JP2006174826A - Temperature-sensitive and swelling resin bead for cell culture - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a resin bead for cell culture designed to directly release/recover proliferated cells without denaturing the cells. <P>SOLUTION: The temperature-sensitive and swelling resin bead for the cell culture comprises a cross-linked polymer (A) having an inflection point in a temperature-water absorption coefficient curve and the temperature corresponding to the inflection point is 0-80°C. The cross-linked polymer (A) is at least one kind selected from the group consisting of a cross-linked vinyl polymer (A1) comprising a vinyl monomer containing a carbamoyl group and/or a polyoxyalkylene group as an essential constituent monomer, a cross-linked alkylene oxide polymer (A2), a cross-linked methyl cellulose (A3) and a cross-linked polyvinyl alcohol partially saponified material (A4). Furthermore, a polypeptide (P) having at least one cell-adhesive minimum amino acid sequence (X) in one molecule is contained. Fine particles (b) are further contained. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a temperature-sensitive swelling resin bead for cell culture.

  As cell culture beads, dextran beads that swell when absorbed (Patent Document 1, Non-Patent Document 1) are known. Cells cultured and grown with the beads are treated with a proteolytic enzyme (for example, trypsin), a chelating agent (for example, EDTA) or the like, and are peeled and collected from the surface of the beads (Non-patent Document 2).

JP-A-57-195701 Microcarrier cell culture principles & methods (Pharmacia Biotech Co., Ltd., issued October 10, 1996) 27-29 etc. Microcarrier cell culture principles & methods (Pharmacia Biotech Co., Ltd., issued October 10, 1996) 74-77

  However, when the proliferated cells are peeled and collected by performing the treatment as described above, there is a problem that the proliferated cells are denatured by the treatment and the original function of the cells is impaired. An object of the present invention is to provide resin beads for cell culture that can be detached and recovered as they are without denaturing proliferated cells.

The feature of the temperature-swellable resin beads for cell culture of the present invention is summarized in that it contains a crosslinked polymer (A) having an inflection point in a temperature-water absorption curve.
The method for producing a thermosensitive resin bead of the present invention is a method for producing a thermosensitive resin bead for cell culture comprising a crosslinked polymer (A) having an inflection point in a temperature-water absorption curve. And
A mixture of a vinyl monomer containing a carbamoyl group and / or a polyoxyalkylene group and a crosslinking agent (c1) is dropped into the hydrophobic solvent (W) and simultaneously polymerized while being dispersed to obtain a crosslinked vinyl polymer (A1). Step (1),
A step (2) of obtaining a crosslinked alkylene oxide polymer (A2) by dispersing a mixture of an alkylene oxide polymer and a crosslinking agent (c2) in a hydrophobic solvent (W) and performing a crosslinking reaction;
A step (3) of obtaining a crosslinked methylcellulose (A3) by dispersing a mixture of methylcellulose and a crosslinking agent (c3) in a hydrophobic solvent (W) and performing a crosslinking reaction;
And / or a step (4) in which a mixture of a partially saponified polyvinyl alcohol and a crosslinking agent (c4) is dispersed in a hydrophobic solvent (W) and subjected to a crosslinking reaction to obtain a crosslinked saponified polyvinyl alcohol (A4). Is the gist.

  The temperature-swellable resin beads for cell culture according to the present invention do not require treatment with a proteolytic enzyme or a chelating agent in the cell detachment / collection step after cell culture, so that the cells can be detached and recovered very easily. Therefore, there is no problem that the original function of the cell is impaired.

The temperature-water absorption curve is a curve obtained by plotting the water absorption of the crosslinked polymer (A) and the measurement temperature.
The water absorption is adjusted to a constant temperature by placing 1.00 g of a crosslinked polymer sample in a tea bag (10 cm long, 5 cm wide) made of a nylon net having an opening of 53 μm according to JIS Z8801-1: 2000. Soaked in 500 ml of deionized water, left at a constant temperature for 3 days, suspended the tea bag for 15 minutes, drained, measured the weight (AG) of the crosslinked polymer sample, and calculated the water absorption rate from the following formula: To do.

なお、測定温度は、０〜９５℃の範囲で少なくとも５点、好ましくは１０点、さらに好ましくは２５点、特に好ましくは５０点、最も好ましくは９５点の温度を選択することである。

The measurement temperature is to select a temperature of at least 5 points, preferably 10 points, more preferably 25 points, particularly preferably 50 points, and most preferably 95 points in the range of 0 to 95 ° C.

The inflection point means a point (a, f (a)) when the function f (x) can be differentiated twice and the unevenness of the curve y = f (x) changes before and after x = a. . That is, it means that the curve changes from an upwardly convex state to an upwardly concave state, or from an upwardly concave state to an upwardly convex state.
In the vicinity of the inflection point, the change in the water absorption rate of the crosslinked polymer (A) is maximized.

  The temperature (° C.) corresponding to the inflection point of the crosslinked polymer (A) is preferably from 0 to 80, more preferably from 5 to 70, particularly preferably from 10 to 50, and most preferably from 15 to 40. Since normal cells are vulnerable to heat, this temperature (° C.) is more preferably 5 to 40, next preferably 10 to 38, and most preferably 15 to 35. Within this range, cell detachment / recovery treatment is further improved.

  As the crosslinked polymer (A) having such an inflection point, a crosslinked vinyl polymer (A1) comprising a vinyl monomer containing a carbamoyl group and / or a polyoxyalkylene group as an essential constituent monomer, a crosslinked alkylene oxide polymer ( Preferred examples include at least one selected from the group consisting of A2), cross-linked methylcellulose (A3), and cross-linked saponified polyvinyl alcohol (A4). These cross-linked polymers may be one kind or a mixture of two or more kinds.

The crosslinked vinyl polymer (A1) may contain a vinyl monomer containing a carbamoyl group and / or a polyoxyalkylene group as an essential constituent monomer, but preferably contains a crosslinking agent (c1) as a constituent unit.
Examples of vinyl monomers containing a carbamoyl group and / or a polyoxyalkylene group include (i) N-alkyl- or N-alkoxyalkyl- (meth) acrylic acid amide, (b) N, N-dialkyl-, N, N -Di (alkoxyalkyl)-or N, N-alkylene- (meth) acrylic acid amide, (c) (meth) acrylic acid amide having a polyiminoethylene group (degree of polymerization 2 to 50), (d) tertiary amino (Meth) acrylic acid amide having a group (5 to 50 carbon atoms), (e) polyoxyalkylene having an alkylene group of 2 to 4 carbon atoms (degree of polymerization 3 to 40) monool or diol (meth) acrylic acid Monoester, (he) polyoxyalkylene having 2 to 4 carbon atoms of alkylene group (degree of polymerization 3 to 40) monool or diol monovinyl pheny Ethers, include vinyl monomers represented by the vinyl monomer and represented by (g) General formula (1) (h) formula (2), in addition to these (meth) acrylamide may also be used.
In addition, (meth) acryl ... means acrylic ... and / or methacryl ...

(I) As N-alkyl- or N-alkoxyalkyl- (meth) acrylic acid amide, N-mono-substituted (meth) acrylic acid amide having 4 to 10 carbon atoms can be used, and N-methyl (meth) acrylic can be used. Acid amide, N-ethyl (meth) acrylic acid amide, N-propyl (meth) acrylic acid amide, N-isopropyl (meth) acrylic acid amide, N-methoxypropyl (meth) acrylic acid amide, N-ethoxypropyl (meth) ) Acrylic acid amide and N-ethoxyethyl (meth) acrylic acid amide.

(B) N, N-dialkyl-, N, N-di (alkoxyalkyl)-or N, N-alkylene- (meth) acrylic acid amide may be an N, N-disubstituted (meta) having 5 to 12 carbon atoms. ) Acrylic acid amide, etc. can be used, N, N-diethyl (meth) acrylic acid amide, N, N-dimethyl (meth) acrylic acid amide, N, N-dimethoxypropyl (meth) acrylic acid amide, (meth) acryloyl Examples include piperidine and N- (meth) acryloylpyrrolidine.

(C) Examples of the (meth) acrylic acid amide having a polyiminoethylene group (degree of polymerization of 2 to 50) include tetraethyleneimine mono (meth) acrylic acid amide and triethyleneimine mono (meth) acrylic acid amide. .

(D) (Meth) acrylic acid amide having a tertiary amino group (5 to 50 carbon atoms) includes N-morpholinoethyl (meth) acrylic acid amide, N-morpholinopropyl (meth) acrylic acid amide, dimethylaminopropyl Examples include (meth) acrylic acid amide and dimethylaminoethyl (meth) acrylic acid amide.

(E) (Meth) acrylic acid monoester of polyoxyalkylene (degree of polymerization 3 to 40) monool or diol having 2 to 4 carbon atoms of alkylene group, tetraethylene glycol monoethyl ether mono (meth) acrylate, Pentaethylene glycol monobutyl ether mono (meth) acrylate, methoxytrioxypropylene tetraoxyethylene (meth) acrylate, tetrapropylene glycol ethylene oxide 6 mol adduct (meth) acrylic acid monoester and butanol ethyleonoxide 20 mol. (Meth) acrylic acid ester of propylene oxide 20 mol block adduct, etc. are mentioned.

(F) Polyoxyalkylene having a alkylene group of 2 to 4 carbon atoms (degree of polymerization 3 to 40) monool or diol monovinyl phenyl ether, tetraethylene glycol monomethyl ether monovinyl phenyl ether, pentaethylene glycol monoethyl ether monovinyl Examples include phenyl ether, methoxypentaoxypropylene tetraoxyethylene vinyl phenyl ether, and monovinyl phenyl ether of an ethylene oxide 8-mole adduct of tetrapropylene glycol.

ＯＡは炭素数２〜４のオキシアルキレン基、ｎは１〜２００の整数、Ｘは−ＣＯ₂Ｈ、−ＣＯ₂Ｒ³、−ＣＯ₂Ｍ、−ＣＯ₂ＮＲ³ ₄、−ＣＯＮＲ³ ₂又は−ＣＮで表される基、Ｍは金属原子、Ｒ¹は水素原子又はメチル基、Ｒ²及びＲ³は水素原子又は炭素数１〜６の有機基を示す。 (G) The vinyl monomer represented by the general formula (1) will be described.

OA is an oxyalkylene group having 2 to 4 carbon atoms, n is an integer of 1 to 200, X is —CO ₂ H, —CO ₂ R ³ , —CO ₂ M, —CO ₂ NR ³ ₄ , —CONR ³ ₂ or A group represented by -CN, M is a metal atom, R ¹ is a hydrogen atom or a methyl group, R ² and R ³ are a hydrogen atom or an organic group having 1 to 6 carbon atoms.

In the general formula (1), examples of the oxyalkylene group (OA) having 2 to 4 carbon atoms include oxyethylene, oxypropylene, and oxybutylene.
Of these, oxyethylene and oxypropylene are preferable from the viewpoint of ease of production, and more preferably oxyethylene.
These oxyalkylene groups may be one kind or a mixture of two or more kinds. When two or more types are mixed, the binding mode may be any of block, random, and combinations thereof (blocks and combinations thereof are preferred).
Further, n is preferably an integer of 1 to 200, but may exceed this range.

Among R ² , the organic group having 1 to 6 carbon atoms includes alkyl, alkenyl, acyl and the like.
Examples of alkyl include methyl, ethyl, isopropyl, n-butyl and n-hexyl.
Examples of alkenyl include vinyl, 1-propenyl, 2-butenyl and 2,2-dimethyl-3-butenyl.
Examples of acyl include formyl, acetyl, propionyl, isobutyryl, valeryl, pivaloyl, acryloyl, methacryloyl, crotonoyl, propioyl and the like.
Among R ² , a hydrogen atom and acyl are preferable from the viewpoint of ease of production, but from the viewpoint of hydrolyzability, a hydrogen atom, alkyl and alkenyl are preferable, and a hydrogen atom and alkyl are more preferable. .

Among R ³ , the organic group having 1 to 6 carbon atoms includes alkyl and alkenyl.
Of R ³ , a hydrogen atom and alkyl are preferable, and a hydrogen atom is more preferable from the viewpoint of hydrolyzability.
M (metal atom) is not limited as long as it can form a carboxylic acid metal, but can form a salt {alkali metal (lithium, potassium, sodium, etc.), alkaline earth metal (magnesium, calcium, etc.) ) And transition metals (such as copper and zinc)} are preferred.
Of X, a group represented by —CO ₂ H, —CO ₂ R ³ , and —CONR ³ ₂ is preferable, and a group represented by —CONR ³ ₂ is more preferable.

Examples of the vinyl monomer represented by the general formula (1) include the following (1) carboxylic acid (X is —CO ₂ H), (2) carboxylic acid ester (X is —CO ₂ R ³ ), (3) Carboxylic acid metal salt (X is —CO ₂ M), (4) Carboxylic acid ammonium salt (X is —CO ₂ NR ³ ₄ ), (5) Amide (X is —CONR ³ ₂ ) and (6) Carboxylic acid nitrile Preferred examples include (X is -CN).

(1) Carboxylic acid (X is —CO ₂ H)
α- (hydroxyethyloxymethyl) acrylic acid, α- (hydroxyethyl (polyoxyethylene) oxymethyl) acrylic acid, α- (hydroxyethyloxypropyloxymethyl) acrylic acid, α- (hydroxypropyloxyethyloxypropyloxy) Methyl) acrylic acid, α- (hydroxyethyloxyethyloxypropyloxymethyl) acrylic acid, α- (hydroxyethyloxypropyloxypropyloxymethyl) acrylic acid, and α- (hydroxyethyl (polyoxyethylene polyoxypropylene) oxy Methyl) acrylic acid and the like.

(2) Carboxylic acid ester (X is —CO ₂ R ³ )
α- (hydroxyethyloxymethyl) methyl acrylate, α- (hydroxyethyl (polyoxyethylene) oxymethyl) methyl acrylate, α- (hydroxypropyloxyethyloxymethyl) methyl acrylate and α- (hydroxyethyl (poly) Oxyethyleneoxypropylene) oxymethyl) methyl acrylate and the like.

(3) Carboxylic acid metal salt (X is —CO ₂ M)
α- (hydroxyethyloxymethyl) acrylic acid sodium salt, α- (hydroxyethyl (polyoxyethylene) oxymethyl) acrylic acid potassium salt, and α- (hydroxyethyl (polyoxyethylenepolyoxypropylene) oxymethyl) acrylic acid Sodium salt etc.

(4) Carboxylic acid ammonium salt (X is —CO ₂ NR ³ ₄ )
α- (hydroxyethyloxymethyl) acrylic acid ammonium salt, α- (hydroxyethyloxymethyl) acrylic acid tetramethylammonium salt, α- (hydroxyethyl (polyoxyethylene) oxymethyl) acrylic acid tetramethylammonium salt, α- (Hydroxyethyloxypropyloxymethyl) acrylic acid tetramethylammonium salt and α- (hydroxyethyl (polyoxyethylenepolyoxypropylene) oxymethyl) acrylic acid tetramethylammonium salt and the like.

(5) Amide (X is —CONR ³ ₂ )
α- (hydroxyethyloxymethyl) acrylic acid amide, N, N-dimethyl-α- (hydroxyethyloxymethyl) acrylic acid amide, α- (hydroxyethyl (polyoxyethylene) oxymethyl) acrylic acid amide, N, N -Dimethyl-α- (hydroxyethyl (polyoxyethylene) oxymethyl) acrylic acid amide, N-methyl-α- (hydroxyethyl (polyoxyethylene) oxymethyl) acrylic acid amide, N-propyl-α- (hydroxyethyl) (Polyoxyethylene) oxymethyl) acrylic acid amide, N-methoxypropyl-α- (hydroxyethyl (polyoxyethylene) oxymethyl) acrylic acid amide, N, N-diethyl-α- (hydroxyethyl (polyoxyethylene) Oxymethyl) acrylic acid amide, α- ( Droxypropyloxymethyl) acrylic acid amide, N, N-dimethyl-α- (hydroxypropyloxymethyl) acrylic acid amide, α- (hydroxyethyl (polyoxypropylene) oxymethyl) acrylic acid amide, N, N-dimethyl -Α- (hydroxyethyl (polyoxypropylene) oxymethyl) acrylic acid amide, α- (hydroxypropyloxyethyloxymethyl) acrylic acid amide, N, N-dimethyl-α- (hydroxyethyloxypropyloxymethyl) acrylic acid Amides, α- (hydroxyethyl (polyoxyethylene polyoxypropylene) oxymethyl) acrylic acid amide, N, N-dimethyl-α- (hydroxyethyl (polyoxyethylene polyoxypropylene) oxymethyl) acrylic acid amide, and the like.

(6) Carboxylic acid nitrile (X is —CN)
α- (hydroxyethyloxymethyl) acrylic nitrile, α- (hydroxyethyl (polyoxyethylene) oxymethyl) acrylic nitrile, α- (hydroxyethyl (polyoxyethylenepolyoxypropylene) oxymethyl) acrylic nitrile and the like.

The vinyl monomer represented by the general formula (1) can be obtained by combining the following known production methods and the like.
(1) Production of α-hydroxymethyl acrylate ester, α-hydroxymethyl acrylate nitrile or N, N-dialkyl-α-hydroxymethyl acrylamide Third class of acrylate ester, acrylonitrile or N, N-dialkyl acrylamide By reacting in the presence of an amine, α-hydroxymethyl acrylate ester, α-hydroxymethyl acrylate nitrile or N, N-dialkyl-α-hydroxymethyl acrylamide is obtained (for example, JP-A-61-134353, US Pat. No. 4,654,432, JP-A-5-70408).

(2) Introduction of oxyalkylene group into α-hydroxymethyl group α-hydroxymethyl acrylate ester, α-hydroxymethyl acrylate nitrile or N, N-dialkyl-α-hydroxymethyl acrylamide, and alkylene having 2 to 4 carbon atoms An oxide (ethylene oxide, propylene oxide, butylene oxide, etc.) is reacted (a known reaction catalyst can be used).
Alternatively, α-hydroxymethyl acrylate ester, α-hydroxymethyl acrylate nitrile or N, N-dialkyl-α-hydroxymethyl acrylamide is reacted with an aliphatic alcohol alkylene oxide adduct in the presence of an alkali (Williamson synthesis). Law).
Examples of the aliphatic alcohol include alkanols (such as methanol, ethanol, isopropanol, n-butanol and n-hexanol) and alkenols (such as propenol, butenol and 2,2-dimethyl-3-butenol).

(3) If necessary, hydrolysis leads to carboxylic acid (X is —CO ₂ H), carboxylic acid metal salt (X is —CO ₂ M), carboxylic acid ammonium salt (X is —CO ₂ NR ³ ₄ ) α -Hydroxymethyl acrylate ester, α-hydroxymethyl acrylate nitrile or N, N-dialkyl-α-hydroxymethyl acrylamide, an oxyalkylene compound introduced with an oxyalkylene group is hydrolyzed to give carboxylic acid, carboxylic acid metal salt or carboxylic acid An acid ammonium salt is used (for example, 4th edition, Experimental Chemistry Course 22, Organic Synthesis IV-Acid / Amino Acid / Peptide-, November 30, 1992, edited by The Chemical Society of Japan, published by Maruzen Co., Ltd.).
It can also be hydrolyzed from a carboxylic nitrile (X is —CN) to an amide (X is —CONR ³ ₂ ) (for example, the above-mentioned Experimental Chemistry Course 22, pages 151 to 156).
In addition, carboxylic acid ester (X is —CO ₂ R ³ ) can be converted to amide (X is —CONR ³ ₂ ) (for example, Experimental Chemistry Course 22, pages 148 to 151).
In addition, the carboxylic acid nitrile (X is —CN) can be converted to a carboxylic acid ester (X is —CO ₂ R ³ ) (for example, the above-mentioned Experimental Chemistry Course 22, pages 53).

(H) The vinyl monomer represented by the general formula (2) will be described.

In the general formula (2), OA is an oxyalkylene group having 2 to 4 carbon atoms, like OA in the general formula (1), m is an integer of 1 to 200, and R is the same as n in the general formula (1). ⁴ is a hydrogen atom or a methyl group, and R ⁵ is a hydrogen atom or an organic group having 1 to 6 carbon atoms, which is the same as R ^{2 in the} general formula (1).

  Examples of the vinyl monomer represented by the general formula (2) include N- (hydroxyethyloxymethyl) (meth) acrylic acid amide, N- (hydroxyethyl (polyoxyethylene) oxymethyl) (meth) acrylic acid amide, N -(Hydroxypropyloxymethyl) (meth) acrylic acid amide, N- (hydroxypropyloxyethyloxymethyl) (meth) acrylic acid amide, N- (hydroxyethyl (polyoxyethylenepolyoxypropylene) oxymethyl) (meth) Acrylic amide and N- (methoxyethyloxymethyl) (meth) acrylic amide, and the like.

  In the same manner as the vinyl monomer represented by the general formula (1), the vinyl monomer represented by the general formula (2) has an oxyalkylene group added to the hydroxymethyl group of N-hydroxymethyl (meth) acrylic acid amide. It can be obtained by introduction. N-hydroxymethyl (meth) acrylamide may be a commercially available one, or a known method {for example, reacting (meth) acrylamide and formalin in the presence of a basic catalyst} or the like. May be obtained.

  Among vinyl monomers containing a carbamoyl group and / or a polyoxyalkylene group, (i) N-alkyl- or N-alkoxyalkyl- (meth) acrylic acid amide from the viewpoint of ease of cell detachment / recovery, ( (B) N, N-dialkyl-, N, N-di (alkoxyalkyl)-or N, N-alkylene- (meth) acrylic acid amide, (c) a polyiminoethylene group (degree of polymerization 2 to 50) ( (Meth) acrylic acid amide, (d) a (meth) acrylic acid amide having a tertiary amino group (5 to 50 carbon atoms), (g) a vinyl monomer represented by the general formula (1), and (h) a general formula ( 2) is preferable, more preferably (A) and (B), particularly preferably N-methyl (meth) acrylic acid amide, N-ethyl (meth) acrylic acid amide. N-propyl (meth) acrylic acid amide, N-isopropyl (meth) acrylic acid amide, N-methoxypropyl (meth) acrylic acid amide, N-ethoxypropyl (meth) acrylic acid amide, N-ethoxyethyl (meth) acrylic Acid amide, N, N-diethyl (meth) acrylic acid amide, N, N-dimethyl (meth) acrylic acid amide, N, N-dimethoxypropyl (meth) acrylic acid amide and N- (meth) acryloylpyrrolidine, most preferred Are N-isopropylacrylic acid amide and N, N-diethylacrylic acid amide.

  Examples of the crosslinking agent (c1) include a monomer (c11) having at least two vinyl groups, a compound (c12) having at least two epoxy groups, a compound (c13) having at least two isocyanate groups, and a vinyl monomer having an epoxy group. (C14), a vinyl monomer (c15) having an isocyanate group, and the like are included.

  As the monomer (c11) having two or more vinyl groups, monomers described in JP-A No. 2002-284803, JP-A No. 2002-20468 or JP-A No. 9-143897 can be used. Among these, ethylene glycol di (meth) acrylate, propylene glycol di (meth) acrylate, polypropylene glycol di (meth) acrylate, N, N′-methylenebis (meth) acrylamide, glycerin tri (meth) acrylate and pentaerythritol tetra ( A (meth) acrylate etc. are preferable.

As the compound (c12) having at least two epoxy groups, compounds having at least two epoxy groups described in JP-A No. 2002-284803 or JP-A No. 2004-263147 can be used, and the following compounds are preferable. .
(1) Aromatic compounds having at least two epoxy groups: bisphenol F diglycidyl ether, bisphenol A diglycidyl ether, bisphenol B diglycidyl ether, bisphenol AD diglycidyl ether, bisphenol S diglycidyl ether, halogenated bisphenol A diglycidyl, Tetrachlorobisphenol A diglycidyl ether, catechin diglycidyl ether, resorcinol diglycidyl ether, hydroquinone diglycidyl ether, pyrogallol triglycidyl ether, 1,5-dihydroxynaphthalene diglycidyl ether, dihydroxybiphenyl diglycidyl ether, octachloro-4,4 ' -Dihydroxybiphenyl diglycidyl ether, phenol or cresol novola Poly (glycidyl ether) of the lacquer resin, diglycidyl ether obtained from the reaction of 2 mol of bisphenol A and 3 mol of epichlorohydrin, polyphenol poly obtained by condensation reaction of phenol and glyoxal, glutaraldehyde, or formaldehyde (Glycidyl ether) and poly (glycidyl ether) of polyphenol obtained by condensation reaction of resorcin and acetone.

(2) Heterocyclic compounds having at least two epoxy groups: trisglycidyl melamine, trisepoxypropyl isocyanurate, 2,5-dihydroxy-1,4-dithiandiglycidyl ether, citrate diglycidyl ether, and the like.

(3) Alicyclic compounds having at least two epoxy groups: vinylcyclohexene dioxide, limonene dioxide, dicyclopentadiene dioxide, bis (2,3-epoxycyclopentyl) ether, ethylene glycol bisepoxy dicyclopentyl ale, 3 , 4-Epoxy-6-methylcyclohexylmethyl-3 ′, 4′-epoxy-6′-methylcyclohexanecarboxylate, bis (3,4-epoxy-6-methylcyclohexylmethyl) adipate, bis (3,4-epoxy -6-methylcyclohexylmethyl) butylamine and the like.

(4) Aliphatic compound having at least two epoxy groups: polyglycidyl ether of polyhydric aliphatic alcohol, polyglycidyl ester of polyhydric fatty acid, glycidyl aliphatic amine, and the like.
Polyglycidyl ether of polyhydric aliphatic alcohol: ethylene glycol diglycidyl ether, propylene glycol diglycidyl ether, tetramethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, trimethylolpropane triglycidyl ether, pentaerythritol Tetraglycidyl ether, sorbitol polyglycidyl ether, etc.
Polyglycidyl ester of polyvalent fatty acid: diglycidyl adipate, diglycidyl succinate, diglycidyl oxalate, diglycidyl tartrate, diglycidyl malonate, etc.
Glycidyl aliphatic amine: N, N, N ′, N′-tetraglycidylhexamethylenediamine, N, N, N ′, N′-tetraglycidylethylenediamine, N, N, N ′, N′-tetraglycidyltrimethylenediamine N, N, diglycidyl butylamine, etc.

As the compound (c13) having at least two isocyanate groups, compounds having at least two isocyanate groups described in JP-A No. 2002-284803 or JP-A No. 2004-263147 can be used, and the following compounds are preferable. .
(1) Aliphatic diisocyanate: ethylene diisocyanate, tetramethylene diisocyanate, hexamethylene diisocyanate (HDI), dodecamethylene diisocyanate, 2,2,4-trimethylhexamethylene diisocyanate, lysine diisocyanate, 2,6-diisocyanatomethylcaproate, Bis (2-isocyanatoethyl) fumarate, bis (2-isocyanatoethyl) carbonate, 2-isocyanatoethyl-2,6-diisocyanatohexanoate and the like.

(2) Cycloaliphatic diisocyanate: isophorone diisocyanate (IPDI), dicyclohexylmethane-4,4′-diisocyanate (hydrogenated MDI), cyclohexylene diisocyanate, methylcyclohexylene diisocyanate (hydrogenated TDI), bis (2-isocyanatoethyl) ) -4-cyclohexene and the like.

(3) Aromatic diisocyanate: 1,3- or 1,4-phenylene diisocyanate, 2,4- or 2,6-tolylene diisocyanate (TDI), crude TDI, 2,4'- or 4,4'-diphenylmethane Diisocyanate (MDI), 4,4′-diisocyanatobiphenyl, 3,3′-dimethyl-4,4′-diisocyanatobiphenyl, 3,3′-dimethyl-4,4′-diisocyanatodiphenylmethane, crude MDI, 1,5-naphthylene diisocyanate, etc.

(4) Aroaliphatic diisocyanate: m- or p-xylylene diisocyanate (XDI), α, α, α ', α'-tetramethylxylylene diisocyanate (TMXDI) or the like.

(5) Modified products of these diisocyanates: HDI isocyanurate, HDI burette, IPDI isocyanurate, IPDI burette and the like.

  The vinyl monomer (c14) having an epoxy group is not particularly limited as long as it is a compound containing an epoxy group and a vinyl group in the molecule. Unsaturated carboxylic acid glycidyl ester {(meth) acrylic acid glycidyl, α-ethylacrylic Glycidyl acid, glycidyl α-n-propyl acrylate, glycidyl α-n-butyl acrylate, (meth) acrylic acid-3,4-epoxybutyl, (meth) acrylic acid-4,5-epoxypentyl, (meth) Acrylic acid-6,7-epoxyheptyl, α-ethylacrylic acid-6,7-epoxyheptyl and (meth) acrylic acid-2,3-epoxypropyl, etc.}, and unsaturated glycidyl ether {p-glycidyloxystyrene, 2,3-di (glycidyloxy) styrene, 4-vinyl-1-cyclohexene-1,2- Poxide, 3,4-diglycidyloxystyrene, 2,4-diglycidyloxystyrene, 3,5-diglycidyloxystyrene, 2,6-diglycidyloxystyrene, 2,3-bis (glycidyloxymethyl) styrene, 3,4-bis (glycidyloxymethyl) styrene, 2,4-bis (glycidyloxymethyl) styrene, 3,5-bis (glycidyloxymethyl) styrene, 2,6-bis (glycidyloxymethyl) styrene, 2, 3,4-tris (glycidyloxymethyl) styrene, 2,4,6-tris (glycidyloxymethyl) styrene, 1,2,3-triglycidyloxy-5-vinylbenzene and 1,2,3-triglycidyloxy -4-vinylbenzene, etc.}.

The vinyl monomer (c15) having an isocyanate group is not particularly limited as long as it is a compound containing an isocyanate group and a vinyl group in the molecule. Isocyanatoethyl (meth) acrylate, isocyanatopropyl (meth) acrylate, isocyanato Examples include butyl (meth) acrylate, isocyanatohexyl (meth) acrylate, 3-isopropenyl-α, α-dimethylbenzyl isocyanate and 3-ethylenyl-α, α-dimethylbenzyl isocyanate.
Among the crosslinking agents (c1), from the viewpoint of ease of handling, the monomer (c11) having at least two vinyl groups is preferable, and ethylene glycol dimethacrylate is more preferable.

  When the crosslinking agent is included as a constituent unit, the content (% by weight) of the crosslinking agent unit is preferably 0.001 to 10, based on the weight of the vinyl monomer containing a carbamoyl group and / or a polyoxyalkylene group, Preferably it is 0.005-7, Most preferably, it is 0.01-5. Within this range, cell detachment / recovery is further facilitated.

In addition to the vinyl monomer containing a carbamoyl group and / or polyoxyalkylene group and the crosslinking agent (c1), another vinyl monomer may be copolymerized in the crosslinked vinyl polymer (A1).
The other vinyl monomer is not particularly limited as long as it is a vinyl monomer that can be copolymerized with the vinyl monomer containing the carbamoyl group and / or the polyoxyalkylene group, and the following vinyl monomers can be used.

Alicyclic vinyl monomers: cyclohexene, (di) cyclopentadiene, pinene, limonene, indene, vinylcyclohexene, ethylidenebicycloheptene, etc.
Aromatic vinyl monomers: styrene, α-methylstyrene, vinyltoluene, 2,4-dimethylstyrene, ethylstyrene, isopropylstyrene, butylstyrene, phenylstyrene, cyclohexylstyrene, benzylstyrene, crotylbenzene, vinylnaphthalene and the like.
Aliphatic vinyl monomers: methyl (meth) acrylate, ethyl (meth) acrylate, propyl (meth) acrylate, butyl (meth) acrylate, ethylene, propylene and the like.
Halogen element-containing vinyl monomers: vinyl chloride, vinyl bromide, vinylidene chloride, allyl chloride, chlorostyrene, bromostyrene, dichlorostyrene, chloromethylstyrene, tetrafluorostyrene, chloroprene and the like.
Carboxy group-containing vinyl monomers and alkali metal salts and ammonium salts thereof: (meth) acrylic acid, (anhydrous) maleic acid, maleic acid monoalkyl ester, fumaric acid, fumaric acid monoalkyl ester, crotonic acid, itaconic acid, itaconic acid mono Alkyl esters, itaconic acid glycol monoether, citraconic acid, citraconic acid monoalkyl esters, cinnamic acid and the like, and alkali metal salts and ammonium salts thereof.
Hydroxy group-containing vinyl monomer: hydroxystyrene, hydroxyethyl (meth) acrylate, hydroxypropyl (meth) acrylate, (meth) allyl alcohol, 1-buten-3-ol, 2-buten-1-ol, 2-butene-1 , 4-diol, propargyl alcohol, 2-hydroxyethylpropenyl ether and the like.

  When other vinyl monomer is included as a constituent unit, the content (% by weight) of the other vinyl monomer unit is 0.005 to 50 based on the weight of the vinyl monomer containing a carbamoyl group and / or a polyoxyalkylene group. Is preferable, more preferably 0.01 to 30, and particularly preferably 0.1 to 20. Within this range, cell detachment / recovery is further facilitated.

The crosslinked vinyl polymer (A1) can be produced by the following methods (1) to (4).
(1) A mixture of a vinyl monomer containing a carbamoyl group and / or a polyoxyalkylene group and a crosslinking agent (c1) is dropped into the hydrophobic solvent (W) and simultaneously polymerized while being dispersed to cause a crosslinked vinyl polymer (A1). ) To obtain (1) (reverse phase suspension polymerization method).
(2) A mixture of a vinyl monomer containing a carbamoyl group and / or a polyoxyalkylene group and a crosslinking agent (c1) is dispersed in a hydrophobic solvent (W) and then subjected to a polymerization reaction to obtain a crosslinked vinyl polymer (A1). A process comprising a obtaining step (5) (reverse phase suspension polymerization method).
(3) A hydrophobic solvent (W) is dispersed while being dropped into a mixture of a vinyl monomer containing a carbamoyl group and / or a polyoxyalkylene group and a crosslinking agent (c1), phase-inverted, and then subjected to a polymerization reaction for crosslinking. A method comprising a step (6) (reverse phase suspension polymerization method) of obtaining a vinyl polymer (A1).
(4) A step of obtaining a crosslinked vinyl polymer (A1) by mixing a vinyl monomer containing a carbamoyl group and / or a polyoxyalkylene group and a crosslinking agent (c1) and polymerizing using a reaction solvent if necessary ( 7) A method comprising (bulk polymerization method or solution polymerization method).
Among these methods, the methods (1), (2) and (3) are preferred from the viewpoint of production efficiency (fine particles can be produced directly), and the method (1) is more preferred.

The method (1) including the step (1) will be described in further detail.
If necessary, the mixture of the vinyl monomer containing a carbamoyl group and / or a polyoxyalkylene group and the crosslinking agent (c1) is preferably dissolved in the hydrophilic solvent (U).
As the hydrophilic solvent (U), any liquid containing water (deionized water or the like) as an essential constituent can be used without limitation, and water or an aqueous solution containing a polar solvent can be used.
The hydrophilic solvent (U) is a term corresponding to the hydrophobic solvent (W), means a solvent that is difficult to dissolve with the hydrophobic solvent (W), and is used together with the hydrophobic solvent (W) to have a reverse phase. It is a solvent for suspension polymerization.
As the polar solvent, a known organic solvent (for example, a solvent described in JP-A-2002-284881) can be used, and alcohol (methanol, ethanol, etc.), ketone (acetone, methyl ethyl ketone, etc.), ester (ethyl acetate, Butyl acetate), ether (tetrahydrofuran, dioxane, etc.), amide (dimethylformamide, dimethylformamide, etc.) and the like.
When a polar solvent is contained, the content (% by weight) is preferably 1 to 80, more preferably 2 to 70, and particularly preferably 3 to 30 based on the weight of the hydrophilic solvent (U). Within this range, resin beads can be prepared more easily.
When the hydrophilic solvent (U) is used, the amount (% by weight) of (U) used is based on the weight of the mixture of the vinyl monomer containing the carbamoyl group and / or polyoxyalkylene group and the crosslinking agent (c1). 1 to 2000, more preferably 10 to 1000, and particularly preferably 20 to 500. Within this range, resin beads can be prepared more easily.

The hydrophobic solvent (W) is not soluble in water and can be used without limitation as long as it is inert to the polymerization reaction, and includes aliphatic hydrocarbons, alicyclic hydrocarbons, aromatic hydrocarbons, and the like.
Examples of the aliphatic hydrocarbon include n-pentane, n-hexane, n-heptane and n-octane.
Examples of the alicyclic hydrocarbon include cyclohexane and methylcyclohexane.
Aromatic hydrocarbons include benzene, toluene and xylene.
The hydrophobic solvent preferably has a boiling point lower than that of water and forms an azeotrope with water. Examples of such a hydrophobic solvent include n-pentane, n-hexane, n-heptane, cyclohexane and benzene.
The used amount (% by weight) of the hydrophobic solvent (W) is preferably 100 to 2000, more preferably 300 to 1700, and particularly preferably 500 to 1500 based on the weight of the crosslinked vinyl polymer and, if necessary, the polar solvent. . If it is less than this range, the resin beads tend to coalesce during the polymerization reaction, so the particle size of the resin beads tends to be too large. On the other hand, if this range is exceeded, economic efficiency (reactor size) Moreover, economy based on energy consumption etc.) is reduced.

The hydrophobic solvent (W) and / or the hydrophilic solvent (U) preferably contains a surfactant (S) as necessary.
As the surfactant (S), known surfactants (for example, surfactants described in JP-A Nos. 2004-1224059 and 2002-284803) can be used, and anionic surfactants can be used. Agent (S1), cationic surfactant (S2), amphoteric surfactant (S3) and nonionic surfactant (S4) are included.
Examples of the anionic surfactant (S1) include octyl alcohol sulfate sodium salt, lauryl alcohol ethylene oxide 2-mol adduct sulfate sodium salt, and dodecylbenzenesulfonic acid sodium salt.
Examples of the cationic surfactant (S2) include lauryltrimethylammonium chloride, didecyldimethylammonium chloride and dioctyldimethylammonium bromide.
Examples of the amphoteric surfactant (S3) include sodium stearylaminopropionate, sodium laurylaminoacetate, and stearyldimethylaminoacetate betaine.
Nonionic surfactants (S4) include sorbitan monostearate, sorbitan distearate, sorbitan monooleate, sorbic acid sorbitan ester ethylene oxide adduct, lauryl alcohol polyoxyethylene ether, polyoxyethylene nonylphenyl ether and N, N-dihydroxyethyl lauryl amide etc. are mentioned.
Of these, nonionic surfactant (S4) or a combination of nonionic surfactant (S4) and another ionic surfactant is preferred.
When the surfactant (S) is used, the amount (% by weight) used is preferably from 0.001 to 10, more preferably from 0.005 to 5, particularly preferably based on the weight of the hydrophobic solvent (W). Preferably it is 0.01-3. Within this range, the particle size of the resin beads can be adjusted more easily.

In the polymerization reaction, a polymerization initiator may be used as necessary. The polymerization initiator is not particularly limited, and includes (1) a peroxide polymerization initiator and (2) an azo polymerization initiator. Further, (1) a peroxide polymerization initiator and a reducing agent may be combined to form (3) a redox polymerization initiator. Furthermore, you may use 2 or more types together from (1)-(3).
(1) Examples of the peroxide polymerization initiator include benzoyl peroxide, di-t-butyl peroxide, hydrogen peroxide, and ammonium persulfate.
(2) As the azo polymerization initiator, 2,2′-azobisisobutyronitrile, 1,1′-azobiscyclohexane 1-carbonitrile, 2,2′-azobis [2-methyl-N- (2 -Hydroxyethyl) propionamide and azobisamidinopropane hydrochloride] and the like.
(3) Examples of the redox polymerization initiator include a combination of a peroxide such as hydrogen peroxide or hydroperoxide and a reducing agent (divalent iron salt, sodium bisulfite, alcohol, polyamine, etc.).

  When a polymerization initiator is used, the amount used (% by weight) is 0.01 to based on the weight of the mixture of the vinyl monomer containing a carbamoyl group and / or polyoxyalkylene group and the crosslinking agent (c1). 5 is preferable, more preferably 0.02 to 4.5, and particularly preferably 0.03 to 4. Within this range, the particle size of the resin beads can be adjusted more easily.

  Dispersing devices that can be used when dispersing a mixture of a vinyl monomer containing a carbamoyl group and / or a polyoxyalkylene group and a crosslinking agent (c1) in a hydrophobic solvent include homomixers, propeller stirrers, dispersers, anchors. Mold mixer, paddle mixer, ultra mixer, pipeline mixer and the like.

The temperature (° C.) of the polymerization reaction is not particularly limited, but is preferably 10 to 95, more preferably 20 to 90, and particularly preferably 30 to 80. Within this range, the particle size of the resin beads can be adjusted more easily.
The polymerization reaction time (hour) is not particularly limited, but is preferably 0.1 to 50, more preferably 1 to 30, and particularly preferably 2 to 20.

  The time (unit: hour) during which the mixture of the vinyl monomer and the crosslinking agent (c1) is dropped is preferably 0.5 to 20, more preferably 0.7 to 15, and particularly preferably 1 to 10. Within this range, the particle size of the resin beads can be adjusted more easily.

  A step of obtaining a crosslinked vinyl polymer (A1) by dispersing a mixture of a vinyl monomer containing a carbamoyl group and / or a polyoxyalkylene group and a crosslinking agent (c1) in a hydrophobic solvent (W) and then performing a polymerization reaction ( 5) For the method (2) including the hydrophobic solvent, the hydrophilic solvent (U) to be used if necessary, the surfactant (S), the polymerization initiator, the temperature and time of the polymerization reaction, and preferred ranges thereof, This is the same as in the case of method (1).

  A hydrophobic solvent (W) is dispersed dropwise into a mixture of a vinyl monomer containing a carbamoyl group and / or a polyoxyalkylene group and a crosslinking agent (c1), phase-inverted and then subjected to a polymerization reaction to form a crosslinked vinyl polymer ( For the method (3) including the step (6) of obtaining A1), a hydrophobic solvent, a hydrophilic solvent (U) to be used if necessary, a surfactant (S), a polymerization initiator, a polymerization reaction temperature and time, and these The preferred range is the same as in the case of the method (1).

Next, a step of obtaining a crosslinked vinyl polymer (A1) by mixing a vinyl monomer containing a carbamoyl group and / or a polyoxyalkylene group and a crosslinking agent (c1) and polymerizing using a reaction solvent if necessary ( The method (4) including 7) will be described in more detail.
As a reaction solvent to be used if necessary, a hydrophilic solvent (U) or the like can be used.
In the polymerization reaction, the same polymerization initiator as in the method (1) may be used.
The temperature and time of the polymerization reaction and preferred ranges thereof are the same as in the method (1).

The crosslinked alkylene oxide polymer (A2) is not limited as long as it is an alkylene oxide polymer having a crosslinked structure, but preferably has a crosslinked structure composed of an alkylene oxide polymer and a crosslinking agent (c2).
As the alkylene oxide polymer, known alkylene oxide polymers can be used.
Although there is no restriction | limiting in particular as an oxyalkylene group (OA group) in an alkylene oxide polymer, From a viewpoint of peeling and collection | recovery of a cell, an oxyethylene group and an oxypropylene group are preferable, More preferably, it is an oxyethylene group.
The OA group can contain two or more oxyalkylene groups, and when it contains two or more oxyalkylene groups, it preferably contains an oxyethylene group.
In this case (when an oxyethylene group is included), the content (% by weight) of the oxyethylene group is preferably 10 to 99, more preferably 20 to 95, particularly preferably 40 to 90, based on the weight of the OA group. It is. Within this range, cell detachment / recovery is further facilitated.
The number average molecular weight of the alkylene oxide polymer is preferably from 200 to 200,000, more preferably from 300 to 100,000, particularly preferably from 400 to 50,000. Within this range, cell detachment / recovery is further facilitated.
The number average molecular weight of the alkylene oxide polymer is determined by gel permeation chromatography (GPC) using polyoxyethylene glycol as a standard substance.

  As the alkylene oxide polymer, an alkylene oxide polymer described in "Alkylene oxide polymer" (issued on November 20, 1990, manufactured by Kaibundo Publishing Co., Ltd.) can be used. (1) Polyalkylene glycol, (2 ) Alcohol alkylene oxide adduct, (3) phenol alkylene oxide adduct, and (4) carboxylic acid alkylene oxide adduct.

(1) As polyalkylene glycol, diethylene glycol, triethylene glycol, dipropylene glycol, polyoxyethylene glycol (for example, number average molecular weight 2000), polyoxypropylene glycol (for example, number average molecular weight 1000), polyoxyethylene polyoxypropylene glycol (Eg oxyethylene / oxypropylene: weight ratio 90/10, block, number average molecular weight 4000) and polyoxyethylene polyoxybutylene glycol (eg oxyethylene / oxybutylene: weight ratio 50/50, random, number average molecular weight 2000) Etc.

(2) As the alcohol alkylene oxide adduct, methanol tetraethylene oxide adduct, methanol tripropylene oxide adduct, ethanol polyethylene oxide polypropylene oxide adduct (for example, oxyethylene / oxypropylene: weight ratio 90/10, number average molecular weight 4000, Random) and glycerin polyethylene oxide adducts (for example, number average molecular weight 2000).

(3) As the phenol alkylene oxide adduct, phenol polyethylene oxide adduct (for example, number average molecular weight 3000), phenol polyethylene oxide polypropylene oxide adduct (for example, oxyethylene / oxypropylene: weight ratio 80/20, number average molecular weight 4000, Random) and bisphenol polyethylene polypropylene oxide oxide adducts (for example, oxyethylene / oxypropylene: weight ratio 90/10, number average molecular weight 6000, random) and the like.

(4) Examples of the carboxylic acid alkylene oxide adduct include lauric acid polyethylene oxide adduct (for example, number average molecular weight 8000), stearic acid polypropylene oxide adduct (for example, number average molecular weight 3000), oleic acid polyethylene oxide polypropylene oxide adduct (for example, Oxyethylene / oxypropylene: weight ratio 85/15, number average molecular weight 4000, random).
Of the alkylene oxide polymers, polyalkylene glycol is preferable from the viewpoint of ease of handling, and more preferably polyoxyethylene polyoxypropylene glycol.

The cross-linking agent (c2) is not particularly limited as long as it is a compound having at least two functional groups (isocyanato, epoxy, etc.) capable of reacting with the hydroxyl group of the alkylene oxide polymer. -284803 or JP-A-2004-263147) can be used. Of these, the crosslinking agent (c12) and the crosslinking agent (c13) are preferable, and trimethylolpropane triglycidyl ether, pentaerythritol tetraglycidyl ether, HDI isocyanurate, HDI burette, and IPDI isocyanurate are more preferable.
When the crosslinking agent is included as a constituent unit, the content (% by weight) of the crosslinking agent (c2) unit is preferably 0.001 to 10, more preferably 0.005 to 7, particularly preferably 0.01 to 5. is there.

The crosslinked alkylene oxide polymer (A2) can be produced by the following methods (5) to (8).
(5) A method comprising a step (2) of obtaining a crosslinked alkylene oxide polymer (A2) by dispersing a mixture of an alkylene oxide polymer and a crosslinking agent (c2) in a hydrophobic solvent (W) and performing a crosslinking reaction.
(6) A method comprising a step (8) of dispersing an alkylene oxide polymer in a hydrophobic solvent (W) and then adding a crosslinking agent (c2) to cause a crosslinking reaction to obtain a crosslinked alkylene oxide polymer (A2).
(7) A method comprising the step (9) of obtaining a crosslinked alkylene oxide polymer (A2) by dropping a mixture of an alkylene oxide polymer and a crosslinking agent (c2) into the hydrophobic solvent (W) and simultaneously carrying out a crosslinking reaction while dispersing the mixture. .
(8) A method comprising the step (10) of mixing the alkylene oxide polymer and the crosslinking agent (c2) and directly subjecting to a crosslinking reaction to obtain a crosslinked alkylene oxide polymer (A2).

The method (5) including the step (2) will be described.
The mixture of the alkylene oxide polymer and the cross-linking agent (c2) is preferably in a liquid state when dispersed (a solid product may be dispersed while being in a liquid state).
When the mixture of the alkylene oxide polymer and the cross-linking agent (c2) is solid, it can be made liquid by dissolving it in the hydrophilic solvent (U) and / or melting it by heating.
The liquid state preferably has a viscosity (mPa · s) of 1 to 100,000, more preferably 10 to 80,000, and particularly preferably 20 to 50,000. Within this range, the particle size of the resin beads can be adjusted more easily.
In the present invention, the viscosity is measured according to JIS K7117-1: 1999 “Plastic—Liquid, Emulsion or Dispersed Resin—A Method for Measuring Apparent Viscosity Using a Brookfield Rotational Viscometer”. It is measured using a B type viscometer manufactured by Keiki Seisakusho.

In the method (5), a catalyst (K) can be used if necessary.
The catalyst (K) is not particularly limited, but is an amine catalyst {diazabicyclooctane, diazabicyclooctane and 1,8-diazabicyclo [5,4,0] -undecene-7 etc.}, acid catalyst {phosphoric acid and p-toluenesulfonic acid and the like} and metal catalysts {dibutyltin dilaurate, zirconium octylate and the like}.
When the catalyst (K) is used, the amount (% by weight) of (K) is preferably 0.01 to 5, more preferably based on the weight of the mixture of the alkylene oxide polymer and the crosslinking agent (c2). It is 0.02-4.5, Most preferably, it is 0.03-4.

  Dispersing apparatus for dispersing a mixture of an alkylene oxide polymer and a crosslinking agent (c2) in a hydrophobic solvent (W), the kind of the hydrophobic solvent (W), the surfactant (s) to be used if necessary, and preferred ranges thereof Is the same as in the method (1) for producing a crosslinked vinyl polymer.

The temperature (° C.) of the crosslinking reaction is not particularly limited, but is preferably 10 to 80, more preferably 15 to 70, and particularly preferably 20 to 50. Within this range, the particle size of the resin beads can be adjusted more easily.
The crosslinking reaction time (time) is not particularly limited, but is preferably 0.01 to 20, more preferably 0.1 to 15, particularly preferably 0.2 to 10.

Next, a method (6) comprising a step (8) of dispersing an alkylene oxide polymer in a hydrophobic solvent (W) and then adding a crosslinking agent (c2) to cause a crosslinking reaction to obtain a crosslinked alkylene oxide polymer (A2) (6). ).
The alkylene oxide polymer is preferably in a liquid state when dispersed (a solid product may be dispersed while being in a liquid state).
When the alkylene oxide polymer is solid, it can be made liquid by dissolving it in the hydrophilic solvent (U) and / or melting it by heating.
The catalyst (K) can be used if necessary in the same manner as in the method (5).
The dispersing apparatus for dispersing the alkylene oxide polymer in the hydrophobic solvent (W), the type of the hydrophobic solvent (W), the surfactant (s) to be used if necessary, and preferred ranges thereof are the method for producing a crosslinked vinyl polymer ( The same as in the case of 1). The temperature and time for the crosslinking reaction are the same as in method (5).

Next, a method comprising the step (9) of obtaining a crosslinked alkylene oxide polymer (A2) by dropping a mixture of the alkylene oxide polymer and the crosslinking agent (c2) into the hydrophobic solvent (W) and simultaneously performing a crosslinking reaction while dispersing the mixture. (7) will be described.
The mixture of the alkylene oxide polymer and the cross-linking agent (c2) is preferably in a liquid state when dispersed (a solid product may be dispersed while being in a liquid state).
When the mixture of the alkylene oxide polymer and the cross-linking agent (c2) is solid, it can be made liquid by dissolving it in the hydrophilic solvent (U) and / or melting it by heating.
The catalyst (K) can be used if necessary in the same manner as in the method (5).
Dispersing apparatus for dispersing a mixture of alkylene oxide polymer and crosslinking agent (c2) in hydrophobic solvent (W), kind of hydrophobic solvent (W), surfactant (s) used if necessary, and preferred ranges thereof are This is the same as in the method (1) for producing a crosslinked vinyl polymer. The temperature and time for the crosslinking reaction are the same as in method (5).

Next, the method (8) including the step (10) of obtaining the crosslinked alkylene oxide polymer (A2) by mixing the alkylene oxide polymer and the crosslinking agent (c2) and causing the crosslinking reaction as it is will be described.
The mixture of the alkylene oxide polymer and the crosslinking agent (c2) is preferably liquid.
When the mixture of the alkylene oxide polymer and the crosslinking agent (c2) is in a solid state, it can be made liquid by dissolving it in a reaction solvent and / or melting it by heating, if necessary.
The reaction solvent used as necessary includes a hydrophilic solvent (U).
The catalyst (K) can be used if necessary in the same manner as in the method (5).
The temperature and time of the crosslinking reaction are the same as in method (5).

The crosslinked methylcellulose (A3) is not limited as long as it is a methylcellulose having a crosslinked structure, but preferably has a crosslinked structure composed of methylcellulose and a crosslinking agent (c3).
As methylcellulose, known methylcellulose {for example, “Polymer Experiments Vol. 8 Natural Polymer” (issued on August 15, 1984, manufactured by Kyoritsu Shuppan Co., Ltd .; for example, methylcellulose (methylation rate 38.6%) , Methylcellulose (methylation rate 29.2%), methylcellulose (methylation rate 41.9%), etc.} can be used.
The weight average molecular weight of methylcellulose is preferably 200 to 20000, more preferably 300 to 15000, and particularly preferably 400 to 10,000. Within this range, cell detachment / recovery is further facilitated.
The weight average molecular weight of methylcellulose is determined by gel permeation chromatography (GPC) using polyoxyethylene glycol as a standard substance {see, for example, Polymer Journal Vol. 4, page 293 (1982)}.
The methylation rate (number%) of methylcellulose (ratio of the number of methoxy groups after methylation to the hydroxyl groups before methylation) is preferably 10 to 60, more preferably 20 to 55, and particularly preferably 30 to 50. is there. Within this range, cell detachment / recovery is further facilitated.
The methylation rate is based on the 7.3 pyridine-acetyl chloride method of JIS K0070: 1992 “Testing method for acid value, saponification value, ester value, iodine value, hydroxyl value and unsaponified product of chemical products”. Then, the hydroxyl value before and after methylation is measured, and is obtained from the following formula.

As the crosslinking agent (c3), the same crosslinking agent as the crosslinking agent (c2) can be used, and the preferred range is also the same.
When the crosslinking agent is included as a structural unit, the content of the crosslinking agent (c3) unit is the same as that in the crosslinked alkylene oxide polymer (A2).

  The crosslinked methylcellulose (A3) can be produced in the same manner as the crosslinked alkylene oxide polymer (A2) except that methylcellulose is used in place of the alkylene oxide polymer. Among these production methods, a production method including a step (3) of obtaining a crosslinked methylcellulose (A3) by dispersing a mixture of methylcellulose and a crosslinking agent (c3) in a hydrophobic solvent (W) and performing a crosslinking reaction is preferable.

The crosslinked polyvinyl alcohol partially saponified product (A4) is not limited as long as it is a polyvinyl alcohol partially saponified product having a crosslinked structure, but preferably has a crosslinked structure composed of the polyvinyl alcohol partially saponified product and the crosslinking material (c4).
As the partially saponified polyvinyl alcohol, a known partially saponified polyvinyl alcohol can be used.
The weight average molecular weight of the partially saponified polyvinyl alcohol is preferably 200 to 20000, more preferably 300 to 15000, and particularly preferably 400 to 10,000. Within this range, cell detachment / recovery is further facilitated.
The weight average molecular weight of the partially saponified polyvinyl alcohol is determined by gel permeation chromatography (GPC) using polyoxyethylene glycol as a standard substance. 10 to 98, more preferably 30 to 97, and particularly preferably 50 to 95. Within this range, cell detachment / recovery is further facilitated.
The saponification value is based on the 4.1 neutralization titration method of JIS K0070: 1992 “Testing method for acid value, saponification value, ester value, iodine value, hydroxyl value and unsaponified product of chemical products”. Measured.
Examples of the crosslinking agent (c4) include the same crosslinking agents as the crosslinking agent (c2), and the preferred range is also the same.
When the crosslinking agent is included as a structural unit, the content of the crosslinking agent (c4) unit is the same as that in the crosslinked polyalkylene oxide polymer (A2).

  The crosslinked polyvinyl alcohol partially saponified product (A4) can be produced in the same manner as the crosslinked alkylene oxide polymer (A2) except that a polyvinyl alcohol partially saponified product is used instead of the alkylene oxide polymer. Among these production methods, a step of obtaining a crosslinked polyvinyl alcohol partially saponified product (A4) by dispersing a mixture of a partially saponified polyvinyl alcohol and a crosslinking agent (c4) in a hydrophobic solvent (W) and causing a crosslinking reaction (4) ) Is preferred.

  Among these cross-linked polymers (A1) to (A4), a cross-link comprising a vinyl monomer containing a carbamoyl group and / or a polyoxyalkylene group as an essential constituent monomer from the viewpoint of ease of cell peeling and recovery. Vinyl polymer (A1) is preferred.

  When the reverse phase suspension polymerization method is applied as a method for producing the crosslinked polymer (A), the temperature-sensitive swellable resin beads of the present invention can be obtained as it is by producing the crosslinked polymer (A). On the other hand, when a bulk polymerization method or a solution polymerization method is applied as a production method of the crosslinked polymer (A), it is necessary to pulverize the obtained crosslinked polymer (A).

In any of the reverse phase suspension polymerization method, bulk polymerization method, and solution polymerization method, the size of the temperature-sensitive swellable resin beads of the present invention {the volume average of the “deposited resin beads after leaching into physiological saline” The particle diameter (μm: ra)} is preferably 20 to 3000, more preferably 40 to 2000, and particularly preferably 50 to 1200 from the viewpoint of cell culture efficiency.
In addition, in the “deposited resin beads after the resin beads of the present invention are soaked in physiological saline” (hereinafter referred to as the “deposited resin beads”), 1 part by weight of the resin beads is added to physiological saline (0.9% by weight). It is adjusted by immersing in 10 parts by weight for 30 minutes at a temperature 10 ° C. higher than the inflection point of the crosslinked polymer (A).
And a volume average particle diameter is based on JISZ8825-1: 2001, a laser-type particle size distribution measuring apparatus, for example, LA-920 by Horiba, Ltd. (dispersion medium: ion-exchange water, measurement temperature: crosslinked polymer (A)). The temperature is 10 ° C. higher than the inflection point.
When the bulk polymerization method or the solution polymerization method is applied, examples of the crusher for crushing or crushing the crosslinked polymer (A) include a colloid mill, a hammer mill, and a ball mill.
The temperature (° C.) when crushing or pulverizing is preferably −20 to 150, more preferably −10 to 130, and particularly preferably −5 to 80. Within this range, resin beads can be prepared more easily (can be more easily crushed or crushed).
After crushing or crushing, classification can be performed as necessary. As classification, a method of sieving with a classifier (dry vibration sieve, wet vibration sieve, wind classifier, etc.) can be applied.

  When a hydrophobic solvent (W), a hydrophilic solvent (U) and / or a surfactant (S) are used in obtaining the crosslinked polymer (A), these may adversely affect cell proliferation, It is preferable to remove these. As a method for removing the hydrophobic solvent (W), the hydrophilic solvent (U) and / or the surfactant (S), resin beads are obtained by a solid-liquid separation method (such as a centrifuge, a spakura filter and / or a filter press). A method of washing with water after the separation is applicable.

The temperature-sensitive swellable resin beads for cell culture of the present invention may contain fine particles (b).
The fine particles (b) are not particularly limited as long as they are fine particles. The fine particles (b) may be uniform fine particles in which the entire fine particles are uniformly formed, or may have voids in a part of the fine particles.
Further, when a part of the fine particles have voids, the fine particles may be porous fine particles in which the voids are distributed almost uniformly, hollow fine particles including a hollow part at the center of the fine particles, etc. preferable.
The outer shape of the fine particles (b) may be spherical, needle-like, flat (elliptical), flaky, irregularly crushed, or fibrous, but from the viewpoint of the surface smoothness of the resin beads, An (elliptical) shape and a flake shape are preferable, a spherical shape and a flat (elliptical) shape are more preferable, and a spherical shape is particularly preferable.

  The size of the fine particles (b) is such that the ratio (rb / ra) between the volume average particle size (rb) of the fine particles (b) and the volume average particle size (ra) of the precipitated resin beads is 0.0015 to 0.00. 3 is preferable, more preferably 0.01 to 0.25, and particularly preferably 0.015 to 0.2. Within this range, the average surface area of the leaching resin beads is further improved (increased).

Although it does not specifically limit as a material of microparticles | fine-particles (b), An organic substance, an inorganic substance, etc. are contained.
As the organic substance, known resins (for example, JP 2002-284881 A and WO 99/43758 pamphlet) can be used, and thermoplastic resins, thermosetting resins, and the like are used.
Examples of the thermosetting resin include a crosslinked vinyl resin, a crosslinked urethane resin, a crosslinked epoxy resin, a crosslinked ester resin, a crosslinked phenol resin, and a crosslinked urea resin.
Examples of the thermoplastic resin include vinyl resin, amide resin, olefin resin, and carbonate resin.
These resins may be used in combination of two or more.
Examples of inorganic substances include glass, shirasu, alumina, and carbon.

  Among these, from the viewpoint of the apparent specific gravity and average surface area of the leaching resin beads, a cross-linked vinyl resin, a vinyl resin, an olefin resin, and a glass are preferable, a cross-linked vinyl resin, an olefin resin, and a glass are particularly preferable, and an olefin resin is particularly preferable. It is.

The fine particles (b) can be produced by a known method (for example, a production method described in JP-A-2002-284881 or WO99 / 43758 pamphlet). The fine particles (b) can be easily obtained from the market. For example, the following products can be preferably exemplified. The values in parentheses are the apparent specific gravity and the volume average particle size.
The apparent specific gravity is measured according to the 8.2.1 Le Chatelier specific gravity bottle method of K0061: 2001 “Method for measuring density and specific gravity of chemical products”.

<Uniform organic fine particles>
“Flow Bead CL Series” (0.94, 1 to 40 μm) manufactured by Sumitomo Seika Co., Ltd., “Flowsen UF Series” (0.94, 25 μm); “Miperon XM221U” (0.94) manufactured by Mitsui Chemicals, Inc. 25 μm), “Miperon XM220” (0.94, 30 μm); “Torefill E-500” (0.97, 3 μm) manufactured by Toray Dow Corning Silicone Co., Ltd.

<Hollow organic fine particles>
“Matsumoto Microsphere MFL80GCA” (0.20, 20 μm) manufactured by Matsumoto Yushi Seiyaku Co., Ltd .; “Expansel 551DE40d42” (0.04, 40 μm) manufactured by Expansel Co., Ltd. “APM” manufactured by Sakai Kogyo Co., Ltd. “PHENOSE BJO-0840” (0.30, 70 μ); “SX866 (a)” (0.76, 0.3 μm) manufactured by JSR Corporation

<Hollow inorganic fine particles>
“Scotch Light Glass Bubbles S60HS” (0.60, 27 μm), Scotch Light Glass Bubbles K46 (0.46, 40 μm) manufactured by Sumitomo 3M; “Ceramics BALOON AR-190” manufactured by Sakai Kogyo Co., Ltd. (0.60, 43μm)

<Porous inorganic fine particles>
“Sunsphere H-31” (2.2, 3 μm), Asahi Glass Co., Ltd., “Sunsphere H-33” (2.2, 3 μm), “Escalon Series” (2.5) 1.2-3.9 μm), “SCP series” (2.5, 1.4-3.9 μm), “Calcy F series” (2.5, 3.5-6.0 μm)
These fine particles may be used in combination of two or more.

A method for incorporating the fine particles (b) into the resin beads will be described.
As a method for incorporating fine particles (b) into resin beads comprising a crosslinked vinyl polymer (A1) having a vinyl monomer containing a carbamoyl group and / or a polyoxyalkylene group as an essential constituent monomer, a carbamoyl group and / or A method of mixing fine particles (b) with a vinyl monomer containing a polyoxyalkylene group {if necessary, a mixture of this vinyl monomer and a crosslinking agent (c1)} can be applied.
As a method for incorporating fine particles (b) into the resin beads comprising the crosslinked alkylene oxide polymer (A2), fine particles (if necessary, a mixture of this polymer and the crosslinking agent (c2)) may be used. A method of mixing b) can be applied.
In addition, as a method for containing fine particles (b) in resin beads made of crosslinked methylcellulose (A3), or a method for containing fine particles (b) in resin beads made of partially saponified polyvinyl alcohol (A4), cross-linking This is the same as the method in which the fine particles (b) are contained in the resin beads made of the alkylene oxide polymer (A2).

  When the fine particles (b) are contained in the resin beads, the content (% by weight) of the fine particles (b) is preferably 0.01 to 50 based on the weight of the crosslinked polymer (A), more preferably 0.8. It is 05-45, Most preferably, it is 0.10-40. Within this range, adjustment of the apparent specific gravity, average surface area, etc. of the leaching resin beads is further facilitated.

  The apparent specific gravity (d) of the precipitated resin beads after the temperature-sensitive swellable resin beads for cell culture of the present invention are immersed in physiological saline is preferably 1.000 to 1.050, more preferably 1.005. To 1.045, particularly preferably 1.010 to 1.040. Within this range, the cells can be cultured better without the resin beads being biased in the medium. Methods for adjusting the apparent specific gravity of the infiltrated resin beads within this range include a method of adjusting using fine particles, a method of microencapsulating the resin beads themselves (such as hollow beads), and a method of swelling the resin beads (hydrophilic And a method of adjusting the monomer composition such as increasing the amount of a strong monomer used).

Here, a method for measuring the apparent specific gravity (d) of the impregnated resin beads will be described.
10 g of resin beads are immersed in 100 g of physiological saline (0.9% by weight) at a temperature 10 ° C. higher than the inflection point of the crosslinked polymer (A) for 30 minutes to obtain a precipitated resin bead. Next, each of 0 to 15% by weight of saline (16 types whose temperature is 10 ° C. higher than the inflection point of the cross-linked polymer (A) and whose concentration is changed by 1% by weight) is introduced to cross-link. It is allowed to stand for 30 minutes at a temperature 10 ° C. higher than the inflection point of the polymer (A), and it is visually determined whether or not the precipitated resin beads are settled.
The arithmetic average value (dav) of the specific gravity of the saline solution on which the precipitated resin beads settled and the specific gravity of the saline solution having a concentration of 1% by weight is obtained, and the same operation is repeated three times to obtain the arithmetic average of three times. The arithmetic average value of the values (dav) is assumed to be the apparent specific gravity (d). In addition, the specific gravity of each concentration of saline is measured in accordance with 7.1 levitation method of JIS K0061: 2001 “Method for measuring density and specific gravity of chemical products”.

  The water content (% by weight) of the resin beads of the present invention is preferably 1 or less, more preferably 0.5 or less, and particularly preferably 0.1 or less, based on the weight of the resin beads. The water content (% by weight) is calculated from the weight loss (g) before and after drying 1 g of resin beads in a 105 ° C. normal air dryer for 1 hour.

Examples of the shape of the temperature-sensitive swellable resin beads for cell culture of the present invention include a spindle shape, a spherical shape, a plate shape, a rectangular parallelepiped shape, a needle shape, and the like. Spherical shape and plate shape are preferable, spindle shape and spherical shape are more preferable, and spherical shape is particularly preferable.
The temperature-sensitive swellable resin beads for cell culture of the present invention may have ribs or the like for the purpose of increasing the surface area. From the viewpoint of ease of cell recovery, it is preferable not to have ribs.

The temperature-sensitive swellable resin beads for cell culture of the present invention preferably contain a polypeptide (P) having at least one cell adhesion minimal amino acid sequence (X) in one molecule. When the polypeptide (P) is contained, the cells easily adhere to each other, and more efficient cell culture can be realized.
Here, the “cell-adhesive minimum amino acid sequence” means a minimum amino acid sequence that has a property of being easily recognized by a cell integrin receptor and allowing cells to adhere to a substrate.
The number (pieces) of the cell adhesion minimal amino acid sequence (X) in the polypeptide (P) is preferably 1 to 50, more preferably 3 to 1 molecule (P) from the viewpoint of cell adhesion. 30, particularly preferably 4-20.

  Any cell adhesion minimal amino acid sequence (X) may be used as long as it functions as an adhesion signal. For example, “Pathophysiology” published by Nagai Publishing Co., Ltd., Vol. 9, no. 7, 1990, page 527, etc. can be used. Of these, the Arg Gly Asp sequence, Leu Asp Val sequence, Leu Arg Glu sequence, His Ala Val sequence, and SEQ ID NOs: (1) to (8) are represented from the viewpoint that there are many types of cells that are likely to adhere. More preferred are Arg Gly Asp sequence, His Ala Val sequence and the sequence represented by SEQ ID NO: (7), and Arg Gly Asp sequence is particularly preferred.

The polypeptide (P) preferably has an auxiliary amino acid sequence (Y) in addition to the cell adhesion minimal amino acid sequence (X) from the viewpoint of improving the thermal stability of (P).
As the auxiliary amino acid sequence (Y), an amino acid sequence other than the minimum amino acid sequence (X) can be used, and a sequence having Gly and / or Ala is preferable from the viewpoint of improving the heat resistance of the polypeptide (P).
The auxiliary amino acid sequence (Y) includes (Gly Ala) a sequence, (Gly Ala Gly Ala Gly Ser) b sequence, (Gly Ala Gly Ala Gly Tyr) c sequence, (Gly Ala Gly Val Gly Tyr) d sequence, ( Gly Ala Gly Tyr Gly Val) e sequence, {Asp Gly Gly (Ala) f Gly Gly Ala} g sequence, (Gly Val Pro Gly Val) h sequence, (Gly) i sequence, (Ala) j sequence, (Gly Gly Ala) k sequence, (Gly Val Gly Val Pro) m sequence, (Gly Pro Pro) n sequence, (Gly Ala Gln Gly Pro Ala Gly Pro Gly) o sequence, (Gly Ala Pro Gly Ala Pro Gly Ser Gln Gly Ala Pro Gly Leu Gln) p sequence and / or (Gly Ala Pro Gly Thr Pro Gly Pro Gln Gly Leu Pro Gly Ser Pro) sequence having q sequence and the like are included. Among these, (Gly Ala) a sequence, (Gly Ala Gly Ala Gly Ser) b sequence, (Gly Ala Gly Ala Gly Tyr) c sequence, (Gly Ala Gly Val Gly Tyr) d sequence, (Gly Ala Gly Tyr Gly Val) e, {Asp Gly Gly (Ala) f Gly Gly Ala} g sequence, (Gly Val Pro Gly Val) h sequence, (Gly Val Gly Val Pro) m sequence and / or (Gly Pro Pro) n sequence Those having a (Gly Ala Gly Ala Gly Ser) b sequence, (Gly Val Pro Gly Val) h sequence, (Gly Val Gly Val Pro) m sequence and / or (Gly Pro Pro) n sequence are preferred. Particularly preferred are those having the (Gly Ala Gly Ala Gly Ser) b sequence.
A is an integer of 5 to 100, b, c, d, and e are integers of 2 to 33, f is an integer of 1 to 194, and g is rounded down to the nearest decimal point of {1} to {200 / (6 + f)}. , H is an integer of 2 to 40, i and j are integers of 10 to 200, k is an integer of 3 to 66, m is an integer of 2 to 40, n is an integer of 3 to 66, o is 1 to 22 , P and q are integers of 1-13.

The auxiliary amino acid sequence (Y) preferably contains glycine (Gly) and / or alanine (Ala). When glycine (Gly) and alanine (Ala) are included, the total content (%) thereof is preferably 10 to 100, more preferably 20 to 95, based on the total number of amino acids in the auxiliary amino acid sequence (Y). Especially preferably, it is 30-90, Most preferably, it is 40-85. Within this range, the heat resistance is further improved.
When both glycine (Gly) and alanine (Ala) are included, the content ratio (Gly / Ala) is preferably 0.03 to 40, more preferably 0.08 to 13, and particularly preferably 0.2. ~ 5. Within this range, the heat resistance is further improved.

  The content of the auxiliary amino acid sequence (Y) in the polypeptide (P) is preferably 2 to 51, more preferably 3 to 35, and particularly preferably 4 in 1 molecule of (P) from the viewpoint of improving heat resistance. ~ 20. The polypeptide (P) may also contain two or more auxiliary amino acid sequences (Y).

Examples of the auxiliary amino acid sequence having the (Gly Ala) a sequence include amino acid sequences represented by SEQ ID NOs: (9) to (11).
Examples of the auxiliary amino acid sequence having (Gly Ala Gly Ala Gly Ser) b sequence include amino acid sequences represented by SEQ ID NOs: (12) to (14).
Examples of the auxiliary amino acid sequence having (Gly Ala Gly Ala Gly Tyr) c sequence include amino acid sequences represented by SEQ ID NOs: (15) to (17).
Examples of the auxiliary amino acid sequence having the (Gly Ala Gly Val Gly Tyr) d sequence include amino acid sequences represented by SEQ ID NOs: (18) to (20).
Examples of the auxiliary amino acid sequence having the (Gly Ala Gly Tyr Gly Val) e sequence include amino acid sequences represented by SEQ ID NOs: (21) to (23).
Examples of the auxiliary amino acid sequence having the {Asp Gly Gly (Ala) f Gly Gly Ala} g sequence include amino acid sequences represented by SEQ ID NOs: (24) to (26).
Examples of the auxiliary amino acid sequence having (Gly Val Pro Gly Val) h sequence include amino acid sequences represented by SEQ ID NOs: (27) to (30).
Examples of the auxiliary amino acid sequence having the (Gly) i sequence include amino acid sequences represented by SEQ ID NOs: (31) to (33).
Examples of the auxiliary amino acid sequence having the (Ala) j sequence include the amino acid sequences represented by SEQ ID NOs: (34) to (36).
Examples of the auxiliary amino acid sequence having the (Gly Gly Ala) k sequence include amino acid sequences represented by SEQ ID NOs: (37) to (39).
Examples of the auxiliary amino acid sequence having (Gly Val Gly Val Pro) m sequence include amino acid sequences represented by SEQ ID NOs: (40) to (42).
Examples of the auxiliary amino acid sequence having (Gly Pro Pro) n sequence include amino acid sequences represented by SEQ ID NOs: (43) to (45).
Examples of the auxiliary amino acid sequence having (Gly Ala Gln Gly Pro Ala Gly Pro Gly) o sequence include amino acid sequences represented by SEQ ID NOs: (46) to (48).
(Gly Ala Pro Gly Ala Pro Gly Ser Gln Gly Ala Pro Gly Leu Gln) Examples of the auxiliary amino acid sequence having the p sequence include amino acid sequences represented by SEQ ID NOs: (49) to (51).
(Gly Ala Pro Gly Thr Pro Gly Pro Gln Gly Leu Pro Gly Ser Pro) Examples of the auxiliary amino acid sequence having the q sequence include amino acid sequences represented by SEQ ID NOs: (52) to (54).

  Among these amino acid sequences, SEQ ID NOs: (9), (10), (12), (13), (14), (15), (16), (18), (19), (21), ( 22), (24), (25), (26), (27), (28), (30), (31), (32), (34), (35), (37), (38) , (40), (41), (43), (44), (46), (47), (49), (50), (52) or (53) is preferred, Preferably, SEQ ID NOs: (10), (12), (13), (14), (16), (19), (22), (26), (27), (28), (29), (30 ), (32), (35), (38), (41), (44), (47), (50) or (53), particularly preferably SEQ ID NO: (12), the amino acid sequence represented by (13) or (30).

  The polypeptide (P) may contain a branched chain, may be partially crosslinked, or may contain a cyclic structure. However, the polypeptide (P) is preferably not cross-linked, more preferably a non-cross-linked linear structure, particularly preferably a non-cross-linked linear structure having no cyclic structure. The linear structure also includes a β structure (secondary structure in which linear peptides are bent and the portions are arranged in parallel and a hydrogen bond is formed therebetween).

The polypeptide (P) preferably has a structure in which the minimum amino acid sequence (X) and the auxiliary amino acid sequence (Y) are alternately chemically bonded from the viewpoint of cell adhesion and heat resistance. In this case, the number (units) of repeating units (XY) of the minimum amino acid sequence (X) and the auxiliary amino acid sequence (Y) is preferably 1 to 50, more preferably 2 from the viewpoint of cell adhesion. -40, particularly preferably 3-30, most preferably 4-20.
Further, the number of the minimum amino acid sequence (X) and the auxiliary amino acid sequence (Y) may be the same or different. When they are different from each other, it is preferable that one of the contained numbers is smaller than the other contained number {in this case, the auxiliary amino acid sequence (Y) is preferably small}. The content ratio (X / Y) between the minimum amino acid sequence (X) and the auxiliary amino acid sequence (Y) in the polypeptide (P) is preferably 0.66 to 1.5, more preferably 0.9 to 1. .4, particularly preferably 1-1.3.
In addition, other amino acids may be contained in the terminal portion of the cell adhesion polypeptide (P) (from the minimum amino acid sequence (X) or auxiliary amino acid sequence (Y) to the peptide end). When other amino acids are included, the content thereof is preferably 1 to 1000, more preferably 3 to 300, and particularly preferably 10 to 100 per cell adhesive polypeptide.

The number average molecular weight (Mn) of the polypeptide (P) is preferably 1,000 to 1,000,000, more preferably 2,000 to 700,000, particularly preferably 3,000 to 400,000, and most preferably. Is 4,000 to 200,000.
The number average molecular weight (Mn) of the polypeptide is known as a method of separating a measurement sample {polypeptide etc.} by SDS-PAGE (SDS polyacrylamide gel electrophoresis) and comparing the migration distance with a standard substance. (Hereinafter the same).

A part of preferable cell adhesion polypeptide (P) is illustrated below.
(1) When the minimum amino acid sequence (X) is Arg Gly Asp sequence (x1), it has 13 (x1) and (Gly Ala Gly Ala Gly Ser) ₉ sequence (13) (13) (y1) A polypeptide having an Mn of about 110,000 having a structure in which these are alternately chemically bonded (“Pronectin F”, Pronectin is a registered trademark of Sanyo Chemical Industries, Ltd. (Japan and the United States), manufactured by Sanyo Chemical Industries, Ltd. The same applies below));
A polypeptide having an Mn of about 20,000 (5) of (x1) and 5 of (GlyAlaGlyAlaGlySer) ₃ sequence (12) (y2) and having a structure in which these are alternately chemically bonded ( “Pronectin F2”);
(X1) 3 and (Gly Val Pro Gly Val) ₂ Gly Gly (Gly Ala Gly Ala Gly Ser) ₃ Sequence (30) (3) (3), which are alternately chemically bonded A polypeptide having a Mn of about 10,000 (“pronectin F3”) and the like.

(2) When the minimum amino acid sequence (X) is the Ile Lys Val Ala Val sequence (x2) The Arg Gly Asp sequence (x1) of pronectin F, pronectin F2 or pronectin F3 is converted into the Ile Lys Val Ala Val sequence (7) (x2) “Pronectin L”, “pronectin L2”, “pronectin L3”, etc.

(3) When the minimum amino acid sequence (X) is the Tyr Ile Gly Ser Arg sequence (x3) The Arg Gly Asp sequence (x1) of pronectin F, pronectin F2 or pronectin F3 was changed to the Tyr Ile Gly Ser Arg sequence (x3) “Pronectin Y”, “Pronectin Y2”, “Pronectin Y3” and the like.

  In addition to the polypeptides (1) to (3), RetroNectin (recombinant human fibronectin CH-296) manufactured by Takara Shuzo Co., Ltd. {Arg Gly Asp sequence (x1) and Leu Asp Val sequence as the minimum amino acid sequence (X) Containing Mn of about 60,000 polypeptide} and RGDS-Protein A {polypeptide having Mn of about 30,000 containing Arg Gly Asp sequence (x1) as the minimum amino acid sequence (X)} can be preferably used. This polypeptide is derived from nature and does not contain the auxiliary amino acid sequence (Y). Therefore, heat resistance etc. are inferior to said (1)-(3) (autoclave sterilization etc. cannot be performed). The amino acid sequences of these polypeptides are disclosed in JP-A-2-31498. }.

  The production method of the polypeptide (P) is not particularly limited, and can be produced in the same manner as a conventionally known method for synthesizing a peptide. For example, an organic synthesis method (solid phase synthesis method, liquid phase synthesis method, etc.) and It can be synthesized by a biochemical synthesis method [genetically modified microorganism (yeast, bacteria, Escherichia coli, etc.)] or the like. Regarding the organic synthesis method, for example, in the Biochemistry Society of Japan, “Sequel Biochemistry Experiment Course 2, Protein Chemistry (below)” pages 641-694 (May 20, 1987; published by Tokyo Chemical Co., Ltd.) The described methods are used. As for the biochemical synthesis method, for example, the method described in JP-T-3-502935 is used. A biochemical synthesis method using a genetically modified microorganism is preferable in that a high molecular weight polypeptide (P) can be easily synthesized, and a method using a genetically modified Escherichia coli is particularly preferable.

  When the polypeptide (P) is contained in the temperature-sensitive swellable resin beads for cell culture of the present invention, (P) may be contained on the surface of the resin beads, and (P) is a chemical bond (ion bond, It is bonded to the surface of the resin beads by hydrogen bonding and / or covalent bonding) and / or physical adsorption (adsorption by van der Waals force). Of these, chemical bonds are preferred, and covalent bonds are more preferred.

The reaction of covalently binding the polypeptide (P) to the temperature-sensitive swelling resin beads for cell culture can be performed by a known method. For example, the method described in “Basics and Experiments of Peptide Synthesis, October 5, 1997, published by Maruzen Co., Ltd.” and the like are specifically mentioned. .
(1) A polypeptide having a primary amino group or a secondary amino group is reacted with a resin bead containing no polypeptide (P) (hereinafter referred to as P-free resin beads) having a carboxyl group. In this case, the carboxyl group of the P-free resin beads was previously reacted with a carbodiimide compound to obtain acylisourea {R′—N═C (OCOR) —NH—R ′ (the portion where —OCOR is derived from the resin beads)}. Thereafter, P-containing resin beads and the polypeptide can be amide-bonded by adding a polypeptide having a primary amino group or a secondary amino group to the acylisourea.
Examples of the carbodiimide compound include N, N′-dicyclohexylcarbodiimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride.

(2) When reacting a polypeptide having a primary amino group or a secondary amino group with a P-free resin bead having a hydroxyl group, the hydroxyl group of the P-free resin bead is previously converted to carbonyl After reacting with an imidazole compound to obtain an imidazole derivative {R-Im, Im is an imidazoline ring, R is derived from a resin bead}, a polypeptide having a primary amino group or a secondary amino group is obtained from this imidazole derivative. In addition, the P-free resin beads and the polypeptide can be N-C bonded.
Examples of the carbonyldiimidazole compound include N, N′-carbonyldiimidazole.

  (3) When reacting a polypeptide having a hydroxyl group with a P-free resin bead having a carboxyl group, the carboxyl group of the P-free resin bead is reacted with a carbodiimide compound in advance, After being obtained, by adding a polypeptide having a hydroxyl group to the acylisourea, the P-free resin beads and the polypeptide can be ester-bonded.

Examples of the method for physically adsorbing, ion-bonding and / or hydrogen-bonding the polypeptide to the P-free resin beads include a method in which the polypeptide and the P-free resin beads are put into a solvent and mixed to prepare. It is done. Although there is no restriction | limiting in particular as a solvent, An inorganic salt, organic acid salt, an acid, and / or a base are 0.001 to 50 weight% (preferably 0.005 to 30 weight%, More preferably, 0.01 to 10 weight% ) Containing aqueous solution (for example, described in JP-A No. 2003-189848) can be used.
Among these solvents, an aqueous solution containing an inorganic salt, an acid and / or a base, and water are preferred, an aqueous solution containing an inorganic salt, an acid and / or a base, and ion-exchanged distilled water, particularly preferably. An aqueous solution containing an inorganic salt, acid and / or base.

When the polypeptide (P) is contained in the temperature-sensitive swelling resin beads for cell culture of the present invention, the content (μg / cm ² ) of the polypeptide (P) is determined by immersing the resin beads of the present invention in physiological saline. It is preferably 0.0001 to 100,000, more preferably 0.001 to 10,000, and particularly preferably 0.01 to 1,000 per 1 cm ² of the average surface area of the precipitated resin beads after analysis. Within this range, the efficiency of cell culture is further increased.

The content of the polypeptide (P) is determined as follows.
(1) The content (μg / g) of the polypeptide (P) per unit weight of the precipitated resin beads after the resin beads of the present invention are immersed in physiological saline, for example, an immunological measurement method (special Quantitative determination according to the description of Open 2004-049921 and the like.
Specifically, the reaction between the leaching resin beads and the antibody (hereinafter referred to as enzyme-labeled antibody 1) that binds to the antibody that binds to the polypeptide (P), and the amount of enzyme in the reacted enzyme-labeled antibody 1 is determined. By measuring, content of polypeptide (P) per unit weight is measured.

(2) Next, using a surface shape measuring microscope {a 3D shape measuring microscope using the confocal principle, for example, VK-9500 manufactured by Keyence Corporation}, the infiltrated resin beads are fixed to the slide glass with an adhesive or the like. Three-dimensional data of the surface shape is obtained for the upper surface (for example, 20 μm × 20 μm) of the upper part (hereinafter referred to as immobilized resin beads). Then, from this three-dimensional data, the pores (with a diameter of less than 1 μm) are uniformly excluded (corrected as a flat surface), and the partial surface area (A) of the infiltrated resin beads having ribs or the like is obtained. . Also, from the three-dimensional data of the surface shape, pores (with a diameter of less than 1 μm) and ribs (畝) are uniformly excluded (corrected as a flat surface), and the partial surface area of the infiltrated resin beads with a flat surface (B) is obtained. Further, three-dimensional data is measured in the same manner for nine of the leaching resin beads, and the partial surface area (A) of the dip resin beads and the partial surface area (B) of the dip resin beads are obtained. Then, after calculating the average partial surface area (HA) and the average partial surface area (HB) for 10 leaching resin beads, the formula {average surface area per unit weight (cm ² / g) = [4 × π × (ra / 2/10000) ² ] / [4/3 × π × (ra / 2/10000) ³ × d] × [(HA) / (HB)]}, the average surface area per unit weight of the leaching resin beads Is calculated.

(3) Then, the content (μg / g) of the polypeptide (P) per unit weight is divided by the average surface area (cm ² / g) per unit weight, so that the resin beads of the present invention are treated with physiological saline. The content (μg / cm ² ) per 1 cm ² of the average surface area of the precipitated resin beads after leaching is calculated.

  The average surface area of the leaching resin beads means the surface area of the surface of the resin beads where cells to be cultured can adhere, and the pores (with a diameter of less than 1 μm) where the cells do not enter are flat surfaces. However, in the case where ribs (ribs) or the like are provided for the purpose of increasing the surface area, the surface area of the ribs (ribs) is included in the surface area of the resin beads.

The temperature-sensitive swellable resin beads for cell culture of the present invention can be dried if necessary. Drying is performed under reduced pressure as necessary.
As the dryer, for example, a fluidized bed dryer, a vacuum dryer, and a circulating dryer are used.
In addition, when not drying, it is preferable to saturate the resin beads in physiological saline or a phosphate buffer aqueous solution.

Next, the process of culturing cells using temperature-sensitive swellable resin beads will be described.
The temperature-sensitive swellable resin beads for cell culture of the present invention may be sterilized as necessary before being used for cell culture. The sterilization method is not particularly limited, and examples thereof include radiation sterilization, ethylene oxide gas sterilization, autoclave sterilization, and dry heat sterilization.

Cells (CE) that can adhere to the resin beads of the present invention are not limited as long as they are cells, but if the resin beads of the present invention are used, the proliferated cells can be peeled and collected without being denatured, and thus useful substances such as pharmaceuticals Suitable are normal cells derived from mammals, established cell lines derived from mammals, and insect cells used for production and treatment.
Normal cells derived from mammals include cells involved in the skin (epithelial cells, fibroblasts, vascular endothelial cells, smooth muscle cells, etc.), cells involved in blood vessels (vascular endothelial cells, smooth muscle cells, fibroblasts, etc.) ), Cells involved in muscle (muscle cells, etc.), cells involved in fat (fat cells, etc.), cells involved in nerves (neurons, etc.), cells involved in liver (liver parenchymal cells, etc.), involved in pancreas Cells (such as pancreatic islet cells), cells involved in the kidney (renal epithelial cells, proximal tubular epithelial cells and mesangial cells, etc.), cells involved in the lungs and bronchi (epithelial cells, fibroblasts, vascular endothelial cells) And smooth muscle cells), cells involved in the eyes (photocells, corneal epithelial cells, corneal endothelial cells, etc.), cells involved in the prostate (epithelial cells, stromal cells, smooth muscle cells, etc.), cells involved in bone (Osteoblasts, bone cells and osteoclasts ), Cells involved in cartilage (such as chondroblasts and chondrocytes), cells involved in teeth (such as periodontal ligament cells and osteoblasts), cells involved in blood (such as leukocytes and erythrocytes), and stem cells {eg, Bone marrow undifferentiated mesenchymal stem cells, skeletal muscle stem cells, hematopoietic stem cells, neural stem cells, hepatic stem cells (oval cells, small hepatocytes, etc.), adipose tissue stem cells, embryonic stem (ES) cells, epidermal stem cells, intestinal stem cells, sperm stem cells Embryonic germ stem (EG) cells, pancreatic stem cells (pancreatic duct epithelial stem cells, etc.), leukocyte stem cells, lymphocyte stem cells, corneal stem cells, progenitor cells (adipose precursor cells, vascular endothelial precursor cells, cartilage precursor cells, lymphocyte cells) Progenitor cells, NK progenitor cells etc.)} and the like.

  Cell lines derived from mammals include 3T3 cells, A549 cells, AH130 cells, B95-8 cells, BHK cells, BOSC23 cells, BS-C-1 cells, C3H10T1 / 2 cells, C-6 cells, CHO cells, COS cells, CV-1 cells, F9 cells, FL cells, FL5-1 cells, FM3A cells, G-361 cells, GP + E-86 cells, GP + envAm12 cells, H4-II-E cells, HEK293 cells, HeLa cells, HEp− 2 cells, HL-60 cells, HTC cells, HUVEC cells, IMR-32 cells, IMR-90 cells, K562 cells, KB cells, L cells, L5178Y cells, L-929 cells, MA104 cells, MDBK cells, MDCK cells, MIA PaCa-2 cells, N18 cells, Namalwa cells, NG108-15 cells, NRK cells , OC10 cells, OTT6050 cells, P388 cells, PA12 cells, PA317 cells, PC-12 cells, PG13 cells, QGH cells, Raji cells, RPMI-1788 cells, SGE1 cells, Sp2 / O-Ag14 cells, ST2 cells, THP- 1 cell, U-937 cell, V79 cell, VERO cell, WI-38 cell, ψ2 cell, ψCRE cell, etc. {Cell culture technology (edited by the Japanese Society for Tissue Culture, published by Asakura Shoten Co., Ltd., 1999) }.

  Insect cells include silkworm cells (BmN cells, BoMo cells, etc.), mulberry cells, sakusan cells, synjusan cells, mushroom cells (Sf9 cells, Sf21 cells, etc.), stag beetle hitori cells, caterpillar cells, Drosophila cells, sentinic fly, Cells, human striped swallowtail cells, swallowtail butterfly cells, American cockroach cells, and nettle cottonweed cells (Tn-5 cells, HIGH FIVE cells, MG1 cells, etc.) {Insect Bio Factory (edited by Shigeru Kimura, published by Industrial Research Institute, Inc., 2000 Year).

As a medium (ME) used for the cell culture method using the resin beads of the present invention, a serum-free medium (Grace medium, IPL-41 medium, Schneider's medium, OPTIPRO _™ SFM medium, VP-SFM medium, CD293 medium, 293SFMII). Medium, CD-CHO medium, CHO-S-SFMII medium, FreeStyle _™ 293 medium, CD-CHO ATG _™ medium, and mixed media thereof, etc .; General medium (RPMI medium, MEM medium, BME medium, DME medium, αMEM) Medium, IMEM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, ASF103 medium, ASF104 medium, ASF301 medium, TC-100 medium, Sf-900II medium, Ex-cell405 medium, Express-Five medium , Drosoph ila medium and mixed medium thereof); and mixed medium thereof.
Among these, a serum-free medium is preferable from the viewpoint of preventing contamination with substances that may infect humans (such as viruses derived from serum), and more preferably, OPTIPRO _™ SFM medium, VP-SFM medium, CD293. Medium, 293SFMII medium, CD-CHO medium, CHO-S-SFMII medium, FreeStyle _™ 293 medium, CD-CHO ATG _™ medium and mixed media thereof, particularly preferably OPTIPRO _™ SFM medium, VP-SFM medium, CD293 medium, 293SFMII medium, FreeStyle _™ 293 medium, and mixed media thereof.

In addition, serum can be added to these media, but it is preferable not to add serum from the viewpoint of preventing contamination with substances (such as viruses derived from serum) that may infect humans. .
Serum includes human serum and animal serum (bovine serum, horse serum, goat serum, sheep serum, pig serum, rabbit serum, chicken serum, rat serum, mouse serum, etc.).
Of these, human serum, bovine serum, and horse serum are preferred when serum is added. The origin of animal serum includes adult-derived serum, offspring-derived serum, newborn-derived serum, fetal-derived serum, and the like. When adding serum, among these, serum derived from offspring, serum derived from newborn, and serum derived from fetus are more preferable, serum derived from newborn, and serum derived from fetus, particularly preferably serum derived from fetus. is there. When serum is added, the serum may be further subjected to inactivation treatment, antibody removal treatment, or the like.
When using serum, the amount of serum used (% by weight) is preferably 0.1 to 50, more preferably 0.3 to 30, and particularly preferably 1 to 20, based on the weight of the medium.

In the culture medium, a cell growth factor can be contained as necessary. By containing a cell growth factor, the cell growth rate can be increased and the cell activity can be increased.
Cell growth factors include fibroblast growth factor, transforming growth factor, epithelial growth factor, hepatocyte growth factor, platelet-derived growth factor, insulin-like growth factor, vascular endothelial growth factor, nerve growth factor, stem cell factor, leukemia Inhibitory factors, osteogenic factors, heparin-binding epithelial cell growth factor, neurotrophic factor, connective tissue growth factor, angiopoietin, cytokines, interleukins, adrenamodulin and natriuretic peptides are included. Of these, fibroblast growth factor, transforming growth factor, insulin-like growth factor and osteogenic factor are preferable, and more preferably fibroblasts, from the viewpoint that the range of applicable cells is wide and the healing period can be further shortened. Growth factors, transforming growth factors and insulin-like growth factors.
When a cell growth factor is used, the content (% by weight) of the cell growth factor varies depending on the type of cell growth factor, but is preferably 10 ₋₁₆ to 10 ₋₃ , more preferably 10 ₋ based on the weight of the medium. ₁₄ to 10 ₋₅ , particularly preferably 10 ₋₁₂ to 10 ₋₇ .

These media can further contain an antibacterial agent (amphotericin B, gentamicin, penicillin, streptomycin, etc.). When the antibacterial agent is contained, the content (% by weight) varies depending on the type of the antibacterial agent, but is preferably 10 ₋₄ to 10, more preferably 10 ₋₅ to 1, particularly preferably based on the weight of the medium. 10 _{−6 to} 0.1.

The cell seeding amount (cells / cm ² ) varies depending on the type of cells used, but is preferably 10 to 10 million, more preferably 1 to 1,000,000 per 1 cm ₂ of the average surface area of the leaching resin beads. Preferably it is 1000-300,000.
The amount of seeded cells can be measured by counting the number of cell nuclei by a cell nucleus counting method using crystal violet by Wezel.
Moreover, there is no restriction | limiting in particular as a density | concentration (cell / mL) of the cell disperse | distributed to a culture medium, However, 100-100 million per 1 mL of culture media are preferable, More preferably, it is 1000-10 million, Most preferably, it is 10,000-1 million. It is.
Further, the amount (g) of the resin beads of the present invention can be appropriately determined depending on the type of cells to be cultured, etc., but is preferably 0.0001 to 1, more preferably 0.001 to 0.8, per 1 mL of the medium. Most preferably, it is 0.005-0.5.

There are no particular limitations on the culture conditions, and carbon dioxide (CO ₂ ) concentration is 1 to 20% by volume, 5 to 45 ° C. for 1 hour to 100 days, and culture conditions are changed if the medium is changed every 1 to 10 days as necessary. Etc. are applicable. Preferable conditions are conditions in which the culture is performed if the medium is changed every 2 to 3 days, at a CO ₂ concentration of 3 to 10% by volume, 30 to 40 ° C., 1 to 20 days, and 2 to 3 days.

A process of peeling and recovering cells from the resin beads of the present invention after culturing will be described.
When cells are cultured using the resin beads of the present invention, the method of cooling the periphery of the resin beads (the method of moving the resin beads together with the medium to a low temperature environment, the method of replacing the medium with a low temperature medium, etc.) Cultured cells can be easily detached and collected.
The temperature (° C.) for detaching / collecting cells is not limited as long as it is lower than the temperature corresponding to the inflection point in the temperature-water absorption curve, but efficiently detaches / collects without denaturing the grown cells. In view of the above, the temperature is preferably 5 to 30 lower than the culture temperature, more preferably 10 to 27, and particularly preferably 15 to 25.
In addition, known peeling / recovery methods {chelating agents (such as EDTA) and / or non-animal derived proteolytic enzymes {plant-derived proteolytic enzymes (such as papain)}; chelating agents (such as EDTA) and / or gene recombination Synthetic enzymes (trade name: TrypLE Select, manufactured by Invitrogen Corporation, etc.); chelating agents (EDTA, etc.) and animal-derived proteolytic enzymes (trypsin, collagenase, etc.); A method of collecting etc.} may be used in combination.

Examples of the cells (CE2) obtained by cell culture using the resin beads of the present invention include the same cells (CE) that can adhere to the resin beads of the present invention. Note that the cell (CE) may be differentiated to obtain a cell (CE2) different from the original cell (CE). For example, when the original cell (CE) is an undifferentiated bone marrow mesenchymal stem cell and the cell (CE2) is an osteoblast, a chondrocyte or an adipocyte (tissue engineering, Minoru Ueda, foundation) Nagoya University Press, October 10, 1999).
As described above, the cell production method of the present invention includes the step of culturing cells using temperature-sensitive swellable resin beads, and detaching and collecting the cells at a temperature 5-30 ° C. lower than the cell culture temperature. It is preferable to include a process.

EXAMPLES Next, the present invention will be specifically described with reference to examples, but the present invention is not limited to the examples without departing from the gist of the present invention. Unless otherwise specified, “part” means “part by weight” and “%” means “% by weight”.
<Example 1>
A mixed solution of 100 parts of N-isopropylacrylamide (trade name NIPAM, manufactured by Kojin Co., Ltd.), 1 part of acrylic acid, 1 part of ethylene glycol dimethacrylate and 50 parts of ion-exchanged water at room temperature (20-25 ° C.). After sufficiently purging with nitrogen (gas phase oxygen concentration of 10 ppm or less), 2.7 parts of a 5% aqueous solution of azobisamidinopropane hydrochloride was added to this mixed solution to obtain a uniform monomer aqueous solution.
Separately, 4 parts of a surfactant (sorbitan monostearate) and 1300 parts of cyclohexane were charged into a reaction vessel equipped with a stirrer, a dropping funnel, a nitrogen gas introduction tube, a thermometer, and a reflux condenser, and the system was stirred (rotation speed 250 rpm). The inside was replaced with nitrogen gas (gas phase oxygen concentration of 10 ppm or less), and the temperature was raised to 57 ° C.
After reaching 57 ° C., the aqueous monomer solution was dropped into the reaction vessel over 2 hours. Subsequently, stirring was continued at 57 ° C. for 1 hour, and then the precipitated crude resin beads were separated by filtration, and dried at 50 ° C. for 4 hours (in a smooth wind dryer) to obtain crude resin beads.
Subsequently, the crude resin beads were classified using sieves with openings of 250 μm and 150 μm (JIS Z8801-1: 2000) (particles that passed through a 150 μm sieve but not through a 250 μm sieve were obtained), and 100 ml of ion-exchanged water. After washing with autoclave for 20 minutes at 121 ° C. in 100 parts of phosphate buffer solution (PBS), the resin beads are taken out from the PBS and irradiated with UV at 25 ° C. and 20% RH for 1 hour. Resin beads (1) were obtained. The resin beads (1) had an inflection point of 32 ° C. The volume average particle diameter of the precipitated resin beads prepared from the resin beads (1) was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.05.

<Example 2>
The resin of the present invention is the same as in Example 1 except that 100 parts of N, N-diethylacrylic acid amide (trade name DEAA, manufactured by Kojin Co., Ltd.) is used instead of 100 parts of N-isopropylacrylic acid amide. Beads (2) were obtained. The resin beads (2) had an inflection point of 32 ° C. The volume average particle diameter of the precipitated resin beads prepared from the resin beads (2) was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.05.

<Example 3>
Instead of 100 parts of N-isopropylacrylic acid amide, Example 1 is used except that 100 parts of Nn-propylacrylic acid amide obtained according to the method described in Examples in Japanese Patent Application Laid-Open No. 2000-273074 is used. Similarly, resin beads (3) of the present invention were obtained. The resin beads (3) had an inflection point of 21 ° C. The volume average particle diameter of the precipitated resin beads prepared from the resin beads (3) was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.05.

<Example 4>
Instead of 100 parts of N-isopropylacrylic acid amide, Example 1 was used except that 100 parts of Nn-propyl methacrylic acid amide obtained according to the method described in Examples in Japanese Patent Application Laid-Open No. 2000-273074 was used. Similarly, resin beads (4) of the present invention were obtained. The resin beads (4) had an inflection point of 27 ° C. The volume average particle diameter of the precipitated resin beads prepared from the resin beads (4) was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.05.

<Example 5>
N, N-diethylacrylic acid amide 99 parts (1 mole part), 37% formaldehyde aqueous solution (methanol content 7%) 122 parts (1.5 mole part), 1,4-diazabicyclo [2,2,2] octane (DABCO) 7 parts (63 mmol parts), methoxyhydroquinone 0.03 parts and acetonitrile 100 parts were uniformly mixed, and then reacted at 80 to 82 ° C. for 1 hour. The reaction solution was cooled to 40 ° C., adjusted to pH 5.0 with concentrated hydrochloric acid, and extracted with 200 parts of toluene. Subsequently, toluene was distilled off from the extract to obtain a crude product of N, N-diethyl-α-hydroxymethylacrylic acid amide (HMAA).
After 30 parts of a crude product of MHAA was dissolved in 150 parts of water, impurities were extracted and removed three times using 60 parts of petroleum ether. Subsequently, sodium chloride was dissolved in an aqueous MHAA solution until saturation, followed by extraction with toluene to purify MHAA (purity 98.5%).
116 parts (1 mole part) of MHAA and 2 parts of potassium hydroxide were mixed and adjusted to 100 ° C., and then 44 parts (1 mole part) of ethylene oxide was added dropwise at 100 ° C. to cause a reaction. Diethyl-α- (hydroxyethyloxymethyl) acrylic acid amide was obtained.
Resin beads (5) of the present invention were prepared in the same manner as in Example 1 except that 100 parts of N, N-diethyl-α- (hydroxyethyloxymethyl) acrylic acid amide was used instead of 100 parts of N-isopropylacrylic acid amide. ) The resin beads (5) had an inflection point of 30 ° C. The volume average particle diameter of the precipitated resin beads prepared from the resin beads (5) was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.05.

<Example 6>
N-hydroxyethylacrylic acid amide (trade name HEAA, manufactured by Kojin Co., Ltd.) 115 parts (1 mol part) and THF 200 parts were mixed and adjusted to 5 ° C., and then adjusted to 5 ° C. at 135 ° C. phosphorus tribromide 135 parts ( N-ethyl bromide acrylic acid amide was obtained by reacting by dropping 0.5 mol part).
After mixing 46 parts (1 mole part) of ethanol and 112 parts (2 mole parts) of potassium hydroxide and adjusting to 75 ° C., the N-ethyl bromide acrylic acid amide obtained above was dropped and reacted. A reaction product was obtained. Thereafter, the reaction product was neutralized with hydrochloric acid, and the precipitated potassium chloride was filtered off to obtain N-ethoxyethylacrylic acid amide.
Resin beads (6) of the present invention were obtained in the same manner as in Example 1 except that 100 parts of N-ethoxyethylacrylamide was used instead of 100 parts of N-isopropylacrylamide. The resin beads (6) had an inflection point of 35 ° C. The volume average particle diameter of the precipitated resin beads prepared from the resin beads (6) was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.05.

<Example 7>
100 parts of polyoxyethylene polyoxybutylene glycol (oxyethylene / oxybutylene: weight ratio 50/50, random, number average molecular weight 2000) and 7 parts of HDI isocyanurate are heated and mixed at 60 ° C., and this mixture is 1000 parts of decane. And 4 parts of a surfactant (sorbitan monostearate), and then mixed for 3 minutes with a disperser (TK homomixer MARKII20: Tokushu Kika Co., Ltd.) {80 ° C., rotation speed 5000 rpm} for dispersion Got.
Thereafter, this dispersion was put into another reaction vessel equipped with a stir bar and a thermometer, and reacted at 60 ° C. for 10 hours. Then, the precipitated crude resin beads were separated by filtration, and 50 ° C. for 4 hours (forward wind). Drying in the dryer) gave crude resin beads.
Next, the crude resin beads are classified using sieves with openings of 250 μm and 150 μm (JIS Z8801-1: 2000), washed 4 times with 100 ml of ion-exchanged water, and autoclaved at 121 ° C. for 20 minutes in 100 parts of PBS. The resin beads were removed from the PBS and irradiated with UV at 25 ° C. and 20% RH for 1 hour to obtain the resin beads (7) of the present invention. The resin beads (7) had an inflection point of 30 ° C. The volume average particle diameter of the precipitated resin beads prepared from the resin beads (7) was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.05.

<Example 8>
Instead of heating and mixing 100 parts of polyoxyethylene polyoxybutylene glycol and 7 parts of HDI isocyanurate, 100 parts of methylcellulose (methylation rate 38.6%, weight average molecular weight 1000) and 5 parts of HDI isocyanurate are mixed. Except for the above, resin beads (8) of the present invention were obtained in the same manner as in Example 7. Resin beads (8) had an inflection point of 32 ° C. The volume average particle diameter of the precipitated resin beads prepared from the resin beads (8) was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.05.

<Example 9>
Instead of heating and mixing 100 parts of polyoxyethylene polyoxybutylene glycol and 7 parts of HDI isocyanurate, 100 parts of polyvinyl alcohol partially saponified product (saponification value 60 mol%, weight average molecular weight 1000), and 5 parts of HDI isocyanurate The resin beads (9) of the present invention were obtained in the same manner as in Example 7 except for mixing. Resin beads (9) had an inflection point of 30 ° C. The volume average particle diameter of the precipitated resin beads prepared from the resin beads (9) was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.05.

<Example 10>
In accordance with the method described in the examples in JP-T-3-502935, a polypeptide having about 110,000 Mn (pronectin F) containing 13 (Gly Ala Gly Ala Gly Ser) ₉ sequences and 13 Arg Gly Asp sequences. ) Next, a 4.5N lithium perchlorate aqueous solution of pronectin F (pronectin F concentration: 1 mg / ml) was diluted with a phosphate buffer solution (PBS) so that the concentration of pronectin F was 300 μg / ml. F solution was prepared.
10 ml of a water-soluble carbodiimide solution {1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, manufactured by Dozindo Co., Ltd., 300 mM aqueous solution), 5 g of resin beads (1) obtained in the same manner as in Example 1 In addition, after stirring for 2 hours with a stirrer made of polyfluorinated ethylene resin (Teflon (registered trademark)), 10 ml of phosphate buffer (PH = 7.2) was added thereto, and the solution was removed by suction 5 times. (Washing) to obtain carbodiimide-bound beads (1).
Subsequently, the carbodiimide-bound beads (1) were immersed in 10 ml of Pronectin F solution and stirred for 2 hours, and 10 ml of phosphate buffer (PH = 7.2) was added and the solution was removed by suction three times ( After washing), the mixture was stirred in an aqueous ammonia solution (300 mM, 10 ml) for 2 hours. Thereafter, the operation of adding 10 ml of ion-exchanged water and removing the solution by suction was performed three times (washing), then transferred to a stainless steel vat set on a shaker, and dried by shaking for 30 minutes while blowing hot air at 100 ° C. As a result, dried beads (1) were obtained.
This dry bead (1) was washed twice with 50 ml of PBS, sterilized in autoclave at 121 ° C. for 20 minutes in PBS, and then the resin beads were taken out from PBS and irradiated with UV at 25 ° C. and 20% RH for 1 hour. Resin beads (10) were obtained. The resin beads (10) had an inflection point of 32 ° C. The volume average particle diameter of the precipitated resin beads prepared from the resin beads was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.05.

About resin bead (10), content of pronectin F was measured in the following procedures.
(1) 10 ml of PBS (PH7.4) containing bovine serum albumin at 1% by weight was added to 0.1 g of resin beads (10), and allowed to stand at room temperature (25 ° C.) for 2 hours. Thereafter, the solution was removed using an aspirator to obtain bovine serum albumin-treated resin beads.
(2) Next, 10 ml of PBS (PH7.4) containing 0.001% by weight of peroxidase-labeled anti-pronectin F antibody, 1% by weight of bovine serum albumin, and 0.2% by weight of Tween 20 on the resin beads treated with bovine serum albumin And reacted at 37 ° C. for 2 hours. After the reaction, the solution was removed three times with 10 ml of PBS (PH7.4) containing 0.2% by weight of Tween 20 to obtain reaction resin beads.
The peroxidase-labeled anti-pronectin F antibody was prepared by immunizing rabbits with pronectin F according to a polyclonal antibody production method (enzyme immunoassay, translated by Junji Ishikawa, Tokyo Chemical Co., Ltd., 1989). By binding the anti-pronectin F antibody and peroxidase (manufactured by Toyobo Co., Ltd.) by the maleimide method (Enzyme immunoassay, translated by Junji Ishikawa, Tokyo Chemical Co., Ltd., 1989), Obtained.
(3) Next, 10 ml of a mixed solution containing 20% by weight of OLYDAS color developing solution set (manufactured by Sanyo Kasei Kogyo Co., Ltd.) and 80% by weight of ion-exchanged water was added to the obtained reaction resin beads. The reaction was carried out at 0 ° C. for 1 hour. After the reaction, the absorbance was measured at a wavelength of 380 nm.
(4) Standard resin beads 1 to 5 were prepared in the same manner as the resin beads (10). The pronectin F content 1 to 5 of the standard resin beads 1 to 5 is obtained by dialyzing the pronectin F solution after the treatment with pronectin F (dialysis membrane trade name: Spectra / Por131204 (manufactured by Spectrum)), 000, using ion-exchanged water, 4 times)) and freeze-drying (−20 ° C., 130 Pa, 24 hours) to determine the weight of unattached pronectin F. From the weight of pronectin F before attachment, It was determined by subtracting the weight of pronectin F.
For standard resin beads 1 to 5, absorbances 1 to 5 were measured in the same manner as described above, and a calibration curve was prepared using the obtained absorbances 1 to 5 and pronectin F contents 1 to 5.
From the calibration curve and the absorbance of the resin beads (10), the content of pronectin F per unit weight of the resin beads (10) (118 μg / g) was obtained.
From this content (118 μg / g) and the average surface area per unit weight (294 cm ² / g), the content (μg / cm ² ) of pronectin F per 1 cm ² of the average surface area of the resin beads (10) is 0.00. It was calculated to be 4 μg / cm ² (the content of pronectin F or F2 was determined in the same manner below).

<Example 11>
Resin beads (11) of the present invention were obtained in the same manner as in Example 10, except that the resin beads (2) obtained in the same manner as in Example 2 were used instead of the resin beads (1). The resin beads (11) had an inflection point of 32 ° C. and a content of pronectin F of 0.4 μg / m ² . The volume average particle diameter of the precipitated resin beads prepared from the resin beads (11) was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.05.

<Example 12>
According to the process of Example described in JP Hei 3-502935, Arg Gly Asp sequence 5 and (Gly Ala Gly Ala Gly Ser) 3 sequences 5 and Mn of about 20,000 polypeptide comprising (pronectin F2) was obtained. Next, a 4.5N lithium perchlorate aqueous solution of pronectin F2 (pronectin F2 concentration: 1 mg / ml) was diluted with a phosphate buffer solution (PBS) so that the concentration of pronectin F2 was 300 μg / ml. An F2 solution was prepared.
Resin beads (12) of the present invention were obtained in the same manner as in Example 10 except that pronectin F2 was used instead of pronectin F. The resin beads (12) had an inflection point of 32 ° C. and a content of pronectin F2 of 0.4 μg / m ² . The volume average particle diameter of the precipitated resin beads prepared from the resin beads (12) was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.05.

<Example 13>
Other than changing 100 parts of N-isopropylacrylic acid amide to 10 parts of N-isopropylacrylic acid amide and 10 parts of polyethylene fine particles (Miperon XM221U: Mitsui Chemicals, apparent specific gravity 0.94, volume average particle diameter 20 μm). Obtained the resin beads (13) of the present invention in the same manner as in Example 1. The resin beads (13) had an inflection point of 32 ° C. The volume average particle diameter of the precipitated resin beads prepared from the resin beads (13) was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.03.

<Example 14>
After adding 86 parts of methyl acrylate and 43 parts of dimethylamine to the reaction vessel and reacting at 35 ° C. for 5 hours, 85 parts of piperidine and 0.5 part of sodium methoxide were charged and reacted at 50 ° C. for 8 hours. The obtained reaction product was distilled under reduced pressure (4 Pa, about 110 ° C.) to obtain acryloylpiperidine.
Resin beads (14) of the present invention were obtained in the same manner as in Example 1 except that 100 parts of acryloylpiperidine was used instead of 100 parts of N-isopropylacrylamide. The resin beads (14) had an inflection point of 5 ° C. The volume average particle diameter of the precipitated resin beads prepared from the resin beads (14) was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.05.

<Example 15>
In a reaction vessel, 86 parts of methyl acrylate and 43 parts of dimethylamine were charged and reacted at 35 ° C. for 5 hours. Then, 102 parts of dipropylamine and 0.5 part of sodium methoxide were charged and reacted at 50 ° C. for 8 hours. The obtained reaction product was distilled under reduced pressure (4 Pa, about 160 ° C.) to obtain N, N-dipropylacrylamide.
Resin beads (15) of the present invention were obtained in the same manner as in Example 1 except that 100 parts of N, N-dipropylacrylamide was used instead of 100 parts of N-isopropylacrylamide. The resin beads (15) had an inflection point of 12 ° C. The volume average particle diameter of the precipitated resin beads prepared from the resin beads (15) was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.05.

<Example 16>
Similar to Example 1 except that 100 parts of N-3-morpholinopropylacrylamide obtained according to the method described in Examples in JP-A-2000-273074 is used instead of 100 parts of N-isopropylacrylic acid amide. Thus, resin beads (16) of the present invention were obtained. The resin beads (16) had an inflection point of 17 ° C. The volume average particle diameter of the precipitated resin beads prepared from the resin beads (16) was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.05.

<Example 17>
Instead of 100 parts of N-isopropylacrylamide, in the same manner as in Example 1, except that 100 parts of N-ethylacrylamide obtained according to the method described in Examples in JP 2000-273074 A is used. Resin beads (17) of the present invention were obtained. The resin beads (17) had an inflection point of 40 ° C. The volume average particle diameter of the precipitated resin beads prepared from the resin beads (17) was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.05.

<Example 18>
Instead of using 100 parts of N-isopropylacrylamide, 70 parts of N-isopropylacrylamide and 30 parts of N-ethylacrylamide obtained according to the method described in Examples in Japanese Patent Application Laid-Open No. 2000-273074 are used. Were the same as in Example 1 to obtain the resin beads (18) of the present invention. The resin beads (18) had an inflection point of 38 ° C. The volume average particle diameter of the precipitated resin beads prepared from the resin beads (18) was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.05.

<Example 19>
Instead of using 100 parts of N-isopropylacrylamide, 50 parts of N-isopropylacrylamide and 50 parts of N-ethylacrylamide obtained according to the method described in Examples in JP-A-2000-273074 are used. Were the same as in Example 1 to obtain the resin beads (19) of the present invention. The resin beads (19) had an inflection point of 35 ° C. The volume average particle diameter of the precipitated resin beads prepared from the resin beads (19) was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.05.

<Example 20>
Instead of 100 parts of N-isopropylacrylamide, 50 parts of N-isopropylacrylamide and 50 parts of N, N-dipropylacrylamide were used in the same manner as in Example 1, except that ) The resin beads (20) had an inflection point of 25 ° C. The volume average particle diameter of the precipitated resin beads prepared from the resin beads (20) was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.05.

<Example 21>
In a reaction vessel, 100 parts of methyl methacrylate and 43 parts of dimethylamine were charged and reacted at 35 ° C. for 5 hours, and then 73 parts of diethylamine and 0.5 part of sodium methoxide were charged and reacted at 50 ° C. for 8 hours. The obtained reaction product was distilled under reduced pressure (4 Pa, about 100 ° C.) to obtain N, N-diethylmethacrylamide.
Resin beads (21) of the present invention were obtained in the same manner as in Example 1 except that 100 parts of N, N-diethylmethacrylamide was used instead of 100 parts of N-isopropylacrylamide. The resin beads (21) had an inflection point of 25 ° C. The volume average particle diameter of the precipitated resin beads prepared from the resin beads (21) was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.05.

<Example 22>
Resin beads (22) of the present invention were obtained in the same manner as in Example 1 except that 0.01 part of pentaerythritol tetraacrylate was used instead of 1 part of ethylene glycol diacrylate. The resin beads (22) had an inflection point of 32 ° C. The volume average particle diameter of the precipitated resin beads prepared from the resin beads (22) was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.05.

<Example 23>
Resin beads (23) of the present invention were obtained in the same manner as in Example 1 except that 5 parts of ethylene glycol diacrylate was used instead of 1 part of ethylene glycol diacrylate. The resin beads (23) had an inflection point of 32 ° C. The volume average particle diameter of the precipitated resin beads prepared from the resin beads (23) was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.05.

<Example 24>
Resin beads (24) of the present invention were obtained in the same manner as in Example 1 except that the concentration of the Pronectin F solution was 30 μg / m ² and the water-soluble carbodiimide solution was a 30 mM aqueous solution. The resin beads (24) had an inflection point of 32 ° C. and a content of pronectin F of 0.01 μg / m ² . The volume average particle diameter of the precipitated resin beads prepared from the resin beads (24) was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.05.

<Example 25>
Resin beads (25) of the present invention were obtained in the same manner as in Example 1 except that the concentration of the Pronectin F solution was 30000 μg / m ² and the water-soluble carbodiimide solution was changed to a 30000 mM aqueous solution. The resin beads (25) had an inflection point of 32 ° C. and a content of pronectin F of 1000 μg / m ² . The volume average particle diameter of the precipitated resin beads prepared from the resin beads (25) was 200 μm, and the apparent specific gravity of the precipitated resin beads was 1.05.

<Comparative Example 1>
In a reaction vessel equipped with a stirrer, dropping funnel, nitrogen gas inlet tube, thermometer, reflux condenser, 4 parts of surfactant (PVA235, manufactured by Kuraray Co., Ltd .: Ken value 87-89 mol%, polymerization degree 3500), ion Exchanged water 200 parts, polymerization initiator (Niper BW, manufactured by Nippon Oil & Fats Co., Ltd.) 0.45 parts, polymerization initiator (Kaya Ester HTP-65W, manufactured by Kayaku Akzo Co., Ltd.) 0.15 parts, chain transfer agent (Dodecyl mercaptan) 0.26 parts and sodium chloride 20 parts were charged, and the system was replaced with nitrogen gas under stirring, and the temperature was raised to 30 ° C.
Next, a mixture of 3 parts of divinylbenzene and 97 parts of styrene was dropped into the reaction vessel over 1 hour while maintaining 30 ° C, and then the temperature was raised to 82 ° C and reacted at 82 ° C for another 5 hours. Next, the temperature was raised to 92 ° C., and the mixture was further reacted for 1 hour at 92 ° C., and then the aqueous medium (ion exchange water, etc.) was removed by filtration, followed by drying in an 80 ° C. vacuum dryer (pressure 100 Pa) for 12 hours. As a result, crude resin beads were obtained.
Next, the crude resin beads were classified using sieves with openings of 250 μm and 150 μm (JIS Z8801-1: 2000), washed 4 times with 100 ml of ion-exchanged water, autoclaved at 121 ° C. for 20 minutes in PBS, Resin beads (26) (polystyrene beads) for comparison were obtained by removing resin beads from PBS and irradiating with UV at 25 ° C. and 20% RH for 1 hour. The resin beads (26) had a volume average particle diameter of 200 μm and an apparent specific gravity of 1.055.

<Comparative example 2>
Comparative resin beads (27) were obtained in the same manner as in Example 10 except that 5 g of resin beads (26) obtained in the same manner as in Comparative Example 1 was used instead of 5 g of resin beads (1). The resin beads (27) had a volume average particle diameter of 200 μm, an apparent specific gravity of 1.06, and a content of pronectin F of 0.4 μm.

<Comparative Example 3>
Commercially available beads for cell culture {Cytodex I: manufactured by Pharmacia Biotech Co., Ltd. (volume average particles after swelled by immersing in physiological saline at 37 ° C. in the same manner as the preparation of dextran beads and leaching resin beads) The diameter was 180 μm, and similarly the specific gravity after swelling was 1.03)} was used as a comparative resin bead (28).

<Comparative example 4>
Commercially available beads for cell culture {CytodexIII: manufactured by Pharmacia Biotech Co., Ltd. (Collagen-bound dextran beads, volume after being swelled by immersing in physiological saline at 37 ° C. in the same manner as preparation of the leaching resin beads) The average particle size was 175 μm, and the specific gravity after swelling was 1.04))} was used as a comparative resin bead (29).

<Cell culture evaluation>
In each of 29 20 ml spinner flasks, resin beads {0.6 g / flask for resin beads 1 to 15; 0.06 g / flask for resin beads 28 and 29 (note that resin beads 28 and 29 absorb water) Since the volume expands to about 10 times, the surface area for cell culture is set to 1/10)) and VP-SFM (serum-free medium manufactured by Invitrogen Corporation) is added at 10 mL / flask. After adjusting the temperature to 37 ° C., 1 ml of Vero cells (Dainippon Pharmaceutical Co., Ltd.) (cell 1) dispersed in the above medium at a cell concentration of 2.0 × 10 ⁶ cells / mL was added and stirred ( Cultivation was performed at 37 ° C for 6 days in a mixture of carbon dioxide and air (carbon dioxide / air = 5/95 volume ratio) with 60 rpm, manufactured by Waken Pharmaceutical Co., Ltd., 4-POSITION MAGNETIC STIRRER MODEL1104. Was Tsu. The medium was changed on the 3rd, 4th and 5th days of culture.
On the 6th day, the sample was sampled from the bead suspension without stopping stirring, and the number of cell nuclei per unit volume was counted by the cell nuclei counting method using crystal violet by Wezel. The counting results are shown in Table 1. .

<Evaluation of cell detachment / recovery>
After the cell culture evaluation, the bead suspension using resin beads 1 to 25 (Examples 1 to 25) was temperature-controlled at 15 ° C. in a 20 ml spinner flask, the resin beads were removed by centrifugation, and the cell suspension was suspended. A liquid was obtained.
On the other hand, in the bead suspension using resin beads 26 to 29 (Comparative Examples 1 to 4), after removing the medium, 10 mL / flask of PBS (PH7.4) was added, and the mixture was gently stirred to remove the PBS. A degrading enzyme-EDTA solution (TrypLE Select, manufactured by Invitrogen Corporation) was added at 10 mL / flask, and the mixture was stirred at 37 ° C. for 10 minutes (60 rpm). Thereafter, the resin beads were removed by centrifugation to obtain a cell suspension.
Next, a part of these cell suspensions were collected and treated with trypan blue to develop the color of dead cells per ml, and the number of developed dead cells and the total number of cells were visually observed under a microscope using a hemocytometer. The cell viability was calculated according to the following formula. These results are shown in Table 1.

  Next, cell culture was performed in the same manner as the above cell culture except that serum medium (DMEM containing 5% FBS) was used instead of VP-SFM, and evaluation was performed in the same manner. These results are shown in Table 2.

  Next, using resin beads (1), (10), (26), (27), (28), and (29), instead of Vero cells [Dainippon Pharmaceutical Co., Ltd.] (cell 1) The above VP-SFM medium was used except that normal human skin capillary endothelial cells [Dainippon Pharmaceutical Co., Ltd.] (cell 2) or Sf9 insect cells [Invitrogen Corp.] (cell 3) were used. Cell culture was performed in the same manner as the cell culture performed and evaluated in the same manner. These results are shown in Table 3.

  Next, the resin beads (1), (10), (26), (27), (28), and (29) were used except that the temperature at the time of cell recovery was 15 ° C. was changed to 22 ° C. or 12 ° C. In the same manner as in cell culture using VP-SFM medium, cell culture was performed using cell 1 and evaluated in the same manner. These results are shown in Table 4.

From the above evaluation results, the resin beads of the present invention are extremely simple (just by lowering the temperature) in comparison with the resin beads for comparison in both cell culture using serum-free medium and cell culture using serum medium. The cultured cells could be detached and collected, and the cells could be detached and collected with extremely high cell viability. There was no problem with cell proliferation.
Furthermore, the resin beads (10), (11), (12), (24), and (25) of the present invention using the polypeptide (P) are those in which no polypeptide is used in cell culture in a serum-free medium. Compared to the above, cell proliferation was excellent.

The bead for cell culture of the present invention can be peeled and collected as it is without denaturing the proliferated cells, and thus is extremely useful for research and development, production of useful substances, treatment and the like related to cells.
For research and development, it can be used for culturing cells for animal experiments (toxicity test, stimulation test, metabolic function test, etc.), culturing cells for gene transfer, and the like.
For the production of useful substances, it can be used for culturing cells for production of cytokines, thrombolytic agents, blood coagulation factor preparations, vaccines, hormones, antibiotics, antibodies, growth factors and the like.
For treatment, skin, skull, muscle, skin tissue, bone, cartilage, blood vessel, nerve, tendon, ligament, follicular tissue, mucosal tissue, periodontal tissue, dentin, bone marrow, retina, serosa, gastrointestinal tract and fat Can be used for cell culture of tissues such as lung, liver, pancreas and kidney.

Claims (8)

A temperature-sensitive swellable resin bead for cell culture, comprising a crosslinked polymer (A) having an inflection point in a temperature-water absorption curve. The temperature-swellable resin beads according to claim 1, wherein the temperature corresponding to the inflection point is 0 to 80 ° C. The crosslinked polymer (A) comprises a vinyl monomer containing a carbamoyl group and / or a polyoxyalkylene group as an essential constituent monomer, a crosslinked vinyl polymer (A1), a crosslinked alkylene oxide polymer (A2), a crosslinked methylcellulose (A3), The temperature-swellable resin beads according to claim 1, which are at least one selected from the group consisting of a partially saponified product of crosslinked polyvinyl alcohol (A4). The temperature-swellable resin bead according to any one of claims 1 to 3, further comprising a polypeptide (P) having at least one cell adhesion minimal amino acid sequence (X) in one molecule. The temperature-swellable resin beads according to any one of claims 1 to 4, further comprising fine particles (b). A method for producing a thermosensitive swellable resin bead for cell culture, comprising a crosslinked polymer (A) having an inflection point in a temperature-water absorption curve,
A mixture of a vinyl monomer containing a carbamoyl group and / or a polyoxyalkylene group and a crosslinking agent (c1) is dropped into the hydrophobic solvent (W) and simultaneously polymerized while being dispersed to obtain a crosslinked vinyl polymer (A1). Step (1),
A step (2) of obtaining a crosslinked alkylene oxide polymer (A2) by dispersing a mixture of an alkylene oxide polymer and a crosslinking agent (c2) in a hydrophobic solvent (W) and performing a crosslinking reaction;
A step (3) of obtaining a crosslinked methylcellulose (A3) by dispersing a mixture of methylcellulose and a crosslinking agent (c3) in a hydrophobic solvent (W) and performing a crosslinking reaction;
And / or a step (4) of obtaining a crosslinked polyvinyl alcohol partially saponified product (A4) by dispersing a mixture of the partially saponified polyvinyl alcohol and the crosslinking agent (c4) in the hydrophobic solvent (W) and performing a crosslinking reaction. A method for producing a temperature-sensitive resin bead. It includes a step of culturing cells using the temperature-sensitive swellable resin beads according to any one of claims 1 to 5 and a step of detaching and collecting cells at a temperature 5-30 ° C lower than the cell culture temperature. A cell production method. The method for producing cells according to claim 7, wherein the cells to be cultured are normal cells derived from mammals, established cell lines derived from mammals, or insect cells.