Carrier for cell culture

Abstract

To provide a carrier for cell culture which exhibits cell proliferative properties comparable to or better than those of a conventional product and which further allows high detachability of cells after proliferation from the carrier.SOLUTION: A carrier for cell culture contains resin particles (A) and a first polypeptide (B), in which the first polypeptide (B) is at least one selected from the group consisting of a polypeptide (B1) composed of a sequence represented by Gly-X-Y (Gly is glycine, and X and Y are each independently the same or different amino acids), and a polypeptide (B2) that does not have a predetermined amino acid sequence (Z) in a molecule.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a carrier for cell culture. [Background technology]

[0002] Crosslinked poly(meth)acrylic acid particles having cell adhesion factors are known as carriers for cell culture (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2007-275056 Summary of the Invention [Problem to be solved by the invention]

[0004] The cell culture carrier described in Patent Document 1 has the effect of providing excellent cell growth properties. However, it takes a long time to detach the grown cells from the carrier, and therefore improvement has been desired. An object of the present invention is to provide a carrier for cell culture which has cell growth properties equal to or greater than those of conventional carriers and which allows the grown cells to be easily detached from the carrier.

[0005] The present inventors have conducted extensive research to achieve the above object and have arrived at the present invention, which is as follows. [1] A cell culture carrier comprising resin particles (A) and a first polypeptide (B), wherein the first polypeptide (B) is at least one selected from the group consisting of a polypeptide (B1) consisting of a sequence represented by Gly-XY (Gly is glycine, and X and Y are each independently the same or different amino acids), and a polypeptide (B2) not having an amino acid sequence (Z) in its molecule, wherein the amino acid sequence (Z) is at least one selected from the group consisting of an RGD sequence, an LDV sequence, a REDV sequence (1), a YIGSR sequence (2), a PDSGR sequence (3), a RYVVLPR sequence (4), a LGTIPG sequence (5), a RNIAEIIKDI sequence (6), a IKVAV sequence (7), a LRE sequence, a DGEA sequence (8), a GVKGDKGNPGWPGAP sequence (9), a GEFYFDLRLKGDK sequence (10), a HAV sequence, and a YKLNVNDS sequence (11). [2] The cell culture carrier according to claim 1, further comprising a second polypeptide (C), wherein the second polypeptide (C) is at least one selected from the group consisting of a cell adhesive peptide (C1) having the amino acid sequence (Z), fibronectin, collagen (excluding the polypeptide (B1)), and laminin. [3] The carrier for cell culture according to [1] or [2], wherein the content of the first polypeptide (B) is 10 ng / g to 50 mg / g based on the dry weight of the carrier for cell culture. [4] The carrier for cell culture according to [2] or [3], wherein the content of the second polypeptide (C) is 10 ng / g to 50 mg / g based on the dry weight of the carrier for cell culture. [Effects of the Invention]

[0006] According to the carrier for cell culture of the present invention, it is possible to provide a carrier for cell culture in which cell growth rate is equal to or higher than that of conventional products and the grown cells are easily detached from the carrier. DETAILED DESCRIPTION OF THE INVENTION

[0007] The carrier for cell culture of the present invention contains resin particles (A) and a first polypeptide (B). The first polypeptide (B) is at least one selected from the group consisting of a polypeptide (B1) consisting of a sequence represented by Gly-XY (Gly is glycine, and X and Y are each independently the same or different amino acids), and a polypeptide (B2) that does not have the following amino acid sequence (Z) in its molecule: The amino acid sequence (Z) is at least one selected from the group consisting of the RGD sequence, the LDV sequence, the REDV sequence (1), the YIGSR sequence (2), the PDSGR sequence (3), the RYVVLPR sequence (4), the LGTIPG sequence (5), the RNIAEIIKDI sequence (6), the IKVAV sequence (7), the LRE sequence, the DGEA sequence (8), the GVKGDKGNPGWPGAP sequence (9), the GEFYFDLRLKGDK sequence (10), the HAV sequence, and the YKLNVNDS sequence (11).

[0008] [Resin particles (A)] The resin particles (A) contained in the cell culture carrier of the present invention include, for example, particles of a water-absorbent resin (A1) having a carboxyl group and a primary to tertiary amino group. The water-absorbent resin (A1) having a carboxyl group and a primary to tertiary amino group includes, for example, a water-absorbent resin obtained by modifying a water-absorbent resin (A0) having a carboxyl group with a compound (S) having a primary to tertiary amino group.

[0009] The water-absorbing resin (A0) having a carboxyl group is preferably a crosslinked polymer obtained by polymerizing a monomer having a carboxyl group, and more preferably a crosslinked poly(meth)acrylic acid (salt). In this specification, "(meth)acrylic acid" means acrylic acid and / or methacrylic acid, and "acid (salt)" means acid and / or acid salt.

[0010] Examples of (meth)acrylic acid salts include alkali metal (lithium, potassium, sodium, etc.) salts of (meth)acrylic acid, polyvalent metal (alkaline earth metal (magnesium, calcium, etc.), boron group metal (aluminum, gallium, indium, etc.), transition metal (titanium, vanadium, chromium, manganese, iron, nickel, cobalt, copper, zinc, zirconium, molybdenum, ruthenium, rhodium, silver, cadmium, osmium, platinum, etc.) salts of (meth)acrylic acid, and ammonium salts of (meth)acrylic acid.

[0011] Of these, from the viewpoint of low cytotoxicity, alkali metal salts of (meth)acrylic acid and polyvalent metal salts of (meth)acrylic acid are preferred, more preferably alkali metal salts of (meth)acrylic acid, alkaline earth metal salts of (meth)acrylic acid and transition metal salts of (meth)acrylic acid, particularly preferably alkali metal salts of (meth)acrylic acid and alkaline earth metal salts of (meth)acrylic acid, and even more preferably sodium salts of (meth)acrylic acid, potassium salts of (meth)acrylic acid, magnesium salts of (meth)acrylic acid and calcium salts of (meth)acrylic acid.

[0012] As long as the crosslinked poly(meth)acrylic acid (salt) contains (meth)acrylic acid (salt) as a main constituent unit, it may contain other monomers copolymerizable with (meth)acrylic acid (salt) as constituent units.

[0013] Examples of other monomers include esters of unsaturated carboxylic acids (e.g., acrylic acid, methacrylic acid, etc.) [e.g., alkyl esters such as methyl ester, ethyl ester, propyl ester, butyl ester, pentyl ester, hexyl ester, heptyl ester, octyl ester, 2-ethylhexyl ester, etc.]; unsaturated carboxylic acid amides [e.g., free amides; N-substituted amides such as N-monoalkylamides and N,N-dialkylamides, etc.]; unsaturated dicarboxylic acid imides [e.g., maleimide, citraconic imide, itaconimide, N-alkyl-substituted, N-cycloalkyl-substituted, or N-aryl-substituted versions thereof, etc.]; alkenyl esters of saturated carboxylic acids (e.g., acetic acid, propionic acid, etc.) [e.g., vinyl esters, allyl esters, etc.]; esters or amides of unsaturated sulfonic acids [e.g., the above-mentioned Examples of the vinyl vinyl compound include esters or amides of unsaturated sulfonic acids corresponding to the esters or amides of the exemplified unsaturated carboxylic acids; unsaturated alcohols (e.g., allyl alcohol, propenyl alcohol, etc.); unsaturated ethers (e.g., alkyl vinyl ethers such as methyl vinyl ether and ethyl vinyl ether, alkyl allyl ethers such as methyl allyl ether and ethyl allyl ether, cycloalkyl vinyl ethers such as cyclohexyl vinyl ether, glycidyl vinyl ether, etc.); unsaturated nitriles (e.g., acrylonitrile, methacrylonitrile, etc.); olefins (e.g., ethylene, propylene, butene, pentene, hexene, etc.); aromatic vinyl compounds (e.g., styrene, α-methylstyrene, vinyltoluene, hydroxystyrene, etc.); heterocyclic vinyl compounds (e.g., N-vinylpyrrolidone, etc.), etc. Furthermore, as the other monomer, a crosslinkable monomer such as a polyhydric alcohol glycidyl ether (for example, an alkylene glycol diglycidyl ether such as ethylene glycol diglycidyl ether, or an alkanetriol di- or triglycidyl ether such as glycerin diglycidyl ether), and those described in JP 2018-126134 A may be used. The other monomer may be used alone or in combination of two or more. As the other monomer, a polyhydric alcohol glycidyl ether is preferred, and ethylene glycol diglycidyl ether is more preferred.

[0014] Crosslinked poly(meth)acrylic acid (salt) can be produced by (1) a method in which (meth)acrylic acid (salt), and optionally other copolymerizable monomers, a polymerization initiator, a chain transfer agent, and / or a graft base material, are continuously fed to a hydrophobic organic solvent under stirring to carry out reverse-phase suspension polymerization; or (2) a method in which (meth)acrylic acid (salt), and optionally other copolymerizable monomers, a polymerization initiator, a chain transfer agent, and / or a graft base material, are aqueous solution polymerized. Examples of method (1) include those described in JP-A Nos. 11-5808, 2001-2935, 2003-165883, 2005-247931, and 2005-186015. Examples of the method (2) include the methods described in JP-A Nos. 2005-075982, 2005-095759, 2005-097569, 2005-186015, and 2005-186016.

[0015] Examples of a method for producing the water absorbent resin (A1) having a carboxyl group and a primary to tertiary amino group include a method of chemically modifying a water absorbent resin (A0) having a carboxyl group with a compound (S) having a primary to tertiary amino group.

[0016] Specific examples of the method for modifying the water-absorbent resin (A0) having a carboxyl group with the compound (S) include the following methods (1) and (2). (1) A method of reacting a compound (S1) having a functional group capable of reacting with a carboxyl group in the water-absorbent resin (A0) and a primary to tertiary amino group with the water-absorbent resin (A0). (2) A method of reacting a compound (S2) having a functional group reactive with a carboxyl group in the water-absorbent resin (A0) and a hydroxyl group with the water-absorbent resin (A0), and then reacting the resulting product with a compound (S3) having a functional group reactive with a hydroxyl group and a primary to tertiary amino group. Of these methods, (2) is preferred from the viewpoint of cell adhesion.

[0017] Examples of the functional group which the compound (S1) and the compound (S2) have, respectively, and which can react with a carboxyl group in the water absorbent resin (A0), include an amino group, a halogenated alkyl group, a halogenated acyl group, a glycidyl ether group, a hydroxyl group, a carbodiimide group, a carbonate group, an oxazoline group, and an aziridine group. Examples of the functional group capable of reacting with a hydroxyl group contained in the compound (S3) include a halogenated alkyl group, a halogenated acyl group, a glycidyl ether group, a carbodiimide group, a carbonate group, and an aziridine group.

[0018] In the compound (S2) having a functional group reactive with a carboxyl group and a hydroxyl group, the functional group reactive with a carboxyl group is preferably an amino group, a glycidyl ether group, or a halogenated alkyl group, from the viewpoint of bond strength, and more preferably an amino group.

[0019] Examples of compound (S2) include compounds having an amino group and a hydroxyl group (e.g., alkanolamines having an alkylene group having 1 to 4 carbon atoms (specifically, 2-aminoethanol, 4-amino-1-butanol, 2-amino-2-methyl-1-propanol, 3-amino-1-propanol, etc.)), compounds having a glycidyl ether group and a hydroxyl group (e.g., 2,3-epoxy-1-propanol, glycerol-2,10-diglycidyl ether), and compounds having a halogenated alkyl group and a hydroxyl group (e.g., halogenated methanol, 2-halogenated ethanol, 3-halogenated propanol, 4-halogenated butanol). Examples of halogens constituting the halogenated alkyl group in the compound having a halogenated alkyl group and a hydroxyl group include fluorine, chlorine, and bromine. Among these halogens, chlorine is preferred from the viewpoints of reactivity, ease of handling, and the safety of by-products.

[0020] From the viewpoint of reactivity, the compound (S2) is preferably a compound having an amino group and a hydroxyl group, more preferably an alkanolamine, and even more preferably ethanolamine.

[0021] A known method can be used to react the water-absorbent resin (A0) having a carboxyl group with the compound (S2). For example, when the compound (S2) has an amino group as a reactive group, the water-absorbent resin (A0) and the compound (S2) can be subjected to an amidation reaction in the presence of a condensing agent such as a carbodiimide. Specifically, the water-absorbent resin (A0) having a carboxyl group is reacted with a carbodiimide compound in advance to obtain an acylisourea {R'-N=C(OCOR)-NH-R' (-OCOR is a moiety derived from (A0))}, and then the compound (S2) (a compound having an amino group) is added to form an amide bond between the water-absorbent resin (A0) and the compound (S2).

[0022] Examples of the primary to tertiary amino groups in the compound (S1) having a primary to tertiary amino group and the compound (S3) having a primary to tertiary amino group include a primary amino group (-NH2), a secondary amino group (-NHR), and a tertiary amino group (-NR2). In the tertiary amino group, the two Rs may be the same or different. In the secondary amino group and the tertiary amino group, R is a monovalent hydrocarbon having 1 to 4 carbon atoms. From the viewpoint of cell adhesiveness, R is preferably a monovalent aliphatic hydrocarbon having 1 to 3 carbon atoms, more preferably an alkyl group having 1 to 2 carbon atoms, and particularly preferably a methyl group or an ethyl group.

[0023] Examples of the primary amino group include amino groups, aminoalkyl groups such as aminomethyl groups, aminoethyl groups and aminopropyl groups, and aminoalkoxyalkyl groups such as 3-amino-1-ethoxypropyl groups and 1-amino-ethoxymethyl groups.

[0024] Examples of secondary amino groups include amino groups substituted with one hydrocarbon group, such as N-alkylaminoalkyl groups, including N-methylaminoethyl and N-ethylaminoethyl groups, and imidazoyl groups.

[0025] Examples of the tertiary amino group include an amino group substituted with two hydrocarbon groups. Examples of functional groups having a tertiary amino group include an N-dimethylaminoethyl group, an N-dimethylaminopropyl group, an N-diethylaminoethyl group, and an N-dibutylaminoethyl group.

[0026] The primary to tertiary amino groups may be in the form of a salt with an acid, such as hydrochloric acid, hydrobromic acid, iodic acid, acetic acid, sulfuric acid, nitric acid, or phosphoric acid.

[0027] In the compound (S1), the functional group capable of reacting with a carboxyl group is preferably a halogenated alkyl group from the viewpoint of reactivity. Specific examples of the compound (S1) include halogenated alkyl compounds having a primary to tertiary amino group {including those having a divalent alkylene group having 2 to 4 carbon atoms, where the halogen includes fluorine and chlorine, such as 2-chloro-N,N-diethylethylamine hydrochloride and (3-chloro-2-hydroxypropyl)trimethylammonium chloride}. From the viewpoint of cytotoxicity, compound (S1) is preferably a halogenated alkyl compound having a primary to tertiary amino group, more preferably a halogenated alkyl compound having a divalent alkylene group having 2 to 3 carbon atoms and a primary to tertiary amino group, and even more preferably 2-chloro-N,N-diethylethylamine hydrochloride.

[0028] In the compound (S3), the functional group capable of reacting with a hydroxyl group is preferably a halogenated alkyl group from the viewpoint of reactivity. Specific examples and preferred examples of the compound (S3) are the same as those of the compound (S1).

[0029] The number average particle size of the resin particles (A) is preferably 0.1 to 500 μm, more preferably 0.2 to 400 μm. The number average particle size of the resin particles can be adjusted by the polymerization conditions such as stirring, classification after polymerization, etc.

[0030] [First polypeptide (B)] The first polypeptide (B) is at least one selected from the group consisting of a polypeptide (B1) consisting of a sequence represented by Gly-XY (Gly is glycine, and X and Y are each independently the same or different amino acids), and a polypeptide (B2) not having the amino acid sequence (Z) in its molecule.

[0031] In the sequence Gly-XY constituting polypeptide (B1), Gly is glycine (G). Examples of X and Y in the sequence Gly-XY include P (proline), E (glutamic acid), B (asparagine), Q (glutamine), S (serine), K (lysine), A (alanine), V (valine), L (leucine), I (isoleucine), T (threonine), D (aspartic acid), R (arginine), C (cysteine), M (methionine), F (phenylalanine), Y (tyrosine), W (tryptophan), and H (histidine). Polypeptide (B1) may also be used, such as the "Biollagen (registered trademark)" sequence (39) manufactured by Mitsui Chemicals Fine Co., Ltd.

[0032] Polypeptide (B2) is a polypeptide that does not have at least one amino acid sequence (Z) selected from the group consisting of the RGD sequence, the LDV sequence, the REDV sequence (1), the YIGSR sequence (2), the PDSGR sequence (3), the RYVVLPR sequence (4), the LGTIPG sequence (5), the RNIAEIIKDI sequence (6), the IKVAV sequence (7), the LRE sequence, the DGEA sequence (8), the GVKGDKGNPGWPGAP sequence (9), the GEFYFDLRLKGDK sequence (10), the HAV sequence, and the YKLNVNDS sequence (11). Although the amino acid sequence (Z) is an amino acid sequence that can express adhesive properties, polypeptide (B2) does not have such an amino acid sequence (Z).

[0033] Examples of the polypeptide (B2) include polypeptides having at least one amino acid sequence (X) selected from the group consisting of the following pentapeptide sequence, the following hexapeptide sequence, the AGVPGFGVG sequence (12), and the AGVPGLGVG (13). Pentapeptide sequences: GVGVP sequence (14), VGVPG sequence (15), GVPGV sequence (16), VPGVG sequence (17) and PGVGV sequence (18). Hexapeptide sequences: GVGVAP sequence (19), VGVAPG sequence (20), GVAPGV sequence (21), VAPGVG sequence (22), APGVGV sequence (23), and PGVGVA sequence (24).

[0034] The polypeptide (B2) preferably contains the GVGVP sequence (14). Specific examples of polypeptide (B2) include a polypeptide having a sequence (27) of Mn 35,164, in which a sequence of four repeats of the GVGVP sequence (14) is chemically bonded to one GKGVP sequence (25), a sequence of three repeats of the GVGVP sequence (14), and a sequence of two repeats of the GAGAGS sequence (26) are chemically bonded to eight repeats of the amino acid block (L1-1); a polypeptide of sequence (28) of Mn 76,581, chemically bonded to a sequence of four repeats of the GVGVP sequence (14), one GKGVP sequence (25), a sequence of three repeats of the GVGVP sequence (14), and a sequence of four repeats of the GAGAGS sequence (26) chemically bonded to seven repeats of the amino acid block (L2-1); a sequence (29) of Mn 35,863, chemically bonded to a sequence of four repeats of the GVGVP sequence (14), one GKGVP sequence (25), a sequence of three repeats of the GVGVP sequence (14), and a sequence of four repeats of the GAGAGS sequence (26) chemically bonded to seven repeats of the amino acid block (L2-1); A polypeptide of Mn 69,772 sequence (30) chemically bonded to a sequence of two repeats of the GAGAGS sequence (26), a sequence of four repeats of the GVGVP sequence (14) chemically bonded to a sequence of the GKGVP sequence (25), a sequence of 11 repeats of the GVGVP sequence (14), and a sequence of Mn 71,445 chemically bonded to eight repeats of the amino acid block (L3-1) chemically bonded to a sequence of two repeats of the GAGAGS sequence (26). Polypeptide (31) is a polypeptide having a sequence (32) of Mn 64,694, which comprises two repeats of the GAGAGS sequence (26) chemically bonded to a sequence of four repeats of the GVGVP sequence (14), a GKGVP sequence (25), a sequence of eleven repeats of the GVGVP sequence (14), and six repeats of an amino acid block (L4-1) chemically bonded to a sequence of four repeats of the GAGAGS sequence (26). Polypeptide (B2) may also be a polypeptide described in JP 2013-151489 A.

[0035] [Second polypeptide (C)] The carrier for cell culture of the present invention may further contain a second polypeptide (C), which is at least one selected from the group consisting of a cell adhesive peptide (C1) having the amino acid sequence (Z), fibronectin, collagen (excluding the polypeptide (B1)), and laminin.

[0036] The amino acid sequence (Z) of the cell adhesive peptide (C1) is at least one selected from the group consisting of the RGD sequence, the LDV sequence, the REDV sequence (1), the YIGSR sequence (2), the PDSGR sequence (3), the RYVVLPR sequence (4), the LGTIPG sequence (5), the RNIAEIIKDI sequence (6), the IKVAV sequence (7), the LRE sequence, the DGEA sequence (8), the GVKGDKGNPGWPGAP sequence (9), the GEFYFDLRLKGDK sequence (10), the HAV sequence, and the YKLNVNDS sequence (11).

[0037] As the cell adhesive peptide (C1), the following polypeptides (C1-1) to (C1-3) are preferred. a means a sequence in which the GAGAGS sequence is repeated a times. (GVPGV) b means a sequence in which the GVPGV sequence is repeated b times.

[0038] (C1-1) Polypeptide having an RGD sequence A polypeptide having a molecular weight of approximately 110,000, which has a structure in which 13 RGD sequences and 12 (GAGAGS) sequences (33) are alternately arranged (sequence (34)) {"Pronectin F" manufactured by Sanyo Chemical Industries, Ltd.; Pronectin is a registered trademark of Sanyo Chemical Industries, Ltd. (the same applies hereinafter)}; a polypeptide obtained by modifying Pronectin F with compound (AM) [a polypeptide modified using N,N-dimethylaminoethyl chloride hydrochloride as compound (AM) ("Pronectin F Plus"); a polypeptide having a molecular weight of approximately 20,000, which has a structure in which 5 RGD sequences and 5 (GAGAGS) sequences (35) are alternately arranged (sequence (36)) ("Pronectin F2"); a polypeptide having a molecular weight of approximately 10,000, which has a structure in which 3 RGD sequences and 3 (GVPGV)2GG(GAGAGS)3 sequences (37) are alternately arranged (sequence (38)) ("Pronectin F3"); The "alternately arranged structure" may be a structure in which two types of alternately arranged sequences are directly linked together, or may be a structure in which they are linked together via another amino acid (sequence). (C1-2) Polypeptide having the IKVAV sequence (7) "Pronectin L," "Pronectin L2," or "Pronectin L3," etc., in which the RGD sequence of Pronectin F, Pronectin F2, or Pronectin F3 has been changed to the IKVAV sequence (7). (C1-3) Polypeptide having the YIGSR sequence (2) "Pronectin Y," "Pronectin Y2," or "Pronectin Y3," etc., in which the RGD sequence of Pronectin F, Pronectin F2, or Pronectin F3 has been changed to the YIGSR sequence (2).

[0039] In addition to the polypeptides (C1-1) to (C1-3), the cell adhesive peptide (C1) may also include RetroNectin (recombinant human fibronectin CH-296) (a polypeptide with a molecular weight of approximately 60,000 containing the RGD sequence and the LDV sequence as the amino acid sequence (Z)) and RGDS-Protein A (a polypeptide with a molecular weight of approximately 30,000 containing the RGD sequence as the amino acid sequence (Z)) manufactured by Takara Shuzo Co., Ltd. The amino acid sequences of these polypeptides are disclosed in Japanese Patent Application Laid-Open No. 2-311498.

[0040] Collagen that can be used as the second polypeptide is not particularly limited as long as it does not fall under the category of polypeptide (B1), and may be any of types I, II, III, IV, and V, including those degraded by enzymes, those obtained by cleaving telopeptides, and those synthesized by genetic engineering. Commercially available products include Cellma trix Type IA (type I, manufactured by Nitta Gelatin Co., Ltd.) and collagen type II derived from bovine dermis (type III, manufactured by Nippi Corporation). Examples of fibronectin that can be used as the second polypeptide include "Fibronectin Solution, from Human Plasma" manufactured by Wako Pure Chemical Industries, Ltd.

[0041] <Cell culture carrier> The carrier for cell culture of the present invention contains resin particles (A) and a first polypeptide (B) that does not have an adhesive peptide, and optionally contains a second polypeptide (C). The content of the first polypeptide (B) is preferably 10 ng / g to 50 mg / g, more preferably 20 ng / g to 40 mg / g, based on the dry weight of the cell culture carrier, from the viewpoints of cell proliferation and detachment from the carrier. When the carrier for cell culture contains a second polypeptide (C), the content of the second polypeptide (C) is preferably 10 ng / g to 50 mg / g, more preferably 20 ng / g to 40 mg / g, based on the dry weight of the carrier for cell culture, from the viewpoints of cell growth and detachment from the carrier.

[0042] The cell culture carrier of the present invention may be in a state in which resin particles (A) are coated with a first polypeptide (B), or in which the resin particles (A) and the first polypeptide (B) are chemically bonded, preferably by chemical bonding, and more preferably by amide bonding.

[0043] When resin particles (A) are coated with a first polypeptide (B), the first polypeptide (B) is dissolved in a solvent (e.g., water, phosphate buffer solution (PBS), etc.), and the resin particles (A) are immersed in the solution, filtered, and dried.

[0044] A method for forming an amide bond between resin particles (A) and first polypeptide (B) is to first react the carboxyl groups of resin particles (A) with a carbodiimide compound to obtain acylisourea {R'-N=C(OCOR)-NH-R' (the portion where -OCOR is derived from (A))}, and then add a first polypeptide (B) having a primary amino group or a secondary amino group to this acylisourea, thereby forming an amide bond between resin particles (A) and first polypeptide (B). Examples of carbodiimide compounds include N,N'-dicyclohexylcarbodiimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. In addition, 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride can also be used.

[0045] The carrier for cell culture of the present invention may contain a cell growth factor in order to enhance cell proliferation. Examples of cell growth factors include substances that promote cell proliferation, such as fibroblast growth factor, transforming growth factor, epidermal growth factor, hepatocyte growth factor, platelet-derived growth factor, insulin-like growth factor, vascular endothelial growth factor, nerve growth factor, stem cell factor, leukemia inhibitory factor, bone morphogenetic factor, heparin-binding epidermal growth factor, neurotrophic factor, connective tissue growth factor, angiopoietin, chondromodulin, tenomodulin, interferon, interleukin, tumor necrosis factor, colony-stimulating factor, adrenomodulin, and physiologically active polypeptides such as natriuretic peptides (for example, as described in "Tissue Engineering" edited by Ueda Minoru, published by Nagoya University Press, 1999). Among these cell growth factors, from the viewpoint of being applicable to a wide range of tissue cells and achieving higher cell proliferation, fibroblast growth factor, transforming growth factor, epidermal growth factor, hepatocyte growth factor, platelet-derived growth factor, insulin-like growth factor, vascular endothelial growth factor, bone morphogenetic factor, interleukin, and tumor necrosis factor are preferred, and fibroblast growth factor, epidermal growth factor, insulin-like growth factor, vascular endothelial growth factor, interleukin, and tumor necrosis factor are more preferred.

[0046] The cell growth factor is preferably bound to the resin particles (A).

[0047] When a cell growth factor is contained, the content thereof is preferably 10 pg / g to 1000 μg / g, more preferably 100 pg / g to 100 μg / g, and particularly preferably 1000 pg / g to 10 μg / g per 1 g of the carrier for cell culture from the viewpoint of cell proliferation and the like.

[0048] The cell culture carrier of the present invention may be sterilized as needed. Sterilization methods that can be used include radiation, ethylene oxide gas, plasma, gamma rays, alcohol, autoclave, dry heat, etc. These methods may be used alone or in combination of two or more.

[0049] There is no limitation on the cells (CE) that can adhere to the carrier for cell culture of the present invention, but because the carrier for cell culture of the present invention allows for high cell proliferation, normal mammalian cells, established mammalian cell lines, and insect cells that are used for the production of useful substances such as pharmaceuticals and for treatment are suitable.

[0050] Normal cells derived from mammals include cells involved in the skin (epithelial cells, fibroblasts, vascular endothelial cells, smooth muscle cells, etc.), cells involved in blood vessels (vascular endothelial cells, smooth muscle cells, fibroblasts, etc.), cells involved in muscle (muscle cells, etc.), cells involved in fat (adipocytes, etc.), cells involved in nerves (neuronal cells, etc.), cells involved in the liver (hepatocytes, etc.), cells involved in the pancreas (pancreatic islet cells, etc.), cells involved in the kidney (renal cells, renal epithelial cells, proximal tubular epithelial cells, mesangial cells, etc.), cells involved in the lungs and bronchi (epithelial cells, etc.). cells, fibroblasts, vascular endothelial cells, and smooth muscle cells, etc.), cells involved in the eye (photoreceptors, corneal epithelial cells, and corneal endothelial cells, etc.), cells involved in the prostate (epithelial cells, stromal cells, and smooth muscle cells, etc.), cells involved in bone (osteoblasts, osteocytes, and osteoclasts, etc.), cells involved in cartilage (chondroblasts and chondrocytes, etc.), cells involved in teeth (period ligament cells and osteoblasts, etc.), cells involved in blood (white blood cells and red blood cells, etc.), and stem cells {e.g., bone marrow undifferentiated mesenchymal stem cells, skeletal muscle stem cells, hematopoietic stem cells, neural stem cells, hepatic stem cells (oval cells, small hepatocytes, etc.), adipose tissue stem cells, embryonic stem (ES) cells, induced pluripotent stem cells, epidermal stem (iPS) cells, intestinal stem cells, spermatogonial stem cells, embryonic germ stem (EG) cells, pancreatic stem cells (pancreatic duct epithelial stem cells, etc.), leukocyte stem cells, lymphocyte stem cells, corneal stem cells, progenitor cells (adipose precursor cells, vascular endothelial precursor cells, chondrocyte precursor cells, lymphocyte precursor cells, NK precursor cells, etc.), etc.

[0051] Mammalian-derived cell lines include CRFK cells, 3T3 cells, A549 cells, AH130 cells, B95-8 cells, BHK cells, BOSC23 cells, BS-C-1 cells, C3H10T1 / 2 cells, C-6 cells, CHO cells, COS cells, CV-1 cells, F9 cells, FL cells, FL5-1 cells, FM3A cells, G-361 cells, GP+E. -86 cells, GP+envAm12 cells, H4-II-E cells, HEK293 cells, HeLa cells, HEp-2 cells, HL-60 cells, HTC cells, HUVEC cells, IMR-32 cells, IMR-90 cells, K562 cells, KB cells, L cells, L5178Y cells, L-929 cells, MA104 cells, MDBK cells, MDCK cells, MIA Examples of such cells include PaCG-2 cells, N18 cells, Namalwa cells, NG108-15 cells, NRK cells, OC10 cells, OTT6050 cells, P388 cells, PA12 cells, PA317 cells, PC-12 cells, PER.C6 cells, PG13 cells, QGH cells, Raji cells, RPMI-1788 cells, SGE1 cells, Sp2 / O-Ag14 cells, ST2 cells, THP-1 cells, U-937 cells, V79 cells, VERO cells, WI-38 cells, ψ2 cells, and ψCRE cells {Cell Culture Techniques (edited by the Japanese Society for Tissue Culture, published by Asakura Shoten, 1999)}.

[0052] Examples of insect cells include silkworm cells (BmN cells, BoMo cells, etc.), mulberry cells, Anemone sieboldii cells, Anemone shinjuii cells, armyworm cells (Sf9 cells, Sf21 cells, etc.), mulberry fly cells, leafroller cells, Drosophila cells, flesh fly cells, Aedes albopictus cells, swallowtail butterfly cells, American cockroach cells, and nettle looper cells (Tn-5 cells, HIGH FIVE cells, MG1 cells, etc.) (Insect Bio Factory (Kimura Shigeru, ed., published by Kogyo Chosakai, 2000)).

[0053] Among these cells, mammalian-derived normal cells and mammalian-derived established cell lines are preferred from the viewpoints of treatment and the production of useful substances such as pharmaceuticals. Furthermore, kidney cells, smooth muscle cells, hepatocytes, osteoblasts, epithelial cells, fibroblasts, vascular endothelial cells, and stem cells are more preferred because of their therapeutic utility. Furthermore, CRFK cells, 3T3 cells, BHK cells, CHO cells, HEK293 cells, HeLa cells, L-929 cells, MDCK cells, PER.C6 cells, VERO cells, and WI-38 cells are more preferred because of their usefulness in the production of useful substances such as pharmaceuticals.

[0054] Media to be used in the cell culture method using the carrier for cell culture of the present invention include serum-free media (Grace's medium, IPL-41 medium, Schneider's medium, Opti-PROTMSFM medium, Opti-MEMTMI medium, VP-SFM medium, CD293 medium, 293SFMII medium, CD-CHO medium, CHO-S-SFMII medium, FreeStyle™293 medium, CD-CHO ATG™ medium, and mixed media thereof); general media (RPMI medium, MEM medium, Eagle's MEM medium, BME medium, DME medium, αMEM medium, IMEM medium, ES medium, DM-160 medium, Fisher's medium, F12 medium, WE medium, ASF103 medium, ASF104 medium, ASF301 medium, TC-100 medium, Sf-900II medium, Ex-cell405 medium, Express-Five medium, Drosophila medium, and mixed media thereof); and mixed media thereof.

[0055] These media may also be supplemented with serum. Serum includes human serum and animal serum (bovine serum, horse serum, goat serum, sheep serum, pig serum, rabbit serum, chicken serum, rat serum, mouse serum, etc.). When serum is added, human serum, bovine serum, and horse serum are preferred. Examples of animal serum origin include adult serum, pup serum, newborn serum, and fetal serum. When serum is added, pup serum, newborn serum, and fetal serum are preferred, more preferably newborn serum and fetal serum, and particularly preferably fetal serum. When serum is added, the serum may be subjected to inactivation treatment, antibody removal treatment, etc.

[0056] When serum is used, the amount of serum used (% by weight) is preferably 0.1 to 50, more preferably 0.3 to 30, and particularly preferably 1 to 20, based on the weight of the medium.

[0057] If necessary, the medium may contain a cell growth factor, which can increase the cell growth rate and cell activity.

[0058] Examples of cell growth factors include fibroblast growth factor, transforming growth factor, epidermal growth factor, hepatocyte growth factor, platelet-derived growth factor, insulin-like growth factor, vascular endothelial growth factor, nerve growth factor, stem cell factor, leukemia inhibitory factor, bone morphogenetic factor, heparin-binding epidermal growth factor, neurotrophic factor, connective tissue growth factor, angiopoietin, cytokine, interleukin, adrenomodulin, and physiologically active peptides such as natriuretic peptides. Among these, fibroblast growth factor, transforming growth factor, insulin-like growth factor, and bone morphogenetic factor are preferred, and fibroblast growth factor, transforming growth factor, and insulin-like growth factor are more preferred, from the viewpoints of being applicable to a wide range of cells and being able to shorten the healing period.

[0059] When a cell growth factor is used, the content (wt%) of the cell growth factor varies depending on the type of cell growth factor, but is preferably 10-16 to 10-3, more preferably 10-14 to 10-5, and particularly preferably 10-12 to 10-7 based on the weight of the medium.

[0060] These media may further contain antibacterial agents (amphotericin B, gentamicin, penicillin, streptomycin, etc.). When an antibacterial agent is contained, the content (wt%) varies depending on the type of antibacterial agent, but is preferably 10-6 to 0, more preferably 10-5 to 1, and particularly preferably 10-4 to 0.1, based on the weight of the medium.

[0061] The concentration of cells (cells / mL) dispersed in the medium is not particularly limited, but is preferably 10 to 100 million, more preferably 1,000 to 10 million, and particularly preferably 10,000 to 1 million per mL of medium.

[0062] The number of cells can be counted by known methods, for example, by a cell nucleus counting method using crystal violet {Cell Culture Techniques (edited by the Japanese Tissue Culture Society, published by Asakura Shoten Co., Ltd., 1999)}. The dry weight (g) of the cell culture carriers to be added to the medium can be determined appropriately depending on the type of cells to be cultured, but is preferably 0.005 to 800, more preferably 0.02 to 200, and particularly preferably 0.1 to 40 per liter of medium.

[0063] The culture conditions are not particularly limited, and include culturing at a carbon dioxide (CO2) concentration of 1 to 20% by volume at 5 to 45°C for 1 hour to 100 days, with medium changes every 1 to 10 days as needed. Preferred conditions are culturing at a CO2 concentration of 3 to 10% by volume at 30 to 40°C for 1 to 20 days, with medium changes every 1 to 3 days.

[0064] The cells can be detached from the cell culture carrier by known methods, such as using a chelating agent (such as EDTA), a non-animal-derived protease (such as a plant-derived protease (such as papain)), a recombinant synthetic enzyme (such as TrypLE™ Select, manufactured by Invitrogen Corporation), and / or an animal-derived protease (such as trypsin or collagenase). [Example]

[0065] The present invention will be further described below with reference to examples and comparative examples, but the present invention is not limited thereto. Unless otherwise specified, parts mean parts by weight and % means % by weight.

[0066] <Production Example 1: Production of Resin Particles (A-1)> A reaction vessel equipped with a stirrer, a monomer supply pipe, a nitrogen gas inlet pipe, a thermometer, and a reflux condenser was charged with 624 parts of decane and 3.1 parts of sorbitan monostearate as a polymerization dispersant, and nitrogen bubbling was carried out for 30 minutes or more to expel dissolved air and raise the temperature to 75°C. Into another reactor, 173 parts of an 80% aqueous acrylic acid solution was charged, and 207 parts of a 28% aqueous sodium hydroxide solution was added to neutralize the solution while cooling. To this aqueous solution, 4.52 parts of a crosslinkable monomer (ethylene glycol diglycidyl ether), 0.278 parts of a polymerization initiator (potassium persulfate), and 0.053 parts of a chain transfer agent (sodium hypophosphite) were added, and then nitrogen bubbling was performed to remove dissolved air, yielding a monomer solution. The obtained monomer solution was continuously fed from the monomer feed pipe of the polymerization reactor at a rate of 6.5 ml / min into the decane liquid in the polymerization reactor under stirring (stirring speed: 500 rpm) over about 1 hour, and polymerization was carried out under decane reflux. Next, 160 parts of water was removed by azeotropic dehydration, and the hydrous gel polymer was taken out and further dried at 120°C for 2 hours. The polymer was then classified using sieves with 63 μm and 53 μm openings (JIS Z8801-1:2000) to obtain particles with a particle size of 53 to 63 μm. Next, 7.5 parts of a methanol / ion-exchanged water (volume ratio 70 / 30) solution containing 2% ethylene glycol diglycidyl ether was added to the resulting particles and mixed until uniform. The methanol was then air-dried, and the mixture was placed in a sealed container and held at 80°C for 1 hour to crosslink. The mixture was then dried in a dryer set at 120°C for 30 minutes to obtain crosslinked particles (A0-1). 3.83 g of water-soluble carbodiimide (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, manufactured by Dojindo Laboratories, Inc.) was added to 25 mL of 0.03 M phosphate buffer (pH 5.2) and completely dissolved, after which 3.9 g of 2-aminoethanolamine hydrochloride (Wako Pure Chemical Industries, Ltd.) was added and completely dissolved to prepare solution Ax. One gram of crosslinked particles (A0-1) was added to solution Ax and stirred at room temperature for approximately 20 minutes at 300 rpm using a polyethylene fluoride resin stirring blade to disperse the crosslinked particles (A0-1). The temperature of the solution containing the crosslinked particles was then raised to 40°C and the reaction was allowed to proceed for 4 hours. After the reaction was complete, the reaction solution was filtered through a 95 μm nylon mesh teabag (20 cm long, 10 cm wide) to recover the particles. The recovered particles were removed, suspended in 200 mL of ion-exchanged water, and adjusted to pH 7.2 with 1N NaOH. The solution was then filtered again, the teabag was closed, and the mixture was washed with stirring in 500 mL of ion-exchanged water. The ion-exchanged water was replaced every 3 hours, and this process was repeated three times. After washing, the solution was dried in a dryer at 40°C for at least 24 hours to obtain dried particles (A'-1). 8.56 g of 2-chloro-N,N-diethylethylamine hydrochloride (Sigma-Aldrich) was added to 10 mL of ion-exchanged water and completely dissolved, after which 3.92 mL of 48% (w / v) NaOH was added and stirred until homogenous, to prepare solution Ay. One gram of dried particles (A'-1) was added to solution Ay and stirred at room temperature for approximately 20 minutes at 300 rpm using a polyethylene fluoride resin stirring blade to disperse the dried particles (A'-1). After the dried particles (A'-1) were completely dispersed, the temperature of the solution containing the dried particles was raised to 60°C and the reaction was continued for 5 hours. After the reaction was complete, the reaction solution was filtered through a tea bag (20 cm long, 10 cm wide) made of a nylon mesh with a mesh opening of 95 μm, suspended in 200 mL of ion-exchanged water, and adjusted to pH 7.2 with 1N NaOH. The solution was then filtered again, the tea bag was closed, and the mixture was washed with stirring in 500 mL of ion-exchanged water. The ion-exchanged water was changed every 3 hours, and this process was repeated three times. After washing, the mixture was dried in a dryer at 40°C for at least 24 hours to obtain resin particles (A-1).

[0067] <Production Example 2: Production of Resin Particles (A-2)> An aqueous solution Sy was prepared by adding 139.2 g of sodium chloride and 39.6 g of magnesium chloride hexahydrate to 480.0 g of pure water and dissolving the mixture by stirring. Next, 20.0 g of a solvent (1-hexanol) and 1.0 g of a radical polymerization initiator (dimethyl 2,2'-azobisisobutyrate) were added to a composition consisting of 22.1 g of N-[3-dimethylaminopropyl]acrylamide, 72.1 g of 2-hydroxypropyl methacrylate, 5.1 g of methacrylic acid, and 0.7 g of ethylene glycol dimethacrylate to prepare a monomer composition. The entire amount of the aqueous solution Sy 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 stirred under a nitrogen atmosphere. 101.1 g of a 5 M aqueous solution of sodium hydroxide was added to the separable flask to prepare a magnesium hydroxide hydrocolloid dispersion. The stirring speed was then set to 700 rpm, and the entire amount of the 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. Next, while the internal temperature was maintained at 86°C, the mixture was stirred for 3 hours to carry out suspension polymerization, and then cooled to room temperature. Next, 5M hydrochloric acid was added to the supernatant so that the pH was adjusted to 5.0 or less, and the mixture was stirred. Next, pure water was added to the reaction solution, and the solution was removed by decantation to wash the resin. Next, the mixture was replaced with t-butyl alcohol, frozen at -20°C, and then vacuum dried to obtain a dry resin (A'-2). The resulting dried resin (A'-2) was classified to obtain resin particles (A-2).

[0068] <Production Example 3: Production of Resin Particles (A-3)> Resin particles (A-3) were obtained in the same manner as in Production Example 2, except that 22.1 g of N-[3-dimethylaminopropyl]acrylamide was replaced with 33.1 g of 2-(dimethylamino)ethyl methacrylate and 72.1 g of 2-hydroxypropyl methacrylate (a2-2) was replaced with 61.1 g.

[0069] <Production Example 4: Production of Polypeptide (B2-1)> Using recombinant Escherichia coli and purifying by column chromatography according to the method described in the examples of JP-A-3-502935, we obtained polypeptide (B2-1) with a Mn of 35,164 and sequence (27), which is composed of eight repeats of amino acid block (L1-1) chemically bonded to a sequence of four repeats of GVGVP sequence (14), one GKGVP sequence (25), a sequence of three repeats of GVGVP sequence (14), and a sequence of two repeats of GAGAGS sequence (26). (B2-1) has a structure in which eight repeats of (L1-1) [(GVGVP)4(GKGVP)1(GVGVP)3(GAGAGS)2] are chemically bonded.

[0070] <Production Example 5: Production of Polypeptide (B2-2)> A polypeptide (B2-2) having an Mn of 76,581 and sequence (28), in which 17 repeats of the amino acid block (L1-1) were chemically bonded, was produced by the same procedure as in Production Example 2, except that the number of repeats of the amino acid block (L1-1) in Production Example 4 was changed from 8 to 17. (B2-2) has a structure in which 17 repeats of (L1-1) [(GVGVP)4(GKGVP)1(GVGVP)3(GAGAGS)2] are chemically bonded.

[0071] <Production Example 6: Production of Polypeptide (B2-3)> A similar procedure to that described in the Examples of JP-A-3-502935 was carried out using recombinant Escherichia coli and purified by column chromatography to produce polypeptide (B2-3) having a molecular weight of 35,863 and a sequence (29) in which seven repeats of amino acid block (L2-1) were chemically bonded, each of which comprised four repeats of GVGVP sequence (14) chemically bonded to a GKGVP sequence (25), which in turn comprised three repeats of GVGVP sequence (14), and which in turn comprised four repeats of GAGAGS sequence (26). (B2-3) has a structure in which seven repeats of (L2-1) [(GVGVP)(GKGVP)(GVGVP)(GAGAGS)] are chemically bonded.

[0072] <Production Example 7: Production of Polypeptide (B2-4)> A polypeptide (B2-4) having Mn 69,772 and sequence (30) in which 13 repeats of the amino acid block (L2-1) were chemically bonded was produced by the same procedure as in Production Example 4, except that the number of repeat bonds of the amino acid block (L2-1) was changed from 7 to 13. (B2-4) has a structure in which 13 repeats of (L2-1) [(GVGVP)4(GKGVP)1(GVGVP)3(GAGAGS)4] are chemically bonded.

[0073] <Production Example 8: Production of Polypeptide (B2-5)> The same procedure as in Preparation Example 4 was carried out to produce polypeptide (B2-5) with a molecular weight of 71,445 and a sequence (31) in which a sequence of two repeats of the GAGAGS sequence (26) was chemically bonded to a sequence of four repeats of the GVGVP sequence (14), a GKGVP sequence (25), a sequence of 11 repeats of the GVGVP sequence (14), and a sequence of two repeats of the GAGAGS sequence (26) were chemically bonded to eight repeats of the amino acid block (L3-1). (B2-5) was (L3-1) [(GAGAGS)2(GVGVP)4(GKGVP)1(GVGVP)] 11 It has a structure in which eight repeating units of [(GAGAGS)2] are chemically bonded.

[0074] <Production Example 9: Production of Polypeptide (B2-6)> The same procedure as in Preparation Example 4 was carried out to produce polypeptide (B2-6) having a Mn of 64,694 and a sequence (32) in which two repeats of the GAGAGS sequence (26) were chemically bonded to a sequence of four repeats of the GVGVP sequence (14), a GKGVP sequence (25), a sequence of 11 repeats of the GVGVP sequence (14), and six repeats of the amino acid block (L4-1) chemically bonded to a sequence of four repeats of the GAGAGS sequence (26). (B2-6) is (L4-1) [(GAGAGS)2(GVGVP)4(GKGVP)1(GVGVP)] 11 It has a structure in which the chemical bond of [(GAGAGS)4] is repeated six times.

[0075] <Raw materials> The raw materials listed in Table 1 are as follows: (A-1): Resin particles produced in Production Example 1 (A-2): Resin particles produced in Production Example 2 (A-3): Resin particles produced in Production Example 3 (A-4): Corning untreated microcarriers (manufactured by Corning) (B1-1): Biollagen SSE (Mitsui Chemicals Fine Co., Ltd.) (B2-1): Polypeptide produced in Production Example 4 (B2-2): Polypeptide produced in Production Example 5 (B2-3): Polypeptide produced in Production Example 6 (B2-4): Polypeptide produced in Production Example 7 (B2-5): Polypeptide produced in Production Example 8 (B2-6): Polypeptide produced in Production Example 9 (C-1): ProNectin F (manufactured by Sanyo Chemical Industries, Ltd.) (C-2): ProNectin F+ (Sanyo Chemical Industries, Ltd.) (C-3): Commercially available collagen (Nitta Gelatin Co., Ltd., Cellma trix Type IA (type I))

[0076] Example 1 800 mg of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride was added to 10 mL of 0.03 M phosphate buffer (pH 5.2) (hereinafter referred to as PBS) containing 0.85% sodium chloride, and completely dissolved to obtain a solution X1. 1 g of resin particles (A-1) was added to solution X1 and reacted for 4 hours at room temperature at a stirring speed of 300 rpm using a polyethylene fluoride resin stirring blade. After the reaction was completed, the reaction solution was filtered through a nylon mesh with a mesh size of 95 μm and washed with 2 L of ion-exchanged water. The washed resin particles were removed and suspended in 12 mL of PBS solution (pH 7.2). Next, 8 mL of a PBS solution (pH 7.2) containing polypeptide (B1-1) (Biollagen (registered trademark) SSE, manufactured by Mitsui Fine Chemicals, Inc.) at a concentration of 1 mg / mL was added, and the mixture was allowed to react overnight. After the reaction was completed, the reaction solution was filtered through a nylon mesh with a mesh size of 95 μm and washed with 2 L of ion-exchanged water. After washing, the mixture was suspended in approximately 10 mL of ion-exchanged water and freeze-dried to obtain the cell culture carrier (α-1) of Example 1.

[0077] <Example 2> 8 mg of the polypeptide (B1-1) was dissolved in 8 ml of ion-exchanged water to obtain a solution X2. 1 g of resin particles (A-1) was added to the solution X2, and the mixture was stirred overnight at room temperature at a stirring speed of 300 rpm in an apparatus equipped with stirring blades made of polyethylene fluoride resin to obtain a suspension. The resulting suspension was filtered through a nylon mesh with 95 μm openings and washed with 2 L of ion-exchanged water. After washing, the suspension was suspended in approximately 10 mL of ion-exchanged water and freeze-dried to obtain the cell culture carrier (α-2) of Example 2.

[0078] <Examples 3 to 8> The same procedure as in Example 1 was carried out, except that 8 mg of the first polypeptide of the type shown in Table 1 was used instead of 8 mg of the polypeptide (B1-1) in Example 1, to obtain carriers for cell culture of Examples 3 to 8.

[0079] <Examples 9 to 10 and 12> The same procedures as in Example 1 were carried out to obtain the cell culture carriers of Examples 9 to 12, except that in Example 1, 5 mg of a first polypeptide of the type listed in Table 1 and 3 mg of a second polypeptide of the type listed in Table 1 were used instead of 8 mg of polypeptide (B1-1).

[0080] Example 11 5 mg of polypeptide (B1-1) was dissolved in 7 ml of ion-exchanged water, and 1 mL of second polypeptide (C-3) (3 mg of pure polypeptide in 1 mL) was added to obtain solution X3. 1 g of resin particles (A-1) was added to solution X3, and the mixture was stirred overnight at room temperature at a stirring speed of 300 rpm in an apparatus equipped with stirring blades made of polyethylene fluoride resin to obtain a suspension. The resulting suspension was filtered through a nylon mesh with 95 μm openings and washed with 2 L of ion-exchanged water. After washing, the suspension was suspended in approximately 10 mL of ion-exchanged water and freeze-dried to obtain the cell culture carrier (α-11) of Example 11.

[0081] <Examples 13 and 14> The same procedure as in Example 1 was carried out, except that 1 g of resin particles (A-2) or (A-3) of the type shown in Table 1 was used instead of the resin particles (A-1) in Example 1, to obtain the cell culture carriers of Examples 13 and 14.

[0082] Example 15 The same procedure as in Example 1 was carried out, except that 1 g of resin particles (A-4) of the type shown in Table 1 was used instead of resin particles (A-1) in Example 1, to obtain a cell culture carrier of Example 15.

[0083] <Comparative Example 1> 2.67 mL of the second polypeptide (C-3) (purity of the polypeptide: approximately 8 mg) was diluted with 5 mL of ion-exchanged water to obtain a solution X4. 1 g of resin particles (A-1) was added to solution X4, and the mixture was stirred overnight at room temperature at a stirring speed of 300 rpm in an apparatus equipped with stirring blades made of polyethylene fluoride resin to obtain a suspension. The resulting suspension was filtered through a nylon mesh with 95 μm openings and washed with 2 L of ion-exchanged water. After washing, the suspension was suspended in approximately 10 mL of ion-exchanged water and freeze-dried to obtain cell culture carriers (α'-1) of Comparative Example 1.

[0084] <Evaluation Test 1: Evaluation of Cell Proliferation (Cell Concentration)> 0.06 g of each cell culture carrier was added to each spinner flask, and 20 ml of PBS was added to each container, followed by autoclave sterilization (121°C, 20 minutes). After autoclave sterilization, the PBS was aspirated and removed using an aspirator. 20 ml of serum medium (E-MEM medium with 10% by volume of fetal bovine serum (Invitrogen)) was added to each flask and allowed to stand for 10 minutes. The medium was then aspirated and 20 ml of the same serum medium was added again to each flask. The spinner flask was left in a CO2 incubator at 37°C with a carbon dioxide gas concentration of 5% by volume for 1 hour, after which pre-cultured VERO cells (manufactured by Sumitomo Dainippon Pharma Co., Ltd.) were seeded into the medium to a cell concentration of 200,000 cells / mL. The cells were cultured for 7 days in a CO2 incubator at 37°C with a carbon dioxide gas concentration of 5% by volume, with stirring at 60 rpm. On the third day of culture, half of the medium was replaced. On the seventh day of culture, the cells were sampled, and the number of cell nuclei per unit volume was counted using a cell nuclei counting method using crystal violet, and the cell concentration (cells / mL) in the medium was measured. The results are shown in Table 1. The higher the cell concentration, the higher the cell proliferation rate.

[0085] <Evaluation Test 2: Evaluation of Cell Recovery> 0.06 g of each cell culture carrier was added to each spinner flask, and 20 ml of PBS was added to each container, followed by autoclave sterilization (121°C, 20 minutes). After autoclave sterilization, the PBS was aspirated and removed using an aspirator. 20 ml of serum medium (E-MEM medium with 10% by volume of fetal bovine serum (Invitrogen)) was added to each flask and allowed to stand for 10 minutes. The medium was then aspirated and 20 ml of the same serum medium was added again to each flask. The spinner flask was left in a CO2 incubator at 37°C with a carbon dioxide gas concentration of 5% by volume for 1 hour, after which pre-cultured VERO cells (manufactured by Sumitomo Dainippon Pharma Co., Ltd.) were seeded into the medium to a cell concentration of 200,000 cells / mL. The cells were cultured for 7 days in a CO2 incubator at 37°C with a carbon dioxide gas concentration of 5% by volume, with stirring at 60 rpm. On the third day of culture, half of the medium was replaced. On the seventh day of culture, the supernatant was aspirated and removed. Subsequently, 20 mL of PBS was added and stirred, and the mixture was left to settle, allowing the porous microcarriers to settle. The supernatant was then aspirated and removed, a process repeated three times. 20 mL of a two-fold diluted solution of TrypLE™ Select (10x) (Thermo Fischer Scientific) containing 15 mM EDTA was added, and the mixture was stirred at 60 rpm for 10 minutes at 37°C. The mixture was left to settle, allowing the cell culture carriers to settle, and the supernatant containing the free cells was aspirated and collected. Further, 20 ml of PBS was added and stirred, and then the mixture was left to settle and the porous microcarriers were allowed to settle. The supernatant was then aspirated to recover the remaining free cells. The cell concentration in the combined two recovery solutions was counted using the trypan blue method, and the cell recovery rate was calculated using the following formula. The results are shown in Table 1. The higher the cell recovery rate, the better the detachment of the proliferated cells. Cell recovery rate (%) = recovered cell concentration / cell concentration before harvesting × 100

[0086] [Table 1]

[0087] As shown in Table 1, the cell proliferation rate of the example product was equal to or higher than that of the comparative example product, and it was found that the product had high detachability from the carrier. As a result, it is clear that the carrier of the present invention can provide a carrier for cell culture which has cell growth properties equal to or greater than those of conventional products and which allows the grown cells to be easily detached from the carrier.

Claims

1. A composition comprising resin particles (A) and a first polypeptide (B), the first polypeptide (B) is at least one selected from the group consisting of a polypeptide (B1) consisting of a sequence represented by Gly-X-Y (Gly is glycine, and X and Y are each independently the same or different amino acids), and a polypeptide (B2) not having an amino acid sequence (Z) in its molecule; A carrier for cell culture, wherein the amino acid sequence (Z) is at least one selected from the group consisting of an RGD sequence, an LDV sequence, a REDV sequence (1), a YIGSR sequence (2), a PDSGR sequence (3), a RYVVLPR sequence (4), a LGTIPG sequence (5), a RNIAEIIKDI sequence (6), a IKVAV sequence (7), a LRE sequence, a DGEA sequence (8), a GVKGDKGNPGWPGAP sequence (9), a GEFYFDLRLKGDK sequence (10), a HAV sequence, and a YKLNVNDS sequence (11).

2. Further, it contains a second polypeptide (C), The cell culture carrier according to claim 1, wherein the second polypeptide (C) is at least one selected from the group consisting of a cell adhesive peptide (C1) having the amino acid sequence (Z), fibronectin, collagen [excluding the polypeptide (B1)], and laminin.

3. 3. The carrier for cell culture according to claim 1, wherein the content of the first polypeptide (B) is 10 ng / g to 50 mg / g based on the dry weight of the carrier for cell culture.

4. 3. The carrier for cell culture according to claim 2, wherein the content of the second polypeptide (C) is 10 ng / g to 50 mg / g based on the dry weight of the carrier for cell culture.

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