Carrier for cell culture

Abstract

The present invention pertains to a carrier for cell culture, said carrier including: copolymer resin particles (A) in which a monomer (a1) represented by general formula (1), a monomer (a2) that has a hydroxyl group and is represented by general formula (2), and a crosslinking monomer (b) are used as constituent monomers; and an adhesive protein (B).　(1): CH2=C(R1)–(C=O)–Q–R2–N(R3)(R4) (In general formula (1), R1 is H or CH3; Q is O or NH; R2 is a C2-5 divalent hydrocarbon group; and R3 and R4 are each independently a C1-4 monovalent hydrocarbon group.)　(2): CH2=C(R5)–(C=O)–Q–(R6–O)n–H (In general formula (2), R5 is H or CH3; Q is O or NH; R6 is a C1-4 divalent hydrocarbon group; and n is an integer from 1 to 7.)

Description

Cell culture carriers

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

[0002] Crosslinked poly(meth)acrylic acid particles having cell adhesion factors (Patent Document 1) are known as carriers for cell culture, but they are not fully satisfactory in terms of cell proliferation.

[0003] Japanese Patent Application Laid-Open No. 2007-275056

[0004] An object of the present invention is to provide a carrier for cell culture that is excellent in cell growth and cell adhesiveness.

[0005] The present inventors have conducted extensive research to achieve the above object and have arrived at the present invention. Specifically, the present invention relates to a carrier for cell culture comprising copolymer resin particles (A) having as constituent monomers a monomer (a1) represented by the following general formula (1), a monomer (a2) having a hydroxyl group represented by the following general formula (2), and a crosslinkable monomer (b), and an adhesive protein (B). CH ２ = C(R １ )-(C=O)-QR ２ -N(R ３ ) (R ４ ) (1) [In general formula (1), R １ is H or CH ３ Q is O or NH; R ２ represents a divalent hydrocarbon group having 2 to 5 carbon atoms; R ３ , R ４ are each independently a monovalent hydrocarbon group having 1 to 4 carbon atoms.] CH ２ = C(R ５ )-(C=O)-Q-(R ６ -O) ｎ -H (2) [In general formula (2), R ５ is H or CH ３ Q is O or NH; R ６ is a divalent hydrocarbon group having 1 to 4 carbon atoms; and n is an integer from 1 to 7.

[0006] The carrier for cell culture of the present invention has the following effects: (1) excellent cell growth properties, (2) excellent cell adhesiveness.

[0007] The cell culture carrier of the present invention has excellent cell proliferation properties and cell adhesiveness because it contains copolymer resin particles (A) having, as constituent monomers, a monomer (a1) represented by the following general formula (1), a monomer (a2) having a hydroxyl group represented by the following general formula (2), and a crosslinkable monomer (b), and an adhesive protein (B).

[0008] <Monomer (a1)> The monomer (a1) in the present invention is represented by the following general formula (1): CH ２ = C(R １ )-(C=O)-QR ２ -N(R ３ ) (R ４ ) (1) [In general formula (1), R １ is H or CH ３ Q is O or NH; R ２ represents a divalent hydrocarbon group having 2 to 5 carbon atoms; R ３ , R ４ are each independently a monovalent hydrocarbon group having 1 to 4 carbon atoms.

[0009] In the above general formula (1), R １ is H or CH ３ Q is O or NH (preferably NH from the viewpoint of cell adhesiveness); R ２ represents a divalent hydrocarbon group having 2 to 5 carbon atoms (preferably having 2 to 4 carbon atoms, more preferably having 2 to 3 carbon atoms, from the viewpoint of cell adhesiveness); R ３ , R ４ are each independently a monovalent hydrocarbon group having 1 to 4 carbon atoms (preferably 1 carbon atom from the viewpoint of cell adhesiveness).

[0010] The monomer (a1) is not particularly limited, but examples thereof include N-[3-(dimethylamino)propyl](meth)acrylamide, N-[3-(diethylamino)propyl](meth)acrylamide, N-[3-(dimethylamino)ethyl](meth)acrylamide, N-[3-(diethylamino)ethyl](meth)acrylamide, N-[4-(dimethylamino)butyl](meth)acrylamide, N-[3-(dimethylamino)propyl](meth)acrylate, N-[3-(diethylamino)propyl](meth)acrylate, N-[3-(dimethylamino)ethyl](meth)acrylate, N-[3-(diethylamino)ethyl](meth)acrylate, and N-[4-(dimethylamino)butyl](meth)acrylate. These may be used alone or in combination of two or more.

[0011] From the viewpoint of cell adhesiveness, the monomer (a1) is preferably R ２ is preferably an alkylene group having 2 to 4 carbon atoms, and R ２ is more preferably an alkylene group having 2 to 3 carbon atoms. ２ is an alkylene group having 3 carbon atoms and R ２ It is also preferable to use in combination with a monomer (a1) having an alkylene group having two carbon atoms. From the viewpoint of cell adhesiveness, the above-mentioned monomer (a1) is preferably N-[3-(dimethylamino)ethyl](meth)acrylate or N-[3-(dimethylamino)propyl](meth)acrylamide, and more preferably N-[3-(dimethylamino)propyl](meth)acrylamide. In the present invention, (meth)acrylamide means methacrylamide and / or acrylamide. Furthermore, (meth)acrylate means methacrylate and / or acrylate.

[0012] <Monomer (a2) Having a Hydroxyl Group> The monomer (a2) having a hydroxyl group in the present invention is represented by the following general formula (2): CH ２ = C(R ５ )-(C=O)-Q-(R ６ -O) ｎ -H (2) [In general formula (2), R５ is H or CH ３ Q is O or NH; R ６ is a divalent hydrocarbon group having 1 to 4 carbon atoms; and n is an integer from 1 to 7.

[0013] In the above general formula (2), R ５ is H or CH ３ Q is O or NH; R ６ is a divalent hydrocarbon group having 1 to 4 carbon atoms (preferably 1 to 3 carbon atoms from the viewpoint of cell recognition and cell proliferation); and n is an integer of 1 to 7 (preferably 1 to 5, more preferably 1 to 3 from the viewpoint of cell proliferation).

[0014] The hydroxyl group-containing monomer (a2) is not particularly limited, but examples thereof include 2-hydroxyethyl (meth)acrylate, 2-hydroxypropyl (meth)acrylate, diethylene glycol mono(meth)acrylate, polyethylene glycol (average degree of polymerization 3 to 7) mono(meth)acrylate, dipropylene glycol mono(meth)acrylate, polypropylene glycol (average degree of polymerization 3 to 7) mono(meth)acrylate, polyalkylene glycol (average degree of polymerization 2 to 7) mono(meth)acrylate, and N-(2-hydroxyethyl)acrylamide. These may be used alone or in combination of two or more. Commercially available products may be used as the hydroxyl group-containing monomer (a2). Examples of commercially available products include those in the "Blemmer" series (manufactured by NOF Corporation) in which the average degree of polymerization of alkylene oxide per hydroxyl group-containing monomer (a2) molecule is 2 to 7. Examples of such commercially available products include Blemmer PE-90 (average degree of polymerization of ethylene oxide: 2), Blemmer PE-200 (average degree of polymerization of ethylene oxide: 4.5), Blemmer PP-1000 (average degree of polymerization of propylene oxide: 4 to 6), Blemmer 50PEP-300 (average degree of polymerization of ethylene oxide: 3.5, average degree of polymerization of propylene oxide: 2.5), Blemmer 10PPB-500B (average degree of polymerization of propylene oxide: 1, average degree of polymerization of butylene oxide: 6), Blemmer AE-90U (average degree of polymerization of ethylene oxide: 2), Blemmer AE-200 (average degree of polymerization of ethylene oxide: 4.5), Blemmer AP-200 (average degree of polymerization of propylene oxide: 3.5), and Blemmer AP-400 (average degree of polymerization of propylene oxide: 6) (all manufactured by NOF Corporation). These may be used alone or in combination of two or more.

[0015] From the viewpoint of cell adhesiveness, the hydroxyl group-containing monomer (a2) is preferably 2-hydroxyethyl (meth)acrylate, 2-hydroxypropyl (meth)acrylate, or diethylene glycol mono(meth)acrylate, more preferably 2-hydroxyethyl (meth)acrylate or 2-hydroxypropyl (meth)acrylate, and particularly preferably a combination of 2-hydroxyethyl (meth)acrylate and 2-hydroxypropyl (meth)acrylate.

[0016] <Crosslinkable Monomer (b)> Examples of the crosslinkable monomer (b) include (meth)acrylamides (b1) having two or more (preferably 2 to 4, more preferably 2) unsaturated bonds, and (meth)acrylates (b2) having two or more (preferably 2 to 4, more preferably 2) unsaturated bonds. The crosslinkable monomers (b) may be used alone or in combination of two or more.

[0017] Examples of (meth)acrylamide (b1) having two or more unsaturated bonds (hereinafter, sometimes simply referred to as (meth)acrylamide (b1)) include N,N'-methylenebisacrylamide, etc. The number of carbon atoms in (meth)acrylamide (b1) (hereinafter, the number of carbon atoms may be abbreviated as C) may be, for example, 5 to 30.

[0018] Examples of the (meth)acrylate (b2) having two or more unsaturated bonds (hereinafter, sometimes simply referred to as (meth)acrylate (b2)) include ethylene glycol di(meth)acrylate, pentaerythritol [poly(2-4)] (meth)acrylate, polyethylene glycol di(meth)acrylate, and dipentaerythritol hexa(meth)acrylate, etc. The (meth)acrylate (b2) may be, for example, a C8-30 (meth)acrylate.

[0019] Of the crosslinkable monomers (b), (meth)acrylates (b2) are preferred, ethylene glycol di(meth)acrylate and polyethylene glycol di(meth)acrylate are more preferred, and ethylene glycol di(meth)acrylate is even more preferred.

[0020] <Copolymer Resin Particles (A)> The copolymer resin particles (A) of the present invention contain, as constituent monomers, monomer (a1), monomer (a2) having a hydroxyl group, and crosslinkable monomer (b). In addition to monomer (a1), monomer (a2) having a hydroxyl group, and crosslinkable monomer (b), copolymer resin particles (A) may also contain, as constituent monomers, monomer (a3) ​​having a carboxyl group and other monomers (a4) described below. From the viewpoint of cell adhesiveness, the weight ratio of monomer (a1) based on the total weight of the constituent monomers of copolymer resin particles (A) is preferably 15 to 90 wt%, more preferably 20 to 80 wt%. From the viewpoint of cell adhesiveness, the weight ratio of monomer (a2) having a hydroxyl group based on the total weight of the constituent monomers of copolymer resin particles (A) is preferably 10 to 80 wt%, more preferably 20 to 60 wt%. Furthermore, the weight proportion of the crosslinkable monomer (b) based on the total weight of the constituent monomers of the copolymer resin particles (A) is preferably 0.1 to 3% by weight, more preferably 0.3 to 2% by weight, from the viewpoint of maintaining the particle shape.

[0021] <Monomer (a3) ​​Having a Carboxyl Group> The copolymer resin particles (A) preferably further contain a monomer (a3) ​​having a carboxyl group as a constituent monomer. Examples of the monomer (a3) ​​having a carboxyl group include (meth)acrylic acid, maleic anhydride, maleic acid, fumaric acid, itaconic anhydride, itaconic acid, vinylbenzoic acid, and allyl acetic acid. The monomer (a3) ​​having a carboxyl group may be, for example, a C3 to C30 monomer. These may be used alone or in combination of two or more. In the present invention, (meth)acrylic acid refers to methacrylic acid and / or acrylic acid.

[0022] Of the above-mentioned monomers (a3) ​​having a carboxyl group, (meth)acrylic acid is preferred. When the copolymer resin particles (A) contain the monomer (a3) ​​having a carboxyl group as a constituent monomer, the weight ratio of the monomer (a3) ​​having a carboxyl group based on the total weight of the constituent monomers of the copolymer resin particles (A) is preferably 1 to 20 wt %, more preferably 1 to 10 wt %.

[0023] As the other monomer (a4), known monomers other than the monomer (a1), the monomer (a2) having a hydroxyl group, the crosslinkable monomer (b), and the monomer (a3) ​​having a carboxyl group can be used, such as (meth)acrylamide, N-alkyl(C1-3)(meth)acrylamide, and alkyl (meth)acrylate (wherein the alkyl is C1-2). These may be used alone or in combination of two or more. The weight proportion of the other monomer (a4) based on the total weight of the constituent monomers of the copolymer resin particles (A) is preferably 10% by weight or less, more preferably 5% by weight or less.

[0024] The copolymer resin particles (A) can be produced by various polymerization methods including known aqueous solution polymerization (e.g., adiabatic polymerization, thin film polymerization, spray polymerization, etc. described in JP-A-55-133413) and known reverse phase suspension polymerization (e.g., those described in JP-B-54-30710, JP-A-56-26909, and JP-A-1-5808) [e.g., photopolymerization (e.g., that described in JP-B-6-804), precipitation polymerization (e.g., that described in JP-A-61-123610), reverse phase emulsion polymerization (e.g., that described in JP-A-58-197398), etc.], if necessary, using a known radical polymerization initiator (d) and a known solvent (e).

[0025] The number average particle diameter of the copolymer resin particles (A) is preferably 0.1 to 500 μm, more preferably 1 to 250 μm, and even more preferably 10 to 200 μm. In this specification, the number average particle diameter is measured using an electron microscope (Olympus Corporation, "IX71") on 50 arbitrarily selected copolymer resin particles (A).

[0026] The number average particle size can be appropriately adjusted by, for example, polymerization conditions such as stirring, and classification after polymerization.

[0027] <Adhesive Protein (B)> Examples of the adhesive protein (B) include a polypeptide (B1) (hereinafter also simply referred to as polypeptide (B1)) having at least one cell-adhesive minimal amino acid sequence (X) (hereinafter also simply referred to as minimal amino acid sequence (X)) per molecule, and an extracellular matrix (B2). These may be used alone or in combination of two or more. The adhesive protein (B) is preferably a polypeptide (B1) or an extracellular matrix (B2) having at least one cell-adhesive minimal amino acid sequence (X) per molecule.

[0028] The minimal amino acid sequence (X) may be, for example, one described in "Pathophysiology, Vol. 9, No. 7, pp. 527-535, 1990" or "Osaka Prefectural Maternal and Child Medical Center Journal, Vol. 8, No. 1, pp. 58-66, 1992."

[0029] Among these minimum amino acid sequences (X), the RGD sequence, the LDV sequence, the LRE sequence, the HAV sequence, the REDV sequence (SEQ ID NO: 1), the YIGSR sequence (SEQ ID NO: 2), the PDSGR sequence (SEQ ID NO: 3), the RYVVLPR sequence (SEQ ID NO: 4), the LGTIPG sequence (SEQ ID NO: 5), the RNIAEIIKDI sequence (SEQ ID NO: 6), the IKVAV sequence (SEQ ID NO: 7), the DGEA sequence (SEQ ID NO: 8), the GVKGDKGNPGWPGAP sequence (SEQ ID NO: 9), the GEFYFDLRLKGDK sequence (SEQ ID NO: 10), the YKLNVNDS sequence (SEQ ID NO: 11), the AKPSYPPTYK sequence (SEQ ID NO: 12), Sequences having at least one sequence selected from the group consisting of the NRWHSIYITRFG sequence (SEQ ID NO: 13), the TWYKIAFQRNRK sequence (SEQ ID NO: 14), the RKRLQVQLSIRT sequence (SEQ ID NO: 15), and the PHSRN sequence (SEQ ID NO: 16) are preferred, and from the viewpoint of cell adhesiveness, those having at least one sequence selected from the group consisting of the RGD sequence, the YIGSR sequence (SEQ ID NO: 2), the IKVAV sequence (SEQ ID NO: 7), the RKRLQVQLSIRT sequence (SEQ ID NO: 15), and the PHSRN sequence (SEQ ID NO: 16) are more preferred, and those having the RGD sequence are even more preferred.

[0030] These minimum amino acid sequences (X) may contain other amino acids {A (alanine), G (glycine), S (serine), T (threonine), V (valine), L (leucine), I (isoleucine), C (cysteine), M (methionine), F (phenylalanine), Y (tyrosine), P (proline), W (tryptophan), N (asparagine), Q (glutamine), D (aspartic acid), E (glutamic acid), R (arginine), K (lysine), H (histidine), etc.} at both ends. The minimum amino acid sequence (X) may contain one of the above-mentioned other amino acids alone or two or more of them in combination.

[0031] The polypeptide (B1) may have at least one minimal amino acid sequence (X) per molecule, but from the viewpoint of cell adhesiveness, the polypeptide (B1) preferably has 1 to 50 minimal amino acid sequences (X) per molecule, more preferably 2 to 50 minimal amino acid sequences (X), even more preferably 3 to 30 minimal amino acid sequences (X), particularly preferably 4 to 20 minimal amino acid sequences (X), and most preferably 5 to 15 minimal amino acid sequences (X). The polypeptide (B1) may have one of the minimal amino acid sequences (X) described above alone or a combination of two or more minimal amino acid sequences (X).

[0032] In order to improve the thermal stability of the polypeptide (B1), it is preferable that the polypeptide (B1) has an auxiliary amino acid sequence (Y) in addition to the minimum amino acid sequence (X).

[0033] As the auxiliary amino acid sequence (Y), an amino acid sequence other than the minimum amino acid sequence (X) can be used, and from the viewpoint of the thermal stability of the polypeptide (B1), a sequence having G (glycine) and / or A (alanine) is preferred. Hereinafter, a sequence in which the same sequence is repeated two or more times may be indicated by writing the repeating part in parentheses and writing the number of repeats in the lower right corner outside the parentheses. For example, a sequence in which the GA sequence is repeated a times is referred to as "(GA) ａ It may be referred to as "array."

[0034] The auxiliary amino acid sequence (Y) may be, for example, (GA) ａ Sequence, (GAGAGS) ｂ Sequence, (GAGAGY) ｃ Sequence, (GAGVGY) ｄ Sequence, (GAGYGV) ｅ The sequence, {DGG(A) ｆ G.G.A. ｇ Sequence, (GVPGV) ｈ Sequence, (G) ｉ Sequence (A) ｊ Sequence, (GGA) ｋ Sequence, (GVGVP) ｍ Sequence, (GPP) ｎ The sequence is (GAQGPAGPG) ｏ The sequence is (GAPGAPGSQGAPGLQ) ｐ The sequence is (GAPGTPGPQGLPGSP) ｑ Sequences having sequences etc. are included.

[0035] Among these, from the viewpoint of thermal stability, (GA) ａ Sequence, (GAGAGS) ｂ Sequence, (GAGAGY) ｃ Sequence, (GAGVGY) ｄ Sequence, (GAGYGV) ｅ The sequence, {DGG(A) ｆ G.G.A. ｇ Sequence, (GVPGV) ｈ Sequence, (GVGVP) ｍ Sequence and (GPP) ｎ Preferably, the sequence has at least one selected from the group consisting of (GAGAGS) ｂ Sequence, (GVPGV) ｈ Sequence, (GVGVP) ｍ Sequence and (GPP) ｎ It has at least one selected from the group consisting of sequences, and particularly preferably (GAGAGS) ｂ It has an array.

[0036] Here, a is an integer of 5 to 100, b is an integer of 1 to 33, c, d, and e are each independently an integer of 2 to 33, f is an integer of 1 to 194, g is an integer of 1 to {200 / (6+f)} rounded down to one whole number, h is an integer of 2 to 40, i and j are each independently an integer of 10 to 200, k is an integer of 3 to 66, m is an integer of 2 to 40, n is an integer of 3 to 66, o is an integer of 1 to 22, and p and q are each independently an integer of 1 to 13.

[0037] The auxiliary amino acid sequence (Y) preferably contains G (glycine) and / or A (alanine). When the auxiliary amino acid sequence (Y) contains G (glycine) and A (alanine), the total number of these is not particularly limited, but is preferably 10 to 100%, more preferably 20 to 95%, particularly preferably 30 to 90%, and most preferably 40 to 85% of the total number of amino acids in the auxiliary amino acid sequence (Y). When the total number of G (glycine) and A (alanine) is within the above range, the thermal stability is further improved.

[0038] When the auxiliary amino acid sequence (Y) contains both G (glycine) and A (alanine), the ratio of the number of G (glycine) to the number of A (alanine) (G / A) is not particularly limited, but is preferably 0.03 to 40, more preferably 0.08 to 13, and particularly preferably 0.2 to 5. When the ratio of the number of G (glycine) to the number of A (alanine) (G / A) is within the above range, the thermal stability is further improved.

[0039] In addition to the sequences exemplified above, the auxiliary amino acid sequence (Y) may contain other amino acids {A (alanine), G (glycine), S (serine), T (threonine), V (valine), L (leucine), I (isoleucine), C (cysteine), M (methionine), F (phenylalanine), Y (tyrosine), P (proline), W (tryptophan), N (asparagine), Q (glutamine), D (aspartic acid), E (glutamic acid), R (arginine), K (lysine), H (histidine), etc.}. The auxiliary amino acid sequence (Y) may contain one of the above-mentioned other amino acids alone, or two or more of them in combination.

[0040] (GA) ａ Examples of the auxiliary amino acid sequence (Y) having the sequence include the amino acid sequences represented by SEQ ID NOs: 17 to 19. (GAGAGS) ｂ Examples of the auxiliary amino acid sequence (Y) having a sequence include the amino acid sequences represented by SEQ ID NOs: 20 to 23. (GAGAGY) ｃ Examples of the auxiliary amino acid sequence (Y) having the sequence include the amino acid sequences represented by SEQ ID NOs: 24 to 26. (GAGVGY) ｄ Examples of the auxiliary amino acid sequence (Y) having the sequence include the amino acid sequences represented by SEQ ID NOs: 27 to 29. (GAGYGV) ｅ Examples of the auxiliary amino acid sequence (Y) having the sequence include the amino acid sequences represented by SEQ ID NOs: 30 to 32. ｆ G.G.A. ｇ Examples of the auxiliary amino acid sequence (Y) having the sequence include the amino acid sequences represented by SEQ ID NOs: 33 to 35. (GVPGV) ｈExamples of the auxiliary amino acid sequence (Y) having the sequence include the amino acid sequences represented by SEQ ID NOs: 36 to 39. (G) ｉ Examples of the auxiliary amino acid sequence (Y) having the sequence include the amino acid sequences represented by SEQ ID NOs: 40 to 42. ｊ Examples of the auxiliary amino acid sequence (Y) having the sequence include the amino acid sequences represented by SEQ ID NOs: 43 to 45. (GGA) ｋ Examples of the auxiliary amino acid sequence (Y) having the sequence include the amino acid sequences represented by SEQ ID NOs: 46 to 48. (GVGVP) ｍ Examples of the auxiliary amino acid sequence (Y) having a sequence include the amino acid sequences represented by SEQ ID NOs: 49 to 51. (GPP) ｎ Examples of the auxiliary amino acid sequence (Y) having the sequence include the amino acid sequences represented by SEQ ID NOs: 52 to 54. (GAQGPAGPG) ｏ Examples of the auxiliary amino acid sequence (Y) having the sequence include the amino acid sequences represented by SEQ ID NOs: 55 to 57. (GAPGAPGSQGAPGLQ) ｐ Examples of the auxiliary amino acid sequence (Y) having the sequence include the amino acid sequences represented by SEQ ID NOs: 58 to 60. (GAPGTPGPQGLPGSP) ｑ Examples of the auxiliary amino acid sequence (Y) having a sequence include the amino acid sequences represented by SEQ ID NOs: 61 to 63.

[0041] Here, a is an integer of 5 to 100, b is an integer of 1 to 33, c, d, and e are each independently an integer of 2 to 33, f is an integer of 1 to 194, g is an integer of 1 to {200 / (6+f)} rounded down to one whole number, h is an integer of 2 to 40, i and j are each independently an integer of 10 to 200, k is an integer of 3 to 66, m is an integer of 2 to 40, n is an integer of 3 to 66, o is an integer of 1 to 22, and p and q are each independently an integer of 1 to 13.

[0042] Of these auxiliary amino acid sequences, from the viewpoint of thermal stability, the amino acid sequences represented by SEQ ID NO: 17, 18, 21, 22, 23, 24, 25, 27, 28, 30, 31, 33, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 46, 47, 49, 50, 52, 53, 55, 56, 58, 59, 61 or 62 are preferred, the amino acid sequences represented by SEQ ID NO: 18, 21, 22, 23, 25, 28, 31, 35, 36, 37, 38, 39, 41, 44, 47, 50, 53, 56, 59 or 62 are more preferred, and the amino acid sequences represented by SEQ ID NO: 21, 22 or 39 are particularly preferred.

[0043] When the polypeptide (B1) contains auxiliary amino acid sequences (Y), the number of auxiliary amino acid sequences (Y) contained in one molecule of the polypeptide (B1) is, from the viewpoint of thermal stability, preferably 2 to 50, more preferably 3 to 30, particularly preferably 4 to 20, and most preferably 5 to 15. The polypeptide (B1) may contain one of the above-mentioned auxiliary amino acid sequences (Y) alone or a combination of two or more of them.

[0044] The polypeptide (B1) may contain a branched chain, may be partially cross-linked, or may contain a cyclic structure. However, the polypeptide (B1) is preferably not cross-linked, more preferably has a non-cross-linked linear structure, and particularly preferably has a non-cross-linked linear structure without a cyclic structure. The linear structure also includes a β structure (a secondary structure in which the bent portions of at least two linear peptides are aligned parallel to each other and hydrogen bonds are formed between them).

[0045] From the viewpoints of cell adhesiveness and thermal stability, the polypeptide (B1) preferably comprises a structure in which a minimum amino acid sequence (X) and an auxiliary amino acid sequence (Y) are alternately chemically bonded, if necessary via another amino acid sequence such as an intervening amino acid sequence (Z) described below, and more preferably comprises a structure in which an amino acid sequence containing the intervening amino acid sequence (Z) and an auxiliary amino acid sequence (Y) are alternately chemically bonded to both ends of the minimum amino acid sequence (X). In this case, from the viewpoints of cell adhesiveness, the number of repeating units of the minimum amino acid sequence (X) and the auxiliary amino acid sequence (Y) (the total number of (X-Y) and (X-Z-Y)) is preferably 1 to 50, more preferably 2 to 40, particularly preferably 3 to 30, and most preferably 4 to 20.

[0046] As the intervening amino acid sequence (Z), from the viewpoint of cell adhesiveness, the N-terminus of the minimum amino acid sequence (X) may contain a GAAVTG sequence (SEQ ID NO: 64), a GLPGPKGD sequence (SEQ ID NO: 65), a GPAVTG sequence (SEQ ID NO: 66), an AGPKGADGSPGPAVTG sequence (SEQ ID NO: 67), a GAAVCEPG sequence (SEQ ID NO: 68), a GAALCVSEPG sequence (SEQ ID NO: 69), a SPASAALCVSEPG sequence (SEQ ID NO: 70), a SPASAAVCEPG sequence (SEQ ID NO: 71), an AGPKGADGSPGPAVCEPG sequence (SEQ ID NO: 72), an AGPKGADGSPGPA It is preferable that at least one selected from the group consisting of the LCVSEPG sequence (SEQ ID NO: 73), the GPAVCEPG sequence (SEQ ID NO: 74), the GPALCVSEPG sequence (SEQ ID NO: 75), and the GAAPGAS sequence (SEQ ID NO: 76) binds, it is more preferable that at least one selected from the group consisting of the GAAVTG sequence (SEQ ID NO: 64), the GLPGPKGD sequence (SEQ ID NO: 65), the GPAVTG sequence (SEQ ID NO: 66), and the AGPKGADGSPGPAVTG sequence (SEQ ID NO: 67) binds, and it is particularly preferable that the GAAVTG sequence (SEQ ID NO: 64) binds.

[0047] As the intervening amino acid sequence (Z), from the viewpoint of cell adhesiveness, the C-terminus of the minimum amino acid sequence (X) may contain a SPASAAGY sequence (SEQ ID NO: 77), a SPASAALCVS sequence (SEQ ID NO: 78), a SPASAAVC sequence (SEQ ID NO: 79), an AGPKGADGSPGP sequence (SEQ ID NO: 80), an AGPKGADGSPGPAVC sequence (SEQ ID NO: 81), an AGPKGADGSPGPALCVS sequence (SEQ ID NO: 82), an AGPKGADGSP sequence (SEQ ID NO: 83), a SPASAAGPVGSP sequence (SEQ ID NO: 84), a CDAGY sequence (SEQ ID NO: 85), a CDAGPVGSP sequence (SEQ ID NO: 86), and a SAGPSAGY sequence (SEQ ID NO: 87). It is preferable that at least one selected from the group consisting of a SPASAAGY sequence (SEQ ID NO: 77), a SPASAALCVS sequence (SEQ ID NO: 78), a SPASAAVC sequence (SEQ ID NO: 79), an AGPKGADGSPGP sequence (SEQ ID NO: 80), an AGPKGADGSPGPAVC sequence (SEQ ID NO: 81), an AGPKGADGSPGPALCVS sequence (SEQ ID NO: 82), an AGPKGADGSP sequence (SEQ ID NO: 83), and a SPASAAGPVGSP sequence (SEQ ID NO: 84) is bound, and it is particularly preferable that the SPASAAGY sequence (SEQ ID NO: 77) is bound.

[0048] Furthermore, the number of minimal amino acid sequences (X) and auxiliary amino acid sequences (Y) contained may be the same or different. If they are different, from the viewpoints of cell adhesiveness and thermal stability, it is preferable that the number of either one is one less than the number of the other, and it is more preferable that the number of auxiliary amino acid sequences (Y) contained is one less than the number of minimal amino acid sequences (X). From the viewpoints of cell adhesiveness and thermal stability, the ratio (X / Y) of the number of minimal amino acid sequences (X) contained to the number of auxiliary amino acid sequences (Y) contained in polypeptide (B1) is preferably 0.5 to 2, more preferably 0.9 to 1.4, and particularly preferably 1 to 1.3.

[0049] Furthermore, the polypeptide (B1) may contain other amino acids {A (alanine), G (glycine), S (serine), T (threonine), V (valine), L (leucine), I (isoleucine), C (cysteine), M (methionine), F (phenylalanine), Y (tyrosine), P (proline), W (tryptophan), N (asparagine), Q (glutamine), D (aspartic acid), E (glutamic acid), R (arginine), K (lysine), H (histidine), etc.} in the terminal portion (from the minimum amino acid sequence (X) or auxiliary amino acid sequence (Y) to the peptide terminus). The polypeptide (B1) may contain one of the above-mentioned other amino acids alone or two or more of them in combination. When the polypeptide (B1) contains other amino acids, the number of such amino acids contained is preferably 1 to 1,000 per polypeptide (B1), more preferably 3 to 300, and particularly preferably 10 to 100.

[0050] The weight average molecular weight (Mw) of the polypeptide (B1) is not particularly limited, but is preferably 1,000 to 1,000,000, more preferably 2,000 to 700,000, particularly preferably 3,000 to 400,000, and most preferably 4,000 to 200,000.

[0051] The Mw of the polypeptide (B1) can be determined by a known method, such as separating a measurement sample (polypeptide, etc.) by SDS-PAGE (SDS polyacrylamide gel electrophoresis) and comparing the migration distance with that of a standard substance.

[0052] Some preferred examples of the polypeptide (B1) are shown below. (1) When the minimum amino acid sequence (X) is an RGD sequence (x1), 13 RGD sequences (x1) and (GAGAGS) ９A polypeptide (SEQ ID NO: 88) having a molecular weight of approximately 110,000 and a structure in which these are chemically bonded alternately to each other ("Pronectin F", Pronectin is a registered trademark of Sanyo Chemical Industries, Ltd. (Japan and the United States). Manufactured by Sanyo Chemical Industries, Ltd. (the same applies hereinafter)); a protein in which Pronectin F is modified with compound (AM), specifically, a polypeptide ("Pronectin F Plus") modified with N,N-dimethylaminoethyl chloride hydrochloride as compound (AM); five RGD sequences (x1) and (GAGAGS) ３ A polypeptide (SEQ ID NO: 89) ("Pronectin F2") having a molecular weight of approximately 20,000 and a structure in which five sequences (SEQ ID NO: 21) (y2) are chemically bonded alternately; three RGD sequences (x1) and (GVPGV) ２ GG (GAGAGS) ３ and a polypeptide (SEQ ID NO: 90) ("Pronectin F3") having a molecular weight of approximately 10,000 and a structure in which three of the sequences (SEQ ID NO: 39) (y3) are chemically bonded alternately.

[0053] (2) When the minimum amino acid sequence (X) is an IKVAV sequence (x2), the RGD sequence (x1) of Pronectin F, Pronectin F2, or Pronectin F3 is changed to an IKVAV sequence (SEQ ID NO: 7) (x2), resulting in "Pronectin L," "Pronectin L2," "Pronectin L3," or the like.

[0054] (3) When the minimum amino acid sequence (X) is a YIGSR sequence (x3), the RGD sequence (x1) of Pronectin F, Pronectin F2, or Pronectin F3 is changed to a YIGSR sequence (SEQ ID NO: 2) (x3), resulting in "Pronectin Y," "Pronectin Y2," "Pronectin Y3," or the like.

[0055] In addition to the polypeptides (1) to (3), RetroNectin (recombinant human fibronectin CH-296) manufactured by Takara Shuzo Co., Ltd. (a polypeptide having a molecular weight of approximately 60,000 and containing an RGD sequence (x1) and an LDV sequence as the minimal amino acid sequence (X)) and RGDS-Protein A manufactured by Takara Shuzo Co., Ltd. (a polypeptide having a molecular weight of approximately 30,000 and containing an RGD sequence (x1) as the minimal amino acid sequence (X)) can also be preferably used (however, these polypeptides are naturally derived and do not contain the auxiliary amino acid sequence (Y). Therefore, their thermal stability and other properties are inferior to those of the above polypeptides (1) to (3). The amino acid sequences of these polypeptides are disclosed in Japanese Patent Application Laid-Open No. 2-311498).

[0056] The polypeptide (B1) includes an artificially synthesized polypeptide (artificial polypeptide), and is produced, for example, by organic synthesis (solid-phase synthesis, liquid-phase synthesis, etc.) and biochemical synthesis (genetically modified microorganisms (yeast, bacteria, Escherichia coli, etc.)), etc. In other words, the polypeptide (B1) does not include cell adhesive proteins such as animal-derived collagen and fibronectin.

[0057] As an organic synthesis method, for example, the method described in "Continued Biochemical Experiment Lectures 2, Chemistry of Proteins (Part 2)" edited by the Japanese Biochemical Society, pp. 641-694 (May 20, 1987; published by Tokyo Kagaku Dojin Co., Ltd.) can be used. As a biochemical synthesis method, for example, the method described in JP-A-3-502935 can be used. In terms of ease of synthesis of polypeptide (B1), biochemical synthesis methods using recombinant microorganisms are preferred, and particularly preferred is a synthesis method using recombinant Escherichia coli.

[0058] As the extracellular matrix (B2), for example, collagen, gelatin, fibronectin, gelsolin, vitronectin, laminin, etc. can be used.

[0059] The collagen is not particularly limited and may be any of type I, type II, type III, type IV, type V, etc., and examples thereof include collagen degraded by enzymes, collagen obtained by cleaving telopeptides, collagen synthesized by genetic engineering, etc. Commercially available products include Cellmatrix Type IA (type I, manufactured by Nitta Gelatin Co., Ltd.), collagen type III derived from bovine dermis (type III, manufactured by Nippi Corporation), and iMatrix-111, 221, 332, 411, and 511 (manufactured by Matrixome Co., Ltd.).

[0060] <Carrier for cell culture> The carrier for cell culture of the present invention contains the copolymer resin particles (A) and an adhesive protein (B). The content of the adhesive protein (B) is not particularly limited, but is preferably 100 ng / g to 50 mg / g, and more preferably 200 ng / g to 20 mg / g, based on the dry weight of the carrier for cell culture.

[0061] The cell culture carrier of the present invention may be in a state in which the copolymer resin particles (A) are coated with the adhesive protein (B) or in a state in which the copolymer resin particles (A) and the adhesive protein (B) are chemically bonded to each other, but the latter is preferred. The chemical bond is more preferably an amide bond.

[0062] A cell culture carrier in which copolymer resin particles (A) are coated with adhesive protein (B) can be obtained by dissolving adhesive protein (B) in a solvent (e.g., water, phosphate buffer solution (PBS), etc.), immersing copolymer resin particles (A) in the solution, filtering, and drying.

[0063] A cell culture carrier in which copolymer resin particles (A) and adhesive protein (B) are bonded by amide bonds can be obtained by first reacting the carboxyl groups of the copolymer resin particles (A) with a carbodiimide compound to obtain acylisourea {R'-N=C(OCOR)-NH-R' (wherein -OCOR is derived from the copolymer resin particles (A))}, and then adding a protein (B) having a primary or secondary amino group to the acylisourea. Examples of carbodiimide compounds include N,N'-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride.

[0064] The cell culture carrier of the present invention may contain a cell growth factor 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 (1999) published by Nagoya University Press, Foundation for Incorporated Foundation). 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.

[0065] The cell growth factor is preferably bound to the copolymer resin particles (A).

[0066] When the carrier for cell culture of the present invention contains a cell growth factor, 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 gram of carrier for cell culture, from the viewpoint of cell proliferation and the like.

[0067] 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.

[0068] There are no limitations on the cells (CE) that can adhere to the carrier for cell culture of the present invention, as long as they are cells. However, because the carrier for cell culture of the present invention has high cell proliferation properties, normal cells derived from mammals, established cell lines derived from mammals, and insect cells that are used for the production of useful substances such as pharmaceuticals and for treatment are suitable.

[0069] 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 (islet cells of Langerhans, 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, fibroblasts, vascular endothelial cells, smooth muscle cells, etc.), cells involved in the eye (photoreceptors, corneal epithelial cells, corneal endothelial cells, etc.), cells involved in the prostate (epithelial cells, stromal cells, smooth muscle cells, etc.), cells involved in bone (osteoblasts, osteocytes, osteoclasts, etc.), cells involved in cartilage (chondroblasts, chondrocytes, etc.), cells involved in teeth (period ligament cells, osteoblasts, etc.), cells involved in blood (white blood cells, 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, epidermal stem 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 (adipocyte precursor cells, vascular endothelial precursor cells, chondrocyte precursor cells, lymphocyte precursor cells, NK precursor cells, etc.), etc.

[0070] Mammalian-derived cell lines include CRFK cells, 3T3 cells, A549 cells, AH130 cells, B95-8 cells, BHK cells, BOSC23 cells, and 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 Co., Ltd., 1999)).

[0071] Examples of insect cells include silkworm cells (BmN cells, BoMo cells, etc.), mulberry silkworm cells, Anemone persica cells, Anemone shinju cells, armyworm cells (Sf9 cells, Sf21 cells, etc.), mulberry fly cells, leafroller cells, Drosophila melanogaster 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 (edited by Kimura Shigeru, published by Kogyo Chosakai Co., Ltd., 2000)).

[0072] Among these cells, mammalian-derived normal cells and mammalian-derived established cell lines are preferred from the viewpoint of treatment and production of useful substances such as pharmaceuticals. Furthermore, in terms of their usefulness in treatment, kidney cells, smooth muscle cells, hepatocytes, osteoblasts, epithelial cells, fibroblasts, vascular endothelial cells, and stem cells are more preferred, kidney cells and epithelial cells are particularly preferred, and kidney cells are most preferred. Furthermore, in terms of their usefulness in production of useful substances such as pharmaceuticals, 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, CRFK cells, MDCK cells, and VERO cells are particularly preferred, and VERO cells are most preferred.

[0073] The culture medium used in the cell culture method using the carrier for cell culture of the present invention includes 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 ... ATG™ medium and mixed media thereof, etc.); general media (RPMI medium, MEM medium, Eagle's MEM medium (E-MEM medium), BME medium, DME medium, αMEM medium, IMEM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, ASF103 medium, ASF104 medium, ASF301 medium, TC-100 medium, Sf-900II medium, Ex-cell405 medium, Express-Five medium, Drosophila medium and mixed media thereof, etc.); and mixed media thereof, etc.

[0074] These media may also contain serum. Examples of serum include 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 the medium contains serum, among these, human serum, bovine serum, and horse serum are preferred. Furthermore, the origin of the animal serum includes serum derived from an adult, serum derived from a pup, serum derived from a newborn, and serum derived from a fetus, etc. Among these, serum derived from a pup, serum derived from a newborn, and serum derived from a fetus are preferred, serum derived from a newborn and serum derived from a fetus are more preferred, and serum derived from a fetus is particularly preferred. Furthermore, the serum may be subjected to inactivation treatment, treatment to remove antibodies, etc.

[0075] When the medium contains serum, the content thereof is preferably 0.1 to 50% by weight, more preferably 0.3 to 30% by weight, and particularly preferably 1 to 20% by weight, based on the weight of the medium.

[0076] The medium may contain a cell growth factor as needed, which can increase the cell growth rate and cell activity.

[0077] 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 further shorten the healing period.

[0078] When a cell growth factor is used, the content of the cell growth factor varies depending on the type of cell growth factor. －１６ ~10－３ % by weight is preferred, and 10% by weight is more preferred. －１４ ~10 －５ % by weight, particularly preferably 10 －１２ ~10 －７ Percent by weight.

[0079] The medium may further contain an antibacterial agent (such as amphotericin B, gentamicin, penicillin, and streptomycin). When an antibacterial agent is contained, the content varies depending on the type of antibacterial agent, but is generally 10% or more based on the weight of the medium. －６ Preferably, the content is up to 10% by weight, and more preferably 10 －５ up to 1% by weight, particularly preferably 10 －４ 0.1% by weight.

[0080] The concentration of cells dispersed in the medium is not particularly limited, but is preferably 100 to 100 million cells, more preferably 1,000 to 10 million cells, and particularly preferably 10,000 to 1 million cells per mL of medium.

[0081] The number of cells can be counted by a known method, for example, 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 of the cell culture carrier to be added to the medium can be determined appropriately depending on the type of cells to be cultured, and is preferably 0.005 to 800 g, more preferably 0.02 to 200 g, and particularly preferably 0.1 to 40 g per 1 L of the medium.

[0082] There are no particular limitations on the culture conditions, and carbon dioxide (CO ２ The conditions for culturing are as follows: a concentration of 1 to 20% by volume, at 5 to 45°C for 1 hour to 100 days, with the medium being changed every 1 to 10 days as necessary. ２ The culture conditions are a concentration of 3 to 10% by volume, at 30 to 40° C. for 1 to 20 days, with the medium being changed every 1 to 3 days.

[0083] As a method for detaching cells from a cell culture carrier, a known method can be used, for example, a method of detaching cells using a chelating agent (EDTA, etc.), a non-animal-derived protease (a plant-derived protease (papain, etc.)), a recombinant synthetic enzyme (trade name: TrypLE™ Select, manufactured by Invitrogen Corporation, etc.) and / or an animal-derived protease (trypsin, collagenase, etc.).

[0084] <Others> This specification discloses the following: <1> A carrier for cell culture containing copolymer resin particles (A) having as constituent monomers a monomer (a1) represented by the following general formula (1), a monomer (a2) having a hydroxyl group represented by the following general formula (2), and a crosslinkable monomer (b), and an adhesive protein (B). CH ２ = C(R １ )-(C=O)-QR ２ -N(R ３ ) (R ４ ) (1) [In general formula (1), R １ is H or CH ３ Q is O or NH; R ２ represents a divalent hydrocarbon group having 2 to 5 carbon atoms; R ３ , R ４ are each independently a monovalent hydrocarbon group having 1 to 4 carbon atoms.] CH ２ = C(R ５ )-(C=O)-Q-(R ６ -O) ｎ -H (2) [In general formula (2), R ５ is H or CH ３ Q is O or NH; R ６is a divalent hydrocarbon group having 1 to 4 carbon atoms; and n is an integer of 1 to 7.] <2> The carrier for cell culture according to <1>, wherein the weight ratio of the crosslinkable monomer (b) based on the weight of the constituent monomers of the copolymer resin particles (A) is 0.1 to 3 wt %. <3> The carrier for cell culture according to <1> or <2>, wherein the copolymer resin particles (A) further contain, as a constituent monomer, a monomer (a3) ​​having a carboxyl group. <4> The carrier for cell culture according to any one of <1> to <3>, wherein the number average particle diameter of the copolymer resin particles (A) is 0.1 to 500 μm. <5> The carrier for cell culture according to any one of <1> to <4>, wherein the adhesive protein (B) is a polypeptide (B1) or an extracellular matrix (B2) having at least one cell-adhesive minimal amino acid sequence (X) per molecule. <6> The carrier for cell culture according to any one of <1> to <5>, wherein the content of the adhesive protein (B) is 100 ng / g to 50 mg / g based on the dry weight of the carrier for cell culture.

[0085] Examples of the present invention will be described below, but the present invention is not limited to these. In the following, parts are by weight.

[0086] <Production Example 1> Copolymer Resin Particles (A-1) 139.2 g of sodium chloride and 39.6 g of magnesium chloride hexahydrate were added to 480.0 g of pure water and dissolved by stirring to prepare an aqueous solution S. Next, 20.0 g of 1-hexanol (e-1) as a solvent and 1.0 g of 2,2'-azobisisobutyrate dimethyl (d-1) as a radical polymerization initiator were added to a composition consisting of 50.0 g of N-[3-dimethylaminopropyl]acrylamide (a1-1), 22.1 g of 2-hydroxyethyl methacrylate (a2-1), 22.1 g of 2-hydroxypropyl methacrylate (a2-2), 5.1 g of methacrylic acid (a3-1), and 0.7 g of ethylene glycol dimethacrylate (b2-1), to prepare a monomer composition. The entire amount of the aqueous solution S 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. 101.1 g of a 5M aqueous sodium hydroxide solution was added to the separable flask to prepare a magnesium hydroxide hydrocolloid dispersion. Next, the stirring speed was set to 700 rpm, and the entire amount of the monomer composition was poured into the separable flask, followed by heating in a hot water bath until the internal temperature reached 86°C. Next, suspension polymerization was carried out by stirring for 3 hours while maintaining the temperature at 86°C, and then the mixture was 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 resulting resin particles were classified to obtain copolymer resin particles (A-1). The number average particle diameter of 50 randomly selected copolymer resin particles (A-1) measured using an electron microscope (manufactured by Olympus Corporation, "IX71") was 60 μm.

[0087] <Production Examples 2 to 17, Comparative Production Example 1> Copolymer resin particles (A-2) to (A-17) and (A'-1) were obtained in the same manner as in Production Example 1, except that the composition of the constituent monomers was changed according to the descriptions in Tables 1 and 2. The constituent monomers listed in Tables 1 and 2 are as follows. (a1-2): N-[3-(dimethylamino)ethyl]methacrylate (a2-3): Polyethylene glycol monomethacrylate (Blemmer PE-90, manufactured by NOF Corporation, average degree of polymerization of ethylene oxide: 2) (a2-4): Polyethylene glycol monomethacrylate (Blemmer PE-200, manufactured by NOF Corporation, average degree of polymerization of ethylene oxide: 4.5) (a2-5): Polypropylene glycol monomethacrylate (Blemmer PP-1000, manufactured by NOF Corporation, average degree of polymerization of propylene oxide: 4 to 6) (a2-6): Polyethylene glycol polypropylene glycol monomethacrylate (Blemmer 50PEP-300, manufactured by NOF Corporation, average degree of polymerization of ethylene oxide: 3.5, average degree of polymerization of propylene oxide: 2.5) (a2-7): Polypropylene glycol polybutylene glycol monomethacrylate (Blemmer 10PPB-500B, manufactured by NOF Corporation, average degree of polymerization of propylene oxide: 1, average degree of polymerization of butylene oxide: 6 moles) (a2-8): Polyethylene glycol monoacrylate (Blemmer AE-90U, manufactured by NOF Corporation, average degree of polymerization of ethylene oxide: 2) (a2-9): Polyethylene glycol monoacrylate (Blemmer AE-200, manufactured by NOF Corporation, average degree of polymerization of ethylene oxide: 4.5) (a2-10): Polypropylene glycol monoacrylate (Blemmer AP-200, manufactured by NOF Corporation, average degree of polymerization of propylene oxide: 3.5) (a2-11): Polypropylene glycol monoacrylate (Blemmer AP-400, manufactured by NOF Corporation, average degree of polymerization of propylene oxide: 6) In Production Example 7, the stirring speed was set to 1500 rpm, and in Production Example 8, the stirring speed was set to 400 rpm. The number average particle size of the obtained copolymer resin particles was measured by the method described above. The results are shown in Tables 1 and 2.

[0088]

[0089]

[0090] <Production Example 18> Polypeptide (B1-1) According to the method described in the Examples in JP-A-3-502935, a polypeptide (B1-1) containing 13 RGD sequences and (GAGAGS) ９ "Pronectin F" was produced as a polypeptide (SEQ ID NO: 88) with a molecular weight of approximately 110,000, which has a structure in which 12 amino acids (SEQ ID NO: 22) are chemically bonded alternately. 50 mg of the polypeptide and 150 mg of N,N-dimethylaminoethyl chloride hydrochloride (special grade reagent) were dissolved in 1.5 mL of 4.5 M aqueous lithium perchlorate solution at 20-40°C to obtain a solution. While stirring the solution at 20-40°C, 1.325 mL of 4.5 M aqueous lithium perchlorate solution containing 100 mg of sodium hydroxide (special grade reagent) was added dropwise at a constant rate over 45-50 seconds. The resulting reaction solution was stirred at room temperature (25°C) for 1 hour, and then dialyzed against 10 L of deionized water for 48 hours using a dialysis membrane with a molecular weight cutoff of 12,000-14,000. Note that the deionized water was replaced every 4 hours during the first 12 hours. The resulting aqueous solution was freeze-dried for 24 hours at -20°C and 0.1 kPa or less to obtain water-soluble polypeptide (B1-1). The number of N,N-dimethylaminoethyl chloride hydrochloride groups introduced was measured according to the method described in the Examples of JP-A-10-500701, and was found to be 12 per molecule.

[0091] 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 wt% sodium chloride and completely dissolved. Next, 1 g of copolymer resin particles (A-1) was added, and the mixture was reacted for 4 hours at room temperature using a stirring blade made of polyethylene fluoride resin at a stirring speed of 300 rpm. 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 particles were removed and suspended in 12 mL of PBS solution (pH 7.2). Next, 8 mL of PBS solution (pH 7.2) containing polypeptide (B1-1) at a concentration of 1 mg / mL was added, and the mixture was reacted for 12 hours. 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 carrier for cell culture (α-1) of the present invention was obtained by suspending the carrier in about 10 mL of ion-exchanged water and lyophilizing the carrier. The weight of the polypeptide (B1-1) based on the dry weight of the carrier for cell culture (α-1) obtained was 8.0 mg / g.

[0092] Examples 2, 4 to 19, Comparative Example 1 Cell culture carriers (α-2), (α-4) to (α-19), and (α'-1) were obtained in the same manner as in Example 1, except that the amounts of copolymer resin particles (A) and adhesive protein (B) added were changed according to the descriptions in Tables 3 and 4. In Example 2, Cellmatrix Type IA (Type I, manufactured by Nitta Gelatin Co., Ltd., 3 mg / ml) (extracellular matrix (B2-1)) was used as the adhesive protein (B).

[0093] Example 3 8 mg of "Pronectin F" (manufactured by Sanyo Chemical Industries, Ltd.) (B1-2) was dissolved in 8 ml of "Pronectin F Dilution Solution" (manufactured by Sanyo Chemical Industries, Ltd.). Next, 1 g of copolymer resin particles (A-1) was added, and the mixture was adsorbed at room temperature for 12 hours at a stirring speed of 300 rpm using a stirring blade made of polyethylene fluoride resin. After adsorption, the suspension 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 a cell culture carrier (α-3) of the present invention.

[0094] The cell adhesion rate (cell adhesiveness) and cell proliferation (cell concentration) at the initial stage of cell culture were evaluated using the evaluation methods described below for each of the cell culture carriers obtained in Examples 1 to 19 and Comparative Example 1. The results are shown in Tables 3 and 4.

[0095] <Cell adhesion rate (cell adhesiveness) at the initial stage of cell culture> 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, and then 20 mL of serum medium, which was E-MEM medium supplemented with 10% by volume of fetal bovine serum (manufactured by Invitrogen Corporation), was added to each flask and left to stand for 10 minutes. The medium was aspirated and 20 mL of the same serum medium was added again to each flask. The spinner flasks were then heated at 37°C, 5% by volume of CO 2 gas, and 20 mL of CO 2 gas was added to each flask. ２ After leaving the medium in the incubator for 1 hour, VERO cells (manufactured by Sumitomo Dainippon Pharma Co., Ltd.) that had been pre-cultured in advance were seeded into the medium so that the cell concentration was 200,000 cells / mL. ２ The cells were cultured for 3 hours in an incubator with stirring at 60 rpm. After 3 hours of culture, 100 randomly selected cell culture carriers were photographed (40x magnification) using an electron microscope (Olympus Corporation, "IX71"). Carriers with cells adhering to 50% or more of the photographed surface area of ​​each cell culture carrier were counted as adhered carriers, and carriers with cells adhering to less than 50% were counted as non-adhered carriers. The cell adhesion rate was calculated using the following formula. The results are shown in Tables 3 and 4. Cell adhesion rate (%) = (number of adhered carriers (cells) x 100) / 100 (cells).

[0096] <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, and then 20 mL of serum medium, which was E-MEM medium supplemented with 10% by volume of fetal bovine serum (manufactured by Invitrogen Corporation), was added to each flask and left to stand for 10 minutes. The medium was aspirated and 20 mL of the same serum medium was added again to each flask. The spinner flasks were then heated at 37°C, 121°C, and 20 mL of CO2 gas with a carbon dioxide gas concentration of 5% by volume was added to each flask. ２ After leaving the medium in the incubator for 1 hour, VERO cells (manufactured by Sumitomo Dainippon Pharma Co., Ltd.) that had been pre-cultured in advance were seeded into the medium so that the cell concentration was 200,000 cells / mL. ２ The cells were cultured in an incubator for 7 days with stirring at 60 rpm. Half of the medium was replaced on the third day of culture. 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. The cell concentration (10,000 cells / mL) in the medium was measured and used as an index of cell proliferation. The results are shown in Tables 3 and 4.

[0097]

[0098]

[0099] The results in Tables 3 and 4 show that the carriers for cell culture of the present invention are superior in cell proliferation and cell adhesion properties compared to the comparative carriers.

[0100] The cell culture carrier of the present invention is capable of exhibiting excellent cell adhesiveness and cell proliferation properties, and is therefore extremely useful in cell-related research and development, production of useful substances, etc. For research and development purposes, it can be used to culture cells for evaluating cell functions such as differentiation function, to culture cells for use as substitutes in animal experiments (toxicity tests, irritation tests, metabolic function tests, etc.), and to culture cells for gene or protein introduction. For useful substance production, it can be used to culture cells for producing cytokines, thrombolytic agents, blood coagulation factor preparations, vaccines, hormones, antibiotics, antibodies, growth factors, etc. Among these, it is suitable for culturing cells for producing vaccines.

Claims

1. A carrier for cell culture comprising copolymer resin particles (A) having as constituent monomers a monomer (a1) represented by the following general formula (1), a monomer (a2) having a hydroxyl group represented by the following general formula (2), and a crosslinkable monomer (b), and an adhesive protein (B). CH ２ = C(R １ )-(C=O)-QR ２ -N(R ３ ) (R ４ ) (1) [In general formula (1), R １ is H or CH ３ Q is O or NH; R ２ is a divalent hydrocarbon group having 2 to 5 carbon atoms; R ３ , R ４ are each independently a monovalent hydrocarbon group having 1 to 4 carbon atoms. ２ = C(R ５ )-(C=O)-Q-(R ６ -O) ｎ -H (2) [In general formula (2), R ５ is H or CH ３ Q is O or NH; R ６ is a divalent hydrocarbon group having 1 to 4 carbon atoms; and n is an integer from 1 to 7.

2. The cell culture carrier according to claim 1, wherein the weight ratio of the crosslinkable monomer (b) based on the total weight of the constituent monomers of the copolymer resin particles (A) is 0.1 to 3% by weight.

3. The cell culture carrier according to claim 1 or 2, wherein the copolymer resin particles (A) further contain, as a constituent monomer, a monomer (a3) ​​having a carboxyl group.

4. The cell culture carrier according to claim 1 or 2, wherein the number average particle size of the copolymer resin particles (A) is 0.1 to 500 μm.

5. The cell culture carrier according to claim 1 or 2, wherein the adhesive protein (B) is a polypeptide (B1) or an extracellular matrix (B2) having at least one cell adhesive minimal amino acid sequence (X) in one molecule.

6. The carrier for cell culture according to claim 1 or 2, wherein the content of the adhesive protein (B) is 100 ng / g to 50 mg / g based on the dry weight of the carrier for cell culture.

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