Cell adhesive particle and particle production method

Abstract

The present invention provides particles having excellent cell adhesiveness.　Cell adhesive particles according to the present invention each contain, in a molecule, a structural unit having at least one selected from groups represented by formula (1) and groups represented by formula (2). [In formula (1), R1 represents an alkanediyl group having 2-10 carbon atoms, R2 and R3 each independently represent a hydrogen atom or an alkyl group having 1-10 carbon atoms, and * represents a binding site.] [In formula (2), R4 represents an alkanediyl group having 2-10 carbon atoms, R5-R7 each independently represent a hydrogen atom or an alkyl group having 1-10 carbon atoms, X- represents a counter anion, and * represents a binding site.]

Description

Cell-adhesive particles and method for producing the particles

[0001] The present invention relates to cell-adhesive particles and a method for producing the particles, and more particularly to a method for producing cell-adhesive particles and polymer particles.

[0002] Microcarrier culture is a method of growing cells by attaching them to particles measuring several micrometers to several hundred micrometers in size. This culture method is easy to scale up, and because the adhesion surface area per volume of culture liquid is large and productive, it is easy to efficiently mass-cultivate cells. For this reason, it has attracted attention in recent years in the fields of biopharmaceuticals, regenerative medicine, immunotherapy, cell therapy, gene therapy, as well as the production of useful substances such as enzymes, cell growth factors, and antibodies, and is expected to be used in vaccine production, therapeutic cell generation, and biomaterial synthesis.

[0003] Known examples of microcarriers that can be used in this culture method include a specific carrier in which collagen and polypeptide are bound to water-absorbent resin particles containing tertiary amino groups in the molecule that have been introduced using 2-chloro-N,N-diethylethylamine hydrochloride (Patent Document 1), diethylaminoethyl group-modified cross-linked cellulose particles, and diethylaminoethyl group-modified cross-linked dextran particles (Patent Document 2).

[0004] JP 2018-99115 A JP 2018-68154 A

[0005] However, although efficient cell proliferation requires sufficient cell adhesion to particles, cell adhesiveness was insufficient when chemical structures such as those described in Patent Documents 1 and 2 were simply introduced into particle molecules. The problem to be solved by the present invention is to provide particles with excellent cell adhesiveness.

[0006] The above-mentioned problems have been solved by the following means <1> to <11>: <1> Cell-adhesive particles (hereinafter also referred to as "cell-adhesive particles of the present invention") containing, in the molecule, a structural unit (hereinafter also referred to as "specific structural unit") having one or more types selected from a group represented by the following formula (1) (hereinafter also referred to as "group (1)") and a group represented by the following formula (2) (hereinafter also referred to as "group (2)"):

[0007]

[0008] [In formula (1), R 1 represents an alkanediyl group having 2 to 10 carbon atoms, and R 2 ~R 3 each independently represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, and * represents a bonding site.

[0009]

[0010] [In formula (2), R 4 represents an alkanediyl group having 2 to 10 carbon atoms, and R 5 ~R 7 each independently represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms; - indicates a counter anion, and * indicates a binding site.

[0011] <2> The cell-adhesive particle according to <1>, further comprising a structural unit represented by the following formula (3) (hereinafter also referred to as "structural unit (3)") in the molecule:

[0012]

[0013] [In formula (3), R 8 and R 9 each independently represents a hydrogen atom or a methyl group, R 10 represents a group represented by the following formula (3-A) or a polyoxyalkylene group.

[0014]

[0015] [In formula (3-A), R 11 represents an alkanediyl group having 3 to 100 carbon atoms, * represents a bonding site that bonds to the oxygen atom in formula (3), ** represents the R 9 The binding site is shown.]

[0016] <3> The cell-adhesive particle according to <1> or <2>, further comprising a structural unit represented by the following formula (4) (hereinafter also referred to as "structural unit (4)") in the molecule:

[0017]

[0018] [In formula (4), R 12 and R 13 each independently represents a hydrogen atom or a methyl group, R 14 represents an alkanediyl group having 1 or 2 carbon atoms.

[0019] <4> The cell adhesive particle according to any one of <1> to <3>, wherein the content of structural units having one or more selected from the group represented by formula (1) and the group represented by formula (2) is 5% by mass or more and 85% by mass or less of all structural units contained in the cell adhesive particle.

[0020] <5> A method for producing polymer particles, comprising the following steps (i) to (iii), wherein the aqueous medium in step (iii) has 30 parts by mass or more of salt dissolved therein per unit mass, where the mass of salt per unit mass of the saturated solution obtained by dissolving a salt in the aqueous medium at 23°C and 1 atmospheric pressure is 100 parts by mass (hereinafter also referred to as the "method for producing polymer particles of the present invention"): (i) a step of preparing a hydrocolloid in a salt-containing aqueous medium, (ii) a step of dispersing one or more monomers in the hydrocolloid-containing aqueous medium obtained in step (i), and (iii) a step of suspension polymerizing the monomer dispersed in step (ii) in an aqueous medium containing a hydrocolloid and a salt.

[0021] <6> The manufacturing method according to <5>, wherein at least a monomer that imparts a specific structural unit is used as the monomer, and the polymer particles are cell adhesive particles containing the specific structural unit in the molecule. <7> The manufacturing method according to <5> or <6>, further comprising the following step (iv): (iv) a step of separating the polymer particles obtained in step (iii) from the hydrocolloid. <8> The manufacturing method according to any one of <5> to <7>, wherein the salt-containing aqueous medium is an aqueous medium containing at least water, and step (ii) is a step of mixing the hydrocolloid-containing aqueous medium obtained in step (i) with a liquid composition in which one or more monomers are dissolved or dispersed in the aqueous medium, and the proportion of the liquid composition used is 5 parts by mass or more and 40 parts by mass or less, where the total mass of water contained in the hydrocolloid-containing aqueous medium in step (ii) is 100 parts by mass. <9> The method according to any one of <5> to <8>, wherein the polymer particles have a sedimentation bulk density of 0.08 g / mL or more and 0.13 g / mL or less when dispersed in an aqueous solution of sodium chloride having a concentration of 0.9% by mass under conditions of 25°C and 1 atmospheric pressure. <10> The method according to any one of <5> to <9>, wherein the polymer particles have an average particle size of 50 μm or more and 500 μm or less.

[0022] <11> A cell culture method (hereinafter also referred to as the "cell culture method of the present invention"), characterized by using the cell adhesive particles according to any one of <1> to <4>.

[0023] The cell adhesive particles of the present invention have excellent cell adhesiveness. According to the method for producing polymer particles of the present invention, polymer particles can be produced simply and efficiently. According to the method for cell culture of the present invention, cells can be cultured efficiently.

[0024] 1 shows particle images used as the evaluation standard for visibility. 1 shows particle images of dry particles 1 (Example 1). 1 shows particle images of dry particles 2 (Example 2). 1 shows particle images of particles with cells adhering thereto. 1 shows particle images of particles without cells adhering thereto.

[0025] [Cell-adhesive particles] The cell-adhesive particles of the present invention contain, in the molecule, a structural unit having one or more types selected from group (1) and group (2).

[0026] (Specific structural unit) R in formula (1) 1 , R in formula (2) 4 represents an alkanediyl group having 2 to 10 carbon atoms. If the number of carbon atoms exceeds 10, cell adhesiveness becomes insufficient. Furthermore, the number of carbon atoms in the alkanediyl group is preferably 2 to 8, more preferably 2 to 6, and particularly preferably 2 to 4, in order to improve cell adhesiveness and visibility. The alkanediyl group may be linear or branched. Specific examples of the alkanediyl group include an ethane-1,1-diyl group, an ethane-1,2-diyl group, a propane-1,1-diyl group, a propane-1,2-diyl group, a propane-1,3-diyl group, a propane-2,2-diyl group, a butane-1,1-diyl group, a butane-1,2-diyl group, a butane-1,3-diyl group, a butane-1,4-diyl group, a pentane-1,1-diyl group, a pentane-1,2-diyl group, a pentane hexane-1,3-diyl group, pentane-1,4-diyl group, pentane-1,5-diyl group, hexane-1,1-diyl group, hexane-1,2-diyl group, hexane-1,3-diyl group, hexane-1,4-diyl group, hexane-1,5-diyl group, hexane-1,6-diyl group, heptane-1,7-diyl group, octane-1,8-diyl group, nonane-1,9-diyl group, and decane-1,10-diyl group.

[0027] R in formula (1) 2 ~R 3 , R in formula (2) 5 ~R 7 are each independently a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. Among these, an alkyl group having 1 to 10 carbon atoms is preferred in order to improve cell adhesiveness and visibility. 2 ~R 3 , R in formula (2) 5 ~R 7In order to improve cell adhesiveness and visibility, the number of carbon atoms in the alkyl group represented by the formula (I) is preferably 1 to 7, more preferably 1 to 5, and particularly preferably 1 or 2. The alkyl group may be linear or branched. Specific examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a 2-ethylhexyl group, a nonyl group, and a decyl group.

[0028] X in formula (2) - The counter anion represented by the formula (I) may be a monovalent counter anion or a polyvalent counter anion. It may also be a monoatomic anion or a polyatomic anion. Examples of polyvalent counter anions include those derived from polyvalent anionic compounds. Polyvalent anionic compounds refer to organic or inorganic compounds that ionize when dissolved in water and assume a negative charge of two or more. Examples of polyvalent anionic compounds include polymeric compounds such as gums and polyacrylic acid derivatives, and compounds known as chelating agents such as citric acid and its salts and EDTA. Examples of polyvalent counter anions include those derived from the above polyvalent anionic compounds, as well as Cr2O7 2- , SO4 2- , O(P(=O)(-OH)O - )2, (CN)2CHPhPhCH(CN)2 2- Examples of monovalent counter anions include F - , Cl - ,Br - , I - Halide ions such as; hydroxide ions, NO3 - , HSO4 - , CF3SO3 - , CH3SO4 - , ClO - , BF4 - , PF6 - , HF2 - , H2F3 - , SCN - , HO(C=O)-CH2(OH)-CH2(OH)-(C=O)O - , CH3(C=O)O- , Ph(OH)(C=O)O - , Ph(C=O)O - , CH3PhSO3 - , NO2PhO - , Br3 - , IC12 - , IBr2 - , ClBr - , AuCl - , AuBr2 - , AuI2 - , N3 - , (CF3SO2)2N - , (CN)2N - , (FSO2)2N - , RuO - , ReO4 - , Cu(CF3)4 - , (CH3(C=O)O)3BH - , BH3CN - , BH4 - , (Ph)4B - , (C6H5)3Si - F2, etc. - As the counter anion, monovalent to hexavalent counter anions are preferred, monovalent to trivalent counter anions are more preferred, monovalent counter anions are even more preferred, and halide ions are particularly preferred.

[0029] The cell-adhesive particles of the present invention may be organic particles, organic-organic composite particles, or organic-inorganic composite particles. However, synthetic polymer or natural polymer cell-adhesive polymer particles containing a specific structural unit within the molecule are preferred, and synthetic polymer or polysaccharide cell-adhesive polymer particles containing a specific structural unit within the molecule are more preferred. For improved availability and quality stability (e.g., reduced variability in physical properties), synthetic polymer cell-adhesive polymer particles containing a specific structural unit within the molecule are particularly preferred. Preferred polysaccharide cell-adhesive polymer particles include polysaccharide cell-adhesive polymer particles containing a modified monosaccharide unit within the molecule, chemically modified with group (1) or group (2) directly or via a linker. Examples of polysaccharides include polysaccharides containing carboxyl groups, such as gellan gum, pectin, gum arabic, xanthan gum, and alginic acid, as well as agarose, dextran, cellulose, carrageenan, schizophyllan, and curdlan. Among these, polysaccharides containing carboxyl groups are preferred, with gellan gum being particularly preferred. When the polysaccharide has a carboxy group, the polysaccharide cell-adhesive polymer particles preferably contain, in the molecule, a modified monosaccharide unit in which the carboxy group is chemically modified with group (1) or group (2). The "modified monosaccharide unit" refers to a structural unit provided by a monosaccharide chemically modified with a functional group directly or via a linker. The cell-adhesive particles of the present invention may be porous or non-porous.

[0030] Examples of the specific structural unit include a structural unit represented by the following formula (5) and a modified monosaccharide unit chemically modified with the above group (1) or group (2) directly or via a linker, with the structural unit represented by formula (5) being preferred for ease of availability and quality stability (such as reduced variation in physical properties). The modified monosaccharide unit chemically modified with group (1) or group (2) directly or via a linker is preferably a modified monosaccharide unit derived from a sugar acid in which the carboxy group of the sugar acid is chemically modified with group (1) or group (2), and more preferably a modified monosaccharide unit derived from a uronic acid in which the carboxy group of the uronic acid is chemically modified with group (1) or group (2). Uronic acids include glucuronic acid, galacturonic acid, mannuronic acid, iduronic acid, etc., with glucuronic acid being preferred.

[0031]

[0032] [In formula (5), R 20 represents a hydrogen atom or a methyl group, R 21 represents a single bond or a divalent linking group, Z 1 represents a group represented by the formula (1) or a group represented by the formula (2) (each symbol in formulas (1) and (2) has the same meaning as defined above).)

[0033] R in formula (5) 21 In order to facilitate the introduction of the group (1) or the group (2), a divalent linking group is preferred, and -(C=O)-, -(C=O)-(OR 22 ) m -*, -(C=O)-(NR 23 )-R 24 - * (R 22 and R 24 each independently represents an alkanediyl group having 1 to 10 carbon atoms, m represents an average value of 1 or more, R 23 represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, and * represents Z in formula (5). 1 represents a bonding site that bonds to the atom. —(C═O)— is more preferred, and —(C═O)— is particularly preferred.

[0034] The above R 22 and R 24 The number of carbon atoms in the alkanediyl group represented by the formula (I) is preferably 2 to 6, more preferably 2 to 3. The alkanediyl group may be linear or branched. Specific examples of the alkanediyl group include a methane-1,1-diyl group, an ethane-1,1-diyl group, an ethane-1,2-diyl group, a propane-1,1-diyl group, a propane-1,2-diyl group, a propane-1,3-diyl group, and a propane-2,2-diyl group. When m is 2 or more, m R 22 may be the same or different. The above m is preferably an average value of 1 to 20, more preferably an average value of 1 to 10. In this specification, each "average value" can be measured by NMR. 23 The alkyl group represented by R 2 The alkyl group is the same as the alkyl group represented by the following formula:

[0035] The pKa of the monomer that provides the specific structural unit when made into a homopolymer is preferably 8.0 or more, more preferably 8.5 or more, even more preferably 9.0 or more, particularly preferably 9.5 or more, in order to enhance cell adhesiveness and visibility, and is preferably 13.5 or less, more preferably 13.0 or less, even more preferably 12.5 or less, particularly preferably 12.0 or less, in order to suppress binding with impurities other than the protein to be immobilized. Specifically, as the monomer that provides the specific structural unit, a pKa of 8.0 or more and 13.5 or less when made into a homopolymer is preferred, a pKa of 8.5 or more and 13.0 or less when made into a homopolymer is more preferred, a pKa of 9.0 or more and 12.5 or less when made into a homopolymer is even more preferred, and a pKa of 9.5 or more and 12.0 or less when made into a homopolymer is particularly preferred. Herein, the acid dissociation constant (pKa) is defined as the acid dissociation constant at 25°C, an ionic strength of 0.1 mol dm -3 pKa is the negative common logarithm of the equilibrium constant Ka in the dissociation reaction in which hydrogen ions are released. Specifically, it refers to the value measured by the methods described in Polymer 42 (2001) 5993-6008, Macromol Chem Phys 1996; 197: 2553-64, Macromol Chem 1985; 186: 111-21, and J Phys Chem 1987; 91: 3092-8.

[0036] Examples of monomers that provide specific structural units include modified monosaccharides chemically modified with the above group (1) or group (2) directly or via a linker, as well as N-[2-(dimethylamino)ethyl]acrylamide, N-[2-(dimethylamino)ethyl]methacrylamide, N-[2-(diethylamino)ethyl]acrylamide, N-[2-(diethylamino)ethyl]methacrylamide, N-[3-(dimethylamino)propyl]acrylamide (pKa when made into a homopolymer: 10.0), N-[3-(dimethylamino)propyl]methacrylamide, N-[3-(diethylamino)propyl]acrylamide, N-[3-(diethylamino)propyl]methacrylamide, N-[4-(dimethylamino)butyl]acrylamide, N-[4-(dimethylamino)butyl]methacrylamide, N-[4- (diethylamino)butyl]acrylamide, N-[4-(diethylamino)butyl]methacrylamide, N-[5-(dimethylamino)pentyl]acrylamide, N-[5-(dimethylamino)pentyl]methacrylamide, N-[5-(diethylamino)pentyl]acrylamide, N-[5-(diethylamino)pentyl]acrylamide, N-[5-(diethylamino)pentyl]methacrylamide, N-[6-(dimethylamino)hexyl]acrylamide, N-[6-(dimethylamino)hexyl]methacrylamide, N-[6-(diethylamino)hexyl]acrylamide, N-[6-(diethylamino)hexyl]methacrylamide, N-(2-aminoethyl)acrylamide, N-(2-aminoethyl)methacrylamide, N-(3-aminopropyl)acrylamide, N-(3-aminopropyl)methacrylamide, etc. These monomers can be used alone or in combination of two or more.

[0037] The content of the specific structural unit is preferably 5% by mass or more, more preferably more than 10% by mass, even more preferably 25% by mass or more, even more preferably 30% by mass or more, and particularly preferably 40% by mass or more, relative to the total structural units contained in the cell adhesive particles in order to improve visibility, cell adhesiveness, stability during polymerization, and salt tolerance, and is preferably 85% by mass or less, more preferably 80% by mass or less, even more preferably 70% by mass or less, even more preferably 65% ​​by mass or less, and particularly preferably 55% by mass or less, relative to the total structural units contained in the cell adhesive particles in order to improve fluidity (storage stability). Specific ranges are preferably 5% by mass or more and 85% by mass or less, more preferably more than 10% by mass but 80% by mass or less, even more preferably 25% by mass or more and 70% by mass or less, even more preferably 30% by mass or more and 65% by mass or less, and particularly preferably 40% by mass or more and 55% by mass or less, relative to the total structural units contained in the cell adhesive particles. When the content of the specific structural unit is 30% by mass or more or 40% by mass or more, the visibility and cell adhesiveness are particularly good. Furthermore, when the content of the specific structural unit is 65% by mass or less or 55% by mass or less, the visibility, fluidity (storage stability), and cell adhesiveness are particularly good. In this specification, "visibility" refers to the ease of observing cell adhesion when observed under a microscope; when observing particles under a microscope, the smaller the ratio of the particle edge to the entire particle, the easier it is to observe cells adhered to the particle. In this specification, "fluidity" refers to the fluidity of the particle. The higher the fluidity, the less likely the particles are to form clumps or aggregate, and the resistance to clumping and aggregation when stored is referred to as "storage stability."

[0038] (Structural Unit (3)) In order to improve fluidity (storage stability) and visibility, the cell adhesive particles of the present invention preferably further contain a structural unit represented by the following formula (3) in the molecule in addition to the specific structural unit:

[0039]

[0040] [In formula (3), R 8 and R 9 each independently represents a hydrogen atom or a methyl group, R 10represents a group represented by the following formula (3-A) or a polyoxyalkylene group.

[0041]

[0042] [In formula (3-A), R 11 represents an alkanediyl group having 3 to 100 carbon atoms, * represents a bonding site that bonds to the oxygen atom in formula (3), ** represents the R 9 The binding site is shown.]

[0043] R in formula (3) 9 In order to improve cell adhesiveness and visibility, R is preferably a hydrogen atom. 10 represents a group represented by formula (3-A) or a polyoxyalkylene group, and is preferably a group represented by formula (3-A) in order to improve cell adhesiveness, fluidity (storage stability), and visibility. 10 The polyoxyalkylene group represented by *-(R 30 O) p -**(R 30 represents an alkanediyl group having 2 to 6 carbon atoms, p represents an average value of 2 to 50, * represents a bonding site that bonds to the oxygen atom in formula (3), and ** represents the R 9 The above R 30 The number of carbon atoms in the alkanediyl group represented by the formula (I) is preferably 2 to 3. The alkanediyl group may be linear or branched. Specific examples of the alkanediyl group include an ethane-1,2-diyl group, a propane-1,2-diyl group, and a propane-1,3-diyl group. 30 The above p is preferably 2 to 1000 on average, more preferably 2 to 100 on average.

[0044] R in formula (3-A) 11The number of carbon atoms in the alkanediyl group represented by the formula (I) is preferably 3 to 10, more preferably 3 to 8, even more preferably 3 to 6, and particularly preferably 3 to 4, in order to improve cell adhesiveness, visibility, and fluidity (storage stability). The alkanediyl group may be linear or branched. Specific examples of the alkanediyl group include a propane-1,1-diyl group, a propane-1,2-diyl group, a propane-1,3-diyl group, a propane-2,2-diyl group, a butane-1,1-diyl group, a butane-1,2-diyl group, a butane-1,3-diyl group, a butane-1,4-diyl group, a pentane-1,1-diyl group, a pentane-1,2-diyl group, a pentane-1,3-diyl group, a pentan-1,4-diyl group, a pentan-1,5-diyl group, a pentan-1,6-diyl group, a pentan-1,7-diyl group, a pentan-1,8-diyl group, a pentan-1,9-diyl group, a pentan-2,10-diyl group, a pentan-2,2-diyl group, a pentan-2,3-diyl group, a pentan-2,4-diyl group, a pentan-2,5-diyl group, a pentan-2,6-diyl group, a pentan-2,7-diyl group, a pentan-2,8-diyl group, a pentan-2,9-diyl group, a pentan-2,10-diyl group, a pentan-2,11-diyl group, a pentan-2,12-diyl group, a pentan-2,13-diyl group, a pentan-2,2-diyl group, a butane-1,1-diyl group, a butane-2,2-diyl group, a butane-1,1 Examples thereof include hexane-1,4-diyl group, pentane-1,5-diyl group, hexane-1,1-diyl group, hexane-1,2-diyl group, hexane-1,3-diyl group, hexane-1,4-diyl group, hexane-1,5-diyl group, hexane-1,6-diyl group, heptane-1,7-diyl group, octane-1,8-diyl group, nonane-1,9-diyl group, and decane-1,10-diyl group.

[0045] Examples of monomers that provide the structural unit (3) include 3-hydroxypropyl(meth)acrylate, 3-methoxypropyl(meth)acrylate, 4-hydroxybutyl(meth)acrylate, 4-methoxybutyl(meth)acrylate, 5-hydroxypentyl(meth)acrylate, 5-methoxypentyl(meth)acrylate, 5-hydroxyhexyl(meth)acrylate, 5-methoxyhexyl(meth)acrylate, polyethylene glycol mono(meth)acrylate, methoxypolyethylene glycol(meth)acrylate, etc. These monomers can be used alone or in combination of two or more.

[0046] The content of the structural unit (3) is preferably 5% by mass or more, more preferably 10% by mass or more, even more preferably 15% by mass or more, and particularly preferably 20% by mass or more, relative to all structural units contained in the cell adhesive particles in order to improve visibility, fluidity (storage stability), and cell adhesiveness. Furthermore, the content of the structural unit (3) is preferably 50% by mass or less, more preferably 35% by mass or less, even more preferably 30% by mass or less, and particularly preferably 25% by mass or less, relative to all structural units contained in the cell adhesive particles in order to improve visibility, cell adhesiveness, and fluidity. Specifically, the content of the structural unit (3) is preferably 5% by mass or more and 50% by mass or less, more preferably 10% by mass or more and 35% by mass or less, even more preferably 15% by mass or more and 30% by mass or less, and particularly preferably 20% by mass or more and 25% by mass or less, relative to all structural units contained in the cell adhesive particles. When the content of the structural unit (3) is 15% by mass or more or 20% by mass or more, visibility, fluidity (storage stability), and cell adhesiveness are particularly good. Furthermore, when the content of the structural unit (3) is 30% by mass or less or 25% by mass or less, the visibility and cell adhesiveness are particularly good. The content of the structural unit (3) may be measured in the same manner as the content of the specific structural unit.

[0047] The content ratio of the specific structural unit to the structural unit (3) contained in the cell adhesive particles [(specific structural unit):(structural unit (3))] is preferably 5:95 to 95:5 by mass, more preferably 40:60 to 90:10, and particularly preferably 60:40 to 80:20, in order to improve visibility, fluidity (storage stability), and cell adhesiveness. When the content ratio [(specific structural unit):(structural unit (3))] is 60:40 to 80:20, visibility, fluidity (storage stability), and cell adhesiveness are particularly good.

[0048] (Structural Unit (4)) In order to enhance visibility, the cell adhesive particles of the present invention preferably contain, in addition to the specific structural unit, a structural unit represented by the following formula (4) in the molecule. In order to enhance visibility and fluidity (storage stability), the cell adhesive particles of the present invention preferably contain, in addition to the specific structural unit, both the structural unit (3) and the structural unit (4) in the molecule.

[0049]

[0050] [In formula (4), R 12 and R 13 each independently represents a hydrogen atom or a methyl group, R 14 represents an alkanediyl group having 1 or 2 carbon atoms.

[0051] R in formula (4) 13 In order to improve cell adhesiveness and visibility, R is preferably a hydrogen atom. 14 Examples of the alkyl group include a methane-1,1-diyl group, an ethane-1,1-diyl group, and an ethane-1,2-diyl group.

[0052] Examples of monomers that provide the structural unit (4) include 2-hydroxymethyl (meth)acrylate, 2-methoxymethyl (meth)acrylate, 2-hydroxyethyl (meth)acrylate, 2-methoxyethyl (meth)acrylate, etc. These monomers can be used alone or in combination of two or more.

[0053] The content of the structural unit (4) is preferably 1% by mass or more, more preferably 5% by mass or more, even more preferably 10% by mass or more, and particularly preferably 15% by mass or more, relative to all structural units contained in the cell adhesive particles in order to improve visibility and cell adhesiveness. Furthermore, in order to improve fluidity (storage stability), the content is preferably 60% by mass or less, more preferably 50% by mass or less, even more preferably 40% by mass or less, and particularly preferably 30% by mass or less, relative to all structural units contained in the cell adhesive particles. Specifically, the content is preferably 1% by mass or more and 60% by mass or less, more preferably 5% by mass or more and 50% by mass or less, even more preferably 10% by mass or more and 40% by mass or less, and particularly preferably 15% by mass or more and 30% by mass or less, relative to all structural units contained in the cell adhesive particles. When the content of the structural unit (4) is 15% by mass or more, visibility, fluidity (storage stability), and cell adhesiveness are particularly good. Furthermore, when the content of the structural unit (4) is 30% by mass or less, the visibility, fluidity (storage stability), and cell adhesiveness are particularly good. The content of the structural unit (4) may be measured in the same manner as the content of the specific structural unit.

[0054] The content ratio of the specific structural unit to the structural unit (4) contained in the cell adhesive particles [(specific structural unit):(structural unit (4))] is preferably 5:95 to 95:5 by mass, more preferably 40:60 to 90:10, and particularly preferably 60:40 to 80:20, in order to improve visibility, fluidity (storage stability), and cell adhesiveness. When the content ratio [(specific structural unit):(structural unit (4))] is 60:40 to 80:20, visibility, fluidity (storage stability), and cell adhesiveness are particularly good.

[0055] (Structural Unit Derived from Functional Group-Containing Monomer) In order to further improve adhesiveness, the cell-adhesive particles of the present invention preferably contain, in addition to the specific structural unit, a structural unit derived from a monomer containing a functional group capable of immobilizing a protein (hereinafter simply referred to as a "functional group-containing monomer") within the molecule. In order to further improve adhesiveness, the cell-adhesive particles of the present invention preferably contain, in the molecule, a structural unit derived from the specific structural unit, structural unit (3), structural unit (4), and functional group-containing monomer. Note that the concept of a functional group-containing monomer excludes the monomer that provides the specific structural unit, the monomer that provides structural unit (3), and the monomer that provides structural unit (4). Examples of functional groups contained in the above-mentioned monomers include functional groups selected from the group consisting of a cyclic ether group, a carboxy group, —C(═O)—O—C(═O)—, a succinimideoxycarbonyl group, a formyl group, a hydroxy group, and an isocyanate group. Among these, a cyclic ether group and a carboxy group are preferred. Here, the "cyclic ether group" is preferably a cyclic ether group having 3 to 7 atoms constituting the ring. The cyclic ether group may have an alkyl group as a substituent. Specific examples of the cyclic ether group include cyclic ether groups represented by the following formulas (10) to (15), with the cyclic ether group represented by formula (10), (12) or (13) being preferred, and the cyclic ether group represented by formula (10) being more preferred.

[0056]

[0057] [In the formula, R 40 ~R 43each independently represents a hydrogen atom or an alkyl group, and * represents a bonding site.

[0058] R 40 ~R 43 The number of carbon atoms in the alkyl group represented by the formula (I) is preferably 1 to 4, and more preferably 1 or 2. The alkyl group may be linear or branched, and examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, and a tert-butyl group. 40 ~R 43 is preferably a hydrogen atom.

[0059] The functional group-containing monomer is preferably a monomer having a functional group capable of immobilizing a protein and a polymerizable unsaturated group. Examples of such monomers include carboxy group-containing vinyl monomers such as (meth)acrylic acid and maleic acid; glycidyl (meth)acrylate, 3-oxiranylpropyl (meth)acrylate, 4-oxiranylbutyl (meth)acrylate, 5-oxiranylpentyl (meth)acrylate, 6-oxiranylhexyl (meth)acrylate, 7-oxiranylheptyl (meth)acrylate, 8-oxiranyloctyl (meth)acrylate, (3-methyloxiranyl)methyl (meth)acrylate, 4-hydroxybutyl (meth)acrylate glycidyl ether, glycerin mono(meth)acrylate glycidyl ether, 3,4-epoxycyclohexylmethyl (meth)acrylate ... Examples of suitable monomers include (meth)acrylate monomers having a cyclic ether group, such as 4-epoxycyclohexylethyl (meth)acrylate, 3,4-epoxycyclohexylpropyl (meth)acrylate, α-(meth)acryl-ω-glycidyl polyethylene glycol, and tetrahydrofurfuryl (meth)acrylate; allyl ether monomers having a cyclic ether group, such as allyl glycidyl ether; (meth)acrylate monomers having an isocyanate group, such as isocyanatoethyl (meth)acrylate; unsaturated dicarboxylic acid anhydride monomers, such as maleic anhydride, methylmaleic anhydride, and glutaconic anhydride; and 3,4-epoxy-1-butene and 3,4-epoxy-3-methyl-1-butene. These monomers may be used alone or in combination of two or more. Among these monomers, in order to facilitate and secure protein immobilization, preferred are carboxyl group-containing vinyl monomers and (meth)acrylate monomers having a cyclic ether group, with carboxyl group-containing vinyl monomers being more preferred.

[0060] The content of structural units derived from functional group-containing monomers is preferably 0.1% by mass or more, more preferably 1% by mass or more, relative to the total structural units contained in the cell-adhesive particles in order to facilitate and further strengthen protein immobilization. Furthermore, in order to suppress binding with impurities other than the protein to be immobilized, the content is preferably 60% by mass or less, more preferably 40% by mass or less, even more preferably 20% by mass or less, and particularly preferably 10% by mass or less, relative to the total structural units contained in the cell-adhesive particles. A specific range is preferably 0.1% by mass or more and 60% by mass or less, more preferably 0.1% by mass or more and 40% by mass or less, even more preferably 1% by mass or more and 20% by mass or less, and particularly preferably 1% by mass or more and 10% by mass or less, relative to the total structural units contained in the cell-adhesive particles. The content of structural units derived from functional group-containing monomers may be measured in the same manner as the content of specific structural units.

[0061] The ratio of the specific structural unit to the structural unit derived from the functional group-containing monomer contained in the cell adhesive particles [(specific structural unit):(structural unit derived from the functional group-containing monomer)] is preferably 30:70 to 99:1 by mass, more preferably 50:50 to 98:2, and particularly preferably 70:30 to 98:2, in order to facilitate protein immobilization, achieve stronger immobilization, and suppress binding with impurities other than the protein to be immobilized.

[0062] (Structural Unit Derived from Crosslinkable Monomer) In order to enhance visibility and fluidity, the cell-adhesive particles of the present invention preferably contain, in addition to the specific structural unit, a structural unit derived from a crosslinkable monomer within the molecule. In order to enhance visibility and fluidity, the cell-adhesive particles of the present invention preferably contain, in the molecule, a specific structural unit, structural unit (3), structural unit (4), a structural unit derived from a functional group-containing monomer, and a structural unit derived from a crosslinkable monomer. Note that the crosslinkable monomer is a concept excluding the monomer that provides the specific structural unit, the monomer that provides structural unit (3), the monomer that provides structural unit (4), and the functional group-containing monomer.

[0063] Examples of the crosslinkable monomer include (meth)acrylate crosslinkable monomers and allyl crosslinkable monomers. These may be used alone or in combination of two or more. The crosslinkable monomer is preferably a di- to penta-functional crosslinkable monomer, more preferably a di- or tri-functional crosslinkable monomer. Of the crosslinkable monomers, a (meth)acrylate crosslinkable monomer is preferred.

[0064] Examples of the (meth)acrylate crosslinkable monomer include ethylene glycol di(meth)acrylate, diethylene glycol di(meth)acrylate, triethylene glycol di(meth)acrylate, tetraethylene glycol di(meth)acrylate, polyethylene glycol di(meth)acrylate, propylene glycol di(meth)acrylate, dipropylene glycol di(meth)acrylate, tripropylene glycol di(meth)acrylate, tetrapropylene glycol di(meth)acrylate, polypropylene glycol di(meth)acrylate, 1,4-butanediol di(meth)acrylate, 1,6-hexanediol di(meth)acrylate, glycerin di(meth)acrylate, trimethylolethane di(meth)acrylate, trimethylolpropane di(meth)acrylate, and trimethylolpropane tri(meth)acrylate. acrylate, butanetriol di(meth)acrylate, pentaerythritol di(meth)acrylate, pentaerythritol tri(meth)acrylate, pentaerythritol tetra(meth)acrylate, glucose di(meth)acrylate, glucose tri(meth)acrylate, glucose tetra(meth)acrylate, dipentaerythritol di(meth)acrylate, dipentaerythritol tri(meth)acrylate, dipentaerythritol tetra(meth)acrylate, dipentaerythritol penta(meth)acrylate, inositol di(meth)acrylate, inositol tri(meth)acrylate, inositol tetra(meth)acrylate, mannitol di(meth)acrylate, mannitol tri(meth)acrylate, mannitol tetra(meth)acrylate, mannitol penta(meth)acrylate, etc. These can be used alone or in combination of two or more.

[0065] Examples of the allyl crosslinkable monomer include diallyl phthalate, diallyl isophthalate, diallyl terephthalate, diallyl maleate, diallyl fumarate, diallyl itaconate, diallyl trimellitate, triallyl trimellitate, triallyl cyanurate, diallyl isocyanurate, triallyl isocyanurate, pentaerythritol diallyl ether, pentaerythritol triallyl ether, pentaerythritol tetraallyl ether, allyl pentaerythritol (a mixture of pentaerythritol diallyl ether, pentaerythritol triallyl ether, and pentaerythritol tetraallyl ether), etc. These may be used alone or in combination of two or more.

[0066] The content of structural units derived from crosslinkable monomers is preferably 0.05% by mass or more, more preferably 0.1% by mass or more, relative to the total structural units contained in the cell-adhesive particles in order to enhance fluidity; and is preferably 10% by mass or less, more preferably 5% by mass or less, and particularly preferably 1% by mass or less, relative to the total structural units contained in the cell-adhesive particles in order to enhance visibility and suppress binding with impurities other than the protein to be immobilized. A specific range is preferably 0.05% by mass or more and 10% by mass or less, more preferably 0.05% by mass or more and 5% by mass or less, even more preferably 0.1% by mass or more and 5% by mass or less, and particularly preferably 0.1% by mass or more and 1% by mass or less, relative to the total structural units contained in the cell-adhesive particles. The content of structural units derived from crosslinkable monomers may be measured in the same manner as the content of specific structural units.

[0067] The ratio of the specific structural unit to the structural unit derived from the crosslinkable monomer contained in the cell adhesive particles [(specific structural unit):(structural unit derived from the crosslinkable monomer)] is preferably 80:20 to 99.9:0.1 by mass, more preferably 85:15 to 99.9:0.1, and particularly preferably 90:10 to 99.9:0.1, in order to improve visibility and fluidity and to suppress binding with impurities other than the protein to be immobilized.

[0068] The cell-adhesive particles of the present invention preferably contain at least a specific structural unit, one or more structural units selected from structural unit (3), structural unit (4), and structural units derived from a functional group-containing monomer, and a structural unit derived from a crosslinkable monomer; more preferably, they contain at least a specific structural unit, one or more structural units selected from structural unit (3) and structural unit (4), a structural unit derived from a functional group-containing monomer, and a structural unit derived from a crosslinkable monomer; and particularly preferably, they contain at least a specific structural unit, structural unit (3), structural unit (4), a structural unit derived from a functional group-containing monomer, and a structural unit derived from a crosslinkable monomer.

[0069] The cell-adhesive particles of the present invention may have structural units (hereinafter also referred to as "other structural units") other than the specific structural unit, the structural unit (3), the structural unit (4), the structural unit derived from a functional group-containing monomer, and the structural unit derived from a crosslinkable monomer. Examples include structural units derived from non-crosslinkable monomers other than the specific structural unit, the structural unit (3), the structural unit (4), and the structural unit derived from a functional group-containing monomer. Specific examples of non-crosslinkable monomers that provide other structural units include dialkylaminoalkyl (meth)acrylates such as N-[2-(diethylamino)ethyl](meth)acrylate, as well as methyl (meth)acrylate, ethyl (meth)acrylate, n-butyl (meth)acrylate, 4-tert-butyl (meth)acrylate, isobutyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, n-octyl (meth)acrylate, isononyl (meth)acrylate, lauryl (meth)acrylate, isodecyl (meth)acrylate, stearyl (meth)acrylate, isostearyl (meth)acrylate, (meth)acrylamide, N-methyl (meth)acrylamide, N,N-dimethyl (meth)acrylamide, N-methoxymethyl (meth)acrylamide, and N-ethoxymethyl (meth)acrylamide. Aromatic hydrocarbon group-containing monomers may also be used. These may be used alone or in combination of two or more. In order to improve visibility, fluidity (storage stability), and cell adhesiveness, the content of structural units derived from dialkylaminoalkyl (meth)acrylate is preferably 0 to 50 mass%, more preferably 0 to 20 mass%, more preferably 0 to 5 mass%, and particularly preferably 0 mass%, relative to all structural units contained in the cell adhesive particles.

[0070] In order to enhance stability during polymerization, the content of structural units derived from aromatic hydrocarbon group-containing monomers (e.g., styrene, α-methylstyrene, divinylbenzene, ethylvinylbenzene, etc.) is preferably 0 to 50 mass%, more preferably 0 to 30 mass%, even more preferably 0 to 10 mass%, and particularly preferably 0 mass%, relative to all structural units contained in the cell adhesive particles. When attempting to produce such particles, the hydrophilicity of the monomer composition increases, making it easier for the monomer to leak into the aqueous phase during suspension polymerization, making particle production generally difficult. However, the polymer particle production method of the present invention makes it possible to efficiently produce such particles.

[0071] The particles may be those to which a crosslinked structure has been introduced or surface modified as described in WO2019 / 039545, JP2011-252929A, WO2017 / 155105, JP62-25102A, etc.

[0072] In order to enhance cell adhesiveness, the average particle size of the cell adhesive particles of the present invention is preferably 50 μm or more and 500 μm or less, more preferably 75 μm or more and 375 μm or less, and particularly preferably 125 μm or more and 250 μm or less. When the average particle size of the cell adhesive particles of the present invention is 125 μm or more and 250 μm or less, cell adhesiveness is particularly good. The average particle size refers to the average area-equivalent diameter of 200 particles measured using an optical microscope in accordance with ISO 13322-1:2014. Specifically, the average particle size may be measured by the method described in the Examples below.

[0073] In order to enhance visibility and fluidity (storage stability), the cell adhesive particles of the present invention preferably have a sedimentation bulk density of 0.01 g / mL to 0.2 g / mL, more preferably 0.05 g / mL to 0.15 g / mL, even more preferably 0.08 g / mL to 0.13 g / mL, and particularly preferably 0.09 g / mL to 0.12 g / mL, when dispersed in an aqueous solution of 0.9% by weight sodium chloride at 25°C and 1 atmosphere. Visibility and fluidity (storage stability) are particularly favorable when the sedimentation bulk density is 0.09 g / mL to 0.12 g / mL. The sedimentation bulk density refers to the sedimentation bulk density when dispersed in an aqueous solution of 0.9% by weight sodium chloride at 25°C and 1 atmosphere. Specifically, the sedimentation bulk density may be measured by the method described in the Examples below.

[0074] In order to improve visibility, fluidity (storage stability), and cell adhesiveness, the cell adhesive particles of the present invention preferably contain specific structural units, structural unit (3) and structural unit (4) in their molecules, the content of the specific structural units being preferably 30% by mass or more and 65% by mass or less, more preferably 40% by mass or more and 55% by mass or less, based on the total structural units contained in the cell adhesive particles, and the average particle diameter is preferably 125 μm or more and 250 μm or less; the cell adhesive particles contain a specific structural unit, structural unit (3), and structural unit (4) in the molecule, the content of the specific structural unit being preferably 30% by mass or more and 65% by mass or less, more preferably 40% by mass or more and 55% by mass or less, relative to all structural units contained in the cell adhesive particles, the average particle size being 125 μm or more and 250 μm or less, and the method for producing polymer particles of the present invention is more preferably obtained by a method in which a monomer that provides the specific structural unit, a monomer that provides the structural unit (3), and a monomer that provides the structural unit (4) are used as the monomers used in step (ii) in the method for producing polymer particles of the present invention, and the aqueous medium in step (iii) is an aqueous medium in which, when the mass of salt per unit mass in a saturated solution obtained by dissolving a salt in the aqueous medium at 23° C. and 1 atmospheric pressure to reach a saturated solution is taken as 100 parts by mass, the amount of salt dissolved per unit mass is 30 parts by mass or more, and the hydrocolloid concentration is 0.75% by mass or more;The cell adhesive particles contain specific structural units, structural unit (3) and structural unit (4) in the molecule, and the content of the specific structural units is preferably 30% by mass or more and 65% by mass or less, more preferably 40% by mass or more and 55% by mass or less, based on the total structural units contained in the cell adhesive particles. The cell adhesive particles have an average particle size of 125 μm or more and 250 μm or less, and when dispersed in an aqueous solution of sodium chloride with a concentration of 0.9% by mass under conditions of 25° C. and 1 atmospheric pressure, the sedimentation bulk density is 0.09 g / mL or more and 0.12 g / mL or less. The cell adhesive particles are prepared by the polymer particle production method of the present invention. In the above, it is more preferred that the monomers used in step (ii) are a monomer that provides a specific structural unit, a monomer that provides the structural unit (3), and a monomer that provides the structural unit (4), and that the aqueous medium in step (iii) is an aqueous medium in which, when a salt is dissolved in the aqueous medium at 23°C and 1 atmospheric pressure to reach a saturated solution, the mass of the salt per unit mass in the saturated solution is taken as 100 parts by mass, the amount of the salt dissolved per unit mass is 30 parts by mass or more, and the hydrocolloid concentration is 0.75% by mass or more. The cell adhesive particles contain specific structural units, structural unit (3) and structural unit (4) in the molecule, and the content of the specific structural units is preferably 30% by mass or more and 65% by mass or less, more preferably 40% by mass or more and 55% by mass or less, based on the total structural units contained in the cell adhesive particles. The cell adhesive particles have an average particle size of 125 μm or more and 250 μm or less, and when dispersed in an aqueous solution of sodium chloride with a concentration of 0.9% by mass under conditions of 25° C. and 1 atmospheric pressure, the sedimentation bulk density is 0.09 g / mL or more and 0.12 g / mL or less. The cell adhesive particles are prepared by the polymer particle production method of the present invention. In the above, it is particularly preferred that the aqueous medium in step (iii) is an aqueous medium in which a salt is dissolved in the aqueous medium at 23°C and 1 atmospheric pressure to reach a saturated solution, and the salt dissolved in the aqueous medium per unit mass is 60 parts by mass or more, and the hydrocolloid concentration is 0.75% by mass or more, when the mass of the salt per unit mass in the saturated solution is 100 parts by mass.

[0075] The cell-adhesive particles of the present invention can be produced by an appropriate combination of known methods (e.g., the methods described in WO2015 / 80174, WO2015 / 119255, JP2008-9092A, and JP2018-59972A) except for using a monomer that imparts a specific structural unit. However, they are preferably produced by the following methods 1 and 2, and more preferably by method 1. (Method 1) A method in which a monomer that imparts a specific structural unit is used as the monomer used in step (ii) in the polymer particle production method of the present invention. (Method 2) A method in which a structural unit within a particle molecule is chemically modified with group (1) or group (2) directly or via a linker. For example, when producing polysaccharide-based cell-adhesive polymer particles, methods include (Method 2-1) preparing polysaccharide-based polymer particles containing structural units having carboxy groups in the molecule, and reacting the carboxy groups with an amine that provides group (1) or group (2) to the carboxy groups using a condensing agent (i.e., an amine having a chemical structure in which * in formulas (1) and (2) is replaced with a hydrogen atom), or (Method 2-2) chemically modifying intramolecular structural units of polysaccharide-based polymer particles with group (1) or group (2) via a linker. The polymer particle production method of the present invention will be described below.

[0076] [Method for Producing Polymer Particles] The method for producing polymer particles of the present invention comprises the following steps (i) to (iii), wherein the aqueous medium in step (iii) is an aqueous medium in which 30 parts by mass or more of salt is dissolved per unit mass, where the mass of salt per unit mass of the saturated solution obtained by dissolving a salt in the aqueous medium at 23°C and 1 atmospheric pressure is taken as 100 parts by mass: (i) a step of preparing a hydrocolloid in the salt-containing aqueous medium, (ii) a step of dispersing one or more monomers in the hydrocolloid-containing aqueous medium obtained in step (i), and (iii) a step of suspension polymerizing the monomer dispersed in step (ii) in the aqueous medium containing the hydrocolloid and the salt. Note that the salt in the "salt-containing aqueous medium" and the "aqueous medium containing the hydrocolloid and the salt" is present in an ionized state in the aqueous medium.

[0077] (Step (i)) Step (i) is a step of preparing a hydrocolloid in a salt-containing aqueous medium (preferably containing at least water as the aqueous medium).

[0078] As used herein, the term "hydrocolloid" refers to a colloid of an inorganic or polymeric compound that disperses well in an aqueous medium. The inorganic compound that provides the hydrocolloid is preferably an inorganic salt. Examples of inorganic compounds that provide the hydrocolloid include metal hydroxides such as magnesium hydroxide, calcium hydroxide, aluminum hydroxide, and ferric hydroxide; inorganic acid salts (sulfates such as calcium sulfate and barium sulfate; carbonates such as calcium carbonate, barium carbonate, and magnesium carbonate; and phosphates such as calcium phosphate); metal oxides such as titanium oxide, aluminum oxide, and zinc oxide, as well as magnesium silicate, talc, bentonite, and silica. Examples of polymeric compounds that provide the hydrocolloid include celluloses such as hydroxyethyl cellulose and methyl cellulose, as well as polyvinyl alcohol, polyethylene glycol, gellan gum, xanthan gum, guar gum, carrageenan, sodium alginate, and pullulan. Among hydrocolloids, in order to reduce leakage of monomers into the aqueous medium (aqueous phase) during suspension polymerization, hydrocolloids of poorly water-soluble metal salts, hydrocolloids of celluloses, and polyethylene glycols with a degree of polymerization of 2 to 10 are preferred, hydrocolloids of metal hydroxides and hydrocolloids of celluloses are more preferred, hydrocolloids of metal hydroxides and hydrocolloids of hydroxyalkyl cellulose are even more preferred, and hydrocolloids of magnesium hydroxide and hydrocolloids of hydroxyethyl cellulose are particularly preferred. Note that when the hydrocolloid is a hydrocolloid of poorly water-soluble metal salts, it is easy to separate it from the polymer particles. Note that the hydrocolloids may be used alone or in combination of two or more.

[0079] The salt contained in the aqueous medium used in step (i) is present in an ionized state in the aqueous medium. The salt may be a compound consisting of an anion and a cation. However, in the present invention, step (i) does not include cases where the salt contained in the aqueous medium used in step (i) is the same as the inorganic compound that provides the hydrocolloid, the polyvalent metal salt described below, or the monovalent metal compound. When using a colloid of an inorganic compound as the hydrocolloid, a salt different from the inorganic compound, the polyvalent metal salt that provides the hydrocolloid, or the monovalent metal compound must be used. Examples of salts include inorganic salts as well as organic salts such as citrates, tartrates, acetates (e.g., ammonium acetate), and trifluoroacetates (e.g., methylammonium trifluoroacetate). However, in order to reduce leakage of monomers into the aqueous medium (aqueous phase) during suspension polymerization, inorganic salts are preferred, more preferably alkali metal halides, alkaline earth metal halides, even more preferably alkali metal halides, and particularly preferably alkali metal chlorides. Examples include sodium chloride, potassium chloride, magnesium chloride, and calcium chloride. In addition, when the hydrocolloid is a hydrocolloid of an inorganic compound other than an inorganic acid salt or a hydrocolloid of a polymer compound, phosphates such as potassium dihydrogen phosphate, sulfates such as sodium sulfate, or nitrates may be used. These may be used alone or in combination of two or more.

[0080] The salt concentration of the aqueous medium used in step (i) may be any concentration that allows the salt concentration of the aqueous medium in step (iii) to fall within the desired range (when the mass of salt per unit mass in the saturated solution obtained by dissolving the salt in the aqueous medium at 23 ° C. and 1 atmosphere is taken as 100 parts by mass, the amount of salt dissolved per unit mass is 30 parts by mass or more). For example, when step (ii) involves adding a liquid composition in which a monomer is dissolved or dispersed in an aqueous medium to the hydrocolloid-containing aqueous medium obtained in step (i), the salt concentration relative to the total amount of the aqueous medium used in step (i) and the aqueous medium in the liquid composition may be determined so that the salt concentration falls within the desired range (when the mass of salt per unit mass in the saturated solution obtained by dissolving the salt in the aqueous medium at 23 ° C. and 1 atmosphere is taken as 100 parts by mass, the amount of salt dissolved per unit mass is 30 parts by mass or more).

[0081] As used herein, the term "aqueous medium" preferably refers to a medium containing at least water. Examples include water, alcohol, and a mixture of water and alcohol, with water and a mixture of water and alcohol being preferred. The alcohol is preferably a linear or branched monohydric alcohol having 1 to 10 carbon atoms, more preferably a linear monohydric alcohol having 3 to 8 carbon atoms. Examples include ethanol, isopropanol, n-propanol, butanol, pentanol, hexanol, and octanol. One of these may be used alone, or two or more may be used in combination. When dispersing the monomer in the hydrocolloid-containing aqueous medium in step (ii), the monomer may be added directly to the hydrocolloid-containing aqueous medium. Alternatively, the monomer and other components may be added to an aqueous medium other than the hydrocolloid-containing aqueous medium to form a monomer composition, and then the monomer composition may be added to the hydrocolloid-containing aqueous medium. In the mixture of water and alcohol, the water content is preferably 50% by mass or more but less than 100% by mass.

[0082] The preparation of the hydrocolloid can be carried out by appropriately combining known techniques described in JP 2008-9092 A, JP 2018-59972 A, etc., except that the salt concentration of the aqueous medium in step (iii) is within the desired range (when the mass of salt per unit mass in the saturated solution obtained by dissolving salt in the aqueous medium at 23°C and 1 atmospheric pressure is taken as 100 parts by mass, the aqueous medium contains 30 parts by mass or more of dissolved salt per unit mass). For example, a hydrocolloid of a polymer compound can be prepared by adding a polymer compound that provides a hydrocolloid to an aqueous medium having a salt concentration within the desired range in step (iii) and stirring the mixture. Furthermore, a hydrocolloid of an inorganic compound (particularly a poorly water-soluble metal salt) can be prepared by stirring a polyvalent metal salt and a monovalent metal compound in an aqueous medium having a salt concentration within the desired range in step (iii). For example, in step (iii), a monovalent metal compound is added to an aqueous medium containing a polyvalent metal salt at a salt concentration such that the salt concentration of the aqueous medium in the desired range, followed by stirring; in step (iii), a monovalent metal salt is added to an aqueous medium containing a monovalent metal compound at a salt concentration such that the salt concentration of the aqueous medium in the desired range, followed by stirring; in step (iii), a monovalent metal compound is added to an aqueous medium containing a polyvalent metal salt at a salt concentration such that the salt concentration of the aqueous medium in the desired range, followed by stirring; and in step (iii), a monovalent metal compound is added to an aqueous medium containing a polyvalent metal salt at a salt concentration such that the salt concentration of the aqueous medium in the desired range, followed by stirring. Hydrates of polyvalent metal salts may also be used as the polyvalent metal salt. The stirring time for preparing hydrocolloids of polymeric compounds or hydrocolloids of inorganic compounds is typically 1 to 60 minutes, preferably 5 to 30 minutes. The stirring temperature when preparing a hydrocolloid of a polymer compound or a hydrocolloid of an inorganic compound is usually 10 to 95°C, preferably 20 to 80°C.

[0083] The polyvalent metal salt is preferably one or more selected from polyvalent metal halides and their hydrates, more preferably one or more selected from magnesium halides, calcium halides, aluminum halides, iron halides, and their hydrates, such as magnesium chloride, magnesium bromide, calcium chloride, calcium bromide, aluminum chloride, aluminum bromide, iron chloride, iron bromide, and their hydrates.

[0084] The monovalent metal compound is preferably an alkali metal hydroxide, more preferably one or more selected from sodium hydroxide, lithium hydroxide and potassium hydroxide, and particularly preferably sodium hydroxide.

[0085] The amount of the hydrocolloid prepared in step (i), the amount of the polyvalent metal salt used, and the amount of the monovalent metal compound used may be determined so that the hydrocolloid content in the aqueous medium in step (iii) is the desired amount.

[0086] (Step (ii)) Step (ii) is a step of dispersing one or more monomers in the hydrocolloid-containing aqueous medium obtained in step (i). Specific examples include a method of mixing a liquid composition in which one or more monomers are dissolved or dispersed in an aqueous medium with the hydrocolloid-containing aqueous medium. More specific examples include a method of mixing using a stirrer equipped with stirring blades, a method of passing through the pores of a porous membrane, a method of passing through a microchannel, a method of colliding droplets with each other at high pressure, and a method of irradiating with ultrasound, and the like, and can be appropriately selected depending on the target particle size. Here, in step (i), the salt-containing aqueous medium is an aqueous medium containing at least water, and step (ii) is a step of mixing the hydrocolloid-containing aqueous medium obtained in step (i) with the liquid composition. When the total mass of water contained in the hydrocolloid-containing aqueous medium is 100 parts by mass, the proportion of the liquid composition used in step (ii) is preferably 5 parts by mass to 40 parts by mass, more preferably 10 parts by mass to 30 parts by mass, and particularly preferably 15 parts by mass to 27.5 parts by mass. When the proportion of the liquid composition used in step (ii) is within this range, leakage of monomers into the aqueous phase during suspension polymerization is reduced. Furthermore, when the proportion of the liquid composition used in step (ii) is 10 parts by mass or more or 15 parts by mass or more, visibility and cell adhesiveness are particularly good. When the proportion of the liquid composition used in step (ii) is 30 parts by mass or less or 27.5 parts by mass or less, fluidity (storage stability) is particularly good. Examples of the aqueous medium contained in the liquid composition include the same aqueous medium as that used in step (i).

[0087] The monomer used in step (ii) may be any monomer that provides particles, and examples thereof include one or more selected from non-crosslinking monomers and crosslinking monomers. Examples of non-crosslinking monomers include monomers that provide the specific structural unit described above, monomers that provide structural unit (3), monomers that provide structural unit (4), functional group-containing monomers, and non-crosslinking monomers that provide other structural units. Examples of these non-crosslinking monomers and crosslinking monomers that can be used include those listed as those that provide structural units that may be contained in the cell-adhesive particles of the present invention. These monomers may be used alone or in combination of two or more. When producing the cell-adhesive particles of the present invention, it is preferable to use at least a monomer that provides a specific structural unit. It is more preferable to use at least one or more monomers selected from the group consisting of a monomer that provides the specific structural unit, a monomer that provides the structural unit (3), a monomer that provides the structural unit (4), and a functional group-containing monomer, and a crosslinkable monomer. It is even more preferable to use at least a monomer that provides the specific structural unit, a monomer that provides the structural unit (3), and a monomer that provides the structural unit (4), a functional group-containing monomer, and a crosslinkable monomer. It is particularly preferable to use at least a monomer that provides the specific structural unit, a monomer that provides the structural unit (3), a monomer that provides the structural unit (4), a functional group-containing monomer, and a crosslinkable monomer. The amount of each monomer used may be determined so that the structural units contained in the cell-adhesive particles of the present invention are contained in the desired proportion. Even when such highly hydrophilic monomers are used, the polymer particle production method of the present invention is less likely to result in leakage of the monomer into the aqueous phase during suspension polymerization.

[0088] Furthermore, in order to enhance stability during polymerization, the amount of aromatic hydrocarbon group-containing monomer (e.g., styrene, α-methylstyrene, divinylbenzene, ethylvinylbenzene, etc.) used is preferably 0 to 50 parts by mass, more preferably 0 to 30 parts by mass, even more preferably 0 to 10 parts by mass, and particularly preferably 0 part by mass, per 100 parts by mass of the total monomers. When attempting to produce such particles, the hydrophilicity of the monomer composition increases, making it easier for the monomer to leak into the aqueous phase during suspension polymerization, and thus particle production is generally difficult. However, according to the polymer particle production method of the present invention, such particles can be produced efficiently.

[0089] (Step (iii)) Step (iii) is a step of suspension polymerizing the monomer dispersed in step (ii) in an aqueous medium containing a hydrocolloid and a salt. In this step, suspension polymerization is carried out in an aqueous medium containing the hydrocolloid prepared in step (i), and in which the salt is dissolved per unit mass of 30 parts by mass or more, assuming that the mass of salt per unit mass of the saturated solution obtained by dissolving the salt in the aqueous medium at 23°C and 1 atmosphere conditions to reach a saturated solution, is 100 parts by mass. This makes it difficult for the monomer to leak into the aqueous medium (aqueous phase) during suspension polymerization. In particular, even if the monomer is highly hydrophilic, it makes it difficult for the monomer to leak into the aqueous medium (aqueous phase) during suspension polymerization.

[0090] The total amount of the monomers used is preferably 1 part by mass or more, more preferably 5 parts by mass or more, even more preferably 10 parts by mass or more, particularly preferably 15 parts by mass or more, relative to 100 parts by mass of the aqueous medium in order to enhance the stability during polymerization, and is preferably 100 parts by mass or less, more preferably 49 parts by mass or less, even more preferably 40 parts by mass or less, particularly preferably 30 parts by mass or less, relative to 100 parts by mass of the aqueous medium in order to enhance the uniformity of the polymer particles. The specific range is preferably 1 part by mass or more and 100 parts by mass or less, more preferably 5 parts by mass or more and 49 parts by mass or less, even more preferably 10 parts by mass or more and 40 parts by mass or less, particularly preferably 15 parts by mass or more and 30 parts by mass or less, relative to 100 parts by mass of the aqueous medium.

[0091] The amount of salt dissolved per unit mass of the aqueous medium in step (iii) is 30 parts by mass or more, assuming that the mass of salt per unit mass in the saturated solution obtained by dissolving the salt in the aqueous medium at 23°C and 1 atmospheric pressure is 100 parts by mass. However, in order to improve visibility and cell adhesiveness and to suppress leakage of monomer into the aqueous phase, the amount of salt dissolved per unit mass in the aqueous medium is preferably 40 parts by mass or more, more preferably 50 parts by mass or more, and particularly preferably 60 parts by mass or more, with the upper limit being, for example, 100 parts by mass. Particularly good visibility and cell adhesiveness are obtained when the amount of salt dissolved per unit mass is 60 parts by mass or more, assuming that the mass of salt per unit mass in the saturated solution obtained by dissolving the salt in the aqueous medium at 23°C and 1 atmospheric pressure is 100 parts by mass.

[0092] The hydrocolloid concentration of the aqueous medium in step (iii) is preferably 0.01% by mass or more, more preferably 0.1% by mass or more, even more preferably 0.5% by mass or more, particularly preferably 0.75% by mass or more, relative to the total mass of the aqueous medium containing the hydrocolloid and salt, in order to improve visibility, fluidity (storage stability), and cell adhesiveness, and to suppress leakage of monomers into the aqueous phase. Furthermore, in order to improve stability during polymerization, the hydrocolloid concentration is preferably 10% by mass or less, more preferably 8% by mass or less, even more preferably 5% by mass or less, particularly preferably 3% by mass or less. Specific ranges are preferably 0.01% by mass or more and 10% by mass or less, more preferably 0.1% by mass or more and 8% by mass or less, more preferably 0.5% by mass or more and 5% by mass or less, and particularly preferably 0.75% by mass or more and 3% by mass or less, relative to the total mass of the aqueous medium containing the hydrocolloid and salt. When the hydrocolloid concentration in the aqueous medium in step (iii) is 0.75% by mass or more, the visibility, fluidity (storage stability) and cell adhesiveness become particularly good.

[0093] Here, more specific methods of steps (ii) to (iii) include, for example, a liquid composition obtained by dissolving or dispersing one or more monomers and a polymerization initiator in an aqueous medium, and a hydrocolloid-containing aqueous medium, and a method of polymerizing by heating to a predetermined temperature, a liquid composition obtained by dissolving or dispersing one or more monomers and a polymerization initiator in an aqueous medium, and a method of adding the liquid composition to a hydrocolloid-containing aqueous medium heated to a predetermined temperature and polymerizing it, preferably a liquid composition obtained by dissolving or dispersing one or more monomers and a polymerization initiator in an aqueous medium, and a hydrocolloid-containing aqueous medium, and a method of polymerizing it by heating to a predetermined temperature. Note that the liquid composition may contain a porosifying agent as described in JP 2018-109643 A, etc., as necessary.

[0094] The polymerization initiator is preferably a radical polymerization initiator. Examples of the radical polymerization initiator include azo initiators, peroxide initiators, and redox initiators. More specific examples include azobisisobutyronitrile, methyl azobisisobutyrate, dimethyl azobisisobutyrate, azobis-2,4-dimethylvaleronitrile, benzoyl peroxide, di-tert-butyl peroxide, and benzoyl peroxide-dimethylaniline. The amount of the polymerization initiator used is usually about 0.5 parts by mass or more and 5 parts by mass or less per 100 parts by mass of the total monomers.

[0095] The polymerization temperature for suspension polymerization may be appropriately determined depending on the type of polymerization initiator, etc., but is usually about 2 to 100° C. The polymerization time is usually 5 minutes to 48 hours.

[0096] The suspension polymerization in step (iii) is preferably carried out by stirring, emulsifying dispersion, or ultrasonic irradiation, and is particularly preferably carried out by stirring. The stirring speed is usually 200 rpm or higher, and to enhance cell adhesiveness, it is preferably 300 to 1200 rpm, and particularly preferably 600 to 800 rpm. When the stirring speed in step (iii) is 600 to 800 rpm, cell adhesiveness is particularly good.

[0097] (Step (iv)) In order to obtain high-purity polymer particles, the method for producing polymer particles of the present invention preferably further comprises the following step (iv) in addition to steps (i) to (iii): (iv) A step of separating the polymer particles obtained in step (iii) from the hydrocolloid.

[0098] In step (iv), for example, when the hydrocolloid is highly water-soluble, such as hydroxyethyl cellulose hydrocolloid, washing with water can be performed. Alternatively, when the hydrocolloid is poorly water-soluble, such as magnesium hydroxide hydrocolloid, the hydrocolloid in the liquid phase containing the polymer particles can be treated with an acid or alkali. This method can decompose the hydrocolloid in the liquid phase.

[0099] The resulting polymer particles may be subjected to the introduction of a crosslinked structure or surface modification as described in WO2019 / 039545, JP 2011-252929 A, WO2017 / 155105, JP 62-25102 A, etc. Proteins may be bound to the particles with reference to known techniques described in JP 2023-5966 A, JP 2022-121125 A, JP 2017-83363 A, Biomacromolecules 2007, 8, 6, 1775-1789, WO2015 / 119288, etc. The reaction products obtained in each of the above steps may be purified by separation means such as filtration, washing, dehydration, and drying. A classification treatment may also be performed.

[0100] The average particle size of the particles obtained by the polymer particle production method of the present invention is preferably 50 μm or more and 500 μm or less, more preferably 75 μm or more and 375 μm or less, and particularly preferably 125 μm or more and 250 μm or less, in order to enhance cell adhesiveness. Cell adhesiveness is particularly good when the average particle size of the particles is 125 μm or more and 250 μm or less. The average particle size refers to the average area-equivalent diameter of 200 particles measured using an optical microscope in accordance with ISO 13322-1:2014. Specifically, the average particle size may be measured by the method described in the Examples below.

[0101] The particles obtained by the polymer particle production method of the present invention can be uniformly stirred without damaging the particles and cells, and in order to improve visibility and fluidity (storage stability), when dispersed in an aqueous solution of 0.9% by weight of sodium chloride at 25°C and 1 atmosphere, preferably have a sedimentation bulk density of 0.01 g / mL to 0.2 g / mL, more preferably 0.05 g / mL to 0.15 g / mL, even more preferably 0.08 g / mL to 0.13 g / mL, and particularly preferably 0.09 g / mL to 0.12 g / mL. Visibility and fluidity (storage stability) are particularly good when the sedimentation bulk density is 0.09 g / mL to 0.12 g / mL. The sedimentation bulk density refers to the sedimentation bulk density when dispersed in an aqueous solution of 0.9% by weight of sodium chloride at 25°C and 1 atmosphere. Specifically, it can be measured by the method described in the Examples below.

[0102] Furthermore, in the polymer particle production method of the present invention, the cell-adhesive particles of the present invention can be produced by a method in which a monomer that provides a specific structural unit is used as the monomer used in step (ii).

[0103] Furthermore, the polymer particle production method of the present invention allows for simple and efficient production of polymer particles. In particular, even when the monomer is highly hydrophilic, the monomer is less likely to leak into the aqueous medium (aqueous phase) during suspension polymerization, allowing for simple and efficient production of polymer particles. Furthermore, the cell adhesive particles of the present invention have excellent cell adhesiveness. They also have water absorbency. "Cell adhesiveness" refers to the property of making cells more likely to adhere to a substrate due to chemical or physical action. Furthermore, "water absorbency" refers to the property of swelling upon contact with water. Therefore, the cell adhesive particles of the present invention are suitable for use in microcarrier culture.

[0104] [Cell Culture Method] The cell culture method of the present invention is characterized by using the cell adhesive particles of the present invention. For example, cells can be cultured by adding cells, the cell adhesive particles of the present invention, and, if necessary, components selected from serum, cell growth factors, and antibacterial agents to a medium and culturing the cells. Specifically, known methods such as those described in JP 2018-99115 A can be appropriately combined. The cell culture method of the present invention is suitable for microcarrier culture.

[0105] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0106] Example 1 (1) Aqueous solution S was prepared by adding 63.2 g of sodium chloride and 18.0 g of magnesium chloride hexahydrate to 217.9 g of pure water and stirring to dissolve the mixture. Next, 9.1 g of 1-hexanol as a solvent and 0.45 g of dimethyl 2,2'-azobis(isobutyrate)ate as an initiator were added to a composition consisting of 10.0 g of 2-hydroxyethyl methacrylate, 10.0 g of hydroxypropyl methacrylate, 22.7 g of N-[3-(dimethylamino)propyl]acrylamide, 2.3 g of methacrylic acid, and 0.34 g of ethylene glycol dimethacrylate to prepare a monomer composition. (2) The entire amount of the aqueous solution S was poured into a separable flask, which was then fitted with a thermometer, stirring blade, and condenser. The flask was then placed in a hot water bath and stirring commenced under a nitrogen atmosphere. 45.9 g of a 5 M aqueous sodium hydroxide solution was added to the separable flask to prepare a magnesium hydroxide hydrocolloid dispersion. Here, the total mass of water contained in the magnesium hydroxide hydrocolloid dispersion was calculated from the mass of pure water used in preparing the aqueous solution S, the mass of water introduced from the magnesium chloride hexahydrate, and the mass of water introduced from the 5M sodium hydroxide aqueous solution. The proportion of the monomer composition used was then calculated, assuming the total mass of water contained in this magnesium hydroxide hydrocolloid dispersion was 100 parts by mass. This value is shown in Table 1. Next, the stirring speed was set to 700 rpm, and the entire amount of the monomer composition was charged into a separable flask, which was then heated in a hot water bath until the internal temperature reached 86°C. (3) Next, while maintaining the temperature at 86°C, the mixture was stirred for 3 hours to allow suspension polymerization, and then cooled to room temperature. Next, 5M hydrochloric acid was added and stirred until the pH of the supernatant was 5.0 or less. Next, pure water was added to the reaction solution, and the solution was removed by decantation to wash the particles. The mixture was then replaced with t-butyl alcohol, frozen at -20°C, and vacuum dried. The particles were classified using a sieve so as to have the average particle diameter shown in Table 1. The particles obtained in Example 1 are referred to as dried particles 1.

[0107] Example 2 Example 1 The same steps as steps (1) to (3) of Example 1 were carried out to obtain dried particles 2, except that in the preparation of the monomer composition in step (1), 2-hydroxyethyl methacrylate was not used and the amount of hydroxypropyl methacrylate used was changed to 20.1 g. Note that, in the same manner as in Example 1, the proportion of the monomer composition used was calculated when the total mass of water contained in the magnesium hydroxide hydrocolloid aqueous dispersion was taken as 100 parts by mass. The values ​​are shown in Table 1.

[0108] Example 3 Example 1 In the preparation of the monomer composition in step (1), the amount of 2-hydroxyethyl methacrylate used was changed to 20.1 g, and hydroxypropyl methacrylate was not used, and the same steps as steps (1) to (3) of Example 1 were carried out to obtain dried particles 3. In the same manner as in Example 1, the proportion of the monomer composition used was calculated when the total mass of water contained in the magnesium hydroxide hydrocolloid aqueous dispersion was taken as 100 parts by mass. The values ​​are shown in Table 1.

[0109] Example 4 Example 1 In the preparation of the monomer composition in step (1), the same steps as steps (1) to (3) of Example 1 were carried out, except that the amounts of 2-hydroxyethyl methacrylate, hydroxypropyl methacrylate, and N-[3-(dimethylamino)propyl]acrylamide used were changed to 14.6 g, 14.6 g, and 13.6 g, respectively, to obtain dried particles 4. In the same manner as in Example 1, the proportion of the monomer composition used was calculated when the total mass of water contained in the magnesium hydroxide hydrocolloid aqueous dispersion was taken as 100 parts by mass. The values ​​are shown in Table 1.

[0110] Example 5 Example 1 In the preparation of the monomer composition in step (1), the same steps as steps (1) to (3) of Example 1 were carried out, except that the amounts of 2-hydroxyethyl methacrylate, hydroxypropyl methacrylate, and N-[3-(dimethylamino)propyl]acrylamide used were changed to 5.5 g, 5.5 g, and 31.8 g, respectively, to obtain dried particles 5. In the same manner as in Example 1, the proportion of the monomer composition used was calculated when the total mass of water contained in the magnesium hydroxide hydrocolloid aqueous dispersion was taken as 100 parts by mass. The values ​​are shown in Table 1.

[0111] Example 6 Example 1 In the preparation of the monomer composition in step (1), the amount of N-[3-(dimethylamino)propyl]acrylamide used was changed to 11.4 g, and 11.4 g of N-[2-(diethylamino)ethyl]methacrylate was further used, and the same steps as steps (1) to (3) of Example 1 were carried out to obtain dried particles 6. Note that, in the same manner as in Example 1, the proportion of the monomer composition used was calculated when the total mass of water contained in the magnesium hydroxide hydrocolloid aqueous dispersion was taken as 100 parts by mass. The values ​​are shown in Table 1.

[0112] (Example 7) The same steps as steps (1) to (3) of Example 1 were carried out, except that the stirring speed in step (2) of Example 1 was changed to 900 rpm, to obtain dried particles 7. The proportion of the monomer composition used was calculated in the same manner as in Example 1, assuming that the total mass of water contained in the magnesium hydroxide hydrocolloid aqueous dispersion was 100 parts by mass. The calculated value is shown in Table 2.

[0113] Example 8 The same steps as steps (1) to (3) of Example 1 were carried out, except that the stirring speed in step (2) of Example 1 was changed to 500 rpm, to obtain dried particles 8. The proportion of the monomer composition used was calculated in the same manner as in Example 1, assuming that the total mass of water contained in the magnesium hydroxide hydrocolloid aqueous dispersion was 100 parts by mass. The calculated value is shown in Table 2.

[0114] Example 9 (1) A hydroxyethyl cellulose hydrocolloid dispersion was prepared by adding 77.2 g of sodium chloride and 0.77 g of hydroxyethyl cellulose to 257.2 g of pure water and dissolving the mixture with stirring. Next, 10.7 g of 1-hexanol as a solvent and 0.54 g of dimethyl 2,2'-azobis(isobutyrate)ate as an initiator were added to a composition consisting of 11.9 g of 2-hydroxyethyl methacrylate, 11.9 g of hydroxypropyl methacrylate, 26.8 g of N-[3-(dimethylamino)propyl]acrylamide, 2.7 g of methacrylic acid, and 0.27 g of ethylene glycol dimethacrylate to prepare a monomer composition. The proportion of the monomer composition used was calculated based on the total mass of water contained in the hydroxyethyl cellulose hydrocolloid dispersion, taken as 100 parts by mass. This value is shown in Table 2. (2) The entire hydrocolloid dispersion solution was poured into a separable flask, which was then fitted with a thermometer, stirring blades, and a condenser. The flask was then placed in a hot water bath, and stirring was initiated under a nitrogen atmosphere. The stirring speed was then set to 700 rpm, and the entire monomer composition was poured into the separable flask. The flask was then heated in the hot water bath until the internal temperature reached 86°C. (3) Stirring was continued for 3 hours while maintaining the temperature at 86°C, and the mixture was then cooled to room temperature. Purified water was then added to the slurry, and the solution was removed by decantation to wash the particles. The mixture was then substituted with t-butyl alcohol, frozen at -20°C, and vacuum-dried. Sieving was then performed to obtain the average particle diameter shown in Table 2. The particles obtained in Example 9 are referred to as dried particles 9.

[0115] Example 10 Example 1 The same steps as steps (1) to (3) of Example 1 were carried out to obtain dried particles 10, except that in the preparation of aqueous solution S in step (1), the amount of pure water used was changed to 231.9 g and the amount of sodium chloride used was changed to 46.4 g. Note that, in the same manner as in Example 1, the proportion of the monomer composition used was calculated when the total mass of water contained in the magnesium hydroxide hydrocolloid aqueous dispersion was taken as 100 parts by mass. The values ​​are shown in Table 2.

[0116] Example 11 Example 1 The same steps as steps (1) to (3) of Example 1 were carried out to obtain dried particles 11, except that in the preparation of aqueous solution S in step (1), the amount of pure water used was changed to 249.6 g and the amount of sodium chloride used was changed to 25.0 g. Note that, in the same manner as in Example 1, the proportion of the monomer composition used was calculated when the total mass of water contained in the magnesium hydroxide hydrocolloid dispersion aqueous solution was taken as 100 parts by mass. The values ​​are shown in Table 2.

[0117] Example 12 Example 1 In the preparation of aqueous solution S in step (1), the amounts of pure water, sodium chloride, and magnesium chloride hexahydrate were changed to 253.7 g, 61.5 g, and 5.9 g, respectively, and in step (2) of Example 1, the amount of 5 M aqueous sodium hydroxide solution was changed to 14.9 g. Except for this, steps (1) to (3) of Example 1 were carried out to obtain dried particles 12. Note that, in the same manner as in Example 1, the proportion of the monomer composition used was calculated when the total mass of water contained in the magnesium hydroxide hydrocolloid dispersion aqueous solution was taken as 100 parts by mass. The values ​​are shown in Table 2.

[0118] Example 13 (1) 50.0 g of a 2% by mass gellan gum aqueous solution was dropped into 1 L of 1 M calcium chloride aqueous solution through a tube with an inner diameter of 80 μm to produce particles. The resulting particles were then recovered with a 0.05 M calcium chloride aqueous solution to a total weight of 200 g. (2) 3.8 g of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate was added to the recovered particles and stirred for 1.5 hours. Next, 1.8 g of hexamethylenediamine and 1.6 g of 3-(dimethylamino)-1-propylamine were added and stirred for 3 hours. Pure water was then added to the reaction solution, and the solution was removed by decantation to wash the particles. The mixture was then replaced with t-butyl alcohol, frozen at -20°C, and vacuum-dried to obtain dried particles 13.

[0119] Example 14 In Example 1, the ratio of the monomer composition used was changed relative to 100 parts by mass of the total mass of water contained in the hydrocolloid-containing aqueous medium. The specific procedure was as follows. In the preparation of aqueous solution S in step (1) of Example 1, the amounts of pure water used were changed to 201.3 g, sodium chloride used to 58.4 g, and magnesium chloride hexahydrate used to 16.7 g, respectively. In the preparation of the monomer composition in step (1) of Example 1, the amounts of 2-hydroxyethyl methacrylate used to 14.9 g, hydroxypropyl methacrylate used to 14.9 g, N-[3-(dimethylamino)propyl]acrylamide used to 33.6 g, methacrylic acid used to 3.4 g, ethylene glycol dimethacrylate used to 0.50 g, 1-hexanol used to 13.4 g, and 2,2'-azobis(isobutyrate)dimethyl used to 0.67 g, respectively. In addition, in step (2) of Example 1, the amount of 5 M aqueous sodium hydroxide solution used to 42.4 g was changed. Otherwise, the same steps as steps (1) to (3) of Example 1 were performed to obtain dried particles 14. The proportion of the monomer composition used was calculated in the same manner as in Example 1, assuming that the total mass of water contained in the magnesium hydroxide hydrocolloid dispersion was 100 parts by mass. The values ​​are shown in Table 3.

[0120] Example 15 In Example 1, the ratio of the monomer composition used was changed relative to 100 parts by mass of the total mass of water contained in the hydrocolloid-containing aqueous medium. The specific procedure was as follows. In the preparation of aqueous solution S in step (1) of Example 1, the amounts of pure water used were changed to 237.5 g, sodium chloride used to 68.9 g, and magnesium chloride hexahydrate used to 19.7 g. In the preparation of the monomer composition in step (1) of Example 1, the amounts of 2-hydroxyethyl methacrylate used to 4.4 g, hydroxypropyl methacrylate used to 4.4 g, N-[3-(dimethylamino)propyl]acrylamide used to 9.9 g, methacrylic acid used to 1.0 g, ethylene glycol dimethacrylate used to 0.15 g, 1-hexanol used to 4.0 g, and 2,2'-azobis(isobutyrate)dimethyl used to 0.2 g. In addition, in step (2) of Example 1, the amount of 5 M aqueous sodium hydroxide solution used was changed to 50.1 g. Dry particles 15 were obtained by performing the same steps as in steps (1) to (3) of Example 1. The proportion of the monomer composition used was calculated in the same manner as in Example 1, assuming that the total mass of water contained in the magnesium hydroxide hydrocolloid dispersion was 100 parts by mass. The values ​​are shown in Table 3.

[0121] Reference Example 1 Example 1 In the preparation of the monomer composition in step (1), the same steps as steps (1) to (3) of Example 1 were carried out, except that the amount of 2-hydroxyethyl methacrylate used was changed to 21.4 g, the amount of hydroxypropyl methacrylate used was changed to 21.4 g, and N-[3-(dimethylamino)propyl]acrylamide was not used, to obtain dried particles 16. In the same manner as in Example 1, the proportion of the monomer composition used was calculated when the total mass of water contained in the magnesium hydroxide hydrocolloid aqueous dispersion was taken as 100 parts by mass. The values ​​are shown in Table 4.

[0122] Reference Example 2 The same steps (1) to (3) as in Example 1 were carried out, except that the amount of pure water used in preparing aqueous solution S in Reference Example 1 was changed to 319.5 g and sodium chloride and magnesium chloride hexahydrate were not used. However, the polymerization reaction did not proceed well, and particles could not be prepared. The proportion of the monomer composition used was calculated in the same manner as in Example 1, assuming that the total mass of water contained in the magnesium hydroxide hydrocolloid aqueous dispersion was 100 parts by mass. The values ​​are shown in Table 4.

[0123] Reference Example 3 Example 1 The same steps as steps (1) to (3) of Example 1 were carried out, except that in the preparation of the monomer composition in step (1), 22.7 g of N-[3-(dimethylamino)propyl]acrylamide was changed to 22.7 g of N-[2-(diethylamino)ethyl]methacrylate, to obtain dried particles 17. The proportion of the monomer composition used was calculated in the same manner as in Example 1, assuming that the total mass of water contained in the magnesium hydroxide hydrocolloid aqueous dispersion was 100 parts by mass. The values ​​are shown in Table 4.

[0124] (Test Example 1: Evaluation of Average Particle Diameter) 1 g of dried particles 1 to 17 obtained in each Example and Reference Example was weighed and dispersed in 20 g of PBS(-) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) for 3 hours to prepare a slurry. Next, in accordance with ISO13322-1:2014, the area-equivalent diameters of 400 particles measured using an optical microscope BX53 manufactured by Evident Co., Ltd. were averaged, and this value was taken as the average particle diameter. Note that if particles overlapped during measurement, they were diluted with PBS(-). The results are shown in Tables 1 to 4.

[0125] (Test Example 2 Bulk Density) 1 g of dried particles 1 to 17 obtained in each Example and Reference Example was weighed and dispersed in 20 g of an aqueous solution containing 0.9% by mass of sodium chloride for 3 hours to form a slurry. The slurry was then transferred to a 25 mL measuring cylinder and allowed to stand overnight at 25°C and 1 atmosphere, after which the volume of the particle layer was measured. The bulk density was calculated using the following formula. The results are shown in Tables 1 to 4.

[0126] Bulk density (g / mL) = (weight of dry particles (g) / volume of particle layer read from measuring cylinder (mL))

[0127] Test Example 3: Fluidity of dried particles (storage stability) Dried particles 1 to 17 obtained in each Example and Reference Example were sealed in an airtight container and stored at room temperature for 10 days or more, and then the container was tilted and the fluidity of the particles was visually confirmed. If the particles were not agglomerated but were dispersed, they were judged to have fluidity and given a grade of "A," and if the particles were agglomerated, they were given a grade of "B." The results are shown in Tables 1 to 4.

[0128] (Test Example 4: Visibility) 1 g of dried particles 1 to 17 obtained in each Example and Reference Example was weighed and dispersed in 20 g of E-MEM (containing L-glutamine and phenol red) for 3 hours to form a slurry. Images of the particles were taken using an optical microscope and a camera, as in Test Example 1. When the ratio of the particle edges to the total particle was equal to or less than that of the reference photograph (Figure 1: Cytodex 1 manufactured by Sigma-Aldrich), the particle was rated as "A," and when the ratio of the edges was greater, the particle was rated as "B." The results are shown in Tables 1 to 4. Furthermore, particle images of dried particles 1 (Example 1) are shown in Figure 2, and particle images of dried particles 2 (Example 2) are shown in Figure 3.

[0129] (Test Example 5: Simple Cell Adhesion Evaluation) (1) 0.2 g of dried particles 1 to 17 obtained in each Example and Reference Example was weighed out and dispersed in 30 g of PBS(-) for 3 hours to prepare a slurry. The slurry was then sterilized in an autoclave at 121°C for 20 minutes. Next, the slurry was centrifuged, the supernatant was removed by decantation, and 30 mL of a medium having the following composition was added. Next, centrifugation, removal of the supernatant, and addition of the medium were performed once more to complete the replacement with the medium.

[0130] <Medium composition> Dulbecco's Modified Eagle's Medium (DMEM) with high glucose (4.5g / l), 4mM L-glutamine, and sodium bicarbonate (Sigma-Aldrich Code D5796) ・10% Fetal Bovine Serum (FBS) (Cytiva HyCloneTM, FBS, characterized, SH30071.03)・100 units / ml penicillin G sodium, 100 μg / ml streptomycin sulfate (gibco, 15140-122) ・1mM sodium pyruvate (Sigma-Aldrich Code S8636)

[0131] (2) After the particles were replaced with the medium, they were added to a sterilized 100 mL Erlenmeyer flask, and then 3 x 10 5 AAVpro® 293T Cell (Takara Bio Inc.) cells were added to the medium to a concentration of 100 cells / mL. The cells were placed in an incubator set at 37°C, and the mixture was shaken at 1.25 rpm for 2 minutes, followed by a 30-minute incubation. This process was repeated four times to allow the cells to adhere to the particles. The cells were then cultured with continued shaking at 10 rpm for 24 hours. (3) After culturing, 100 particles were observed using an optical microscope (Evident BX53) and photographed. Cell adhesion was observed in more than half of the 100 particles, as determined by the "A" classification; cell adhesion was observed in more than one-quarter but less than one-half of the particles, as determined by the "B" classification; and cell adhesion was observed in less than one-quarter or no cell adhesion at all, as determined by the "C" classification. The results are shown in Tables 1 to 4. Images of cells adhering to the particles are shown in Figure 4 and Figure 5, respectively.

[0132]

[0133]

[0134]

[0135]

[0136] As shown in Table 4, the particles not containing the specific structural unit (Reference Example 1) and the particles containing a structural unit derived from DEAEMA rather than the specific structural unit (Reference Example 3) did not exhibit cell adhesiveness. Furthermore, when the monomer was subjected to suspension polymerization in the absence of a hydrocolloid (Reference Example 2), the polymerization reaction did not proceed well, and particles could not be prepared.

Claims

1. A cell-adhesive particle comprising, in its molecule, a structural unit having one or more types selected from a group represented by the following formula (1) and a group represented by the following formula (2): [In formula (1), R 1 represents an alkanediyl group having 2 to 10 carbon atoms; R 2 ~R 3 each independently represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, and * represents a bonding site. [In formula (2), R 4 represents an alkanediyl group having 2 to 10 carbon atoms; R 5 ~R 7 each independently represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms; - indicates a counter anion, and * indicates a binding site.

2. The cell adhesive particle according to claim 1, further comprising a structural unit represented by the following formula (3) in the molecule: [In formula (3), R 8 and R 9 each independently represents a hydrogen atom or a methyl group; R 10 represents a group represented by the following formula (3-A) or a polyoxyalkylene group. [In formula (3-A), R 11 represents an alkanediyl group having 3 to 100 carbon atoms, * represents a bonding site that bonds to the oxygen atom in formula (3), ** represents R 9 The binding site that binds to is shown.

3. The cell adhesive particle according to claim 1 or 2, further comprising a structural unit represented by the following formula (4) in the molecule: [In formula (4), R 12 and R 13 each independently represents a hydrogen atom or a methyl group; R 14 represents an alkanediyl group having 1 or 2 carbon atoms.

4. A cell adhesive particle described in any one of claims 1 to 3, wherein the content of structural units having one or more types selected from the group represented by formula (1) and the group represented by formula (2) is 5 mass% or more and 85 mass% or less of all structural units contained in the cell adhesive particle.

5. A method for producing polymer particles, comprising the following steps (i) to (iii), in which the aqueous medium in step (iii) has 30 parts by mass or more of salt dissolved per unit mass, assuming that the mass of salt per unit mass in a saturated solution obtained by dissolving a salt in the aqueous medium at 23°C and 1 atmospheric pressure is 100 parts by mass: (i) a step of preparing a hydrocolloid in the aqueous medium containing a salt, (ii) a step of dispersing one or more monomers in the aqueous medium containing the hydrocolloid obtained in step (i), and (iii) a step of suspension polymerizing the monomer dispersed in step (ii) in the aqueous medium containing the hydrocolloid and a salt.

6. The monomer is represented by the following formula (1); [In formula (1), R 1 represents an alkanediyl group having 2 to 10 carbon atoms; R 2 ~R 3 each independently represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, and * represents a bonding site. [In formula (2), R 4 represents an alkanediyl group having 2 to 10 carbon atoms; R 5 ~R 7 each independently represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms; - represents a counter anion, and * represents a binding site.] is used, and the polymer particles are cell-adhesive particles containing, in their molecules, structural units having one or more types selected from the group represented by formula (1) and the group represented by formula (2).

7. The method according to claim 5 or 6, further comprising the following step (iv): (iv) separating the polymer particles obtained in step (iii) from the hydrocolloid.

8. The method according to any one of claims 5 to 7, wherein at least water is used as the salt-containing aqueous medium, and step (ii) is a step of mixing the hydrocolloid-containing aqueous medium obtained in step (i) with a liquid composition in which one or more monomers are dissolved or dispersed in the aqueous medium, and the proportion of the liquid composition used is 5 parts by mass or more and 40 parts by mass or less when the total mass of water contained in the hydrocolloid-containing aqueous medium in step (ii) is taken as 100 parts by mass.

9. The method according to any one of claims 5 to 8, wherein the polymer particles have a settling bulk density of 0.08 g / mL or more and 0.13 g / mL or less when dispersed in an aqueous solution of sodium chloride having a concentration of 0.9% by mass under conditions of 25°C and 1 atmospheric pressure.

10. The method according to any one of claims 5 to 9, wherein the average particle size of the polymer particles is 50 μm or more and 500 μm or less.

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