examples 2-3 and 2-4. 3 is a graph showing fluorescence intensity values in Examples 3-1 to 3-7 and Comparative Examples 3-1 and 3-2. FIG. 10 is a graph showing fluorescence intensity values in Examples 3-8 to 3-11 and Comparative Examples 3-3 and 3-4. FIG. 4 is a graph showing fluorescence intensity values in Examples 4-1 to 4-7 and Comparative Examples 4-1 and 4-2. FIG. 10 is a graph showing fluorescence intensity values in Examples 4-8 to 4-14 and Comparative Examples 4-3 and 4-4. FIG. FIG. 10 is a graph showing fluorescence intensity values in Examples 4-15 to 4-20 and Comparative Examples 4-5 and 4-6. FIG. FIG. 10 is a graph showing fluorescence intensity values in Examples 4-21 to 4-24 and Comparative Examples 4-7 and 4-8. FIG. FIG. 10 is a graph showing fluorescence intensity values in Examples 5-1 to 5-7 and Comparative Examples 5-1 and 5-2. FIG. FIG. 10 is a graph showing fluorescence intensity values in Examples 5-8 to 5-14 and Comparative Examples 5-3 and 5-4. FIG. FIG. 10 is a graph showing fluorescence intensity values in Examples 5-15 to 5-21 and Comparative Examples 5-5 and 5-6. FIG.

[Coating agent for cell culture]
The coating agent for cell culture of the present invention is selected from the group consisting of an aqueous polyurethane resin (hereinafter also referred to as "(A) component") and a compound having a terpene skeleton (hereinafter also referred to as "(B) component"). contains one or more compounds that are

(A) Component is an aqueous polyurethane resin.
Component (A) is a reaction product obtained by reacting a polyol compound (hereinafter also referred to as "compound (a1)") and an isocyanate compound (hereinafter also referred to as "compound (a2)") by a known method. It is a thing.
When the compound (a1) and the compound (a2) are reacted, in addition to the compound (a1) and the compound (a2), an aliphatic having any one of a hydroxy group, a primary amino group and a secondary amino group At least one of a hydrocarbon compound (hereinafter also referred to as "compound (a3)") and a chain extender may be used in combination. That is, the component (A) may be a reaction product obtained by reacting the compound (a1), the compound (a2), the compound (a3) and at least one of the chain extender.
Further, after reacting at least the compound (a1) and the compound (a2), a compound having one or more hydroxy groups and one or more polymerizable double bonds (hereinafter also referred to as "compound (a4)". ) may be further reacted.

Compound (a1) is a polyol compound.
Examples of the compound (a1) include polyether polyols such as polymers or copolymers of ethylene oxide, propylene oxide and tetrahydrofuran; ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propanediol, 1,3- Propanediol, 1,3-butanediol, 1,4-butanediol, neopentyl glycol, pentanediol, 3-methyl-1,5-pentanediol, 1,6-hexanediol, octanediol, 1,4-butyne Various known saturated or unsaturated low-molecular-weight glycols such as diols and dipropylene glycol, alkyl glycidyl ethers such as n-butyl glycidyl ether and 2-ethylhexyl glycidyl ether, and monocarboxylic acid glycidyl esters such as versatic acid glycidyl ester and dibasic acids such as adipic acid, maleic acid, fumaric acid, phthalic anhydride, isophthalic acid, terephthalic acid, succinic acid, oxalic acid, malonic acid, glutaric acid, pimelic acid, azelaic acid, sebacic acid, and suberic acid. or polyester polyols obtained by dehydration condensation with corresponding acid anhydrides or dimer acids; polyester polyols obtained by ring-opening polymerization of cyclic ester compounds; other polycarbonate polyols, polybutadiene glycols, bisphenol A Various known high-molecular-weight polyols generally used for producing polyurethane resins, such as glycols obtained by adding ethylene oxide or propylene oxide to . These compounds (a1) may be used alone or in combination of two or more.

The compound (a1) may contain a liquid polytetrahydrofuran-based polyether polyol.
The liquid polytetrahydrofuran-based polyether polyol is a copolymer obtained by copolymerizing tetrahydrofuran with an alkylene oxide having an alkyl group in its side chain or an alcohol having an alkyl group in its side chain by various known methods.
The liquid polytetrahydrofuran-based polyether polyol preferably has a number average molecular weight of 500 to 5,000.

Alkylene oxides having an alkyl group in the side chain include, for example, 1,2-propylene oxide, 1,2-butylene oxide, 3-methyltetrahydrofuran, 3-methyloxetane and 3,3-dimethyloxetane. Among these, 1,2-propylene oxide and 3-methyltetrahydrofuran are particularly industrially useful. Examples of alcohols having alkyl groups in side chains include neopentyl glycol, 1,3-butanediol, 3-methyl-1,5-pentanediol and the like.
These alkylene oxides and alcohols may be used singly or in combination of two or more.

As a method of copolymerization, for example, tetrahydrofuran and an alkylene oxide having an alkyl group in a side chain are polymerized in the presence of a ring-opening catalyst such as chlorosulfonic acid or fluorosulfonic acid; and an alcohol having a heteropolyacid, a method of polymerizing in the presence of a catalyst such as toluenesulfonic acid, and the like.
The liquid polytetrahydrofuran-based polyether polyol may be a block copolymer or a random copolymer.

When the liquid polytetrahydrofuran-based polyether polyol is a copolymer of tetrahydrofuran and an alkylene oxide having an alkyl group in the side chain, the ratio of the alkylene oxide having an alkyl group in the side chain is It is preferably 5 to 50% by mass, more preferably 10 to 40% by mass, based on the total mass.

Compound (a2) is an isocyanate compound.
Examples of the compound (a2) include diisocyanate compounds.
Examples of diisocyanate compounds include methylene diisocyanate, isopropylene diisocyanate, butane-1,4-diisocyanate, hexamethylene diisocyanate, 2,2,4-trimethylhexamethylene diisocyanate, 2,4,4-trimethylhexamethylene diisocyanate, and dimer acid. chain aliphatic diisocyanate such as dimer diisocyanate having carboxyl group replaced with isocyanate group; , cycloaliphatic diisocyanates such as methylcyclohexane diisocyanate; dialkyldiphenylmethane diisocyanates such as 4,4′-diphenyldimethylmethane diisocyanate; tetraalkyldiphenylmethane diisocyanates such as 4,4′-diphenyltetramethylmethane diisocyanate; 1,5-naphthylene diisocyanate , 4,4′-diphenylmethane diisocyanate, 4,4′-diphenyldimethylmethane diisocyanate, 4,4′-dibenzyl isocyanate, 1,3-phenylene diisocyanate, 1,4-phenylene diisocyanate, tolylene diisocyanate, xylylene diisocyanate, Aromatic diisocyanates such as m-tetramethylxylylene diisocyanate; amino acid diisocyanates such as lysine diisocyanate; Among these, chain aliphatic diisocyanates and cyclic aliphatic diisocyanates are preferred.
One of these compounds (a2) may be used alone, or two or more thereof may be used in combination.

Compound (a3) is an aliphatic hydrocarbon compound having any one of a hydroxy group, a primary amino group and a secondary amino group.
The number of carbon atoms in the aliphatic hydrocarbon compound is preferably 10-40.
Examples of aliphatic hydrocarbon compounds having one hydroxy group and having 10 to 40 carbon atoms include decyl alcohol, isodecyl alcohol, lauryl alcohol, tridecyl alcohol, myristyl alcohol, cetyl alcohol, stearyl alcohol, and isostearyl. monoalcohols such as alcohol, oleyl alcohol, and behenyl alcohol;
Examples of aliphatic hydrocarbon compounds having one primary amino group and having 10 to 40 carbon atoms include primary monoamines such as decylamine, laurylamine, myristylamine, stearylamine and oleylamine.
Examples of aliphatic hydrocarbon compounds having one secondary amino group and having 10 to 40 carbon atoms include secondary monoamines such as di-2-ethylhexylamine and distearylamine.
One of these compounds (a3) may be used alone, or two or more thereof may be used in combination.

Examples of the chain extender include the low-molecular-weight glycols exemplified above in the description of compound (a1); Amines such as triethylenetetramine, diethylenetriamine, isophoronediamine, dicyclohexylmethane-4,4′-diamine; 2-hydroxyethylethylenediamine, 2-hydroxyethylpropylenediamine, di-2-hydroxyethylethylenediamine, di-2-hydroxyethylpropylene Diamine having a hydroxyl group in the molecule such as diamine, 2-hydroxypropylethylenediamine, di-2-hydroxypropylethylenediamine; dimer diamine, hydrazine, adipic dihydrazide obtained by replacing the carboxyl group of dimer acid with an amino group.
One of these chain extenders may be used alone, or two or more thereof may be used in combination.

A chain extender having an ionic functional group may also be used to impart an ionic functional group to the component (A) for the purpose of improving dispersibility in water.
Examples of ionic functional groups include quaternary amino bases and carboxylic acid bases. Specific examples of chain extenders having a quaternary amino base include N-methyldiethanolamine, N-ethyldiethanolamine, N-propyldiethanolamine, N-isopropyldiethanolamine, N-butyldiethanolamine, N-isobutyldiethanolamine, N-oleyldiethanolamine, N-stearyldiethanolamine, ethoxylated palm oil amine, N-allyldiethanolamine, N-methyldiisopropanolamine, N-ethyldiisopropanolamine, N-propyldiisopropanolamine, N-butyldiisopropanolamine, dimethyldiethoxyhydrazine, propoxy Methyldiethanolamine, N-(3-aminopropyl)-N-methylethanolamine, N,N'-bis(oxyethyl)propylenediamine, diethanolaminoacetamide, diethanolaminopropionamide, N,N-bis(oxymethyl)semicarbazide, etc. Alkoxylated linear aliphatic amines such as N-cyclohexyldiisopropanolamine; N,N-diethoxyaniline, N,N-diethoxytoluidine, N,N-diethoxy-1-aminopyridine , N,N′-bis(2-hydroxyethyl)-N,N′-diethylhexahydro-p-phenylenediamine, N,N′-bis(oxyethyl)phenyl semicarbazide; N,N Alkoxylated heterocyclic amines such as '-diethoxypiperazine, N-2-hydroxyethylpiperazine; N-methyl-N,N-bis(3-aminopropyl)amine, N-(3-aminopropyl)-N,N '-dimethylethylenediamine, N,N'-bis(3-aminopropyl)-N,N'-dimethylethylenediamine, 2-methyl-2-[(N,N-dimethylamino)methyl]propane-1,3-diol Chain aliphatic amines such as; 2,6-diaminopyridine, aromatic amines such as p,p'-bis-aminomethyldibenzylmethylamine;N,N'-bis(3-aminopropyl)piperazine, N- heterocyclic amines such as (2-aminoethyl)piperazine;
These chain extenders having a quaternary amino base may be used singly or in combination of two or more.
In addition, the basic nitrogen possessed by the chain elongating agent is, before dispersing the chain elongating agent in water or after dispersing the chain elongating agent in water, chloride ions, sulfate ions, anions of organic carboxylic acids, etc. It is quaternized using a quaternizing agent.

A chain extender having a carboxylic acid group is used when introducing the carboxylic acid group into the resulting aqueous polyurethane resin.
Specific examples of chain extenders having a carboxylic acid group include glyceric acid, dioxymaleic acid, dioxyfumaric acid, tartaric acid, dimethylolpropionic acid, dimethylolbutanoic acid, 2,2-dimethylolvaleric acid, 2,2-di aliphatic carboxylic acids such as methylolpentanoic acid, 4,4-di(hydroxyphenyl)valeric acid and 4,4-di(hydroxyphenyl)butyric acid; and aromatic carboxylic acids such as 2,6-dioxybenzoic acid. .
These chain extenders having a carboxylic acid group may be used singly or in combination of two or more.
Further, the carboxylic acid base possessed by such a chain extender includes alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; ammonia; trimethylamine, triethylamine, triethanolamine, triisopropanolamine, N-methyldiethanolamine and N-ethyl Neutralizing agent such as N-alkyldiethanolamine such as diethanolamine, N,N-dimethylethanolamine and tertiary amine such as N,N-dialkylethanolamine such as N,N-diethylethanolamine, etc. be reconciled.
Moreover, such a chain extender may be used before the compound (a1), the compound (a2), etc. are dispersed in water, or may be used after they are dispersed in water.

Compound (a4) is a compound having one or more hydroxy groups and one or more polymerizable double bonds.
Examples of the compound (a4) include 2-hydroxyethyl methacrylate, 2-hydroxypropyl (meth)acrylate, 2-hydroxybutyl (meth)acrylate, 4-hydroxybutyl (meth)acrylate, 2-hydroxy-3-phenoxypropyl ( meth)acrylate, 2-hydroxy-3-acryloyloxypropyl (meth)acrylate, glycerin di(meth)acrylate, glycerin (meth)acrylate, trimethylolpropane diallyl ether, glycerin mono(meth)acrylate, glycidol/acrylic acid adduct , pentaerythritol tri(meth)acrylate, pentaerythritol triallyl ether, 2-hydroxyethyl monovinyl ether, 4-hydroxybutyl monovinyl ether, N-methylol acrylamide, and polypropylene glycol (meth) acrylate with a molecular weight of 500 or less, polyethylene glycol (meth) ) acrylate, polycaprolactone (meth)acrylate, and the like. Among these, 2-hydroxyethyl (meth)acrylate, 2-hydroxypropyl (meth)acrylate, and 4-hydroxybutyl (meth)acrylate are preferred from the viewpoint of high polymerization reactivity.
One of these compounds (a4) may be used alone, or two or more thereof may be used in combination. In addition, in this specification, "(meth)acrylate" means at least one of acrylate and methacrylate.

(A) A component is obtained as follows, for example.
The compound (a1) and the compound (a2) and, if necessary, the compound (a3) are added so that the isocyanate group of the compound (a2) is in excess with respect to the total number of active hydrogen groups of the compound (a1) and the compound (a3). to obtain a urethane prepolymer having an isocyanate group. The obtained urethane prepolymer may be used as the component (A) as it is.

Alternatively, the urethane prepolymer may be dissolved in a suitable organic solvent to form a solution, and the chain extender may be added to the solution for reaction. When the chain extender has an ionic functional group, it is quaternized with the quaternizing agent described above after the reaction, or neutralized with a neutralizing agent, and dispersed in water, if necessary. The organic solvent may be removed by squeezing to produce the component (A).
Examples of organic solvents include aromatic solvents such as benzene, toluene and xylene; ester solvents such as ethyl acetate and butyl acetate; ketone solvents such as acetone, methyl ethyl ketone, diethyl ketone and methyl isobutyl ketone; amides such as dimethylformamide. sulfoxide solvents such as dimethyl sulfoxide; ether solvents such as dimethyl ether and diethyl ether; alcohols such as isopropyl alcohol and ethanol; and N-methylpyrrolidone. These organic solvents may be used individually by 1 type, and may use 2 or more types together.

The urethane prepolymer may be produced by copolymerizing the compounds (a1) and (a2), the chain extender, and, if necessary, the compound (a3). Specifically, first, the compound (a1) and the compound (a2), a chain extender containing a chain extender having an ionic functional group, and optionally the compound (a3) are copolymerized in an organic solvent. to produce a urethane prepolymer. Then, after quaternizing with a quaternizing agent or neutralizing with a neutralizing agent, the mixture is dispersed in water and, if necessary, the organic solvent is removed to obtain component (A). Alternatively, first, the compounds (a1) and (a2), a chain extender having an ionic functional group, and optionally the compound (a3) are copolymerized in an organic solvent to produce a urethane prepolymer. . Then, after quaternizing with a quaternizing agent or neutralizing with a neutralizing agent, the mixture is dispersed in water, reacted by adding a chain extender, and if necessary, the organic solvent is removed. (A) A component is obtained.

Further, after reacting the urethane prepolymer and the compound (a4), the double bond of the obtained reaction product is polymerized in the presence of a polymerization initiator to increase the molecular weight, and the component (A) is obtained. may be used.
In addition, before polymerizing the double bond, the reaction product is optionally dispersed in an aqueous solution containing the above-described organic solvent, a chain extender, and a quaternizing agent or a neutralizing agent to generate a reaction product. You may react a thing and a chain extender.
Examples of polymerization initiators include benzoyl peroxide, tert-butylperoxy-2-ethylhexanoate, tert-butylperoctoate, 2,2-azobisisobutyronitrile, 2,2-azobis(2,4 -dimethylvaleronitrile), 2,2′-azobis(2-amidinopropane) dihydrochloride, dimethyl-2,2′-azobisisobutyrate and the like.

The weight average molecular weight of component (A) is preferably 5,000 to 5,000,000.
The acid value of component (A) is preferably 10 to 100 mgKOH/g.
(A) The mass average molecular weight of the component is a value in terms of standard polystyrene measured by gel permeation chromatography (GPC).
The acid value of component (A) is a value determined by titration of mg of potassium hydroxide required to neutralize 1 g of component (A).

(B) A component is a compound which has a terpene skeleton.
Examples of component (B) include rosin resins and terpene resins. Among these, rosin resin is preferred.
These (B) components may be used individually by 1 type, and may use 2 or more types together.

Rosin resins include, for example, natural rosins (gum rosin, tall oil rosin, wood rosin) derived from Mao pine, Slash pine, Mercushi pine, Shichi pine, Teda pine, Daio pine, etc., refined rosin, hydrogenated rosin, and disproportionated rosin. , a polymerized rosin obtained by polymerizing at least one of natural rosin and purified rosin, at least one of natural rosin and purified rosin is added with α,β-unsaturated carboxylic acid such as maleic acid, fumaric acid, acrylic acid (or α, rosins such as α,β-unsaturated carboxylic acid-modified rosins obtained by modifying with β-unsaturated carboxylic anhydrides); rosins obtained by esterifying rosins with alcohols; Examples thereof include rosin derivatives such as rosin-phenol resin obtained by reacting with phenols.
These rosin resins may be used alone or in combination of two or more.
In the present specification, natural rosin, refined rosin, disproportionated rosin, hydrogenated rosin, polymerized rosin and α,β-unsaturated carboxylic acid-modified rosin may be collectively referred to as “rosins”. Also, at least one of natural rosin and purified rosin may be referred to as "natural rosin and/or purified rosin". Also, rosin esters and rosin-phenol resins are sometimes collectively referred to as "rosin derivatives".

Purified rosin can be obtained using various known means, and specifically, can be obtained using various known purification means such as a distillation method, an extraction method, and a recrystallization method.
The distillation method includes, for example, a method of distilling rosins at a temperature of about 200 to 300° C. under a reduced pressure of about 0.01 to 3 kPa.
Examples of the extraction method include a method in which rosins are made into an alkaline aqueous solution, insoluble unsaponifiable substances are extracted with various organic solvents, and then the aqueous layer is neutralized.
Examples of the recrystallization method include a method of dissolving a rosin in an organic solvent as a good solvent, then distilling off the solvent to obtain a concentrated solution, and then adding an organic solvent as a poor solvent.
Examples of good solvents include aromatic hydrocarbon solvents such as benzene, toluene and xylene; chlorinated hydrocarbon solvents such as chloroform; lower alcohols such as methanol and ethanol; ketones such as acetone; is mentioned.
Examples of poor solvents include n-hexane, n-heptane, cyclohexane and isooctane.

Disproportionated rosin can be obtained using various known means, specifically, a method (disproportionation) in which natural rosin and/or purified rosin are heated in the presence of a disproportionation catalyst. be able to.
Examples of disproportionation catalysts include supported catalysts such as palladium-carbon, rhodium-carbon and platinum-carbon; metal powders such as nickel and platinum; iodides such as iodine and iron iodide.
The amount of catalyst used is preferably 0.01 to 5 parts by mass, more preferably 0.01 to 1 part by mass, per 100 parts by mass of natural rosin and/or purified rosin.
The reaction temperature is preferably 100-300°C, more preferably 150-290°C.

Hydrogenated rosin can be obtained using various known means, and specifically, it can be obtained by hydrogenating natural rosin and/or purified rosin under known hydrogenation conditions. The method of hydrogenation includes, for example, a method of heating natural rosin and/or refined rosin in the presence of a hydrogenation catalyst.
As the hydrogenation catalyst, various known catalysts such as supported catalysts and metal powders can be used. Supported catalysts include, for example, palladium-carbon, rhodium-carbon, ruthenium-carbon, and platinum-carbon. Examples of metal powders include nickel and platinum. Among these, palladium, rhodium, ruthenium, and platinum-based catalysts are preferable from the viewpoint of increasing the hydrogenation rate and shortening the hydrogenation time.
The amount of hydrogenation catalyst used is preferably 0.01 to 5 parts by mass, more preferably 0.01 to 2 parts by mass, per 100 parts by mass of natural rosin and/or refined rosin.
The hydrogen pressure during hydrogenation is preferably 2 to 20 MPa, more preferably 5 to 20 MPa. The reaction temperature is preferably 100-300°C, more preferably 150-300°C.

Hydrogenation may be carried out with the natural rosin and/or purified rosin dissolved in a solvent, if desired.
The solvent to be used is not particularly limited, and any solvent that is inert to the reaction and easily dissolves the raw materials and products can be used. Specific examples include cyclohexane, n-hexane, n-heptane, decalin, tetrahydrofuran, dioxane, and the like. mentioned.
These solvents may be used individually by 1 type, and may use 2 or more types together.
The amount of solvent used is not particularly limited, but it may be used so that the solid content is 10% by mass or more, preferably in the range of about 10 to 70% by mass, relative to natural rosin and/or purified rosin.

The α,β-unsaturated carboxylic acid-modified rosin is obtained by addition reaction of α,β-unsaturated carboxylic acid to natural rosin and/or purified rosin.
The α,β-unsaturated carboxylic acid is not particularly limited, and various known ones can be used, such as acrylic acid, methacrylic acid, maleic acid, fumaric acid, itaconic acid, citraconic acid, muconic acid, maleic anhydride, and anhydride. Examples include itaconic acid, citraconic anhydride, and muconic anhydride. Among these, acrylic acid, maleic acid, maleic anhydride, and fumaric acid are preferred.
These α,β-unsaturated carboxylic acids may be used singly or in combination of two or more.
The amount of α,β-unsaturated carboxylic acid to be used is usually about 1 to 20 parts by mass, preferably 1 to 3 parts by mass, per 100 parts by mass of natural rosin and/or purified rosin.

The method for producing the α,β-unsaturated carboxylic acid-modified rosin is not particularly limited. A method of reacting at about 180 to 240° C. for about 1 to 9 hours can be mentioned. Moreover, the above reaction may be carried out while blowing an inert gas such as nitrogen into a closed reaction system.
Furthermore, in the above reaction, known catalysts such as Lewis acids such as zinc chloride, iron chloride and tin chloride, and Bronsted acids such as paratoluenesulfonic acid and methanesulfonic acid may be used. The amount of these catalysts to be used is usually about 0.01 to 10 parts by weight per 100 parts by weight of natural rosin and/or purified rosin.

Also, the α,β-unsaturated carboxylic acid-modified rosin may be obtained by subjecting the α,β-unsaturated carboxylic acid-modified rosin to the hydrogenation described above.

A rosin ester can be obtained using various known methods, and specifically, it can be obtained by subjecting a rosin and an alcohol to an esterification reaction under known esterification conditions.
Examples of alcohols include monohydric alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, tert-butyl alcohol, n-octyl alcohol, 2-ethylhexyl alcohol, decyl alcohol, and lauryl alcohol; ethylene; Glycol, diethylene glycol, triethylene glycol, propylene glycol, neopentyl glycol, dihydric alcohols such as cyclohexanedimethanol; trihydric alcohols such as glycerin, trimethylolethane, trimethylolpropane; pentaerythritol, diglycerin, di(trimethylolpropane) ); pentahydric alcohols such as triglycerin; hexahydric alcohols such as dipentaerythritol. Among these, methanol, glycerin, and pentaerythritol are preferred.
As the alcohol, glycidyl ethers, glycidol, and the like, which react with carboxylic acid to form an ester, may be used.
These alcohols may be used individually by 1 type, and may use 2 or more types together.

The amounts of rosins and alcohol charged are not particularly limited, but usually the ratio of alcohol OH groups to rosin COOH groups (equivalence ratio) is about 0.8 to 8, preferably about 3 to 7. is determined as
The reaction temperature for the esterification reaction is preferably 150 to 320°C, more preferably 150 to 300°C.
The reaction time is preferably 2 to 24 hours, more preferably 2 to 7 hours.

In the method for producing a rosin ester, the esterification reaction may be carried out in the presence of a catalyst for the purpose of shortening the reaction time.
Examples of the catalyst include acid catalysts such as p-toluenesulfonic acid; metal hydroxides such as calcium hydroxide and magnesium hydroxide; and metal oxides such as calcium oxide and magnesium oxide.
Moreover, since water is produced by the esterification reaction, the esterification reaction may be carried out while removing the produced water from the system.
Moreover, considering the color tone of the resulting rosin ester, it is preferable to carry out the esterification reaction under an inert gas stream. Furthermore, the esterification reaction may be carried out under pressure if necessary.

In the method for producing a rosin ester, the rosin and alcohol may be reacted in an organic solvent that is non-reactive with respect to the rosin and alcohol.
Examples of organic solvents include hexane, cyclohexane, toluene and xylene.
When an organic solvent is used, it is preferable to distill off the organic solvent or unreacted raw materials under reduced pressure, if necessary.

The rosin resin is preferably an esterified product of disproportionated rosin (disproportionated rosin ester), an α,β-unsaturated carboxylic acid-modified rosin, or a hydride of α,β-unsaturated carboxylic acid-modified rosin. - Unsaturated carboxylic acid-modified rosin is more preferred.

The acid value of the rosin resin is preferably 350 mgKOH/g or less, more preferably 330 mgKOH/g or less.
The softening point of the rosin resin is preferably 180°C or less, more preferably 50 to 160°C. The acid value of the rosin resin is a value measured according to JIS K 0070.
The softening point of rosin resin is a value measured according to JIS K 5902.

Terpene resins include, for example, α-pinene resin, β-pinene resin; aromatic modified terpene series obtained by copolymerizing terpenes such as α-pinene, β-pinene and limonene with aromatic monomers such as styrene and phenol resins; terpene phenol resins obtained by copolymerizing terpenes and phenols; and hydrides thereof.
These terpene resins may be used alone or in combination of two or more.

The coating agent for cell culture may be composed of only one or more compounds selected from the group consisting of the components (A) and (B), or within a range that does not impair the effects of the present invention. If necessary, components other than the components (A) and (B) (hereinafter also referred to as “optional components”) may be included.
Optional components include, for example, plasticizers, dispersants, surfactants, stabilizers, and the like. The content of the optional component is preferably 10% by mass or less, more preferably 5% by mass or less, and even more preferably 1% by mass or less with respect to the total mass of the cell culture coating agent. Substantially free is particularly preferred.
Here, "substantially free" means not containing optional components other than those unavoidably mixed in the manufacturing process.

Since the components (A) and (B) described above can be mass-produced, the coating agent for cell culture of the present invention can be produced at low cost.
In addition, the components (A) and (B) have low cytotoxicity. In addition, components (A) and (B) are excellent in cell adhesion and coating stability. In particular, component (B) is superior in coating stability.
Therefore, the coating agent for cell culture of the present invention is low in cost and excellent in low cytotoxicity, cell adhesion and coating stability.

The coating agent for cell culture of the present invention is suitable as a coating agent for coating the culture surface of a cell culture vessel used when culturing cells.
Although the details will be described later, when coating a culture surface, the coating agent for cell culture of the present invention is used by dissolving or dispersing it in a solvent or water.

[Cell culture vessel]
FIG. 1 is a perspective view showing an example of the cell culture vessel of the present invention, and FIG. 2 is a cross-sectional view taken along line XX in FIG.
The cell culture vessel 10 shown in FIGS. 1 and 2 has a vessel body 11 which is a substrate for culturing cells, and a cell adhesion layer 12 formed on the culture surface 11 a of the vessel body 11 .

Although the container body 11 in this example is a petri dish, the shape of the container body 11 is not limited to a petri dish, such as a dish, a multiwell plate, a flask, a chamber slide, a glass slide, a culture bag, a test tube, a plant box, and a centrifuge tube. (For example, a conical tube, a microtube, etc.).

The material of the container body 11 is not particularly limited as long as it has low cytotoxicity, water resistance, and is suitable for cell culture. Examples thereof include plastics, synthetic rubbers, inorganic substances, and metals.
Examples of plastics include thermoplastic resins such as polystyrene, polycarbonate, polyvinyl chloride, polyethylene, polypropylene, polymethylpentene, acrylic resins, and fluorine resins; thermosetting resins such as phenolic resins, urea resins, epoxy resins, melamine resins, and silicone resins. curable resins, and the like. Among these, polystyrene is preferable from the viewpoint of excellent transparency.
Examples of synthetic rubber include butadiene styrene rubber, butadiene acrylonitrile rubber, butyl rubber, polysulfide synthetic rubber, fluororubber, and silicone rubber.
Examples of inorganic materials include glass, hydroxyapatite, silicon, and carbon nanotubes.
Examples of metals include stainless steel, copper, iron, nickel, aluminum, titanium, gold, silver, platinum, and oxides thereof.

The cell adhesion layer 12 is a layer formed by coating the culture surface 11a with a coating agent.
The coating agent contains one or more compounds selected from the group consisting of water-based polyurethane resins and compounds having a terpene skeleton. In addition, the coating agent may contain optional components as necessary, as long as they do not impair the effects of the present invention.
That is, the cell adhesion layer 12 is a layer containing one or more compounds selected from the group consisting of water-based polyurethane resins and compounds having a terpene skeleton, and optionally optional components.

Examples of the water-based polyurethane resin contained in the coating agent or the cell adhesion layer 12 include component (A) exemplified above in the description of the coating agent for cell culture of the present invention.
Compounds having a terpene skeleton contained in the coating agent and the cell adhesion layer 12 include the component (B) exemplified above in the description of the coating agent for cell culture of the present invention. Optional components contained in the coating agent and the cell adhesion layer 12 include the optional components exemplified above in the description of the coating agent for cell culture of the present invention.
As the coating agent, it is preferable to use the coating agent for cell culture of the present invention.
The coating method of the coating agent will be described later.

The coating amount is preferably 0.1-35000 μg/cm ² , more preferably 0.2-15 μg/cm ² . In particular, when the cell adhesion layer 12 contains an aqueous polyurethane resin, the coating amount is preferably 0.6 to 35,000 μg/cm ² , more preferably 0.6 to 7,000 μg/cm ² , and more preferably 0.6 to 3,500 μg/cm ² . More preferably, 1 to 15 μg/cm ² , more preferably 5 to 15 μg/cm ² , even more preferably 8 to 15 μg/cm ² . When the cell adhesion layer 12 contains a compound having a terpene skeleton, the coating amount is preferably 0.1 to 4000 μg/cm ² , more preferably 0.1 to 1000 μg/cm ² and even more preferably 0.1 to 100 μg/cm ² . More preferably 0.2 to 5 μg/cm ² , more preferably 0.2 to 2 μg/cm ² , still more preferably 0.2 to 0.5 μg/cm ² .
The mass per unit area of the cell adhesion layer 12 is preferably 0.01-35000 μg/cm ² , more preferably 0.02-15 μg/cm ² . In particular, when the cell adhesion layer 12 contains an aqueous polyurethane resin, the mass per unit area of the cell adhesion layer 12 is preferably 0.06 to 35000 μg/cm ² , more preferably 0.06 to 7000 μg/cm ² . 0.06 to 3500 μg/cm ² is more preferred, 0.1 to 15 μg/cm ² is more preferred, 0.5 to 15 μg/cm ² is more preferred, and 0.8 to 15 μg/cm ² is even more preferred. When the cell adhesion layer 12 contains a compound having a terpene skeleton, the mass per unit area of the cell adhesion layer 12 is preferably 0.01 to 4000 μg/cm ² , more preferably 0.01 to 1000 μg/cm ² , and 0 0.01 to 100 µg/cm ² is more preferred, 0.02 to 5 µg/cm ² is more preferred, 0.02 to 2 µg/cm ² is more preferred, and 0.02 to 0.5 µg/cm ² is even more preferred.

The cell culture vessel 10 may have an anchor coat layer (not shown) formed between the vessel body 11 and the cell adhesion layer 12 as long as the effect of the present invention is not impaired.
The anchor coat layer is formed using, for example, an anchor coat agent.
The anchor coating agent contains, for example, a resin and, if necessary, additives such as a curing agent and a silane coupling agent. That is, the anchor coat layer is a layer containing, for example, a resin and, if necessary, additives.
Examples of resins include alkyd resins, melamine resins, acrylic resins, nitrocellulose, polyesters, phenolic resins, amino resins, fluorine resins, epoxy resins, and carbodiimide group-containing resins.
These resins may be used individually by 1 type, and may use 2 or more types together.
A protein may also be used as the anchor coating agent.

Also, the anchor coat layer may be a layer made of an inorganic material formed by a vapor deposition method (hereinafter also referred to as an “inorganic vapor deposition layer”).
Examples of inorganic materials include aluminum, aluminum oxide, magnesium oxide, silicon oxide, and tin oxide.
These inorganic materials may be used singly or in combination of two or more. When the anchor coat layer is an inorganic deposited layer, the thickness of the inorganic deposited layer is preferably 5 to 300 nm, more preferably 10 to 50 nm.

Also, instead of forming the anchor coat layer, the culture surface 11a of the container body 11 may be plasma-treated.

In the cell culture vessel 10 of the present embodiment, the cell adhesion layer 12 containing one or more compounds selected from the group consisting of compounds having an aqueous polyurethane resin and a terpene skeleton is formed on the culture surface 11a. Excellent cell adhesion.

[Method for producing cell culture vessel]
The cell culture vessel 10 can be manufactured by coating the culture surface 11a with the coating agent for cell culture of the present invention to form the cell adhesion layer 12, for example.
Examples of the method of coating the culture surface 11a with the cell culture coating agent include a method of applying a coating liquid containing the cell culture coating agent to the culture surface 11a of the container body 11 .

The coating liquid is obtained, for example, by dispersing the above component (A) and, if necessary, one or more of the above component (B) and optional components in water.
Moreover, the coating liquid is obtained, for example, by dissolving the above-described component (B) and, if necessary, one or more of the above-described component (A) and optional components in alcohol.
Hereinafter, the coating liquid containing the component (A) is also referred to as "coating liquid (A)", and the coating liquid containing the component (B) is also referred to as "coating liquid (B)".

The content of component (A) is preferably 3×10 ⁻⁴ to 5% by mass, more preferably 3×10 ⁻⁴ to 1% by mass, and 3×10 ⁻⁴ with respect to the total mass of coating liquid (A). ~0.5 mass% is more preferable, 3 × 10 ^-4 to 5 × 10 ^-3 mass% is more preferable, 1 × 10 ^-3 to 5 × 10 ^-3 mass% is more preferable, 3 × 10 ^-3 ~ 5×10 ⁻³ mass % is more preferred. (A) If content of a component is more than the said lower limit, cell adhesiveness will increase more. (A) If the content of the component is equal to or less than the above upper limit, low cytotoxicity is further enhanced.
The component (A) can be obtained in the form of a dispersion in water, for example, by the method described above, but this dispersion may be used as the coating liquid (A) as it is. Further, if necessary, the dispersion may be further diluted with water and used, or one or more of the component (B) and optional components may be added to the dispersion.

The content of component (B) is preferably 5×10 ⁻⁵ to 0.5% by mass, more preferably 5×10 ⁻⁵ to 0.1% by mass, based on the total mass of coating liquid (B). ×10 ^-5 to 0.01% by mass is more preferable, 5×10 ^-5 to 1.5×10 ^-3 % by mass is more preferable, and 5×10 ^-5 to 5.5×10 ^-4 % by mass is more preferable. 5×10 ⁻⁵ to 1.5×10 ⁻⁴ mass % is more preferred. (B) If content of a component is more than the said lower limit, cell adhesiveness will increase more. If the content of component (B) is equal to or less than the above upper limit, low cytotoxicity is further enhanced.

Examples of alcohols contained in the coating liquid (B) include methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, tert-butyl alcohol, n-octyl alcohol, and 2-ethylhexyl alcohol. , decyl alcohol, lauryl alcohol, and the like.
These alcohols may be used individually by 1 type, and may use 2 or more types together.

The method of applying the coating liquid is not particularly limited, but examples include casting, dipping, roll coating, gravure coating, screen printing, reverse coating, spray coating, kit coating, die coating, and metering bar coating. method, chamber doctor combined coating method, curtain coating method, and the like.

The coating temperature is preferably room temperature (20°C) to 50°C, more preferably room temperature to 37°C, and even more preferably room temperature to 27°C.
The coating time (application time) is preferably 0.5 hours to 10 days, more preferably 1 hour to 7 days. Since the components (A) and (B) are excellent in coating stability, excellent cell adhesiveness can be expressed even after coating for a long period of time (about 10 days).
Here, the "coating time" is the time during which the coating solution is in contact with the culture surface 11a. For example, when the coating liquid is applied by dipping, the immersion time is the coating time.

The cell adhesion layer 12 is formed on the culture surface 11a by washing or drying the container body 11 after applying the coating liquid to the culture surface 11a.
Examples of the cleaning method include a method of immersing the container body 11 in water, a method of rinsing the container body 11 with running water, and the like.
The number of washings may be one, or two or more. When washing is performed twice or more, it is preferably 5 times or less, more preferably 4 times or less, and even more preferably 3 times or less.

Examples of drying methods include natural drying, hot air drying, hot roll contact, infrared heating, microwave heating, and vacuum drying.
The drying temperature is preferably room temperature (20°C) to 50°C, more preferably room temperature to 37°C, and even more preferably room temperature to 27°C.
The drying time is preferably 30 minutes to 24 hours, more preferably 1 to 10 hours.

When forming an anchor coat layer (not shown) between the culture surface 11a and the cell adhesion layer 12, an anchor coat agent is applied to the culture surface 11a and dried to form an anchor coat layer on the culture surface 11a. After that, the anchor coat layer is coated with a cell culture coating agent to form the cell adhesion layer 12 on the anchor coat layer.
When the anchor coat layer is an inorganic vapor deposition layer, various known vapor deposition methods can be used as methods for forming the inorganic vapor deposition layer, such as vacuum vapor deposition, sputtering, ion plating, and chemical vapor deposition. etc.

[Cell culture method]
For cell culture, the above-described cell culture vessel of the present invention is used, and cells are seeded on the cell adhesion layer of the cell culture vessel and cultured. Specifically, a cell suspension and a cell culture medium (eg, DME medium, D-MEM medium, MEM medium, HamF12 medium, HamF10 medium, etc.) are added onto the cell adhesion layer of the cell culture vessel.

The type of cells that can be cultured in the cell culture vessel of the present invention is not particularly limited. The cell culture vessel of the present invention includes, for example, mesenchymal cells, hepatocytes, fibroblasts, endothelial cells, Schwann cells, nerve cells, cardiomyocytes, glial cells, corneal epithelial cells, chondrocytes, osteoblasts, adipocytes, and the like. normal cells; cancer cell-derived cell lines (eg, MCF7, HepG2, HT29), immortalized cells, and abnormal cells such as cells with chromosomal aberrations.

The culture environment (culture conditions) for cells can be appropriately selected according to the type of cells to be cultured. Also, the culture time can be arbitrarily selected depending on the cell type, cell number, or desired size.
The culture temperature may also be any condition as long as it is suitable for the object.
The number of cells to be seeded can be arbitrarily set depending on the culture environment, and can be carried out, for example, from 1×10 ³ to 1×10 ⁶ cells.

According to the cell culture method of the present embodiment, since a cell culture vessel provided with a cell adhesion layer having excellent cell adhesion strength is used, cell culture can be effectively performed.
The present invention has the following aspects.
<1-1> A coating liquid or a cell culture coating agent containing one or more compounds selected from the group consisting of an aqueous polyurethane resin and a compound having a terpene skeleton is applied to the culture surface of the cell culture vessel main body. , forming a cell adhesion layer, and seeding and culturing cells on the cell adhesion layer.
<1-2> The method according to <1-1>, wherein the compound having a terpene skeleton contains one or more compounds selected from the group consisting of terpene resins and rosin resins.
<1-3> The method according to <1-1> or <1-2>, wherein the rosin resin comprises an α,β-unsaturated carboxylic acid-modified rosin.
<2-1> Use of one or more compounds selected from the group consisting of water-based polyurethane resins and compounds having a terpene skeleton as a coating agent for cell culture.
<2-2> The use according to <2-1>, wherein the compound having a terpene skeleton contains one or more compounds selected from the group consisting of terpene resins and rosin resins.
<2-3> Use according to <2-1> or <2-2>, wherein the rosin resin comprises an α,β-unsaturated carboxylic acid-modified rosin.
<3-1> Use of one or more compounds selected from the group consisting of water-based polyurethane resins and compounds having a terpene skeleton for producing a coating agent for cell culture.
<3-2> The use according to <3-1>, wherein the compound having a terpene skeleton contains one or more compounds selected from the group consisting of terpene resins and rosin resins.
<3-3> Use according to <3-1> or <3-2>, wherein the rosin resin comprises an α,β-unsaturated carboxylic acid-modified rosin.

The present invention will be specifically described below with reference to Examples, but the present invention is not limited to these.

[Undiluted solution of coating solution]
<(A) Component>
Stock solutions (A-1 to A-3) of coating solutions were prepared using the following as components (A).
A-1: Aqueous dispersion of water-based polyurethane resin (manufactured by Arakawa Chemical Industries, Ltd., trade name “Uriano W302”).
A-1 is an aqueous dispersion of an aqueous polyurethane resin (resin solid content 35% by mass, water 60% by mass, isopropyl alcohol 5% by mass, resin acid value 40 mgKOH/g, pH 7.7 at 25°C, viscosity at 25°C 700 mPa·s), and this was used as it was as the stock solution of the coating solution.

A-2: Aqueous dispersion of water-based polyurethane resin (manufactured by Arakawa Chemical Industries, Ltd., trade name “Uriano W600”).
A-2 is an aqueous dispersion of an aqueous polyurethane resin (resin solid content 35% by mass, water 60% by mass, isopropyl alcohol 5% by mass, resin acid value 20 mgKOH/g, pH 7.6 at 25°C, viscosity at 25°C 140 mPa·s), and this was used as it was as the stock solution of the coating solution.

· A-3: Aqueous polyurethane resin aqueous dispersion (manufactured by Arakawa Chemical Industries, Ltd., trade name "Uriano W601").
A-3 is an aqueous dispersion of an aqueous polyurethane resin (resin solid content 35% by mass, water 60% by mass, isopropyl alcohol 5% by mass, resin acid value 30 mgKOH/g, pH 7.6 at 25°C, viscosity at 25°C 60 mPa·s), and this was used as it was as the stock solution of the coating solution.

<(B) Component>
Stock solutions (B-1 to B-4) of coating solutions were prepared using the following as components (B).
・B-1: Disproportionated rosin methyl ester as rosin resin (manufactured by Arakawa Chemical Industries, Ltd., trade name “DHA-ME95”, acid value of resin 0.4 mgKOH / g, melting point of resin 62 ° C., color tone of resin 40H ) was dissolved in ethanol to prepare a stock solution of a coating solution having a resin solid content of 8.8% by mass.

· B-2: rosin hydride modified with maleic anhydride as rosin resin (manufactured by Arakawa Chemical Industries, Ltd., trade name "KR-120", resin acid value 322.8 mgKOH / g, resin softening point 118.5 ° C. , resin color tone 120H) was dissolved in ethanol to prepare a stock solution of a coating solution having a resin solid content of 10.2% by mass.

B-3: Fumaric acid-modified rosin as a rosin resin (manufactured by Arakawa Chemical Industries, Ltd., trade name "Malquedo No. 33", resin acid value 314 mgKOH / g, resin softening point 152.5 ° C., resin color tone 6 Gardner) was dissolved in ethanol to prepare a stock solution of a coating solution having a resin solid content of 10.4% by mass.

B-4: hydride of acrylic acid-modified rosin as rosin resin (manufactured by Arakawa Chemical Industries, Ltd., trade name “KE-604”, resin acid value 240 mgKOH / g, resin softening point 129.5 ° C., resin Color 110H) was dissolved in ethanol to prepare a stock solution of a coating solution having a resin solid content of 10.2% by mass.

[Cell suspension]
<Preparation of cell suspension (1)>
Fibroblasts were detached from the plastic surface using an enzyme (trypsin) and suspended in a medium, PBS, or the like to prepare a cell suspension (1).

<Preparation of cell suspension (2)>
Vascular endothelial cells were detached from the plastic surface using an enzyme (trypsin) and suspended in a medium, PBS, or the like to prepare a cell suspension (2).

<Preparation of cell suspension (3)>
Schwann cells were detached from the plastic surface using an enzyme (trypsin) and suspended in a medium, PBS, or the like to prepare a cell suspension (3).

[Method for measuring the number of cells]
The number of cells after cell culture was counted as follows.
Fluorescence intensity values were measured by fluorescently staining living cells with calcein, and the fluorescence intensity values were taken as the number of living cells (cell adhesion amount).

[Test 1]
<Example 1-1>
As a coating solution, A-1 (undiluted solution), A-1 diluted 10 times with pure water (10-fold diluted solution), A-1 diluted 100-fold with pure water (100-fold diluent) was used.
0.1 mL of the coating solution was dispensed into a 96-well plate (manufactured by Corning, model number "353072") and allowed to stand at 37°C for 1 hour to form a uniform layer over the entire culture surface. After that, the surplus coating solution in the 96-well plate is removed, 3 mL of pure water is added, and the operation of standing at 37 ° C. for 1 hour and washing is performed once, and the cells are placed on the bottom (culture surface) of the 96-well plate. An adhesive layer was formed.
Cell suspension (1) was seeded on the cell adhesion layer of the washed 96-well plate and cultured at 20°C for 6 days. During culturing, the medium was exchanged once every 2 to 3 days. After culturing, the cells were fluorescently stained and the number of viable cells was counted. The results are shown in FIG.
Separately, cells were similarly cultured using a 96-well plate (uncoated) that was not coated with the coating solution, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 1-2>
As the coating solution, A-2 (undiluted solution), a diluted solution obtained by diluting A-2 10 times with pure water (10-fold diluted solution), a diluted solution obtained by diluting A-2 100-fold with pure water (100-fold Cells were cultured in the same manner as in Example 1-1, except that the diluent) was used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 1-3>
As a coating solution, A-3 (undiluted solution), a diluted solution obtained by diluting A-3 10 times with pure water (10-fold diluted solution), a diluted solution obtained by diluting A-3 100-fold with pure water (100-fold Cells were cultured in the same manner as in Example 1-1, except that the diluent) was used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 1-4>
As the coating solution, B-1 (undiluted solution), a diluted solution obtained by diluting B-1 10 times with ethanol (10-fold diluted solution), a diluted solution obtained by diluting B-1 100 times with ethanol (100-fold diluted solution ), the cells were cultured in the same manner as in Example 1-1, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 1-5>
As a coating solution, B-2 (undiluted solution), a diluted solution obtained by diluting B-2 10 times with ethanol (10-fold diluted solution), a diluted solution obtained by diluting B-2 100 times with ethanol (100-fold diluted solution ), the cells were cultured in the same manner as in Example 1-1, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 1-6>
As a coating solution, B-3 (undiluted solution), a diluted solution obtained by diluting B-3 10 times with ethanol (10-fold diluted solution), a diluted solution obtained by diluting B-3 100-fold with ethanol (100-fold diluted solution ), the cells were cultured in the same manner as in Example 1-1, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 1-7>
As a coating solution, B-4 (undiluted solution), B-4 diluted 10 times with ethanol (10-fold diluted solution), B-4 diluted 100 times with ethanol (100-fold diluted solution ), the cells were cultured in the same manner as in Example 1-1, and fluorescence intensity values were measured. The results are shown in FIG.

As shown in Figure 3, the (A) component and (B) component were shown to have low cytotoxicity.

[Test 2]
<Example 2-1>
As a coating solution, A-1 was diluted 20,000 times with pure water (20,000-fold dilution), and A-1 was diluted 10,000-fold with pure water (10,000-fold dilution). was used.
0.1 mL of the coating solution was dispensed into a 96-well plate (manufactured by Corning, model number "353072") and allowed to stand at 37°C for 1 hour to form a uniform layer over the entire culture surface. After that, the surplus coating solution in the 96-well plate is removed, 3 mL of pure water is added, and the operation of standing at 37 ° C. for 1 hour and washing is performed once, and the cells are placed on the bottom (culture surface) of the 96-well plate. An adhesive layer was formed.
The cell suspension (2) was seeded on the cell adhesion layer of the washed 96-well plate and cultured at 20°C for 5 days. The culture medium was not exchanged during the culture.
After culturing, the cells were fluorescently stained and the number of viable cells was counted. The results are shown in FIG.

<Example 2-2>
As a coating solution, A-2 was diluted 100,000 times with pure water (100,000-fold dilution), and A-2 was diluted 20,000-fold with pure water (20,000-fold dilution). , Cells were cultured in the same manner as in Example 2-1, except that a diluted solution obtained by diluting A-2 10,000 times with pure water (10,000-fold diluted solution) was used, and the fluorescence intensity value was measured. . The results are shown in FIG.

<Example 2-3>
As a coating solution, A-3 was diluted 100,000 times with pure water (100,000-fold dilution), and A-3 was diluted 20,000-fold with pure water (20,000-fold dilution). , Cells were cultured in the same manner as in Example 2-1, except that a diluted solution (10,000-fold diluted solution) obtained by diluting A-3 with pure water 10,000 times was used, and the fluorescence intensity value was measured. . The results are shown in FIG.

<Example 2-4>
As a coating solution, B-1 diluted 100,000 times with ethanol (100,000-fold diluted solution), B-1 diluted 20,000-fold with ethanol (20,000-fold diluted solution), B Cells were cultured in the same manner as in Example 2-1 except that a diluted solution (10,000-fold diluted solution) obtained by diluting -1 with ethanol to 10,000 times was used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 2-5>
As a coating solution, a diluted solution of B-2 diluted 100,000 times with ethanol (100,000-fold diluted solution), a diluted solution of B-2 diluted 20,000-fold with ethanol (20,000-fold diluted solution), B Cells were cultured in the same manner as in Example 2-1, except that a diluted solution (10,000-fold diluted solution) obtained by diluting -2 with ethanol to 10,000 times was used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 2-6>
As a coating solution, B-3 diluted 100,000 times with ethanol (100,000-fold diluted solution), B-3 diluted 20,000-fold with ethanol (20,000-fold diluted solution), B Cells were cultured in the same manner as in Example 2-1, except that a diluted solution (10,000-fold diluted solution) obtained by diluting -3 with ethanol to 10,000 times was used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 2-7>
As a coating solution, B-4 diluted 100,000 times with ethanol (100,000-fold diluted solution), B-4 diluted 20,000-fold with ethanol (20,000-fold diluted solution), B Cells were cultured in the same manner as in Example 2-1, except that a diluted solution (10,000-fold diluted solution) obtained by diluting -4 with ethanol to 10,000 times was used, and fluorescence intensity values were measured. The results are shown in FIG.

<Comparative Example 2-1>
Cells were cultured and fluorescence intensity values were measured in the same manner as in Example 2-1, except that a 96-well plate (uncoated) not coated with the coating solution was used. The results are shown in FIG.

<Comparative Example 2-2>
Cells were coated in the same manner as in Example 2-1, except that an aqueous solution obtained by dissolving poly-L-lysine (manufactured by ScienceCell Research Laboratories) in water to a concentration of 0.001% by mass was used as the coating solution. After culturing, fluorescence intensity values were measured. The results are shown in FIG.

<Example 2-8>
As a coating solution, A-1 was diluted 20,000 times with pure water (20,000-fold dilution), and A-1 was diluted 10,000-fold with pure water (10,000-fold dilution). was used.
0.1 mL of the coating solution was dispensed into a 96-well plate (manufactured by Corning, model number "353072") and allowed to stand at 37°C for 8 hours to form a uniform layer over the entire culture surface. Thereafter, excess coating solution in the 96-well plate was removed, and the plate was allowed to stand at 37° C. for 1 hour to dry to form a cell adhesion layer on the bottom surface (culture surface) of the 96-well plate.
The cell suspension (2) was seeded on the cell adhesion layer of the dried 96-well plate and cultured at 20°C for 5 days. The culture medium was not exchanged during the culture.
After culturing, the cells were fluorescently stained and the number of viable cells was counted. The results are shown in FIG.

<Example 2-9>
As a coating solution, A-2 was diluted 100,000 times with pure water (100,000-fold dilution), and A-2 was diluted 20,000-fold with pure water (20,000-fold dilution). , Except for using a 10,000-fold dilution of A-2 with pure water (10,000-fold dilution), the cells were cultured in the same manner as in Example 2-8, and the fluorescence intensity value was measured. . The results are shown in FIG.

<Example 2-10>
As a coating solution, A-3 was diluted 100,000 times with pure water (100,000-fold dilution), and A-3 was diluted 20,000-fold with pure water (20,000-fold dilution). , Except for using a 10,000-fold dilution of A-3 with pure water (10,000-fold dilution), the cells were cultured in the same manner as in Example 2-8, and the fluorescence intensity value was measured. . The results are shown in FIG.

<Example 2-11>
As a coating solution, B-1 diluted 100,000 times with ethanol (100,000-fold diluted solution), B-1 diluted 20,000-fold with ethanol (20,000-fold diluted solution), B Cells were cultured in the same manner as in Example 2-8, except that a diluted solution (10,000-fold diluted solution) obtained by diluting -1 with ethanol to 10,000 times was used, and the fluorescence intensity value was measured. The results are shown in FIG.

<Example 2-12>
As a coating solution, a diluted solution of B-2 diluted 100,000 times with ethanol (100,000-fold diluted solution), a diluted solution of B-2 diluted 20,000-fold with ethanol (20,000-fold diluted solution), B Cells were cultured in the same manner as in Example 2-8, except that a diluted solution (10,000-fold diluted solution) obtained by diluting -2 with ethanol to 10,000 times was used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 2-13>
As a coating solution, B-3 diluted 100,000 times with ethanol (100,000-fold diluted solution), B-3 diluted 20,000-fold with ethanol (20,000-fold diluted solution), B Cells were cultured in the same manner as in Example 2-8, except that a diluted solution (10,000-fold diluted solution) obtained by diluting -3 with ethanol to 10,000 times was used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 2-14>
As a coating solution, B-4 diluted 100,000 times with ethanol (100,000-fold diluted solution), B-4 diluted 20,000-fold with ethanol (20,000-fold diluted solution), B Cells were cultured in the same manner as in Example 2-8, except that a diluted solution obtained by diluting -4 with ethanol to 10,000 times (10,000-fold diluted solution) was used, and fluorescence intensity values were measured. The results are shown in FIG.

<Comparative Example 2-3>
Cells were cultured and fluorescence intensity values were measured in the same manner as in Example 2-8, except that a 96-well plate (uncoated) not coated with the coating solution was used. The results are shown in FIG.

<Comparative Example 2-4>
Cells were coated in the same manner as in Example 2-8, except that an aqueous solution obtained by dissolving poly-L-lysine (manufactured by ScienCell Research Laboratories) in water to a concentration of 0.001% by mass was used as the coating solution. After culturing, fluorescence intensity values were measured. The results are shown in FIG.

As shown in FIGS. 4 and 5, when cell culture is performed using a cell culture vessel coated with component (A) or component (B), uncoated cell culture vessels or poly-L-lysine-coated cell culture Compared to the case of cell culture using a vessel, the fluorescence intensity value was higher, and the effect of cell proliferation was recognized even for vascular endothelial cells, which are said to have weak cell adhesion.

[Test 3]
<Example 3-1>
As a coating solution, A-1 was diluted 20,000 times with pure water (20,000-fold dilution), and A-1 was diluted 10,000-fold with pure water (10,000-fold dilution). was used.
0.1 mL of the coating solution was dispensed into a 96-well plate (manufactured by Corning, model number "353072") and allowed to stand at 37°C for 1 hour to form a uniform layer over the entire culture surface. After that, the surplus coating solution in the 96-well plate is removed, 3 mL of pure water is added, and the operation of standing at 37 ° C. for 1 hour and washing is performed once, and the cells are placed on the bottom (culture surface) of the 96-well plate. An adhesive layer was formed.
The cell suspension (2) was seeded on the cell adhesion layer of the washed 96-well plate and cultured at 20°C for 7 days. During culturing, the medium was exchanged once every 2 to 3 days. After culturing, the cells were fluorescently stained and the number of viable cells was counted. The results are shown in FIG.

<Example 3-2>
As a coating solution, A-2 was diluted 20,000 times with pure water (20,000-fold dilution), and A-2 was diluted 10,000-fold with pure water (10,000-fold dilution). Cells were cultured in the same manner as in Example 3-1 except that , and fluorescence intensity values were measured. The results are shown in FIG.

<Example 3-3>
As a coating solution, A-3 was diluted 100,000 times with pure water (100,000-fold dilution), and A-3 was diluted 20,000-fold with pure water (20,000-fold dilution). Cells were cultured in the same manner as in Example 3-1 except that , and fluorescence intensity values were measured. The results are shown in FIG.

<Example 3-4>
As the coating solution, a diluted solution of B-1 diluted 100,000 times with ethanol (100,000-fold diluted solution) and a diluted solution of B-1 diluted 20,000-fold with ethanol (20,000-fold diluted solution) were used. Cells were cultured in the same manner as in Example 3-1, except that they were used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 3-5>
As a coating solution, a diluted solution of B-2 diluted 20,000 times with ethanol (20,000-fold diluted solution) and a diluted solution of B-2 diluted 10,000-fold with ethanol (10,000-fold diluted solution) were used. Cells were cultured in the same manner as in Example 3-1, except that they were used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 3-6>
As the coating solution, a diluted solution of B-3 diluted 20,000 times with ethanol (20,000-fold diluted solution) and a diluted solution of B-3 diluted 10,000 times with ethanol (10,000-fold diluted solution) were used. Cells were cultured in the same manner as in Example 3-1, except that they were used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 3-7>
As the coating solution, a diluted solution of B-4 diluted 20,000 times with ethanol (20,000-fold diluted solution) and a diluted solution of B-4 diluted 10,000-fold with ethanol (10,000-fold diluted solution) were used. Cells were cultured in the same manner as in Example 3-1, except that they were used, and fluorescence intensity values were measured. The results are shown in FIG.

<Comparative Example 3-1>
Cells were cultured and fluorescence intensity values were measured in the same manner as in Example 3-1, except that a 96-well plate (uncoated) not coated with the coating solution was used. The results are shown in FIG.

<Comparative Example 3-2>
Cells were coated in the same manner as in Example 3-1, except that an aqueous solution obtained by dissolving poly-L-lysine (manufactured by ScienceCell Research Laboratories) in water to a concentration of 0.001% by mass was used as the coating solution. After culturing, fluorescence intensity values were measured. The results are shown in FIG.

<Example 3-8>
As the coating solution, a diluted solution of B-1 diluted 100,000 times with ethanol (100,000-fold diluted solution) and a diluted solution of B-1 diluted 20,000-fold with ethanol (20,000-fold diluted solution) were used. board. 0.1 mL of the coating solution was dispensed into a 96-well plate (manufactured by Corning, model number "353072") and allowed to stand at 37°C for 1 week to form a uniform layer over the entire culture surface. Thereafter, excess coating solution in the 96-well plate was removed, and the plate was allowed to stand at 37° C. for 1 hour to dry to form a cell adhesion layer on the bottom surface (culture surface) of the 96-well plate.
The cell suspension (2) was seeded on the cell adhesion layer of the dried 96-well plate and cultured at 20°C for 7 days. During culturing, the medium was exchanged once every 2 to 3 days. After culturing, the cells were fluorescently stained and the number of viable cells was counted. The results are shown in FIG.

<Example 3-9>
As the coating solution, a diluted solution of B-2 diluted 20,000 times with ethanol (20,000-fold diluted solution) and a diluted solution of B-2 diluted 10,000 times with ethanol (10,000-fold diluted solution) were used. Cells were cultured in the same manner as in Example 3-8, except that they were used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 3-10>
As the coating solution, a diluted solution of B-3 diluted 20,000 times with ethanol (20,000-fold diluted solution) and a diluted solution of B-3 diluted 10,000 times with ethanol (10,000-fold diluted solution) were used. Cells were cultured in the same manner as in Example 3-8, except that they were used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 3-11>
As the coating solution, a diluted solution of B-4 diluted 20,000 times with ethanol (20,000-fold diluted solution) and a diluted solution of B-4 diluted 10,000-fold with ethanol (10,000-fold diluted solution) were used. Cells were cultured in the same manner as in Example 3-8, except that they were used, and fluorescence intensity values were measured. The results are shown in FIG.

<Comparative Example 3-3>
Cells were cultured and fluorescence intensity values were measured in the same manner as in Example 3-8, except that 96-well plates (uncoated) that were not coated with the coating solution were used. The results are shown in FIG.

<Comparative Example 3-4>
Cells were coated in the same manner as in Example 3-8, except that an aqueous solution obtained by dissolving poly-L-lysine (manufactured by ScienCell Research Laboratories) in water to a concentration of 0.001% by mass was used as the coating solution. After culturing, fluorescence intensity values were measured. The results are shown in FIG.

As shown in FIGS. 6 and 7, when cell culture is performed using a cell culture vessel coated with component (A) or component (B), uncoated cell culture vessels or poly-L-lysine-coated cell culture Compared to the case of cell culture using a vessel, the fluorescence intensity value was higher, and the effect of cell proliferation was recognized even for vascular endothelial cells, which are said to have weak cell adhesion.

[Test 4]
<Example 4-1>
As a coating solution, A-1 was diluted 100,000 times with pure water (100,000-fold diluted solution), and A-1 was diluted 20,000-fold with pure water (20,000-fold diluted solution). , A-1 diluted 10,000 times with pure water (10,000 times diluted solution).
0.1 mL of the coating solution was dispensed into a 96-well plate (manufactured by Corning, model number "353072") and allowed to stand at 37°C for 1 hour to form a uniform layer over the entire culture surface. After that, the surplus coating solution in the 96-well plate is removed, 3 mL of pure water is added, and the operation of standing at 37 ° C. for 1 hour and washing is performed once, and the cells are placed on the bottom (culture surface) of the 96-well plate. An adhesive layer was formed.
The cell suspension (2) was seeded on the cell adhesion layer of the washed 96-well plate and cultured at 20°C for 5 days. During culturing, the medium was exchanged once every 2 to 3 days. After culturing, the cells were fluorescently stained and the number of viable cells was counted. The results are shown in FIG.

<Example 4-2>
As a coating solution, A-2 was diluted 100,000 times with pure water (100,000-fold dilution), and A-2 was diluted 20,000-fold with pure water (20,000-fold dilution). , Except for using a 10,000-fold dilution of A-2 with pure water (10,000-fold dilution), the cells were cultured in the same manner as in Example 4-1, and the fluorescence intensity value was measured. . The results are shown in FIG.

<Example 4-3>
As a coating solution, A-3 was diluted 100,000 times with pure water (100,000-fold dilution), and A-3 was diluted 20,000-fold with pure water (20,000-fold dilution). , Except for using a 10,000-fold dilution of A-3 with pure water (10,000-fold dilution), the cells were cultured in the same manner as in Example 4-1, and the fluorescence intensity value was measured. . The results are shown in FIG.

<Example 4-4>
As the coating solution, a diluted solution of B-1 diluted 100,000 times with ethanol (100,000-fold diluted solution) and a diluted solution of B-1 diluted 20,000-fold with ethanol (20,000-fold diluted solution) were used. Cells were cultured in the same manner as in Example 4-1, except that they were used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 4-5>
As a coating solution, a diluted solution of B-2 diluted 20,000 times with ethanol (20,000-fold diluted solution) and a diluted solution of B-2 diluted 10,000-fold with ethanol (10,000-fold diluted solution) were used. Cells were cultured in the same manner as in Example 4-1, except that they were used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 4-6>
As a coating solution, B-3 diluted 100,000 times with ethanol (100,000-fold diluted solution), B-3 diluted 20,000-fold with ethanol (20,000-fold diluted solution), B Cells were cultured in the same manner as in Example 4-1, except that a diluted solution (10,000-fold diluted solution) obtained by diluting -3 with ethanol to 10,000 times was used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 4-7>
As a coating solution, B-4 diluted 100,000 times with ethanol (100,000-fold diluted solution), B-4 diluted 20,000-fold with ethanol (20,000-fold diluted solution), B Cells were cultured in the same manner as in Example 4-1, except that a diluted solution obtained by diluting -4 with ethanol to 10,000 times (10,000-fold diluted solution) was used, and fluorescence intensity values were measured. The results are shown in FIG.

<Comparative Example 4-1>
Cells were cultured and fluorescence intensity values were measured in the same manner as in Example 4-1, except that a 96-well plate (uncoated) not coated with the coating solution was used. The results are shown in FIG.

<Comparative Example 4-2>
Cells were coated in the same manner as in Example 4-1, except that an aqueous solution obtained by dissolving poly-L-lysine (manufactured by ScienceCell Research Laboratories) in water to a concentration of 0.001% by mass was used as the coating solution. After culturing, fluorescence intensity values were measured. The results are shown in FIG.

<Example 4-8>
A diluted solution (10,000-fold diluted solution) obtained by diluting A-1 with pure water to 10,000-fold was used as the coating solution.
0.1 mL of the coating solution was dispensed into a 96-well plate (manufactured by Corning, model number "353072") and allowed to stand at 37°C for 8 hours to form a uniform layer over the entire culture surface. Thereafter, excess coating solution in the 96-well plate was removed, and the plate was allowed to stand at 37° C. for 1 hour to dry to form a cell adhesion layer on the bottom surface (culture surface) of the 96-well plate.
The cell suspension (2) was seeded on the cell adhesion layer of the dried 96-well plate and cultured at 20°C for 5 days. During culturing, the medium was exchanged once every 2 to 3 days. After culturing, the cells were fluorescently stained and the number of viable cells was counted. The results are shown in FIG.

<Example 4-9>
Cells were cultured in the same manner as in Example 4-8 except that a 10,000-fold dilution of A-2 with pure water (10,000-fold dilution) was used as the coating solution, and the fluorescence intensity value was measured. It was measured. The results are shown in FIG.

<Example 4-10>
Cells were cultured in the same manner as in Example 4-8 except that a 10,000-fold dilution of A-3 with pure water (10,000-fold dilution) was used as the coating solution, and the fluorescence intensity value was measured. It was measured. The results are shown in FIG.

<Example 4-11>
Cells were cultured in the same manner as in Example 4-8 except that a 20,000-fold dilution of B-1 with ethanol (20,000-fold dilution) was used as the coating solution, and the fluorescence intensity value was measured. did. The results are shown in FIG.

<Example 4-12>
As a coating solution, a diluted solution of B-2 diluted 100,000 times with ethanol (100,000-fold diluted solution) and a diluted solution of B-2 diluted 20,000-fold with ethanol (20,000-fold diluted solution) , and B-2 were diluted 10,000 times with ethanol (10,000-fold diluted solution), and the cells were cultured in the same manner as in Example 4-8, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 4-13>
As a coating solution, B-3 diluted 100,000 times with ethanol (100,000-fold diluted solution), B-3 diluted 20,000-fold with ethanol (20,000-fold diluted solution), B Cells were cultured in the same manner as in Example 4-8, except that a diluted solution obtained by diluting -3 with ethanol to 10,000 times (10,000-fold diluted solution) was used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 4-14>
As the coating solution, a diluted solution of B-4 diluted 20,000 times with ethanol (20,000-fold diluted solution) and a diluted solution of B-4 diluted 10,000-fold with ethanol (10,000-fold diluted solution) were used. Cells were cultured in the same manner as in Example 4-8, except that they were used, and fluorescence intensity values were measured. The results are shown in FIG.

<Comparative Example 4-3>
Cells were cultured and fluorescence intensity values were measured in the same manner as in Example 4-8 except that a 96-well plate (uncoated) not coated with a coating solution was used. The results are shown in FIG.

<Comparative Example 4-4>
Cells were coated in the same manner as in Example 4-8, except that an aqueous solution obtained by dissolving poly-L-lysine (manufactured by ScienCell Research Laboratories) in water to a concentration of 0.001% by mass was used as the coating solution. After culturing, fluorescence intensity values were measured. The results are shown in FIG.

<Example 4-15>
A diluted solution (10,000-fold diluted solution) obtained by diluting A-1 with pure water to 10,000-fold was used as the coating solution.
0.1 mL of the coating solution was dispensed into a 96-well plate (manufactured by Corning, model number "353072") and allowed to stand at 37°C for 1 week to form a uniform layer over the entire culture surface. Thereafter, excess coating solution in the 96-well plate was removed, and the plate was allowed to stand at 37° C. for 1 hour to dry to form a cell adhesion layer on the bottom surface (culture surface) of the 96-well plate.
The cell suspension (2) was seeded on the cell adhesion layer of the dried 96-well plate and cultured at 20°C for 5 days. During culturing, the medium was exchanged once every 2 to 3 days. After culturing, the cells were fluorescently stained and the number of viable cells was counted. The results are shown in FIG.

<Example 4-16>
Cells were cultured in the same manner as in Example 4-15 except that a 10,000-fold dilution of A-2 with pure water (10,000-fold dilution) was used as the coating solution, and the fluorescence intensity value was measured. It was measured. The results are shown in FIG.

<Example 4-17>
Cells were cultured in the same manner as in Example 4-15, except that a 100,000-fold dilution of B-1 with ethanol (100,000-fold dilution) was used as the coating solution, and the fluorescence intensity value was measured. did. The results are shown in FIG.

<Example 4-18>
As a coating solution, a diluted solution of B-2 diluted 20,000 times with ethanol (20,000-fold diluted solution) and a diluted solution of B-2 diluted 10,000-fold with ethanol (10,000-fold diluted solution) Cells were cultured in the same manner as in Example 4-15, except that , was used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 4-19>
As the coating solution, a diluted solution of B-3 diluted 20,000 times with ethanol (20,000-fold diluted solution) and a diluted solution of B-3 diluted 10,000 times with ethanol (10,000-fold diluted solution) were used. Cells were cultured in the same manner as in Example 4-15, except that they were used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 4-20>
Cells were cultured in the same manner as in Example 4-15 except that a 10,000-fold dilution of B-4 with ethanol (10,000-fold dilution) was used as the coating solution, and the fluorescence intensity value was measured. did. The results are shown in FIG.

<Comparative Example 4-5>
Cells were cultured and fluorescence intensity values were measured in the same manner as in Examples 4-15, except that a 96-well plate (uncoated) not coated with a coating solution was used. The results are shown in FIG.

<Comparative Example 4-6>
Cells were coated in the same manner as in Example 4-15, except that an aqueous solution obtained by dissolving poly-L-lysine (manufactured by ScienCell Research Laboratories) in water to a concentration of 0.001% by mass was used as the coating solution. After culturing, fluorescence intensity values were measured. The results are shown in FIG.

<Example 4-21>
As the coating solution, a diluted solution of B-1 diluted 100,000 times with ethanol (100,000-fold diluted solution) and a diluted solution of B-1 diluted 20,000-fold with ethanol (20,000-fold diluted solution) were used. board. 0.1 mL of the coating solution was dispensed into a 96-well plate (manufactured by Corning, model number "353072") and allowed to stand at 37°C for 1 week to form a uniform layer over the entire culture surface. After that, excess coating solution in the 96-well plate was removed, and the plate was vacuum-dried at room temperature for 1 week to form a cell adhesion layer on the bottom (culture surface) of the 96-well plate.
The cell suspension (2) was seeded on the cell adhesion layer of the dried 96-well plate and cultured at 20°C for 5 days. During culturing, the medium was exchanged once every 2 to 3 days. After culturing, the cells were fluorescently stained and the number of viable cells was counted. The results are shown in FIG.

<Example 4-22>
As a coating solution, a diluted solution of B-2 diluted 20,000 times with ethanol (20,000-fold diluted solution) and a diluted solution of B-2 diluted 10,000-fold with ethanol (10,000-fold diluted solution) , and B-2 were diluted 10,000 times with ethanol (10,000-fold diluted solution), and the cells were cultured in the same manner as in Example 4-21, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 4-23>
As a coating solution, B-3 diluted 100,000 times with ethanol (100,000-fold diluted solution), B-3 diluted 20,000-fold with ethanol (20,000-fold diluted solution), B Cells were cultured in the same manner as in Example 4-21, except that a diluted solution (10,000-fold diluted solution) obtained by diluting -3 with ethanol to 10,000 times was used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 4-24>
As the coating solution, a diluted solution of B-4 diluted 20,000 times with ethanol (20,000-fold diluted solution) and a diluted solution of B-4 diluted 10,000-fold with ethanol (10,000-fold diluted solution) were used. Cells were cultured in the same manner as in Example 4-21, except that they were used, and fluorescence intensity values were measured. The results are shown in FIG.

<Comparative Example 4-7>
Cells were cultured and fluorescence intensity values were measured in the same manner as in Example 4-21, except that a 96-well plate (uncoated) not coated with the coating solution was used. The results are shown in FIG.

<Comparative Example 4-8>
Cells were coated in the same manner as in Example 4-21, except that an aqueous solution obtained by dissolving poly-L-lysine (manufactured by ScienCell Research Laboratories) in water to a concentration of 0.001% by mass was used as the coating solution. After culturing, fluorescence intensity values were measured. The results are shown in FIG.

As shown in FIGS. 8 to 11, when cell culture is performed using a cell culture vessel coated with component (A) or component (B), uncoated cell culture vessels or poly-L-lysine-coated cell culture Compared to the case of cell culture using a vessel, the fluorescence intensity value was higher, and the effect of cell proliferation was recognized even for vascular endothelial cells, which are said to have weak cell adhesion. In particular, component (B) exhibited excellent cell adhesiveness even after a coating time of one week, and was superior in coating stability.

[Test 5]
<Example 5-1>
As a coating solution, A-1 was diluted 100,000 times with pure water (100,000-fold diluted solution), and A-1 was diluted 20,000-fold with pure water (20,000-fold diluted solution). , A-1 diluted 10,000 times with pure water (10,000 times diluted solution).
0.1 mL of the coating solution was dispensed into a 96-well plate (manufactured by Corning, model number "353072") and allowed to stand at 37°C for 1 hour to form a uniform layer over the entire culture surface. After that, the surplus coating solution in the 96-well plate is removed, 3 mL of pure water is added, and the operation of standing at 37 ° C. for 1 hour and washing is performed once, and the cells are placed on the bottom (culture surface) of the 96-well plate. An adhesive layer was formed.
The cell suspension (3) was seeded on the cell adhesion layer of the washed 96-well plate and cultured at 20°C for 7 days. During culturing, the medium was exchanged once every 2 to 3 days. After culturing, the cells were fluorescently stained and the number of viable cells was counted. The results are shown in FIG.

<Example 5-2>
As a coating solution, A-2 was diluted 20,000 times with pure water (20,000-fold dilution), and A-2 was diluted 10,000-fold with pure water (10,000-fold dilution). Cells were cultured in the same manner as in Example 5-1 except that , and fluorescence intensity values were measured. The results are shown in FIG.

<Example 5-3>
As a coating solution, A-3 was diluted 100,000 times with pure water (100,000-fold dilution), and A-3 was diluted 20,000-fold with pure water (20,000-fold dilution). , Cells were cultured in the same manner as in Example 5-1, except that a diluted solution (10,000-fold diluted solution) obtained by diluting A-3 with pure water 10,000 times was used, and the fluorescence intensity value was measured. . The results are shown in FIG.

<Example 5-4>
As the coating solution, a diluted solution of B-1 diluted 100,000 times with ethanol (100,000-fold diluted solution) and a diluted solution of B-1 diluted 20,000-fold with ethanol (20,000-fold diluted solution) were used. Cells were cultured in the same manner as in Example 5-1, except that they were used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 5-5>
As a coating solution, a diluted solution of B-2 diluted 20,000 times with ethanol (20,000-fold diluted solution) and a diluted solution of B-2 diluted 10,000-fold with ethanol (10,000-fold diluted solution) were used. Cells were cultured in the same manner as in Example 5-1, except that they were used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 5-6>
As a coating solution, B-3 diluted 100,000 times with ethanol (100,000-fold diluted solution), B-3 diluted 20,000-fold with ethanol (20,000-fold diluted solution), B Cells were cultured in the same manner as in Example 5-1, except that a diluted solution obtained by diluting -3 with ethanol to 10,000 times (10,000-fold diluted solution) was used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 5-7>
As the coating solution, a diluted solution of B-4 diluted 20,000 times with ethanol (20,000-fold diluted solution) and a diluted solution of B-4 diluted 10,000-fold with ethanol (10,000-fold diluted solution) were used. Cells were cultured in the same manner as in Example 5-1, except that they were used, and fluorescence intensity values were measured. The results are shown in FIG.

<Comparative Example 5-1>
Cells were cultured and fluorescence intensity values were measured in the same manner as in Example 5-1, except that a 96-well plate (uncoated) not coated with a coating solution was used. The results are shown in FIG.

<Comparative Example 5-2>
Cells were coated in the same manner as in Example 5-1, except that an aqueous solution obtained by dissolving poly-L-lysine (manufactured by ScienceCell Research Laboratories) in water to a concentration of 0.001% by mass was used as the coating solution. After culturing, fluorescence intensity values were measured. The results are shown in FIG.

<Example 5-8>
As a coating solution, A-1 was diluted 100,000 times with pure water (100,000-fold diluted solution), and A-1 was diluted 20,000-fold with pure water (20,000-fold diluted solution). , A-1 diluted 10,000 times with pure water (10,000 times diluted solution).
0.1 mL of the coating solution was dispensed into a 96-well plate (manufactured by Corning, model number "353072") and allowed to stand at 37°C for 1 hour to form a uniform layer over the entire culture surface. After that, the surplus coating solution in the 96-well plate is removed, 3 mL of pure water is added, and the operation of standing at 37° C. for 1 hour and washing is performed twice, and the cells are placed on the bottom (culture surface) of the 96-well plate. An adhesive layer was formed.
The cell suspension (3) was seeded on the cell adhesion layer of the washed 96-well plate and cultured at 20°C for 7 days. During culturing, the medium was exchanged once every 2 to 3 days. After culturing, the cells were fluorescently stained and the number of viable cells was counted. The results are shown in FIG.

<Example 5-9>
As a coating solution, A-2 was diluted 20,000 times with pure water (20,000-fold dilution), and A-2 was diluted 10,000-fold with pure water (10,000-fold dilution). Cells were cultured in the same manner as in Example 5-8 except that , and fluorescence intensity values were measured. The results are shown in FIG.

<Example 5-10>
As a coating solution, A-3 was diluted 100,000 times with pure water (100,000-fold dilution), and A-3 was diluted 20,000-fold with pure water (20,000-fold dilution). , Except for using a 10,000-fold dilution of A-3 with pure water (10,000-fold dilution), the cells were cultured in the same manner as in Example 5-8, and the fluorescence intensity value was measured. . The results are shown in FIG.

<Example 5-11>
As the coating solution, a diluted solution of B-1 diluted 100,000 times with ethanol (100,000-fold diluted solution) and a diluted solution of B-1 diluted 20,000-fold with ethanol (20,000-fold diluted solution) were used. Cells were cultured in the same manner as in Example 5-8, except that they were used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 5-12>
As a coating solution, a diluted solution of B-2 diluted 20,000 times with ethanol (20,000-fold diluted solution) and a diluted solution of B-2 diluted 10,000-fold with ethanol (10,000-fold diluted solution) were used. Cells were cultured in the same manner as in Example 5-8, except that they were used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 5-13>
As a coating solution, B-3 diluted 100,000 times with ethanol (100,000-fold diluted solution), B-3 diluted 20,000-fold with ethanol (20,000-fold diluted solution), B Cells were cultured in the same manner as in Example 5-8 except that a diluted solution obtained by diluting -3 with ethanol to 10,000 times (10,000-fold diluted solution) was used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 5-14>
As a coating solution, a diluted solution of B-4 diluted 20,000 times with ethanol (20,000-fold diluted solution) and a diluted solution of B-4 diluted 10,000-fold with ethanol (10,000-fold diluted solution) were used. Cells were cultured in the same manner as in Example 5-8, except that they were used, and fluorescence intensity values were measured. The results are shown in FIG.

<Comparative Example 5-3>
Cells were cultured and fluorescence intensity values were measured in the same manner as in Examples 5-8, except that a 96-well plate (uncoated) not coated with a coating solution was used. The results are shown in FIG.

<Comparative Example 5-4>
Cells were coated in the same manner as in Example 5-8, except that an aqueous solution obtained by dissolving poly-L-lysine (manufactured by ScienceCell Research Laboratories) in water to a concentration of 0.001% by mass was used as the coating solution. After culturing, fluorescence intensity values were measured. The results are shown in FIG.

<Example 5-15>
As a coating solution, A-1 was diluted 100,000 times with pure water (100,000-fold diluted solution), and A-1 was diluted 20,000-fold with pure water (20,000-fold diluted solution). , A-1 diluted 10,000 times with pure water (10,000 times diluted solution).
0.1 mL of the coating solution was dispensed into a 96-well plate (manufactured by Corning, model number "353072") and allowed to stand at 37°C for 1 hour to form a uniform layer over the entire culture surface. After that, the excess coating solution in the 96-well plate is removed, 3 mL of pure water is added, and the operation of standing at 37° C. for 1 hour and washing is performed three times, and the cells are placed on the bottom (culture surface) of the 96-well plate. An adhesive layer was formed.
Cell suspension (3) was seeded on the cell adhesion layer of the washed 96-well plate and cultured at 20°C for 7 days. During culturing, the medium was exchanged once every 2 to 3 days. After culturing, the cells were fluorescently stained and the number of viable cells was counted. The results are shown in FIG.

<Example 5-16>
As a coating solution, A-2 was diluted 20,000 times with pure water (20,000-fold dilution), and A-2 was diluted 10,000-fold with pure water (10,000-fold dilution). Cells were cultured in the same manner as in Example 5-15, except that , was used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 5-17>
As a coating solution, A-3 was diluted 100,000 times with pure water (100,000-fold dilution), and A-3 was diluted 20,000-fold with pure water (20,000-fold dilution). , Cells were cultured in the same manner as in Example 5-15, except that a 10,000-fold dilution of A-3 with pure water (10,000-fold dilution) was used, and the fluorescence intensity value was measured. . The results are shown in FIG.

<Example 5-18>
As the coating solution, a diluted solution of B-1 diluted 100,000 times with ethanol (100,000-fold diluted solution) and a diluted solution of B-1 diluted 20,000-fold with ethanol (20,000-fold diluted solution) were used. Cells were cultured in the same manner as in Example 5-15, except that they were used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 5-19>
As a coating solution, a diluted solution of B-2 diluted 20,000 times with ethanol (20,000-fold diluted solution) and a diluted solution of B-2 diluted 10,000-fold with ethanol (10,000-fold diluted solution) were used. Cells were cultured in the same manner as in Examples 5-15, except that they were used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 5-20>
As a coating solution, B-3 diluted 100,000 times with ethanol (100,000-fold diluted solution), B-3 diluted 20,000-fold with ethanol (20,000-fold diluted solution), B Cells were cultured in the same manner as in Example 5-15 except that a diluted solution obtained by diluting -3 with ethanol to 10,000 times (10,000-fold diluted solution) was used, and fluorescence intensity values were measured. The results are shown in FIG.

<Example 5-21>
As the coating solution, a diluted solution of B-4 diluted 20,000 times with ethanol (20,000-fold diluted solution) and a diluted solution of B-4 diluted 10,000-fold with ethanol (10,000-fold diluted solution) were used. Cells were cultured in the same manner as in Example 5-15, except that they were used, and fluorescence intensity values were measured. The results are shown in FIG.

<Comparative Example 5-5>
Cells were cultured and fluorescence intensity values were measured in the same manner as in Examples 5-15, except that a 96-well plate (uncoated) not coated with the coating solution was used. The results are shown in FIG.

<Comparative Example 5-6>
Cells were coated in the same manner as in Example 5-15, except that an aqueous solution obtained by dissolving poly-L-lysine (manufactured by ScienCell Research Laboratories) in water to a concentration of 0.001% by mass was used as the coating solution. After culturing, fluorescence intensity values were measured. The results are shown in FIG.

As shown in FIGS. 12 to 14, when cell culture is performed using a cell culture vessel coated with component (A) or component (B), uncoated cell culture vessels or poly-L-lysine-coated cell culture The effect of cell proliferation was observed even for Schwann cells, whose fluorescence intensity is higher than when cells are cultured using a vessel, and whose cell adhesion is considered to be very weak. In addition, the effect of cell proliferation could be maintained even after washing several times after coating with the coating agent.

10 cell culture vessel 11 vessel body 11a culture surface 12 cell adhesive layer

Claims

A coating agent for cell culture containing one or more compounds selected from the group consisting of water-based polyurethane resins and compounds having a terpene skeleton.
The coating agent for cell culture according to claim 1, wherein the compound having a terpene skeleton contains one or more compounds selected from the group consisting of terpene resins and rosin resins.
The coating agent for cell culture according to claim 1 or 2, wherein the rosin resin contains an α,β-unsaturated carboxylic acid-modified rosin.
A method for manufacturing a cell culture vessel, wherein the culture surface is coated with the cell culture coating agent according to any one of claims 1 to 3.
A cell culture vessel having a culture surface coated with a coating agent,
A cell culture vessel, wherein the coating agent contains one or more compounds selected from the group consisting of water-based polyurethane resins and compounds having a terpene skeleton.
A cell culture method for culturing cells using the cell culture vessel according to claim 5.