JP2014023508A - Manufacture method of plate for cell cultivation, plate for cell cultivation made by this manufacture method, cell cultivation method, cell sheet-manufacture method, cell sheet and photosensitive resin composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a photosensitive resin composition for forming a resin pattern on a substrate, a manufacture method of a plate for cell cultivation by using this photosensitive resin composition, a plate for cell cultivation made by this manufacture method, a cell cultivation method and a cell sheet-manufacture method by using this plate for cell cultivation, and a cell sheet made by this cell sheet-manufacture method.SOLUTION: The photosensitive resin composition according to the present invention comprises an alcoholic organic solvent (S) and is for forming a resin pattern on a substrate. The manufacture method of a plate for cell cultivation according to the present invention comprises using the photosensitive resin composition.

Description

  The present invention is a cell culture plate production method, a cell culture plate produced by the production method, a cell culture method and a cell sheet production method using the cell culture plate, and the cell sheet production method. The present invention relates to a cell sheet and a photosensitive resin composition.

  In recent years, cell sheets and cell micropatterns are often used in regenerative medicine and the like. Here, the cell sheet refers to a sheet-like cell aggregate in which cells are connected to each other in at least a single layer by intercellular bonding, and a cell pattern is a cell sheet formed as a specific shape desired. It is. A micro pattern refers to a pattern having a width of a micron (μm) scale, and a cell micro pattern is a cell sheet of a micro pattern. These cell sheets can be obtained by culturing cells on a support such as a petri dish, but the cell sheets formed on the support are firmly bonded to the support surface via adhesion molecules, etc. When used in applications such as regenerative medicine, it is necessary to peel from the support.

  Various methods for detaching the cell sheet from the cell culture support have been studied so far. Conventionally, a method of weakening the bond between the support and the cell using an enzyme reaction, a support having a weak cell adhesion, or a cell adhesion A method using a support having a variety of diameters is used.

  More specifically, the method using an enzyme reaction includes a method of degrading a protein constituting an intercellular adhesion molecule using a protease (proteolytic enzyme) or the like. However, this method weakens not only cell-support surface binding but also cell-cell binding. For this reason, this method has a problem that the cell sheet is damaged to some extent.

In addition, as a method of using a support that changes cell adhesive force, for example, in Patent Document 1, a base material is disposed on the base material, has cell adhesiveness, and is non-adhesive by stimulation. A micropatterning comprising a stimulus-responsive layer containing a stimulus-responsive material capable of expressing a cell, and a cell adhesion-inhibiting layer containing a cell adhesion-inhibiting material that is arranged in a pattern on the substrate and has cell non-adhesiveness A method using a vascular endothelial cell transfer substrate is disclosed. In Patent Document 1, a cell adhesion inhibition layer arranged in a pattern on a substrate is formed by patterning a coating film containing a cell adhesion inhibition material by a photolithographic method, or patterning a cell adhesion inhibition material by a known pattern printing method. It is formed by applying to.
In the field of cell culture, including the production of cell sheets and cell micropatterns, it is common to use a glass substrate or a polystyrene substrate as a substrate for culturing cells.

JP 2011-223944 A

  In forming a cell sheet or a cell micropattern, high accuracy and reproducibility are required. In order to realize such accuracy and reproducibility, the present inventors examined using a micropatterning technique using a photosensitive resin composition used as a photoresist. As a result, a substrate used for cell culture, for example, a resin substrate containing a resin such as polystyrene, is damaged by deformation, discoloration, dissolution, etc. due to contact with the photosensitive resin composition, and cells with high accuracy and reproducibility. It has proven difficult to form micropatterns.

  The present invention has been made in view of such conventional circumstances, and a photosensitive resin composition for forming a resin pattern on a substrate, and production of a plate for cell culture using the photosensitive resin composition It is an object to provide a method, a cell culture plate produced by the production method, a cell culture method and a cell sheet production method using the cell culture plate, and a cell sheet produced by the cell sheet production method To do.

  The inventors of the present invention have made extensive studies to solve the above problems. As a result, it has been found that the above problem can be solved by using an alcohol-based organic solvent as a solvent in the photosensitive resin composition, and the present invention has been completed. Specifically, the present invention provides the following.

The first aspect of the present invention is a method for producing a cell culture plate, which comprises using a photosensitive resin composition containing an alcohol-based organic solvent (S).
The second aspect of the present invention is a cell culture plate produced by the method for producing a cell culture plate according to the present invention.
The third aspect of the present invention is a cell culturing method including a cell culturing step of culturing cells on the cell culturing plate produced by the method for producing a cell culturing plate according to the present invention.
A fourth aspect of the present invention is a cell culture plate produced by the method for producing a cell culture plate according to the present invention, which has cell adhesiveness and exhibits cell non-adhesiveness due to temperature change. A cell culturing step for culturing cells at a temperature higher than a critical dissolution temperature at which the temperature responsive material changes from hydrophobic to hydrophilic on a cell culture plate having a temperature responsive material layer made of a temperature responsive material And a recovery step of recovering the cell sheet by separating the cell from the temperature-responsive material layer by changing the temperature of the cell culture plate to the critical lysis temperature or lower. It is.
The fifth aspect of the present invention is a cell sheet produced by the cell sheet production method according to the present invention.
The 6th aspect of this invention is the photosensitive resin composition for forming a resin pattern on a board | substrate containing alcohol type organic solvent (S).

  According to the present invention, a photosensitive resin composition for forming a resin pattern on a substrate, a method for producing a cell culture plate using this photosensitive resin composition, and a cell culture plate produced by this production method In addition, a cell culture method and a cell sheet production method using the cell culture plate, and a cell sheet produced by the cell sheet production method can be provided.

It is a cross-sectional view which shows one Embodiment (The manufacturing method 1 of a cell culture plate) of the manufacturing method of the cell culture plate which concerns on this invention. Thereby, a plate for cell culture provided with substrate 1 and cell adhesion inhibition material layer 3 provided in a part on substrate 1 is obtained. It is a cross-sectional view showing another embodiment of the cell culture plate production method according to the present invention (cell culture plate production method 2). As a result, a cell culture plate including the substrate 1 and the temperature-responsive material layer 4 provided on a part of the substrate 1 is obtained. It is a cross-sectional view showing still another embodiment (Method 3 for producing a cell culture plate) of the method for producing a cell culture plate according to the present invention. Accordingly, the substrate 1, the cell adhesion inhibiting material layer 3 provided on a part of the substrate 1, and the temperature responsive material layer provided on a part of the substrate 1 where the cell adhesion inhibiting material layer 3 is not provided. 4 is obtained.

  Hereinafter, embodiments of the present invention will be described in detail. In the following, first, a photosensitive resin composition for forming a resin pattern on a substrate according to the present invention will be described, and then a method for producing a cell culture plate, a cell culture plate, and a cell culture according to the present invention A method, a cell sheet preparation method, and a cell sheet will be described sequentially.

<< Photosensitive resin composition for forming a resin pattern on a substrate >>
The photosensitive resin composition for forming a resin pattern on a substrate according to the present invention (hereinafter simply referred to as “photosensitive resin composition”) contains at least an alcohol-based organic solvent (S). . This photosensitive resin composition is suitably used for forming a resin pattern on a substrate, particularly on a resin substrate containing a resin such as polystyrene. The photosensitive resin composition may be either a positive type or a negative type, but is preferably a positive type. Further, when the photosensitive resin composition is a positive type, it may be a non-chemical amplification type or a chemical amplification type. Hereinafter, each component contained in the non-chemically amplified positive photosensitive resin composition and the chemically amplified positive photosensitive resin composition will be described in detail.

<Non-chemical amplification type positive photosensitive resin composition>
The non-chemically amplified positive photosensitive resin composition contains at least a quinonediazide group-containing compound (A), an alkali-soluble resin (B), and an alcohol organic solvent (S).

[Quinonediazide group-containing compound (A)]
Although it does not specifically limit as a quinone diazide group containing compound (A), The complete esterification thing and partial esterification thing of the compound which has one or more phenolic hydroxyl groups, and a quinone diazide group containing sulfonic acid are preferable. Such a quinonediazide group-containing compound is obtained by combining a compound having one or more phenolic hydroxyl groups and a quinonediazide group-containing sulfonic acid in an appropriate solvent such as dioxane with an alkali such as triethanolamine, alkali carbonate, or alkali hydrogencarbonate. It can be obtained by condensation in the presence and complete esterification or partial esterification.

Examples of the compound having one or more phenolic hydroxyl groups include polyhydroxybenzophenones such as 2,3,4-trihydroxybenzophenone and 2,3,4,4′-tetrahydroxybenzophenone;
Tris (4-hydroxyphenyl) methane, bis (4-hydroxy-3-methylphenyl) -2-hydroxyphenylmethane, bis (4-hydroxy-2,3,5-trimethylphenyl) -2-hydroxyphenylmethane, Bis (4-hydroxy-3,5-dimethylphenyl) -4-hydroxyphenylmethane, bis (4-hydroxy-3,5-dimethylphenyl) -3-hydroxyphenylmethane, bis (4-hydroxy-3,5- Dimethylphenyl) -2-hydroxyphenylmethane, bis (4-hydroxy-2,5-dimethylphenyl) -4-hydroxyphenylmethane, bis (4-hydroxy-2,5-dimethylphenyl) -3-hydroxyphenylmethane, Bis (4-hydroxy-2,5-dimethylphenyl) -2-hydroxyl Nylmethane, bis (4-hydroxy-3,5-dimethylphenyl) -3,4-dihydroxyphenylmethane, bis (4-hydroxy-2,5-dimethylphenyl) -3,4-dihydroxyphenylmethane, bis (4- Hydroxy-2,5-dimethylphenyl) -2,4-dihydroxyphenylmethane, bis (4-hydroxyphenyl) -3-methoxy-4-hydroxyphenylmethane, bis (5-cyclohexyl-4-hydroxy-2-methylphenyl) ) -4-hydroxyphenylmethane, bis (5-cyclohexyl-4-hydroxy-2-methylphenyl) -3-hydroxyphenylmethane, bis (5-cyclohexyl-4-hydroxy-2-methylphenyl) -2-hydroxyphenyl Methane, bis (5-cyclohexyl-4-hydro Shi-2-methylphenyl) -3,4-trisphenol compounds such dihydroxyphenyl methane;
Linear type 3 such as 2,4-bis (3,5-dimethyl-4-hydroxybenzyl) -5-hydroxyphenol, 2,6-bis (2,5-dimethyl-4-hydroxybenzyl) -4-methylphenol Nuclear phenolic compounds;
1,1-bis [3- (2-hydroxy-5-methylbenzyl) -4-hydroxy-5-cyclohexylphenyl] isopropane, bis [2,5-dimethyl-3- (4-hydroxy-5-methylbenzyl) ) -4-hydroxyphenyl] methane, bis [2,5-dimethyl-3- (4-hydroxybenzyl) -4-hydroxyphenyl] methane, bis [3- (3,5-dimethyl-4-hydroxybenzyl)- 4-hydroxy-5-methylphenyl] methane, bis [3- (3,5-dimethyl-4-hydroxybenzyl) -4-hydroxy-5-ethylphenyl] methane, bis [3- (3,5-diethyl- 4-hydroxybenzyl) -4-hydroxy-5-methylphenyl] methane, bis [3- (3,5-diethyl-4-hydroxybenzyl) -4- Loxy-5-ethylphenyl] methane, bis [2-hydroxy-3- (3,5-dimethyl-4-hydroxybenzyl) -5-methylphenyl] methane, bis [2-hydroxy-3- (2-hydroxy-) 5-methylbenzyl) -5-methylphenyl] methane, bis [4-hydroxy-3- (2-hydroxy-5-methylbenzyl) -5-methylphenyl] methane, bis [2,5-dimethyl-3- ( Linear tetranuclear phenolic compounds such as 2-hydroxy-5-methylbenzyl) -4-hydroxyphenyl] methane;
2,4-bis [2-hydroxy-3- (4-hydroxybenzyl) -5-methylbenzyl] -6-cyclohexylphenol, 2,4-bis [4-hydroxy-3- (4-hydroxybenzyl) -5 Linear type 5 such as -methylbenzyl] -6-cyclohexylphenol and 2,6-bis [2,5-dimethyl-3- (2-hydroxy-5-methylbenzyl) -4-hydroxybenzyl] -4-methylphenol Nuclear phenolic compounds;
Bis (2,3,4-trihydroxyphenyl) methane, bis (2,4-dihydroxyphenyl) methane, 2,3,4-trihydroxyphenyl-4′-hydroxyphenylmethane, 2- (2,3,4) -Trihydroxyphenyl) -2- (2 ', 3', 4'-trihydroxyphenyl) propane, 2- (2,4-dihydroxyphenyl) -2- (2 ', 4'-dihydroxyphenyl) propane, 2 -(4-hydroxyphenyl) -2- (4'-hydroxyphenyl) propane, 2- (3-fluoro-4-hydroxyphenyl) -2- (3'-fluoro-4'-hydroxyphenyl) propane, 2- (2,4-dihydroxyphenyl) -2- (4′-hydroxyphenyl) propane, 2- (2,3,4-trihydroxyphenyl) -2- (4′-hydroxyphenyl) ) Propane, 2- (2,3,4-trihydroxyphenyl) -2- (4′-hydroxy-3 ′, 5′-dimethylphenyl) propane, 4,4 ′-[1- [4- [1 Bisphenol type compounds such as-(4-hydroxyphenyl) -1-methylethyl] phenyl] ethylidene] bisphenol;
1- [1- (4-hydroxyphenyl) isopropyl] -4- [1,1-bis (4-hydroxyphenyl) ethyl] benzene, 1- [1- (3-methyl-4-hydroxyphenyl) isopropyl]- Polynuclear branched compounds such as 4- [1,1-bis (3-methyl-4-hydroxyphenyl) ethyl] benzene;
And a condensed phenol compound such as 1,1-bis (4-hydroxyphenyl) cyclohexane.
These compounds may be used alone or in combination of two or more.

  Examples of the quinonediazide group-containing sulfonic acid include naphthoquinone-1,2-diazide-5-sulfonic acid, naphthoquinone-1,2-diazide-4-sulfonic acid, and orthoanthraquinonediazidesulfonic acid.

  The content of the quinonediazide group-containing compound (A) is preferably 5 to 50 parts by mass and more preferably 5 to 25 parts by mass with respect to 100 parts by mass of the alkali-soluble resin (B). By setting it as said range, the sensitivity of the non-chemical amplification type positive photosensitive resin composition obtained can be made favorable.

[Alkali-soluble resin (B)]
It does not specifically limit as alkali-soluble resin (B), Arbitrary alkali-soluble resins can be used. Here, the alkali-soluble resin is a resin film having a resin concentration of 20% by mass (solvent: propylene glycol monomethyl ether acetate), and a 1 μm-thick resin film is formed on the substrate to form a 2.38% by mass TMAH aqueous solution. When immersed for 1 minute, it means one that dissolves by 0.01 μm or more. The alkali-soluble resin (B) is preferably at least one resin selected from the group consisting of a novolak resin (B1), a polyhydroxystyrene resin (B2), and an acrylic resin (B3).

(Novolac resin (B1))
The novolac resin (B1) can be obtained, for example, by addition condensation of an aromatic compound having a phenolic hydroxyl group (hereinafter simply referred to as “phenols”) and an aldehyde in the presence of an acid catalyst.

Examples of the phenols include phenol, o-cresol, m-cresol, p-cresol, o-ethylphenol, m-ethylphenol, p-ethylphenol, o-butylphenol, m-butylphenol, p-butylphenol, 2 , 3-xylenol, 2,4-xylenol, 2,5-xylenol, 2,6-xylenol, 3,4-xylenol, 3,5-xylenol, 2,3,5-trimethylphenol, 3,4,5- Examples include trimethylphenol, p-phenylphenol, resorcinol, hydroquinone, hydroquinone monomethyl ether, pyrogallol, phloroglicinol, hydroxydiphenyl, bisphenol A, gallic acid, gallic acid ester, α-naphthol, β-naphthol and the like.
Examples of the aldehydes include formaldehyde, furfural, benzaldehyde, nitrobenzaldehyde, acetaldehyde and the like.
The catalyst for the addition condensation reaction is not particularly limited. For example, hydrochloric acid, nitric acid, sulfuric acid, formic acid, oxalic acid, acetic acid and the like are used as the acid catalyst.

In addition, it is possible to further improve the flexibility of the novolak resin by using o-cresol, by replacing the hydrogen atom of the hydroxyl group in the resin with another substituent, or by using bulky aldehydes. It is.
The novolak resin (B1) preferably has a mass average molecular weight of 1000 to 50000.

(Polyhydroxystyrene resin (B2))
Examples of the hydroxystyrene-based compound constituting the polyhydroxystyrene resin (B2) include p-hydroxystyrene, α-methylhydroxystyrene, α-ethylhydroxystyrene, and the like.
Furthermore, the polyhydroxystyrene resin (B2) is preferably a copolymer with a styrene resin. Examples of the styrene compound constituting such a styrene resin include styrene, chlorostyrene, chloromethylstyrene, vinyltoluene, α-methylstyrene, and the like.
The polyhydroxystyrene resin (B2) preferably has a mass average molecular weight of 1000 to 50000.

(Acrylic resin (B3))
The acrylic resin (B3) preferably includes a structural unit derived from a polymerizable compound having an ether bond and a structural unit derived from a polymerizable compound having a carboxyl group.

  Examples of the polymerizable compound having an ether bond include 2-methoxyethyl (meth) acrylate, methoxytriethylene glycol (meth) acrylate, 3-methoxybutyl (meth) acrylate, ethyl carbitol (meth) acrylate, phenoxypolyethylene glycol ( Examples include (meth) acrylic acid derivatives having an ether bond and an ester bond such as (meth) acrylate, methoxypolypropylene glycol (meth) acrylate, and tetrahydrofurfuryl (meth) acrylate. The polymerizable compound having an ether bond is preferably 2-methoxyethyl acrylate or methoxytriethylene glycol acrylate. These polymerizable compounds may be used alone or in combination of two or more.

Examples of the polymerizable compound having a carboxyl group include monocarboxylic acids such as acrylic acid, methacrylic acid, and crotonic acid; dicarboxylic acids such as maleic acid, fumaric acid, and itaconic acid; 2-methacryloyloxyethyl succinic acid, and 2-methacryloyloxy. Examples thereof include compounds having a carboxyl group and an ester bond such as ethylmaleic acid, 2-methacryloyloxyethylphthalic acid, and 2-methacryloyloxyethylhexahydrophthalic acid. The polymerizable compound having a carboxyl group is preferably acrylic acid or methacrylic acid. These polymerizable compounds may be used alone or in combination of two or more.
The acrylic resin (B3) preferably has a mass average molecular weight of 50,000 to 800,000.

  It is preferable that content of alkali-soluble resin (B) shall be 5-60 mass% with respect to the total mass of the non-chemical type positive photosensitive resin composition which concerns on this invention.

[Alcohol-based organic solvent (S)]
The non-chemical type positive photosensitive resin composition according to the present invention can be produced and used as a uniform solution in which components other than the alcohol-based organic solvent (S) are dissolved in the alcohol-based organic solvent (S). In the present specification, the “alcohol-based organic solvent” refers to a compound in which at least one of the hydrogen atoms of an aliphatic hydrocarbon is substituted with a hydroxyl group and is a liquid at normal temperature and pressure. Since the alcohol organic solvent (S) does not have a high ability to dissolve a substrate, particularly a resin substrate containing a resin such as polystyrene, the substrate has sufficient resistance to the alcohol organic solvent (S). Therefore, the photosensitive resin composition of the present invention containing the alcohol-based organic solvent (S) can form a resin pattern on the substrate with high accuracy and reproducibility while effectively suppressing damage to the substrate. The alcohol-based organic solvent (S) may be used alone or in combination of two or more.

  Examples of the alcohol-based organic solvent (S) include monohydric alcohols and dihydric alcohols, monohydric alcohols are preferred, primary or secondary monohydric alcohols are more preferred, and secondary monohydric alcohols are preferred. Further, secondary monohydric alcohols having 5 or more carbon atoms (more preferably 7 or more carbon atoms) are particularly preferable. Furthermore, as the alcohol-based organic solvent (S), an alcohol having a cyclic structure having 5 or more carbon atoms (more preferably 7 or more carbon atoms) and 5 or more carbon atoms (more preferably 7 or more carbon atoms) are used. An alcohol having a chain structure is preferred. The cyclic structure or chain structure may include an ether bond in the structure. For a chain structure having 5 or more carbon atoms (more preferably 7 or more carbon atoms), it is further preferred that the longest main chain containing a hydroxyl group has 5 or more carbon atoms. The main chain may contain an ether bond in its structure. Particularly preferred as the alcohol-based organic solvent (S) is a monohydric alcohol having a chain structure having 5 or more (more preferably 7 or more) carbon atoms. Here, “monohydric alcohol” means an alcohol having one hydroxyl group in the molecule, and does not include dihydric alcohols, trihydric alcohols, and derivatives thereof. The upper limit of the carbon number is not particularly limited as long as the alcohol-based organic solvent (S) is a liquid at normal temperature and normal pressure, but is, for example, 30 or less, preferably 20 or less.

  The boiling point of the alcohol-based organic solvent (S) is preferably 80 to 250 ° C. from the viewpoints of coating properties, stability of the composition during storage, and heating temperature in the PAB step or PEB step, and 90 to 240 ° C. It is more preferable that it is 100-235 degreeC. “Boiling point” refers to the standard boiling point measured under normal pressure.

  Specific examples of the alcohol organic solvent (S) include propylene glycol (PG; boiling point 188 ° C.); 1-butoxy-2-propanol (BP; boiling point 170 ° C.), n-hexanol (boiling point 157.1 ° C.), 2 -Heptanol (boiling point 160.4 ° C), 3-heptanol (boiling point 156.2 ° C), 1-heptanol (boiling point 176 ° C), 5-methyl-1-hexanol (boiling point 167 ° C), 6-methyl-2-heptanol (Boiling point 171 ° C), 1-octanol (boiling point 196 ° C) 2-octanol (boiling point 179 ° C), 3-octanol (boiling point 175 ° C), 4-octanol (boiling point 175 ° C), 2-ethyl-1-hexanol (boiling point) 185 ° C), 2- (2-butoxyethoxy) ethanol (boiling point 231 ° C), n-pentyl alcohol (boiling point 138.0 ° C), s- Nthyl alcohol (boiling point 119.3 ° C.), t-pentyl alcohol (101.8 ° C.), isopentyl alcohol (boiling point 130.8 ° C.), isobutanol (also called isobutyl alcohol or 2-methyl-1-propanol) (Boiling point 107.9 ° C), isopropyl alcohol (boiling point 82.3 ° C), 2-ethylbutanol (boiling point 147 ° C), neopentyl alcohol (boiling point 114 ° C), n-butanol (boiling point 117.7 ° C), s- Butanol (boiling point 99.5 ° C), t-butanol (boiling point 82.5 ° C), 1-propanol (boiling point 97.2 ° C), 2-methyl-1-butanol (boiling point 128.0 ° C), 2-methyl- Monohydric alcohols having a chain structure such as 2-butanol (boiling point 112.0 ° C.), 4-methyl-2-pentanol (boiling point 131.8 ° C.); N-methanol (boiling point 162 ° C), 1-cyclopentylethanol (boiling point 167 ° C), cyclohexanol (boiling point 160 ° C), cyclohexanemethanol (CM; boiling point 183 ° C), cyclohexaneethanol (boiling point 205 ° C), 1, 2, 3, 6-tetrahydrobenzyl alcohol (boiling point 191 ° C), exo-norbornol (boiling point 176 ° C), 2-methylcyclohexanol (boiling point 165 ° C), cycloheptanol (boiling point 185 ° C), 3,5-dimethylcyclohexanol (boiling point) 185 ° C.) and cyclic alcohols such as benzyl alcohol (boiling point 204 ° C.). Among these, a monohydric alcohol having a chain structure is preferable, a secondary monohydric alcohol having a chain structure is more preferable, and a longest main chain containing a hydroxyl group has 5 to 9 carbon atoms (preferably 5 to 8 carbon atoms). A secondary monohydric alcohol is more preferable, and 1-butoxy-2-propanol is most preferable.

  Content of alcohol type organic solvent (S) is not specifically limited, For example, the solid content concentration of the non-chemical type positive photosensitive resin composition which concerns on this invention is 5-50 mass%, Preferably it is 10-30 mass. %.

(Other ingredients)
The non-chemical type positive photosensitive resin composition according to the present invention may further contain a polyvinyl resin in order to improve plasticity. Specific examples of the polyvinyl resin include polyvinyl chloride, polystyrene, polyhydroxystyrene, polyvinyl acetate, polyvinyl benzoic acid, polyvinyl methyl ether, polyvinyl ethyl ether, polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl phenol, and copolymers thereof. Is mentioned. The polyvinyl resin is preferably polyvinyl methyl ether because of its low glass transition point.
In addition, the non-chemical positive photosensitive resin composition according to the present invention may further contain an adhesion assistant in order to improve the adhesion to the support.

  In addition, the non-chemical positive photosensitive resin composition according to the present invention may further contain a surfactant in order to improve coating properties, antifoaming properties, leveling properties and the like. Specific examples of the surfactant include BM-1000, BM-1100 (all manufactured by BM Chemie), MegaFuck F142D, MegaFuck F172, MegaFuck F173, MegaFak F183 (all manufactured by Dainippon Ink & Chemicals, Inc.) ), Fluorard FC-135, Fluorard FC-170C, Fluorard FC-430, Fluorard FC-431 (all manufactured by Sumitomo 3M), Surflon S-112, Surflon S-113, Surflon S-131, Surflon S-141, Commercially available fluorosurfactants such as Surflon S-145 (all manufactured by Asahi Glass Co., Ltd.), SH-28PA, SH-190, SH-193, SZ-6032, SF-8428 (all manufactured by Toray Silicone Co., Ltd.) However, it is not limited to these.

Further, the non-chemical positive photosensitive resin composition according to the present invention may further contain an acid or an acid anhydride in order to finely adjust the solubility in a developer. Specific examples of the acid and acid anhydride include monocarboxylic acids such as acetic acid, propionic acid, n-butyric acid, isobutyric acid, n-valeric acid, isovaleric acid, benzoic acid and cinnamic acid; lactic acid, 2-hydroxybutyric acid, Hydroxy monocarboxylic acids such as 3-hydroxybutyric acid, salicylic acid, m-hydroxybenzoic acid, p-hydroxybenzoic acid, 2-hydroxycinnamic acid, 3-hydroxycinnamic acid, 4-hydroxycinnamic acid, 5-hydroxyisophthalic acid and syringic acid Acids: oxalic acid, succinic acid, glutaric acid, adipic acid, maleic acid, itaconic acid, hexahydrophthalic acid, phthalic acid, isophthalic acid, terephthalic acid, 1,2-cyclohexanedicarboxylic acid, 1,2,4-cyclohexanetricarboxylic acid Acid, butanetetracarboxylic acid, trimellitic acid, pyromellitic acid, cyclopentanetetracarbo Polycarboxylic acids such as acid, butanetetracarboxylic acid, 1,2,5,8-naphthalenetetracarboxylic acid; itaconic anhydride, succinic anhydride, citraconic anhydride, dodecenyl succinic anhydride, tricarbanilic anhydride, maleic anhydride, Hexahydrophthalic anhydride, methyltetrahydrophthalic anhydride, hymic anhydride, 1,2,3,4-butanetetracarboxylic anhydride, cyclopentanetetracarboxylic dianhydride, phthalic anhydride, pyromellitic anhydride, anhydrous And acid anhydrides such as trimellitic acid, benzophenone anhydride tetracarboxylic acid, ethylene glycol bistrimellitic anhydride, and glycerin tris anhydrous trimellitate.
In addition, the non-chemical positive photosensitive resin composition according to the present invention may further contain a sensitizer in order to improve sensitivity.
Furthermore, the non-chemical type positive photosensitive resin composition according to the present invention may contain an organic solvent other than the alcohol-based organic solvent (S) as long as the effects of the present invention are not impaired.

<Chemical amplification type positive photosensitive resin composition>
The chemically amplified positive photosensitive resin composition includes an acid generator (C) that generates an acid upon irradiation with actinic rays or radiation, a resin (D) whose solubility in an alkali is increased by the action of an acid, and an alcohol type At least the organic solvent (S) is contained.

[Acid generator (C)]
The acid generator (C) used in the present invention is a compound that generates an acid by irradiation with actinic rays or radiation, and is not particularly limited as long as it is a compound that generates an acid directly or indirectly by light.

  As a first aspect in the acid generator (C), 2,4-bis (trichloromethyl) -6-piperonyl-1,3,5-triazine, 2,4-bis (trichloromethyl) -6- [2 -(2-furyl) ethenyl] -s-triazine, 2,4-bis (trichloromethyl) -6- [2- (5-methyl-2-furyl) ethenyl] -s-triazine, 2,4-bis ( Trichloromethyl) -6- [2- (5-ethyl-2-furyl) ethenyl] -s-triazine, 2,4-bis (trichloromethyl) -6- [2- (5-propyl-2-furyl) ethenyl ] -S-triazine, 2,4-bis (trichloromethyl) -6- [2- (3,5-dimethoxyphenyl) ethenyl] -s-triazine, 2,4-bis (trichloromethyl) -6- [2 -(3,5-diethoxyf Nyl) ethenyl] -s-triazine, 2,4-bis (trichloromethyl) -6- [2- (3,5-dipropoxyphenyl) ethenyl] -s-triazine, 2,4-bis (trichloromethyl)- 6- [2- (3-Methoxy-5-ethoxyphenyl) ethenyl] -s-triazine, 2,4-bis (trichloromethyl) -6- [2- (3-methoxy-5-propoxyphenyl) ethenyl]- s-triazine, 2,4-bis (trichloromethyl) -6- [2- (3,4-methylenedioxyphenyl) ethenyl] -s-triazine, 2,4-bis (trichloromethyl) -6- (3 , 4-methylenedioxyphenyl) -s-triazine, 2,4-bis-trichloromethyl-6- (3-bromo-4-methoxy) phenyl-s-triazine, 2,4-bis- Lichloromethyl-6- (2-bromo-4-methoxy) phenyl-s-triazine, 2,4-bis-trichloromethyl-6- (2-bromo-4-methoxy) styrylphenyl-s-triazine, 2,4- Bis-trichloromethyl-6- (3-bromo-4-methoxy) styrylphenyl-s-triazine, 2- (4-methoxyphenyl) -4,6-bis (trichloromethyl) -1,3,5-triazine, 2- (4-Methoxynaphthyl) -4,6-bis (trichloromethyl) -1,3,5-triazine, 2- [2- (2-furyl) ethenyl] -4,6-bis (trichloromethyl)- 1,3,5-triazine, 2- [2- (5-methyl-2-furyl) ethenyl] -4,6-bis (trichloromethyl) -1,3,5-triazine, 2- [2- (3 , 5- Dimethoxyphenyl) ethenyl] -4,6-bis (trichloromethyl) -1,3,5-triazine, 2- [2- (3,4-dimethoxyphenyl) ethenyl] -4,6-bis (trichloromethyl)- 1,3,5-triazine, 2- (3,4-methylenedioxyphenyl) -4,6-bis (trichloromethyl) -1,3,5-triazine, tris (1,3-dibromopropyl) -1 , 3,5-triazine, halogen-containing triazine compounds such as tris (2,3-dibromopropyl) -1,3,5-triazine, and the following general formulas such as tris (2,3-dibromopropyl) isocyanurate ( and halogen-containing triazine compounds represented by a3).

In the general formula (a3), R ^9a , R ^10a and R ^11a each independently represent a halogenated alkyl group.
In addition, as a second aspect in the acid generator (C), α- (p-toluenesulfonyloxyimino) -phenylacetonitrile, α- (benzenesulfonyloxyimino) -2,4-dichlorophenylacetonitrile, α- (benzene Sulfonyloxyimino) -2,6-dichlorophenylacetonitrile, α- (2-chlorobenzenesulfonyloxyimino) -4-methoxyphenylacetonitrile, α- (ethylsulfonyloxyimino) -1-cyclopentenylacetonitrile, and oxime sulfonate The compound represented by the following general formula (a4) containing a group is mentioned.

In the general formula (a4), R ^12a represents a monovalent, divalent, or trivalent organic group, and R ^13a represents a substituted or unsubstituted saturated hydrocarbon group, unsaturated hydrocarbon group, or aromatic group. N represents the number of repeating units having a structure in parentheses.
In the above general formula (a4), the aromatic compound group refers to a group of a compound exhibiting physical and chemical properties peculiar to an aromatic compound. For example, an aryl group such as a phenyl group or a naphthyl group, or furyl And heteroaryl groups such as a thienyl group and the like. These may have one or more suitable substituents such as a halogen atom, an alkyl group, an alkoxy group, and a nitro group on the ring. R ^13a is particularly preferably an alkyl group having 1 to 6 carbon atoms, and examples thereof include a methyl group, an ethyl group, a propyl group, and a butyl group. In particular, a compound in which R ^12a is an aromatic compound group and R ^13a is an alkyl group having 1 to 4 carbon atoms is preferable.
As the acid generator represented by the general formula (a4), when n = 1, R ^12a is any one of a phenyl group, a methylphenyl group, and a methoxyphenyl group, and R ^13a is a methyl group, Specifically, α- (methylsulfonyloxyimino) -1-phenylacetonitrile, α- (methylsulfonyloxyimino) -1- (p-methylphenyl) acetonitrile, α- (methylsulfonyloxyimino) -1- (p -Methoxyphenyl) acetonitrile, [2- (propylsulfonyloxyimino) -2,3-dihydroxythiophene-3-ylidene] (o-tolyl) acetonitrile, and the like. When n = 2, the acid generator represented by the general formula (a4) specifically includes an acid generator represented by the following formula.

  As a third aspect of the acid generator (C), an onium salt-based generator is provided, and for example, a compound represented by the following general formula (b1) or (b2) is used as the onium salt-based acid generator. be able to.

［一般式（ｂ１）、及び（ｂ２）において、Ｒ^ｂ１〜Ｒ^ｂ３，Ｒ^ｂ５〜Ｒ^ｂ６は、それぞれ独立に、置換基を有していてもよいアリール基又はアルキル基を表す。一般式（ｂ１）におけるＲ^ｂ１〜Ｒ^ｂ３のうち、何れか２つが相互に結合して式中のイオウ原子とともに環を形成してもよい。Ｍ⁻は有機アニオンを表す。Ｒ^ｂ１〜Ｒ^ｂ３のうち少なくとも１つはアリール基を表し、Ｒ^ｂ５〜Ｒ^ｂ６のうち少なくとも１つはアリール基を表す。］

[In General Formulas (b1) and (b2), R ^{b1 to} R ^b3 and R ^{b5 to} R ^b6 each independently represents an aryl group or an alkyl group which may have a substituent. Any two of R ^{b1 to} R ^b3 in the general formula (b1) may be bonded to each other to form a ring together with the sulfur atom in the formula. M ⁻ represents an organic anion. At least one of R ^{b1 to} R ^b3 represents an aryl group, and at least one of R ^{b5 to} R ^b6 represents an aryl group. ]

In general formula (b1), R ^{b1 to} R ^b3 each independently represents an aryl group or an alkyl group which may have a substituent. In addition, any two of R ^{b1 to} R ^b3 in the general formula (b1) may be bonded to each other to form a ring together with the sulfur atom in the formula. Further, at least one of R ^{b1 to} R ^b3 represents an aryl group. Of R ^{b1 to} R ^b3 , two or more are preferably aryl groups, and all of R ^{b1 to} R ^b3 are most preferably aryl groups.

The aryl group of R ^b1 to R ^b3, not particularly limited, for example, an aryl group having 6 to 20 carbon atoms. The aryl group is preferably an aryl group having 6 to 10 carbon atoms because it can be synthesized at a low cost. Specific examples include a phenyl group and a naphthyl group.

The aryl group may have a substituent. Here, “having a substituent” means that part or all of the hydrogen atoms of the aryl group are substituted with a substituent. As the substituent that the aryl group has, an alkyl group, an alkoxy group, a halogen atom, a hydroxyl group, an alkoxyalkyloxy group, —O—R ^b7 —C (═O) — (O) _{n ″} —R ^b8 [wherein R ^b7 is an alkylene group or a single bond, R ^b8 is an acid dissociable group or an acid non-dissociable group, and n ″ is 0 or 1. ] Etc. are mentioned.

The anion portion of such an acid generator is a fluoroalkyl sulfonate ion or an aryl sulfonate ion in which a part or all of hydrogen atoms are fluorinated.
The alkyl group in the fluoroalkylsulfonic acid ion may be linear, branched or cyclic having 1 to 20 carbon atoms, and preferably has 1 to 10 carbon atoms from the bulk of the acid generated and its diffusion distance. In particular, a branched or annular shape is preferable because of its short diffusion distance. Moreover, since it can synthesize | combine cheaply, a methyl group, an ethyl group, a propyl group, a butyl group, an octyl group etc. can be mentioned as a preferable thing.
The aryl group in the aryl sulfonate ion is an aryl group having 6 to 20 carbon atoms, and examples thereof include an alkyl group, a phenyl group which may or may not be substituted with a halogen atom, and a naphthyl group. In particular, an aryl group having 6 to 10 carbon atoms is preferable because it can be synthesized at low cost. Specific examples of preferable ones include a phenyl group, a toluenesulfonyl group, an ethylphenyl group, a naphthyl group, and a methylnaphthyl group.
In the fluoroalkyl sulfonate ion or aryl sulfonate ion, the fluorination rate when part or all of the hydrogen atoms are fluorinated is preferably 10 to 100%, more preferably 50 to 100%, In particular, those in which all hydrogen atoms are substituted with fluorine atoms are preferred because the strength of the acid is increased. Specific examples thereof include trifluoromethane sulfonate, perfluorobutane sulfonate, perfluorooctane sulfonate, and perfluorobenzene sulfonate.
Among these, preferable anion moieties include those represented by the following general formula (a9).

In the general formula (a9), R ^20a is a group represented by the following general formulas (a10) and (a11) or a group represented by the following formula (a12).

In the general formula (a10), x represents an integer of 1 to 4. In the general formula (a11), R ^21a represents a hydrogen atom, a hydroxyl group, a linear or branched alkyl group having 1 to 6 carbon atoms, or a linear or branched alkoxy group having 1 to 6 carbon atoms. Represents a group, and y represents an integer of 1 to 3. Among these, trifluoromethanesulfonate and perfluorobutanesulfonate are preferable from the viewpoint of safety.
Moreover, as an anion part, what contains the nitrogen represented by the following general formula (a13) and (a14) can also be used.

Above general formula (a13), in (a14), X ^a represents a linear or branched alkylene group having at least one hydrogen atom is substituted with a fluorine atom, the carbon number of the alkylene group having 2 to 6 Preferably 3 to 5 and most preferably 3 carbon atoms. Y ^a and Z ^a each independently represents a linear or branched alkyl group in which at least one hydrogen atom is substituted with a fluorine atom, and the alkyl group has 1 to 10 carbon atoms, Is 1-7, more preferably 1-3.
The smaller the carbon number of the alkylene group of X ^a or the carbon number of the alkyl group of Y ^a and Z ^a , the better the solubility in an organic solvent, which is preferable.
Further, in the alkylene group of X ^a or the alkyl group of Y ^a and Z ^a , the larger the number of hydrogen atoms substituted with fluorine atoms, the more preferable the strength of the acid. The proportion of fluorine atoms in the alkylene group or alkyl group, that is, the fluorination rate is preferably 70 to 100%, more preferably 90 to 100%, and most preferably all hydrogen atoms are substituted with fluorine atoms. A perfluoroalkylene group or a perfluoroalkyl group.

  Further, as a fourth aspect in the acid generator (C), bis (p-toluenesulfonyl) diazomethane, bis (1,1-dimethylethylsulfonyl) diazomethane, bis (cyclohexylsulfonyl) diazomethane, bis (2,4- Bissulfonyldiazomethanes such as dimethylphenylsulfonyl) diazomethane; 2-nitrobenzyl p-toluenesulfonate, 2,6-dinitrobenzyl p-toluenesulfonate, nitrobenzyl tosylate, dinitrobenzyl tosylate, nitrobenzyl sulfonate, Nitrobenzyl derivatives such as nitrobenzyl carbonate and dinitrobenzyl carbonate; pyrogallol trimesylate, pyrogallol tritosylate, benzyl tosylate, benzyl sulfonate, N-methylsulfonyloxysuccine Sulfonic acid esters such as amide, N-trichloromethylsulfonyloxysuccinimide, N-phenylsulfonyloxymaleimide and N-methylsulfonyloxyphthalimide; trifluoromethanesulfonic acid esters such as N-hydroxyphthalimide and N-hydroxynaphthalimide; Iodonium hexafluorophosphate, (4-methoxyphenyl) phenyliodonium trifluoromethanesulfonate, bis (p-tert-butylphenyl) iodonium trifluoromethanesulfonate, triphenylsulfonium hexafluorophosphate, (4-methoxyphenyl) diphenylsulfonium Trifluoromethanesulfonate, (p-tert-butylphenyl) diphenylsulfonium trifluoro Onium salts such as Tan sulfonate; benzoin tosylate, benzoin tosylate such as α- methyl benzoin tosylate; other diphenyliodonium salts, triphenylsulfonium salts, phenyldiazonium salts, benzyl carbonate and the like.

  This acid generator (C) may be used alone or in combination of two or more. Moreover, it is preferable that content of an acid generator (C) shall be 0.1-10 mass% with respect to the total mass of the chemical amplification type positive photosensitive resin composition which concerns on this invention, 0.5- It is more preferable to set it as 3 mass%. By setting it as the above range, sufficient sensitivity can be obtained, solubility in an organic solvent is good, a uniform solution is obtained, and storage stability is improved.

[Resin (D) whose solubility in alkali is increased by the action of acid]
The resin (D) whose solubility in an alkali is increased by the action of an acid is not particularly limited, and any resin whose solubility in an alkali is increased by the action of an acid can be used. Among these, it is preferable to contain at least one resin selected from the group consisting of a novolac resin (D1), a polyhydroxystyrene resin (D2), and an acrylic resin (D3).

(Novolac resin (D1))
As the novolac resin (D1), a resin containing a structural unit represented by the following general formula (b1) can be used.

In the general formula (b1), R ^1b represents an acid dissociable, dissolution inhibiting group, and R ^2b and R ^3b each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.
Examples of the acid dissociable, dissolution inhibiting group represented by R ^1b include groups represented by the following general formulas (b2) and (b3), linear, branched or cyclic alkyl groups having 1 to 6 carbon atoms. , A vinyloxyethyl group, a tetrahydropyranyl group, a tetrahydrofuranyl group, or a trialkylsilyl group.

In the general formulas (b2) and (b3), R ^4b and R ^5b each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms, and R ^6b represents a carbon number. Represents a linear, branched or cyclic alkyl group having 1 to 10 carbon atoms, R ^7b represents a linear, branched or cyclic alkyl group having 1 to 6 carbon atoms, and o represents 0 or 1 Represent.

  Examples of the linear or branched alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, and a neopentyl group. . Examples of the cyclic alkyl group include a cyclopentyl group and a cyclohexyl group.

  Here, specific examples of the acid dissociable, dissolution inhibiting group represented by the general formula (b2) include a methoxyethyl group, an ethoxyethyl group, an n-propoxyethyl group, an isopropoxyethyl group, and an n-butoxyethyl group. , Isobutoxyethyl group, tert-butoxyethyl group, cyclohexyloxyethyl group, methoxypropyl group, ethoxypropyl group, 1-methoxy-1-methyl-ethyl group, 1-ethoxy-1-methylethyl group and the like. Specific examples of the acid dissociable, dissolution inhibiting group represented by the general formula (b3) include a tert-butoxycarbonyl group and a tert-butoxycarbonylmethyl group. Examples of the trialkylsilyl group include those having 1 to 6 carbon atoms in each alkyl group such as a trimethylsilyl group and a tri-tert-butyldimethylsilyl group.

(Polyhydroxystyrene resin (D2))
As polyhydroxystyrene resin (D2), resin containing the structural unit represented by the following general formula (b4) can be used.

In the general formula (b4), R ^8b represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and R ^9b represents an acid dissociable, dissolution inhibiting group.
The said C1-C6 alkyl group is a C1-C6 linear, branched, or cyclic alkyl group, for example. Examples of the linear or branched alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, and a neopentyl group. Examples of the cyclic alkyl group include a cyclopentyl group and a cyclohexyl group.
As the acid dissociable, dissolution inhibiting group represented by R ^9b , the same acid dissociable, dissolution inhibiting groups as exemplified in the general formulas (b2) and (b3) can be used.

  Furthermore, the polyhydroxystyrene resin (D2) can contain other polymerizable compounds as constituent units for the purpose of appropriately controlling physical and chemical properties. Examples of such polymerizable compounds include known radical polymerizable compounds and anionic polymerizable compounds. Examples of such a polymerizable compound include monocarboxylic acids such as acrylic acid, methacrylic acid, and crotonic acid; dicarboxylic acids such as maleic acid, fumaric acid, and itaconic acid; 2-methacryloyloxyethyl succinic acid, 2- Methacryloyloxyethylmaleic acid, 2-methacryloyloxyethylphthalic acid, 2-methacryloyloxyethylhexahydrophthalic acid and other methacrylic acid derivatives having a carboxyl group and an ester bond; methyl (meth) acrylate, ethyl (meth) acrylate, butyl (Meth) acrylic acid alkyl esters such as (meth) acrylate; (meth) acrylic acid hydroxyalkyl esters such as 2-hydroxyethyl (meth) acrylate and 2-hydroxypropyl (meth) acrylate; phenyl (meta (Meth) acrylic acid aryl esters such as acrylate and benzyl (meth) acrylate; dicarboxylic acid diesters such as diethyl maleate and dibutyl fumarate; styrene, α-methylstyrene, chlorostyrene, chloromethylstyrene, vinyltoluene, hydroxy Vinyl group-containing aromatic compounds such as styrene, α-methylhydroxystyrene and α-ethylhydroxystyrene; Vinyl group-containing aliphatic compounds such as vinyl acetate; Conjugated diolefins such as butadiene and isoprene; Acrylonitrile, methacrylonitrile Nitrile group-containing polymerizable compounds such as; chlorine-containing polymerizable compounds such as vinyl chloride and vinylidene chloride; amide bond-containing polymerizable compounds such as acrylamide and methacrylamide;

(Acrylic resin (D3))
As acrylic resin (D3), resin containing the structural unit represented by the following general formula (b5)-(b7) can be used.

In the above general formulas (b5) to (b7), R ^{10b to} R ^17b each independently represent a hydrogen atom, a linear or branched alkyl group having 1 to 6 carbon atoms, a fluorine atom, or 1 to 6 carbon atoms. Represents a linear or branched fluorinated alkyl group (wherein R ^11b is not a hydrogen atom), and X ^b is a hydrocarbon having 5 to 20 carbon atoms together with the carbon atom to which it is bonded. forms a ring, Y ^b is substituted represents those aliphatic cyclic group or an alkyl group, p represents an integer of 0 to 4, q represents 0 or 1.

  Examples of the linear or branched alkyl group include methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, and the like. Can be mentioned. The fluorinated alkyl group is one in which part or all of the hydrogen atoms of the alkyl group are substituted with fluorine atoms.

R ^11b is preferably a linear or branched alkyl group having 2 to 4 carbon atoms from the viewpoint of high contrast, good resolution, depth of focus, etc., and the above R ^13b , R ^14b , R ^16b and R ^17b are preferably a hydrogen atom or a methyl group.

^Xb forms an aliphatic cyclic group having 5 to 20 carbon atoms together with the carbon atom to which it is bonded. Specific examples of such an aliphatic cyclic group include groups in which one or more hydrogen atoms have been removed from a polycycloalkane such as monocycloalkane, bicycloalkane, tricycloalkane, and tetracycloalkane. Specifically, one or more hydrogen atoms were removed from monocycloalkanes such as cyclopentane, cyclohexane, cycloheptane, and cyclooctane, and polycycloalkanes such as adamantane, norbornane, isobornane, tricyclodecane, and tetracyclododecane. Groups. In particular, a group obtained by removing one or more hydrogen atoms from cyclohexane or adamantane (which may further have a substituent) is preferable.

Furthermore, when the aliphatic cyclic group of ^Xb has a substituent on the ring skeleton, examples of the substituent include polar groups such as a hydroxyl group, a carboxy group, a cyano group, and an oxygen atom (═O). And a linear or branched alkyl group having 1 to 4 carbon atoms. As the polar group, an oxygen atom (═O) is particularly preferable.

Y ^b is an aliphatic cyclic group or an alkyl group, and examples thereof include groups in which one or more hydrogen atoms have been removed from a polycycloalkane such as monocycloalkane, bicycloalkane, tricycloalkane, and tetracycloalkane. . Specifically, one or more hydrogen atoms were removed from monocycloalkanes such as cyclopentane, cyclohexane, cycloheptane, and cyclooctane, and polycycloalkanes such as adamantane, norbornane, isobornane, tricyclodecane, and tetracyclododecane. Groups and the like. In particular, a group in which one or more hydrogen atoms are removed from adamantane (which may further have a substituent) is preferable.

Furthermore, the alicyclic group of the abovementioned Y ^b is, if they have a substituent on the ring skeleton, examples of the substituent, a hydroxyl group, a carboxyl group, a cyano group, an oxygen atom (= O) polar groups such as And a linear or branched alkyl group having 1 to 4 carbon atoms. As the polar group, an oxygen atom (═O) is particularly preferable.

Also, if Y ^b is an alkyl group, having 1 to 20 carbon atoms, it is preferred that preferably a linear or branched alkyl group having 6 to 15. Such an alkyl group is particularly preferably an alkoxyalkyl group. Examples of the alkoxyalkyl group include 1-methoxyethyl group, 1-ethoxyethyl group, 1-n-propoxyethyl group, 1-isopropoxy group. Ethyl group, 1-n-butoxyethyl group, 1-isobutoxyethyl group, 1-tert-butoxyethyl group, 1-methoxypropyl group, 1-ethoxypropyl group, 1-methoxy-1-methyl-ethyl group, 1 -Ethoxy-1-methylethyl group etc. are mentioned.

  Preferable specific examples of the structural unit represented by the general formula (b5) include those represented by the following formulas (b5-1) to (b5-33).

In the above formulas (b5-1) to (b5-33), R ^18b represents a hydrogen atom or a methyl group.
Preferable specific examples of the structural unit represented by the general formula (b6) include those represented by the following formulas (b6-1) to (b6-24).

In the above formulas (b6-1) to (b6-24), R ^18b represents a hydrogen atom or a methyl group.
Preferable specific examples of the structural unit represented by the general formula (b7) include those represented by the following formulas (b7-1) to (b7-15).

In the above formulas (b7-1) to (b7-15), R ^18b represents a hydrogen atom or a methyl group.
Furthermore, acrylic resin (D3) consists of a copolymer containing the structural unit further derived from the polymeric compound which has an ether bond with respect to the structural unit represented by the said general formula (b5)-(b7). A resin is preferred.

  Examples of the polymerizable compound having an ether bond include radical polymerizable compounds such as a (meth) acrylic acid derivative having an ether bond and an ester bond, and specific examples include 2-methoxyethyl (meth) acrylate. 2-ethoxyethyl (meth) acrylate, methoxytriethylene glycol (meth) acrylate, 3-methoxybutyl (meth) acrylate, ethyl carbitol (meth) acrylate, phenoxypolyethylene glycol (meth) acrylate, methoxypolyethylene glycol (meth) Examples thereof include acrylate, methoxypolypropylene glycol (meth) acrylate, and tetrahydrofurfuryl (meth) acrylate. The polymerizable compound having an ether bond is preferably 2-methoxyethyl (meth) acrylate, 2-ethoxyethyl (meth) acrylate, or methoxytriethylene glycol (meth) acrylate. These polymerizable compounds may be used alone or in combination of two or more.

  Further, the acrylic resin (D3) can contain other polymerizable compounds as structural units for the purpose of appropriately controlling physical and chemical properties. Examples of such polymerizable compounds include known radical polymerizable compounds and anionic polymerizable compounds.

  Examples of such polymerizable compounds include monocarboxylic acids such as acrylic acid, methacrylic acid, and crotonic acid; dicarboxylic acids such as maleic acid, fumaric acid, and itaconic acid; 2-methacryloyloxyethyl succinic acid, and 2-methacryloyloxy. Methacrylic acid derivatives having a carboxyl group and an ester bond such as ethyl maleic acid, 2-methacryloyloxyethyl phthalic acid, 2-methacryloyloxyethyl hexahydrophthalic acid; methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth ) (Meth) acrylic acid alkyl esters such as acrylate and cyclohexyl (meth) acrylate; (meth) acrylic acid hydroxyal such as 2-hydroxyethyl (meth) acrylate and 2-hydroxypropyl (meth) acrylate (Meth) acrylic acid aryl esters such as phenyl (meth) acrylate and benzyl (meth) acrylate; dicarboxylic acid diesters such as diethyl maleate and dibutyl fumarate; styrene, α-methylstyrene, chlorostyrene, chloro Vinyl group-containing aromatic compounds such as methylstyrene, vinyltoluene, hydroxystyrene, α-methylhydroxystyrene, α-ethylhydroxystyrene; vinyl group-containing aliphatic compounds such as vinyl acetate; conjugated diolefins such as butadiene and isoprene Nitrile group-containing polymerizable compounds such as acrylonitrile and methacrylonitrile; chlorine-containing polymerizable compounds such as vinyl chloride and vinylidene chloride; amide bond-containing polymerizable compounds such as acrylamide and methacrylamide; Kill.

  Examples of the polymerizable compound include (meth) acrylic acid esters having a non-acid dissociable aliphatic polycyclic group, and vinyl group-containing aromatic compounds. As the non-acid-dissociable aliphatic polycyclic group, a tricyclodecanyl group, an adamantyl group, a tetracyclododecanyl group, an isobornyl group, a norbornyl group, and the like are particularly preferable in terms of industrial availability. These aliphatic polycyclic groups may have a linear or branched alkyl group having 1 to 5 carbon atoms as a substituent.

  The polystyrene equivalent mass average molecular weight of the resin (D) is preferably 5,000 to 600,000, more preferably 7,000 to 400,000, and still more preferably 10,000 to 300,000. By setting it as such a mass average molecular weight, sufficient intensity | strength of a resin layer can be hold | maintained, without the peelability with a support body falling.

The resin (D) is preferably a resin having a dispersity of 1.05 or more. Here, the degree of dispersion is a value obtained by dividing the mass average molecular weight by the number average molecular weight. By setting such a degree of dispersion, a desired stress resistance can be obtained.
The content of the resin (D) is preferably 5 to 60% by mass with respect to the total mass of the chemically amplified positive photosensitive resin composition according to the present invention.

[Acid diffusion controller (E)]
The chemical amplification type positive photosensitive resin composition according to the present invention may further contain an acid diffusion control agent (E) in order to improve the resin pattern shape, retention stability and the like. As the acid diffusion controller (E), a nitrogen-containing compound (E1) is preferable, and an organic carboxylic acid, or an oxo acid of phosphorus or a derivative thereof (E2) can be further contained as necessary.

(Nitrogen-containing compound (E1))
Examples of the nitrogen-containing compound (E1) include trimethylamine, diethylamine, triethylamine, di-n-propylamine, tri-n-propylamine, tri-n-pentylamine, tribenzylamine, diethanolamine, triethanolamine, and n-hexylamine. , N-heptylamine, n-octylamine, n-nonylamine, ethylenediamine, N, N, N ′, N′-tetramethylethylenediamine, tetramethylenediamine, hexamethylenediamine, 4,4′-diaminodiphenylmethane, 4,4 '-Diaminodiphenyl ether, 4,4'-diaminobenzophenone, 4,4'-diaminodiphenylamine, formamide, N-methylformamide, N, N-dimethylformamide, acetamide, N-methylacetamide, N, N-di Tylacetamide, propionamide, benzamide, pyrrolidone, N-methylpyrrolidone, methylurea, 1,1-dimethylurea, 1,3-dimethylurea, 1,1,3,3-tetramethylurea, 1,3-diphenylurea Imidazole, benzimidazole, 4-methylimidazole, 8-oxyquinoline, acridine, purine, pyrrolidine, piperidine, 2,4,6-tri (2-pyridyl) -S-triazine, morpholine, 4-methylmorpholine, piperazine, Examples include 1,4-dimethylpiperazine, 1,4-diazabicyclo [2.2.2] octane, pyridine and the like. These may be used alone or in combination of two or more.

  The nitrogen-containing compound (E1) is usually used in a range of 0 to 5 parts by mass, particularly 0 to 3 parts by mass with respect to 100 parts by mass of the total mass of the resin (E) and the alkali-soluble resin (B). It is preferably used in a range.

(Organic carboxylic acid or phosphorus oxo acid or derivative (E2))
Among organic carboxylic acids or phosphorus oxo acids or derivatives thereof (E2), specifically, as malonic acid, malonic acid, citric acid, malic acid, succinic acid, benzoic acid, salicylic acid and the like are preferable. In particular, salicylic acid is preferred.

  Examples of phosphorus oxo acids or derivatives thereof include phosphoric acid, phosphoric acid di-n-butyl ester, phosphoric acid diphenyl ester and the like phosphoric acid and derivatives thereof such as phosphonic acid, phosphonic acid dimethyl ester, phosphonic acid- Phosphonic acids such as di-n-butyl ester, phenylphosphonic acid, phosphonic acid diphenyl ester, phosphonic acid dibenzyl ester and derivatives thereof; phosphinic acids such as phosphinic acid and phenylphosphinic acid and their esters Derivatives thereof, and the like. Among these, phosphonic acid is particularly preferable. These may be used alone or in combination of two or more.

The organic carboxylic acid or phosphorus oxo acid or derivative thereof (E2) is usually used in the range of 0 to 5 parts by mass with respect to 100 parts by mass of the total mass of the resin (E) and the alkali-soluble resin (B). In particular, it is preferably used in the range of 0 to 3 parts by mass.
In order to form a salt and stabilize, it is preferable to use an organic carboxylic acid, or an oxo acid of phosphorus or a derivative thereof (E2) in an amount equivalent to the nitrogen-containing compound (E1).

[Alcohol-based organic solvent (S)]
The chemically amplified positive photosensitive resin composition according to the present invention can be produced and used as a uniform solution in which components other than the alcohol-based organic solvent (S) are dissolved in the alcohol-based organic solvent (S). As the alcohol organic solvent (S), those exemplified in the non-chemically amplified positive photosensitive resin composition can be used.

  Content of alcohol type organic solvent (S) is not specifically limited, For example, solid content concentration of the chemical amplification type positive photosensitive resin composition concerning this invention is 5-50 mass%, Preferably it is 10-30 mass %.

[Other ingredients]
The chemically amplified positive photosensitive resin composition according to the present invention may contain other components. As other components, those exemplified in the non-chemically amplified positive photosensitive resin composition can be used.

<Preparation of photosensitive resin composition>
The photosensitive resin composition according to the present invention can be adjusted by simply mixing and stirring the above-described components by a usual method, and if necessary, disperse using a disperser such as a dissolver, a homogenizer, or a three-roll mill. , May be mixed. Moreover, after mixing, you may filter using a mesh, a membrane filter, etc. further.

<Method for forming resin pattern on substrate>
As a method for forming a resin pattern on a substrate using the photosensitive resin composition according to the present invention, for example, the photosensitive resin composition according to the present invention is applied on a substrate and dried to form a resin layer. And a resin layer forming step, an exposure step of selectively exposing the resin layer, and a development step of developing the resin layer after exposure to form a resin pattern.

First, in the resin layer forming step, a desired resin layer is formed by applying a solution of the photosensitive resin composition prepared as described above onto a substrate and removing the solvent by heating. As a coating method on the substrate to be processed, methods such as a spin coating method, a slit coating method, a roll coating method, a screen printing method, and an applicator method can be employed. Although the pre-baking conditions of the resin layer of the composition of the present invention vary depending on the type or blending ratio of each component in the composition or the film thickness of the resin layer, it is usually 70 to 150 ° C., preferably 80 to 140 ° C. 2 to 60 minutes.
The film thickness of the resin layer is preferably in the range of 0.1 to 10 μm, more preferably 0.5 to 5 μm, and even more preferably 0.5 to 3 μm.

The substrate is not particularly limited, and a conventionally known substrate can be used. For example, polystyrene (PS), polyethylene terephthalate (PET), polycarbonate (PC), TAC (triacetyl cellulose), polyimide (PI), nylon (Ny), low density polyethylene (LDPE), medium density polyethylene (MDPE), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyethersulfone, polyethylene naphthalate, polypropylene, acrylic materials (for example, urethane acrylate), cellulose, etc. A resin substrate containing the above resin; a glass substrate. It may be a resin substrate containing a biodegradable polymer such as polylactic acid, polyglycolic acid, polycaprolactan, or a copolymer thereof. The substrate may be porous.
In the present invention, among them, a resin substrate or a glass substrate containing a resin such as polystyrene, polyethylene terephthalate, or polycarbonate can be preferably used. In particular, a resin substrate containing polystyrene has transparency, mechanical properties, electrical properties, Since it is excellent in properties such as biocompatibility, it can be most preferably used.

Next, in the exposure step, actinic rays or radiation, for example, ultraviolet rays or visible rays having a wavelength of 100 to 500 nm is applied to the obtained resin layer through a mask having a predetermined pattern or using a maskless exposure apparatus. Irradiate selectively (exposure).
As a radiation source, a low-pressure mercury lamp, a high-pressure mercury lamp, an ultrahigh-pressure mercury lamp, a metal halide lamp, an argon gas laser, or the like can be used. The radiation includes microwaves, infrared rays, visible rays, ultraviolet rays, X-rays, γ rays, electron beams, proton beams, neutron beams, ion beams, and the like. The amount of radiation irradiation varies depending on the composition of the photosensitive resin composition according to the present invention, the film thickness of the resin layer, and the like, but is, for example, 100 to 10,000 mJ / cm ² when using an ultrahigh pressure mercury lamp. Radiation includes light that activates the acid generator (C) in order to generate an acid.
After the exposure, the acid generated by the exposure changes the alkali solubility of the exposed resin layer.

Next, in the developing step, for example, a predetermined alkaline aqueous solution is used as a developer, and unnecessary portions are dissolved and removed to obtain a predetermined resin pattern.
Examples of the developer include sodium hydroxide, potassium hydroxide, sodium carbonate, sodium silicate, sodium metasilicate, aqueous ammonia, ethylamine, n-propylamine, diethylamine, di-n-propylamine, triethylamine, methyldiethylamine, Dimethylethanolamine, triethanolamine, tetramethylammonium hydroxide, tetraethylammonium hydroxide, pyrrole, piperidine, 1,8-diazabicyclo [5,4,0] -7-undecene, 1,5-diazabicyclo [4,3, [0] An aqueous solution of an alkali such as 5-nonane can be used. Further, an aqueous solution obtained by adding an appropriate amount of a water-soluble alcohol-based organic solvent such as methanol or ethanol or a surfactant to the alkaline aqueous solution can also be used as a developer.

  The development time is usually 1 to 30 minutes, although it varies depending on the composition of the photosensitive resin composition according to the present invention and the film thickness of the resin layer. The developing method may be any of a liquid piling method, a dipping method, a paddle method, a spray developing method and the like.

  After the development, washing with running water is performed for 30 to 90 seconds and dried using an air gun or an oven. In this way, a resin pattern can be formed.

≪Method for preparing cell culture plate≫
The method for producing a plate for cell culture according to the present invention includes using the photosensitive resin composition according to the present invention. In the method for producing a plate for cell culture according to the present invention, the photosensitive resin composition according to the present invention forms a cell adhesion-inhibiting material layer, a temperature-responsive material layer, or a combination thereof described later in a desired pattern. Used to form a resin pattern used in

  Specifically, the method for producing a plate for cell culture according to the present invention includes a resin layer forming step in which the photosensitive resin composition according to the present invention is applied on a substrate and dried to form a resin layer, and the above resin layer An exposure step of selectively exposing the resin layer and a development step of developing the resin layer after exposure to form a resin pattern. The resin layer forming step, the exposure step, the developing step, and the substrate are as described above in <Method for forming resin pattern on substrate>.

Hereinafter, the method for producing a cell culture plate according to the present invention will be described more specifically with reference to the drawings.
<Method 1 for preparing cell culture plate>
FIG. 1 is a cross-sectional view showing one embodiment of a method for producing a cell culture plate according to the present invention (Method 1 for producing a cell culture plate). A method 1 for producing a cell culture plate will be described with reference to FIG.

[Resin layer forming step, exposure step, and development step]
First, as shown in FIG. 1A, the resin pattern 2 is provided on a part of the substrate 1 by the resin layer forming process, the exposure process, and the developing process.

[Cell adhesion inhibiting material layer forming step]
Next, as shown in FIG. 1B, the cell adhesion inhibiting material layer 3 is formed on the portion of the substrate 1 where the resin pattern 2 is not provided (cell adhesion inhibiting material layer forming step). The cell adhesion inhibiting material layer 3 includes a cell adhesion inhibiting material.

  The cell adhesion inhibiting material used in the present invention has cell non-adhesiveness. Such a cell adhesion inhibiting material is not particularly limited as long as it has a cell adhesion inhibiting property that inhibits adhesion to cells. Examples of the cell adhesion inhibiting material include polyethylene glycol materials such as polyethylene glycol, polyethylene glycol diacrylate, and polyethylene glycol methacrylate; long chain alkyl materials such as decylmethoxysilane polymer; fluorine materials such as polyfluoroalkylsilane; Examples include hydrophilic materials such as polyvinyl alcohol (PVA) and polyacrylamide; phospholipid materials such as MPC polymer; BSA protein and the like.

  In the present invention, the cell adhesion inhibiting material layer 3 can be formed, for example, as follows. That is, a solution containing a cell adhesion inhibiting material and a solvent (alcohol organic solvent, water, etc.) capable of dissolving the same is prepared, and this is provided with a resin pattern 2 on the substrate 1 according to a conventional coating method. The cell adhesion inhibiting material layer 3 can be formed in a portion where the resin pattern 2 on the substrate 1 is not provided by applying to the portion not present.

Further, when the cell adhesion inhibiting material is a polymer that can be formed by a photopolymerization reaction, such as polyacrylamide, a raw material monomer of such a polymer, a photopolymerization initiator, and a solvent (alcohol-based solvent) that can dissolve them. A solution containing an organic solvent, water, etc.) is applied to a portion of the substrate 1 where the resin pattern 2 is not provided according to a conventional coating method, and then light is irradiated on the entire surface to obtain a light. By causing the polymerization reaction, the cell adhesion inhibiting material layer 3 can be formed on the portion of the substrate 1 where the resin pattern 2 is not provided. Examples of the photopolymerization initiator that can be used here include water-soluble camphorquinone. As light to irradiate, for example, ultraviolet light or visible light having a wavelength of 150 to 600 nm and an illuminance of 1 to 100 mW / cm ² can be used.

  In the cell culture plate according to the present invention, the width between the regions covered with the cell adhesion inhibiting material is preferably 1 μm to 500 μm, and more preferably 2 to 200 μm. In the cell culture plate according to the present invention, the thickness of the cell adhesion-inhibiting material layer when dried is preferably 0.01 to 15 μm, more preferably 0.05 to 1.5 μm.

[Resin pattern peeling process]
Thereafter, the resin pattern 2 is peeled off (resin pattern peeling step). As shown in FIG. 1C, the substrate 1 and the cell adhesion inhibiting material layer 3 provided on a part of the substrate 1 are used for cell culture. Get the plate. The resin pattern 2 is peeled using a stripping solution or the like according to a conventional method. The peeling method is not particularly limited, and an immersion method, a spray method, a shower method, a paddle method, or the like can be used. Examples of the stripping solution include 3 to 15% by mass of sodium hydroxide aqueous solution, potassium hydroxide aqueous solution, organic amine, tetramethylammonium hydroxide, triethanolamine, and N-methylpyrrolidone. The peeling treatment time is not particularly limited, but is, for example, about 1 to 120 minutes. The stripping solution may be heated to about 25 to 60 ° C.

<Method 2 for preparing cell culture plate>
FIG. 2 is a cross-sectional view showing another embodiment of the cell culture plate production method according to the present invention (cell culture plate production method 2). With reference to FIG. 2, the production method 2 of the cell culture plate will be described.

[Resin layer forming step, exposure step, and development step]
First, in the same manner as described in <Cell Culture Plate Preparation Method 1>, as shown in FIG. 2A, a part of the substrate 1 is formed by the resin layer forming process, the exposure process, and the developing process. The resin pattern 2 is provided on the surface.

[Temperature-responsive material layer forming process]
Next, as shown in FIG. 2 (b), the temperature responsiveness that has cell adhesiveness in a portion where the resin pattern 2 on the substrate 1 is not provided and can express cell non-adhesiveness due to temperature change. The temperature-responsive material layer 4 made of a material is formed (temperature-responsive material layer forming step).

  The temperature-responsive material used in the present invention is not particularly limited as long as it has cell adhesiveness and can express cell non-adhesiveness depending on temperature. In the present invention, the temperature-responsive materials may be used alone or in combination of two or more.

  The change in the degree of cell adhesion of the temperature-responsive material used in the present invention is particularly limited as long as it can be changed from a state having cell adhesion to a state having non-cell adhesion. Instead, it may be reversibly changed or irreversibly changed.

  The temperature-responsive material used in the present invention is not particularly limited as long as the degree of cell adhesion changes by a desired amount due to temperature change. In the present invention, the temperature at which the temperature-responsive material exhibits cell adhesiveness is preferably within a range of 10 ° C to 45 ° C, and more preferably within a range of 33 ° C to 40 ° C. This is because the cells can be stably cultured when the temperature is within the above-mentioned range.

  In the present invention, the temperature at which the temperature-responsive material exhibits cell non-adhesiveness is preferably in the range of 1 ° C to 36 ° C, and more preferably in the range of 4 ° C to 32 ° C. This is because damage to cells can be reduced when the temperature is within the above-mentioned range.

  Specific examples of the temperature-responsive material used in the present invention include poly-N-isopropylacrylamide (PIPAAm), poly-Nn-propylacrylamide, poly-Nn-propylmethacrylamide, poly-N-. Examples include ethoxyethyl acrylamide, poly-N-tetrahydrofurfuryl acrylamide, poly-N-tetrahydrofurfuryl methacrylamide, and poly-N, N-diethyl acrylamide, among which PIPAAm, poly-Nn-propyl methacryl. Amide and poly-N, N-diethylacrylamide can be preferably used, and PIPAAm can be particularly preferably used. Since cells can be cultured under suitable culture conditions and exhibit non-adhesive properties under temperature conditions that have little influence such as reducing cell activity, cells are stably formed, detached, recovered, and transferred Because it can. In the present invention, the temperature-responsive materials may be used alone or in combination of two or more.

The temperature-responsive material layer 4 used in the present invention can change the degree of cell adhesion from a state expressing cell adhesion to a state expressing cell non-adhesion depending on the temperature. is there.
Here, the expression of the cell-responsiveness of the temperature-responsive material layer 4 means that the cells are easy to adhere and extend on the temperature-responsive material layer 4 and have a high cell adhesion extension rate. Is. In the present invention, specifically, such a state where the cell adhesion extension rate is high can be a state where the cell adhesion extension rate is 60% or more. In the present invention, it is preferably 80% or more. This is because cells can be efficiently cultured to form a cell sheet or cell pattern.

In the present invention, the cell adhesion spreading rate is such that bovine vascular endothelial cells are seeded in a seeding density range of 4000 cells / cm ² or more and less than 30000 cells / cm ² and stored in a 37 ° C. incubator (CO ₂ concentration 5% by volume). And the ratio of the cells that have adhered and extended when cultured for 14.5 hours ({(the number of adherent cells) / (the number of cells seeded)} × 100 (%)). The cells were seeded by suspending in 10% by mass FBS (serum) -containing DMEM medium and seeding on a cell culture substrate, and then seeding the cells so that the cells were distributed as uniformly as possible. This is performed by gently shaking the cell culture substrate. Further, the cell adhesion extension rate is measured after exchanging the medium immediately before the measurement to remove the non-adhered cells. In addition, the cell adhesion extension rate is measured at a location where the cell density is likely to be specific (for example, the center of a predetermined area where the density is likely to be high and the periphery of the predetermined area where the density is likely to be low). To measure.

  In addition, when the temperature-responsive material layer 4 expresses cell non-adhesiveness, it means that cells are not easily adhered and spread on the temperature-responsive material layer 4 and the cell adhesion extension rate is low. Is. In the present invention, specifically, such a state where the cell adhesion extension rate is low can be a state where the cell adhesion extension rate is 5% or less. In the present invention, the content is preferably 2% or less. Therefore, when cells are seeded on the temperature-responsive material layer 4, the cells adhere to the temperature-responsive material layer 4 when the cell adhesiveness is expressed, but when the cells are non-adhesive. Since the cells are inhibited from adhering to the temperature-responsive material layer 4, the cells that have adhered to the temperature-responsive material layer 4 can be easily peeled off.

  Moreover, the width of the exposed surface of the temperature-responsive material layer 4, that is, the width that becomes the width of the cell sheet or cell pattern is not particularly limited as long as the cells can be adhered and cultured. In the case of producing a micro pattern, that is, a micron (μm) scale cell pattern, it is preferably within a range of 15 μm to 500 μm, and more preferably within a range of 40 μm to 80 μm. In addition, about the width | variety, height, etc. of each structure in this application, it is a thing at the time of drying unless there is particular notice.

  The film thickness of the temperature-responsive material layer 4 is not particularly limited as long as it can exhibit temperature responsiveness, and specifically, it may be in the range of 0.5 nm to 300 nm. Among these, it is preferable that the thickness is in the range of 1 nm to 100 nm.

  The temperature-responsive material layer 4 contains the above-mentioned temperature-responsive material, but within a range that does not inhibit the stimulus responsiveness, a leveling agent, a plasticizer, a surfactant, an antifoaming agent, and a sensitizer are added as necessary. Additives such as, polyvinyl alcohol, unsaturated polyester, acrylic resin, polyethylene, diallyl phthalate, ethylene propylene diene monomer, epoxy resin, phenol resin, polyurethane, melamine resin, polycarbonate, polyvinyl chloride, polyamide, polyimide, styrene butadiene rubber , Chloroprene rubber, polypropylene, polybutylene, polystyrene, polyvinyl acetate, nylon, polyester, polybutadiene, polybenzimidazole, polyacrylonitrile, epichlorohydrin, polysulfide, polyisoprene, polyethylene Glycol, it may contain a MPC polymer binder resin such as zwitterionic polymer (trade name).

  Further, the temperature-responsive material layer 4 may be subjected to surface treatment as necessary. This is because various functions can be imparted depending on the type of surface treatment. Examples of the surface treatment in the present invention include silane treatment, that is, the surface of the temperature-responsive material layer 4 is treated with silane. Examples of such silane treatment include application of a silane coupling agent.

  In the present invention, examples of the silane coupling agent used for the silane treatment include methacryloyloxysilane, vinyl silane, aminosilane, epoxy silane, etc. Among them, methacryloyloxysilane can be preferably used.

  As a coating method of the silane coupling agent in the present invention, it is dissolved using an alcohol-based organic solvent capable of dissolving the silane coupling agent, and this is dissolved by a conventional coating method such as a spinner method, a die coating method, or a dipping method. And a method of applying the temperature-responsive material layer 4 by gravure printing, CVD (chemical vapor deposition), or the like. For example, when the spinner method is used, the conditions can be 700 rpm to 2000 rpm and about 3 seconds to 20 seconds.

  In addition, in the temperature-responsive material layer 4 whose surface is silane-treated, the silane-treated layer is in an integral or indistinguishable state with the temperature-responsive material, and does not form separate and independent layers. Conceivable. For this reason, in this application, in the case of a temperature-responsive material layer, it may include a silane-treated layer. Note that these are theories or assumptions and do not limit the present invention.

  Such polymer coating amount or silane-treated coating amount can be determined by, for example, analysis by coating or non-coating dyeing or fluorescent substance dyeing, surface analysis by contact angle measurement, X-ray photoelectron spectroscopy measurement (XPS) or flight. The time secondary ion mass spectrometer (TOF-SIMS) can be determined alone or in combination.

  In the present invention, the temperature-responsive material layer 4 can be formed, for example, as follows. That is, a temperature responsive material composition including the temperature responsive material or a monomer component capable of forming the temperature responsive material is prepared, and this is provided with a resin pattern 2 on the substrate 1 according to a conventional coating method. By applying to the part which is not, the temperature-responsive material layer 4 can be formed in the part on which the resin pattern 2 on the substrate 1 is not provided.

[Resin pattern peeling process]
Thereafter, the resin pattern 2 is peeled off (resin pattern peeling step). As shown in FIG. 2C, the substrate 1 and the temperature-responsive material layer 4 provided on a part of the substrate 1 are used for cell culture. Get the plate. The details of the resin pattern peeling step are as described in <Cell Culture Plate Production Method 1>.

<Method 3 for preparing cell culture plate>
FIG. 3 is a cross-sectional view showing still another embodiment of the cell culture plate production method according to the present invention (cell culture plate production method 3). With reference to FIG. 3, the manufacturing method 3 of the cell culture plate is demonstrated.

[Resin layer forming step, exposure step, developing step, cell adhesion inhibiting material layer forming step, and resin pattern peeling step]
First, in the same manner as described in <Cell Culture Plate Preparation Method 1>, as shown in FIG. 3A, a part of the substrate 1 is formed by the resin layer formation step, the exposure step, and the development step. The resin pattern 2 is provided on the substrate 1, and then the cell adhesion inhibiting material layer 3 is formed on the portion of the substrate 1 where the resin pattern 2 is not provided, as shown in FIG. Then, the cell adhesion inhibiting material layer 3 is provided on a part of the substrate 1 as shown in FIG.

[Resin layer lamination process]
Next, as shown in FIG. 3D, the resin layer 5 made of the photosensitive resin composition according to the present invention is laminated on the cell adhesion inhibiting material layer 3 (resin layer lamination).
In the present invention, the resin layer 5 can be formed, for example, as follows. That is, after the coating film of the photosensitive resin composition according to the present invention is formed on the entire surface of the substrate 1 partially provided with the cell adhesion inhibiting material layer 3, the above-mentioned pattern is formed in accordance with the pattern of the cell adhesion inhibiting material layer 3. A patterning method such as gravure printing, flexographic printing, screen printing, inkjet method, etc., for patterning by a photolithographic method so that the photosensitive resin composition remains, and the photosensitive resin composition according to the present invention, A method of coating on the cell adhesion inhibiting material layer 3 can be used.

[Temperature-responsive material layer forming process]
Next, in the same manner as described in <Cell Culture Plate Preparation Method 2>, the cell-responsive inhibitor material layer on the substrate 1 as shown in FIG. A temperature-responsive material layer 4 made of a temperature-responsive material having cell adhesiveness and capable of expressing cell non-adhesiveness due to temperature change is formed in a portion where 3 is not provided. The details of the temperature-responsive material layer forming step are as described in <Method 2 for producing cell culture plate>.

[Resin layer peeling process]
Thereafter, the resin layer 5 is peeled off (resin layer peeling step). As shown in FIG. 2 (f), the substrate 1, the cell adhesion inhibiting material layer 3 provided on a part of the substrate 1, and the substrate 1 A cell culture plate comprising a temperature responsive material layer 4 provided in a portion where the cell adhesion inhibiting material layer 3 is not provided is obtained. The stripping method, stripping solution, stripping conditions, etc. in the resin layer stripping step are the same as those described in the resin pattern stripping step of <Cell Culture Plate Preparation Method 1>.

≪Cell culture plate≫
The plate for cell culture according to the present invention is produced by the method for producing a plate for cell culture according to the present invention.
<Cell culture plate 1>
For example, the cell culture plate produced by the cell culture plate production method 1 includes a substrate and a cell adhesion inhibiting material layer provided on a part of the substrate. On the substrate, the cell adhesion inhibiting material layer is arranged in a pattern. The pattern of the cell adhesion-inhibiting material layer corresponds to the shape of the portion where the resin pattern formed through the resin layer formation step, the exposure step, and the development step of the cell culture plate production method 1 is not provided.

<Cell culture plate 2>
The cell culture plate produced by the cell culture plate production method 2 includes a substrate and a temperature responsive material layer provided on a part of the substrate, and the temperature responsive material layer includes: It consists of a temperature-responsive material that has cell adhesiveness and can express cell non-adhesiveness by temperature change. On the substrate, the temperature-responsive material layer is arranged in a pattern. The pattern of the temperature-responsive material layer corresponds to the shape of the portion where the resin pattern formed through the resin layer forming process, the exposure process, and the developing process of the cell culture plate manufacturing method 2 is not provided.

<Cell culture plate 3>
The cell culture plate produced by the cell culture plate production method 3 includes a substrate, a cell adhesion inhibiting material layer provided on a part of the substrate, and the cell adhesion inhibiting material layer on the substrate. Temperature responsive material layer provided in a portion not provided with a temperature responsive material layer having cell adhesiveness and capable of expressing cell non-adhesiveness due to temperature change Made of material. On the substrate, the cell adhesion inhibiting material layer and the temperature-responsive material layer are arranged in a pattern. The pattern of the cell adhesion inhibiting material layer corresponds to the shape of the part where the resin pattern formed through the resin layer forming process, the exposure process, and the developing process of the cell culture plate production method 3 was not provided, and the temperature response The pattern of the conductive material layer corresponds to the shape of the portion provided with this resin pattern.

≪Cell culture method≫
The cell culture method according to the present invention includes a cell culture step of culturing cells on the cell culture plate produced by the method for producing a cell culture plate according to the present invention. More specifically, the cells can be cultured by seeding the cells on the cell culture plate and incubating under temperature conditions suitable for the culture.

  By the cell culture method according to the present invention, various cells such as epithelial cells and endothelial cells constituting each tissue and organ in a living body; skeletal muscle cells, smooth muscle cells, and cardiomyocytes exhibiting contractility; Comprising neurons and glial cells; fibroblasts; liver parenchymal cells, non-hepatic parenchymal cells, and adipocytes related to biological metabolism; stem cells and bone marrow cells and ES cells present in various tissues have differentiation potential Cells can be cultured.

  In the cell culture method according to the present invention, the method for seeding cells is not particularly limited as long as the cells can be uniformly seeded on the cell culture plate according to the present invention. Various sowing methods can be used. For example, the method of seed | inoculating on the plate for cell cultures in the state suspended in the culture medium can be mentioned.

  In the cell culture method according to the present invention, the culture temperature is not particularly limited as long as it is a temperature suitable for culturing the cells to be used. For example, when a cell culture plate having a temperature-responsive material layer is used, examples of the culture temperature include a temperature higher than a critical dissolution temperature at which the temperature-responsive material changes from hydrophobic to hydrophilic.

  Examples of the cell culture plate used in the cell culture method include cell culture plates 1, 2, and 3. When the cell culture plate 1 is used, the cells adhere only to the portion on the substrate where the cell adhesion inhibiting material layer is not formed, and do not adhere to the cell adhesion inhibiting material layer. When the cell culturing plate 2 is used and the temperature-responsive material is cultured at a temperature higher than the critical dissolution temperature at which it changes from hydrophobic to hydrophilic, the cells are also placed on the temperature-responsive material layer on the substrate. It adheres to the part where the temperature-responsive material layer is not formed. Note that the cells adhered to the temperature-responsive material layer can be detached from the temperature-responsive material layer by changing the temperature of the cell culture plate 2 to the above-described critical dissolution temperature or lower. Further, when the cell culture plate 3 is used and the culture is performed at a temperature higher than the above critical dissolution temperature, the cells adhere only to the temperature-responsive material layer and do not adhere to the cell adhesion-inhibiting material layer. Thus, when the cell culture plate 1 or 3 is used, the cells can be cultured in a pattern on the substrate.

≪Cell sheet preparation method≫
The cell sheet production method according to the present invention is a cell culture plate produced by the method for producing a cell culture plate according to the present invention, which has cell adhesiveness and has cell non-adhesiveness due to temperature change. A cell culturing process for culturing cells at a temperature higher than the critical lysis temperature at which the temperature responsive material changes from hydrophobic to hydrophilic on a cell culture plate having a temperature responsive material layer made of a temperature responsive material that can be expressed. And a recovery step of recovering the cell sheet by separating the cells from the temperature-responsive material layer by changing the temperature of the cell culture plate to the critical lysis temperature or lower. As the cell culture plate, for example, cell culture plates 2 and 3 can be used.

  In the cell culturing step, cell culturing can be performed, for example, in the same manner as described in the above << Cell culturing method >>.

  In the collection step, the cell sheet peeled from the temperature-responsive material layer can be collected by a known method. For example, a cell collection layer such as a collagen gel membrane, cellulose membrane, PVDF membrane, parchment paper, gelatin membrane is placed on the cell sheet, and the cell sheet is transferred to the cell collection layer by bringing the cell collection layer into contact with the cell collection layer. By doing so, the cell sheet can be collected.

  When a collagen gel membrane, cellulose membrane, PVDF membrane, nylon mesh, or parchment paper is used as the cell recovery layer, water is dropped into the cell recovery layer to absorb moisture as a method of peeling the cell sheet from the cell recovery layer. Thus, a method of reducing the adsorptive power between the cell recovery layer and the cell sheet can be used. When a gelatin film is used as the cell recovery layer, a method of keeping the gelatin film to which the cell sheet has been transferred at the culture temperature can be used as a method for peeling the cell sheet from the gelatin film. The gelatin film can be dissolved and removed from the cell sheet by heating to a temperature suitable for culture, specifically within a range of 33 ° C. to 40 ° C.

≪Cell sheet≫
The cell sheet according to the present invention is produced by the cell sheet production method according to the present invention. The cell sheet thus prepared has a desired specific shape (cell pattern), and the surface contact factor is not impaired, and the part in contact with the cell culture surface has a uniform quality. Therefore, it is suitable for use in regenerative medicine and the like. In addition, the cell sheet can be applied to detection devices such as biosensors.

Examples of the present invention will be described below, but the scope of the present invention is not limited to these examples.
<Example 1, Comparative Examples 1 and 2>
According to the formulation shown in Table 1 (unit is part by mass), an alkali-soluble resin, a quinonediazide group-containing compound, and a solvent were mixed to prepare the photosensitive resin compositions of Example 1 and Comparative Examples 1 and 2.
Details of each component in Table 1 are as follows.

Alkali-soluble resin A: cresol novolak resin obtained by mixing m-cresol and p-cresol at m-cresol / p-cresol = 60/40 (mass ratio), and adding and condensing by formalin ( (Mass average molecular weight 16300)
Quinonediazide group-containing compound A: bis (4-hydroxy-2,3,5-trimethylphenyl) -2-hydroxyphenylmethane (1 mol) and 1,2-naphthoquinonediazide-5-sulfonic acid chloride (2.3 mol) ) Condensate (bis (4-hydroxy-2,3,5-trimethylphenyl) -2-hydroxyphenylmethane-1,2-naphthoquinonediazide-5-sulfonic acid ester)
Quinonediazide group-containing compound B: condensate (2,3,4-trihydroxy) of 2,3,4-trihydroxybenzophenone (1 mol) and 1,2-naphthoquinonediazide-5-sulfonic acid chloride (2 mol) Benzophenone-1,2-naphthoquinonediazide-5-sulfonic acid ester)
Solvent A: 1-butoxy-2-propanol Solvent B: Mixed solvent of ethyl lactate and ethyl acetate (mass mixing ratio 9: 1)
Solvent C: Ethyl lactate

<Evaluation of solvent resistance of polystyrene dishes>
30 g of each solvent used in Example 1 and Comparative Examples 1 and 2 was put in a separate polystyrene dish (electron beam sterilized, diameter 8 cm, 8.73 g). After 30 minutes, the solvent was removed, the surface of the polystyrene dish was visually observed, and evaluated according to the following criteria. The results are shown in Table 2.
A: No change was observed on the surface of the polystyrene dish.
B: Part or all of the surface of the polystyrene dish became cloudy.

<Nonvolatile analysis>
A polystyrene dish similar to the above was immersed in 500 g of each solvent used in Example 1 and Comparative Examples 1 and 2. After 30 minutes, the polystyrene dish was taken out from the solvent, and the concentration of the components derived from the polystyrene dish dissolved in the solvent (ppm, based on mass) was determined by measuring the mass of the nonvolatile content in the solvent. The results are shown in Table 2.

<Evaluation of sensitivity of photosensitive resin composition>
The photosensitive resin compositions of Example 1 and Comparative Examples 1 and 2 were coated on a polystyrene dish similar to the above with a spin coater and heated (prebaked) at 80 ° C. for 1 hour to obtain a resin layer having a thickness of 2 μm. It was. Next, the resin layer is irradiated with ultraviolet rays through a mask in which line patterns having line widths of 6, 8, 10, and 12 μm are formed by a parallel exposure apparatus (MAT2501, manufactured by Hakuto Co., Ltd.) using a ghi line. Then, the exposed portion was dissolved and removed by immersing in 2.38 mass% tetramethylammonium hydroxide at 23 ° C. for 65 seconds to shake, thereby forming a line and space pattern. The amount of UV irradiation necessary to form a good line and space pattern was determined and used as the sensitivity of the photosensitive resin composition. The results are shown in Table 2.

<AFM analysis of polystyrene dish surface>
The photosensitive resin composition was applied, exposed, and developed in the same manner as in the above <Evaluation of Sensitivity of Photosensitive Resin Composition>, except that ultraviolet rays were irradiated without passing through a mask on which a line pattern was formed. Then, a polystyrene dish from which the photosensitive resin composition was removed was obtained. The roughness and state of the polystyrene dish surface were measured by AFM analysis. In addition, as a control experiment, AFM analysis was also performed on an untreated polystyrene dish and a polystyrene dish on which only development processing was performed without applying the photosensitive resin composition. The results are shown in Table 3.

<Formation of cell pattern>
A cell culture plate including a substrate and a cell adhesion inhibiting material layer provided on a part of the substrate was obtained, and cells were cultured on the cell culture plate to form a cell pattern. The cell culture plate was produced as shown in FIG.

  The photosensitive resin composition of Example 1 or Comparative Example 1 was coated on a substrate 1, that is, a polystyrene dish similar to the above with a spin coater, heated (prebaked) at 80 ° C. for 1 hour, and a resin having a thickness of 2 μm. A layer was obtained. Next, the resin layer is irradiated with ultraviolet rays through a photomask using a mask aligner exposure apparatus (BA100, manufactured by Nanotech), and then immersed and shaken in 2.38 mass% tetramethylammonium hydroxide at 23 ° C. for 65 seconds. Thus, the exposed portion was dissolved and removed, and a resin pattern 2 was provided on a part of the substrate 1. As the shape of the resin pattern, a line and space pattern having a line width of 100 μm or a square pattern having a side of 100 μm was used.

  On the part where the resin pattern 2 on the substrate 1 is not provided, a solution consisting of 20 parts by mass of acrylamide, 1 part by mass of water-soluble camphorquinone as a polymerization initiator, and 79 parts by mass of water is applied, and the peak wavelength is 470 nm, the illuminance The cell adhesion inhibiting material layer 3 was formed by irradiation with 16000 LX visible light. Irradiation with visible light was performed with a blue LED light source using a research LED light source system (manufactured by Panasonic).

  Subsequently, the resin pattern 2 was peeled off using 2.38 mass% tetramethylammonium hydroxide as a peeling solution. Thus, a cell culture plate including the substrate 1 and the cell adhesion inhibiting material layer 3 provided on a part of the substrate 1 was obtained.

The cell culture plate thus obtained was seeded with bovine vascular endothelial cells suspended in DMEM medium containing 10% by mass FBS (serum) and cultured in a 37 ° C. incubator (CO ₂ concentration 5% by volume). did. As a result, when the photosensitive resin composition of Example 1 was used, cells proliferated along the part where the cell adhesion inhibiting material layer 3 was not provided, that is, the part where polystyrene was exposed, and cell culture A cell pattern was formed on the working plate. On the other hand, when the photosensitive resin composition of Comparative Example 1 was used, no cell pattern was formed.

DESCRIPTION OF SYMBOLS 1 Board | substrate 2 Resin pattern 3 Cell adhesion inhibition material layer 4 Temperature-responsive material layer 5 Resin layer

Claims (13)

  A method for producing a cell culture plate, comprising using a photosensitive resin composition containing an alcohol-based organic solvent (S). A resin layer forming step in which the photosensitive resin composition is applied onto a substrate and dried to form a resin layer;
An exposure step of selectively exposing the resin layer;
A method for producing a plate for cell culture according to claim 1, further comprising a developing step of developing the resin layer after exposure to form a resin pattern.   The method for producing a plate for cell culture according to claim 2, wherein the substrate comprises polystyrene.   The method for producing a cell culture plate according to claim 2 or 3, further comprising a cell adhesion inhibiting material layer forming step of forming a cell adhesion inhibiting material layer on a portion of the substrate where the resin pattern is not provided.   The method for producing a cell culture plate according to claim 4, further comprising a resin pattern peeling step for peeling the resin pattern.   Temperature response for forming a temperature-responsive material layer made of a temperature-responsive material having cell adhesiveness and capable of expressing cell non-adhesiveness due to temperature change in a portion where the resin pattern on the substrate is not provided The method for producing a plate for cell culture according to claim 2 or 3, further comprising a step of forming a functional material layer.   The method for producing a cell culture plate according to claim 6, further comprising a resin pattern peeling step for peeling the resin pattern. A resin layer laminating step of laminating a resin layer made of the photosensitive resin composition on the cell adhesion inhibiting material layer;
A temperature-responsive material layer made of a temperature-responsive material having cell adhesion and capable of expressing cell non-adhesiveness due to temperature change is formed on the substrate where the cell adhesion inhibiting material layer is not provided. A temperature-responsive material layer forming step;
A resin layer peeling step for peeling the resin layer;
The method for producing a cell culture plate according to claim 5, further comprising:   A cell culture plate produced by the method for producing a cell culture plate according to claim 1.   A cell culture method comprising a cell culture step of culturing cells on a cell culture plate produced by the method for producing a cell culture plate according to any one of claims 1 to 8. A temperature responsive material on a cell culture plate produced by the method for producing a cell culture plate according to any one of claims 6 to 8, at a temperature higher than a critical dissolution temperature at which the material changes from hydrophobic to hydrophilic. A cell culture process for culturing cells;
And a recovery step of recovering the cell sheet by separating the cell from the temperature-responsive material layer by changing the temperature of the cell culture plate to the critical lysis temperature or lower.   A cell sheet produced by the method for producing a cell sheet according to claim 11.   A photosensitive resin composition for forming a resin pattern on a substrate, comprising an alcohol-based organic solvent (S).

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