JP2018143211A - Cell culture substrate, cell production method and cell culture kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell culture substrate and a cell culture kit that have a high cell survival rate and a high cell adhesion rate and allow cells to be peeled off in a short time, and a cell production method using the cell culture substrate.SOLUTION: A cell culture substrate contains a crosslinked product (C) of a polysaccharide (A) with a crosslinker (B). The crosslinker (B) is an organic compound having at least two functional groups (b) that can react with an active hydrogen group, and the cell culture substrate has a 37°C storage elastic modulus G' of 1000-1×10Pa.SELECTED DRAWING: None

Description

  The present invention relates to a cell culture substrate, a cell production method, and a cell culture kit.

  Conventionally, cell culture is performed on the surface of glass or the surface of polystyrene subjected to various treatments. As an example, for example, various shapes of containers made of polystyrene coated with cell adhesion proteins such as collagen, fibronectin or polylysine on polystyrene surface-treated by plasma treatment, corona treatment, etc. Widely used as a material. However, when cells are cultured using polystyrene or the like, it is necessary to treat the cells with a proteolytic enzyme such as trypsin, which is highly toxic to cells and further degrades extracellular matrix that is beneficial for subsequent use. There is a problem.

In order to solve these problems, culture dishes (Patent Document 1 and Non-Patent Document 1) onto which poly (N-isopropylacrylamide) (PNIPAM) is surface-grafted and culture dishes (Patent Documents 2 to 4) made of polysaccharides are reported. Has been. In a culture dish on which PNIPAM is surface-grafted, cells are treated at 20 ° C. for about 30 minutes in order to recover the cells, but there is a problem that the cells cannot be applied to cells having high sensitivity to temperature.
Moreover, the culture dish which consists of polysaccharide has the method of using hydrophilic polysaccharides (alginic acid etc.) and the method of using a poorly water-soluble polysaccharide (cellulose etc.). In the method using a hydrophilic polysaccharide, cells are cultured on a gel of a hydrophilic polysaccharide gelled with a metal ion or the like, but unlike a petri dish like polystyrene, the gel has insufficient hardness. There is a problem that causes poor cell adhesion and poor shape of adherent cells. Furthermore, although what hardened the gel with the chelating agent is known, the chelating agent used in order to harden the gel causes the cytotoxicity. In the method using a poorly water-soluble polysaccharide, there are a method of peeling cells with a glycolytic enzyme and a method of degrading modified cellulose with an amino group-containing compound. Since the method using a saccharolytic enzyme is sparingly water-soluble, the base material is too hard, so that there is a problem that polysaccharides are not easily degraded in a water-based system for culturing cells. Moreover, in the method of decomposing | disassembling with an amino group containing compound, there exists a problem which cannot add an amino acid (amino group containing compound) essential to cell culture to a culture medium.

Japanese Patent Publication No. 6-104061 JP 2007-259735 A Japanese Patent Laying-Open No. 2015-48467 JP 2005-110537 A

Tatsuya Shimizu, Mitsuo Okano, Bioscience and Bioindustry, 2000, 58 (12), pp. 851-854

  The present invention provides a cell culture substrate, a cell culture kit, and a cell production method using a cell culture base, which have high cell viability and cell adhesion rate and are capable of peeling cells in a short time. Objective.

As a result of intensive studies to solve these problems, the present inventors have reached the present invention.
That is, the present invention is a cell culture substrate containing a cross-linked product (C) of a polysaccharide (A) with a cross-linking agent (B), wherein the cross-linking agent (B) can react with an active hydrogen group. A cell culture substrate that is an organic compound having at least two (b) and has a storage elastic modulus G ′ of 1000 to 1 × 10 ¹⁰ Pa at 37 ° C. of the cell culture substrate; Cell production method for culturing cells (X); a cell culture kit comprising a cell culture substrate and a degradation agent (E).

  The cell culture substrate and the cell culture kit of the present invention have a high cell viability and cell adhesion rate, and have an effect that cells can be detached in a short time. In addition, the cell production method using the cell culture base of the present invention has a high cell survival rate and cell adhesion rate, and has the effect that cells can be detached in a short time.

The cell culture substrate of the present invention is a cell culture substrate containing a cross-linked product (C) of a polysaccharide (A) cross-linking agent (B), and the cross-linking agent (B) reacts with an active hydrogen group. It is an organic compound having at least two functional groups (b) that can be obtained, and is a cell culture substrate in which the storage elastic modulus G ′ at 37 ° C. of the cell culture substrate is 1000 to 1 × 10 ¹⁰ Pa.

Examples of the polysaccharide (A) include those in which 10 or more monosaccharides (a) are bonded by glycosidic bonds and derivatives thereof.
As the monosaccharide (a), a monosaccharide having 3 to 7 carbon atoms (a1) or the hydroxyl group and / or hydroxymethyl group of the (a1) is a carboxyl group, an amino group, an N-acetylamino group, a sulfooxy group, A monosaccharide (a2) substituted with at least one substituent (E) selected from the group consisting of a methoxycarbonyl group and a carboxymethyl group is included.

Examples of the monosaccharide (a1) having 3 to 7 carbon atoms include triose, tetrose, pentose, hexose and heptose.
Examples of triose include dihydroxyacetone and glyceraldehyde.
Examples of tetrose include erythrose, threose and erythrulose.
Examples of pentose include arabinose, xylose, ribose, xylulose, ribulose and deoxyribose.
Examples of hexose include glucose, mannose, galactose, fructose, sorbose, tagatose, fucose, fucose, and rhamnose.
Examples of heptose include sedoheptulose.

As the monosaccharide (a2), one or more hydroxymethyl groups (—CH ₂ OH) are substituted with carboxyl groups (—COOH), monosaccharides (a2-1), and one or more hydroxyl groups (—OH) Monosaccharide (a2-2) in which is substituted with an amino group (—NH ₂ ), monosaccharide (a2-3) in which one or more hydroxyl groups are substituted with N-acetylamino group (—NHCOCH ₃ ), 1 Monosaccharide (a2-4) in which one or more hydroxyl groups are substituted with sulfooxy group (—OSO ₃ H) Monosaccharide (a2) in which one or more hydroxymethyl groups are substituted with methoxycarbonyl group (—COOCH ₃ ) -5) Monosaccharide (a2-6) in which one or more hydroxymethyl groups are substituted with carboxymethyl group (—CH ₂ COOH), and two or more hydroxyl groups and / or hydroxymethyl groups are substituted in plural types Group (E In substituted monosaccharide (A2-7) and the like.

Examples of (a2-1) include uronic acids such as glucuronic acid, iduronic acid, mannuronic acid and galacturonic acid.
Examples of (a2-2) include amino sugars such as glucosamine, galactosamine, mannosamine and muramic acid.
Examples of (a2-3) include N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, and N-acetylmuramic acid.
Examples of (a2-4) include galactose-3-sulfuric acid.
Examples of (a2-5) include glucose methyl ester and methyl esterified carboxylic acid in (a2-1).
As (a2-7), N-acetylglucosamine-4-sulfuric acid, iduronic acid-2-sulfuric acid, glucuronic acid-2-sulfuric acid, N-acetylgalactosamine-4-sulfuric acid, neuraminic acid and N-acetylneuraminic acid Etc.

Specific examples of the polysaccharide (A) include cellulose, amylose, amylopectin, glycosaminoglycan, alginic acid, carrageenan, hyaluronic acid, glycogen, dextrin, glucan, fructan, chitin, tamarind seed gum, guar gum, locust bean gum, Arabia Examples thereof include gum, karaya gum, pectin, xanthan gum, duran gum, Agrobacterium succinoglycan, diyutan gum, mannan, glucomannan, chitosan, tara gum, psyllium seed gum and derivatives thereof (carboxymethylcellulose and the like).
Among the polysaccharides (A), cellulose, amylose, amylopectin, glycosaminoglycan, alginic acid, carrageenan, hyaluronic acid, glycogen, dextrin, glucan, fructan, chitin, tamarind seed gum, from the viewpoints of industrial utilization and degradability Guar gum, locust bean gum, gum arabic, karaya gum, pectin, xanthan gum, juran gum, Agrobacterium succinoglycan, dieutan gum, mannan, glucomannan, chitosan, tara gum, psyllium seed gum and their derivatives At least one is preferable, and more preferably a small amount selected from the group consisting of cellulose, alginic acid, carrageenan, hyaluronic acid, xanthan, amylose, dextran, and derivatives thereof. A Kutomo one, particularly preferably carboxymethylcellulose.

  In the carboxymethyl cellulose, the substitution rate of the hydroxyl group with the carboxymethyl group is preferably 0.02 to 3, more preferably 0.2 to 2, from the viewpoint of water solubility.

In the present invention, the crosslinking agent (B) is an organic compound having at least two functional groups (b) capable of reacting with active hydrogen groups.
Examples of the functional group (b) include a carboxyl group, an epoxy group, an isocyanate group, a carbodiimide group, an oxazoline group, and an acid halide group.
Among these, at least one selected from the group consisting of a carboxyl group, an epoxy group, and an isocyanate group is preferable from the viewpoint of the reactivity and the strength of the crosslinked product (C).

  The crosslinking agent (B) may have one type as the functional group (b), or may have two or more types.

In the crosslinking agent (B), those having a carboxyl group include polycarboxylic acids.
Specific examples of polycarboxylic acids include those having two carboxyl groups {alkane dicarboxylic acids having 2 to 50 carbon atoms (oxalic acid, malonic acid, succinic acid, adipic acid, reparginic acid, sebacic acid, etc. ) And aromatic dicarboxylic acids having 8 to 36 carbon atoms (phthalic acid, isophthalic acid, terephthalic acid, naphthalenedicarboxylic acid, etc.), unsaturated dicarboxylic acids having 4 to 50 carbon atoms (maleic acid, fumaric acid, and 12 to 46 carbon atoms). Alkenyl succinic acid having a alkenyl group, etc.)} having three or more carboxyl groups {aromatic polycarboxylic acids having 9 to 20 carbon atoms (such as trimellitic acid and pyromellitic acid), carbon number 6-36 aliphatic tricarboxylic acid (hexane tricarboxylic acid etc.) etc. are mentioned.

  As those having a carboxyl group, anhydrides of these carboxylic acids, lower alkyl (1 to 4 carbon atoms) esters (methyl esters, ethyl esters, isopropyl esters, etc.) may be used. Or you may use together with what has a lower alkyl esterification thing and the said carboxyl group.

In addition, the unsaturated monocarboxylic acid may be used as a cross-linking agent, and the carboxyl group in the unsaturated monocarboxylic acid may be reacted with the active hydrogen of the polysaccharide (A) and further cross-linked by reacting with a double bond. Good.
Examples of the unsaturated monocarboxylic acid include unsaturated monocarboxylic acids having 2 to 30 carbon atoms. Specifically, acrylic acid, methacrylic acid, propiolic acid, 2-butyric acid, crotonic acid, isocrotonic acid, 3-butene Acid, angelic acid, tiglic acid, 4-pentenoic acid, 2-ethyl-2-butenoic acid, 10-undecenoic acid, 2,4-hexadienoic acid, myristoleic acid, palmitoleic acid, sapienoic acid, oleic acid, elaidic acid, Examples include vaccenic acid, gadoleic acid, eicosenoic acid, erucic acid, and nervonic acid.

  Among those having a carboxyl group, preferred are adipic acid, alkenyl succinic acid having an alkenyl group having 12 to 46 carbon atoms, terephthalic acid, and isophthalic acid from the viewpoint of the reactivity and strength of the crosslinked product (C). , Maleic acid, fumaric acid, trimellitic acid, pyromellitic acid and combinations thereof. Likewise preferred are anhydrides and lower alkyl esters of these acids.

  Specifically, those having an epoxy group include polyglycidyl ether (ethylene glycol diglycidyl ether, tetramethylene glycol diglycidyl ether, bisphenol A diglycidyl ether, bisphenol F diglycidyl ether, glycerin triglycidyl ether, Pentaerythritol tetraglycidyl ether, phenol novolac glycidyl ether and the like).

  As what has an isocyanate group, an aliphatic diisocyanate compound, an alicyclic diisocyanate, and an aromatic diisocyanate compound are contained.

  Examples of the aliphatic diisocyanate compound include trimethylene diisocyanate, tetramethylene diisocyanate, hexamethylene diisocyanate (hereinafter abbreviated as HDI), pentamethylene diisocyanate, 1,2-propylene diisocyanate, 1,3-butylene diisocyanate, dodecamethylene diisocyanate. And 2,4,4-trimethylhexamethylene diisocyanate.

  Examples of the alicyclic diisocyanate compound include 1,3-cyclopentene diisocyanate, 1,3-cyclohexane diisocyanate, 1,4-cyclohexane diisocyanate, isophorone diisocyanate (hereinafter abbreviated as IPDI), hydrogenated diphenylmethane diisocyanate (hereinafter referred to as IPDI). And hydrogenated xylylene diisocyanate, hydrogenated tolylene diisocyanate, hydrogenated tetramethyl xylylene diisocyanate, and the like.

  Examples of the aromatic diisocyanate compound include phenylene diisocyanate, tolylene diisocyanate (2,4-tolylene diisocyanate and 2,6-tolylene diisocyanate, hereinafter abbreviated as TDI), 2,2 ′ monodiphenylmethane diisocyanate, 4, 4'-diphenylmethane diisocyanate (hereinafter abbreviated as MDI), 4,4'-toluidine diisocyanate, 4,4'-diphenyl ether diisocyanate, 4,4'-diphenyl diisocyanate, 1,5-naphthalene diisocyanate, xylylene diisocyanate and the like Can be mentioned.

  As what has an isocyanate group, from a reactive viewpoint and the viewpoint of the intensity | strength of a crosslinked material (C), a preferable thing is C6-C15 aromatic diisocyanate, C4-C12 aliphatic diisocyanate, and C4. ~ 15 selected from the group consisting of alicyclic diisocyanates, and particularly preferred is at least one selected from the group consisting of TDI, MDI, HDI, hydrogenated MDI and IPDI.

  As the crosslinking agent (B), those having an epoxy group are preferable from the viewpoint of reactivity and the strength of the crosslinked product (C), and polyglycidyl ether is more preferable.

The substrate for cell culture of the present invention is not particularly limited as long as it contains a cross-linked product (C), but has fibronectin, collagen, laminin and the following minimum amino acid sequence (X) from the viewpoint of cell adhesion. It is preferable to contain at least one cell adhesion factor selected from the group consisting of the cell adhesion peptide (D).
Minimum amino acid sequence (X): RGD sequence, LDV sequence, REDV sequence (1), YIGSR sequence (2), PDSGR sequence (3), RYVVLPR sequence (4), LGTIPG sequence (5), RNIAEIIKDI sequence (6), IKVAV At least one selected from the group consisting of a sequence (7), an LRE sequence, a DGEA sequence (8), a GVGKGDKGNPGPGAP sequence (9), a GEFYFDLRLKGGDK sequence (10), a HAV sequence and a YKLNVNDS sequence (11).
In the present invention, “sequence (1)” means “sequence number (1) in the sequence listing”, and the same applies to sequences other than sequence (1).

Fibronectin, collagen and laminin are commonly known as cell adhesion molecules.
In the present invention, “cell adhesion” means a property in which a specific minimum amino acid sequence is recognized by an integrin receptor of a cell, and the cell easily adheres to a base material (Osaka Prefectural Maternal and Child Medical Center magazine, No. 1). 8: No. 1, 58-66, 1992).

  As the minimum amino acid sequence (X), “Pathophysiology, Vol. 9, No. 7, pp. 527-535, 1990” or “Osaka Prefectural Maternal and Child Medical Center Magazine, Vol. 8, No. 1, pp. 58-66, 1992” From the viewpoint of cell adhesion, the above sequences (1) to (11) are preferable, and an RGD sequence is more preferable.

  The cell adhesion peptide (D) may have at least one minimum amino acid sequence (X) in one molecule, but from the viewpoint of cell adhesion, etc., one having 1 to 50 in one molecule is preferable. More preferably 2-50, then 3-30, particularly preferably 4-20, most preferably 5-15. Two or more kinds of minimum amino acid sequences (X) may be included in one molecule.

  In addition to the minimum amino acid sequence (X), the cell adhesive peptide (D) preferably has an auxiliary amino acid sequence (Y) from the viewpoint of improving the thermal stability of the cell adhesive peptide (D).

  As the auxiliary amino acid sequence (Y), an amino acid sequence other than the minimum amino acid sequence (X) can be used. From the viewpoint of the thermal stability of the cell adhesive peptide (D), G (glycine) and / or A (alanine) are used. The sequence having is preferred.

The auxiliary amino acid sequence (Y) includes (GA) _a sequence, (GAGAGS) _b sequence, (GAGAGY) _c sequence, (GAGVGY) _d sequence, (GAGYGV) _e sequence, {DGG (A) _f GGA} _g sequence, (GVPPGV) _h sequence, (G) _i sequence, (A) _j sequence, (GGA) _k sequence, (GVGVP) _m sequence, (GPP) _n sequence, (GAQGPAGPG) _o sequence, (GAPGGAPSQGAPGGLQ) _p sequence and / or (GAPGTPGPQGLPGSP) A sequence having a _q sequence and the like are included.
Among these, from the viewpoint of thermal stability, (GA) _a sequence, (GAGAGS) _b sequence, (GAGAGY) _c sequence, (GAGVGY) _d sequence, (GAGYGV) _e sequence, {DGG (A) _f GGA} _g It preferably has at least one selected from the group consisting of a sequence, (GVPPGV) _h sequence, (GVGVP) _m sequence and (GPP) _n sequence, more preferably (GAGAGS) _b sequence, (GVPPGV) _h sequence, ( It has at least one selected from the group consisting of (GVGVP) _m sequence and (GPP) _n sequence, and particularly preferably has (GAGAGS) _b sequence.
A is an integer of 5 to 100, b is an integer of 1 to 33, c, d and e are integers of 2 to 33, f is an integer of 1 to 194, and g is {1} to {200 / (6 + f). } Is an integer obtained by rounding down the decimal point, h is an integer of 2 to 40, i and j are integers of 10 to 200, k is an integer of 3 to 66, m is an integer of 2 to 40, and n is an integer of 3 to 66. , O is an integer from 1 to 22, and p and q are integers from 1 to 13.

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

  In addition to the above examples, the auxiliary amino acid sequence (Y) includes 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) and the like}.

(GA) Examples of the auxiliary amino acid sequence having _a sequence include amino acid sequences represented by SEQ ID NOs: (12) to (14).
(GAGAGS) The auxiliary amino acid sequence having the _b sequence includes amino acid sequences represented by SEQ ID NOs: (15) to (17).
Examples of the auxiliary amino acid sequence having the (GAGAGY) _c sequence include amino acid sequences represented by SEQ ID NOs: (18) to (20).
(GAGVGY) Examples of the auxiliary amino acid sequence having the _d sequence include amino acid sequences represented by SEQ ID NOs: (21) to (23).
(GAGYGV) Examples of auxiliary amino acid sequences having an _e sequence include amino acid sequences represented by SEQ ID NOs: (24) to (26).
Examples of the auxiliary amino acid sequence having the {DGG (A) _f GGA} _g sequence include amino acid sequences represented by SEQ ID NOs: (27) to (29).
(GVPGV) Examples of the auxiliary amino acid sequence having the _h sequence include amino acid sequences represented by SEQ ID NOs: (30) to (33).
(G) _Examples of the auxiliary amino acid sequence having _i sequence include amino acid sequences represented by SEQ ID NOs: (34) to (36).
(A) The auxiliary amino acid sequence having _j sequence includes the amino acid sequences represented by SEQ ID NOs: (37) to (39).
(GGA) Examples of the auxiliary amino acid sequence having the _k sequence include amino acid sequences represented by SEQ ID NOs: (40) to (42).
Examples of the auxiliary amino acid sequence having (GVGVP) _m sequence include amino acid sequences represented by SEQ ID NOs: (43) to (45).
Examples of the auxiliary amino acid sequence having (GPP) _n sequence include amino acid sequences represented by SEQ ID NOs: (46) to (48).
(GAQGPAGPG) Examples of the auxiliary amino acid sequence having _o sequence include amino acid sequences represented by SEQ ID NOs: (49) to (51).
(GAPGAPGSQGAPGLQ) Examples of the auxiliary amino acid sequence having the _p sequence include amino acid sequences represented by SEQ ID NOs: (52) to (54).
(GAPGTPGPQGLPGSP) Examples of the auxiliary amino acid sequence having the _q sequence include amino acid sequences represented by SEQ ID NOs: (55) to (57).

  Among these auxiliary amino acid sequences, SEQ ID NOs: (12), (13), (15), (16), (17), (18), (19), (21), (22), (24), (25), (27), (28), (29), (30), (31), (33), (34), (35), (37), (38), (40), (41 ), (43), (44), (46), (47), (49), (50), (52), (53), (55) or (56) is preferred, More preferably, SEQ ID NOs: (13), (15), (16), (17), (19), (22), (25), (29), (30), (31), (32), ( 33), (35), (38), (41), (44), (47), (50), (53) or (56), particularly preferably Column number (15), the amino acid sequence represented by (16) or (33).

  When the auxiliary amino acid sequence (Y) is included, the content of (Y) is preferably 2 to 50, more preferably 3 to 30 in one molecule of the cell adhesive peptide (D) from the viewpoint of thermal stability. Particularly preferably 4 to 20, most preferably 5 to 15. Further, the cell adhesive peptide (D) may contain two or more auxiliary amino acid sequences (Y).

  The cell adhesion peptide (D) has a minimum amino acid sequence (X) and an auxiliary amino acid sequence (Y), if necessary, between (X) and (Y) from the viewpoint of cell adhesion and thermal stability. It is preferable that the structure is formed by alternately chemical bonding via the amino acid sequence of (X), and the amino acid sequence including the intervening amino acid sequence (Z) at both ends of (X) and (Y) are alternately chemically bonded. More preferably, it is a structure. In this case, the number (units) of repeating units (XY) of the minimum amino acid sequence (X) and the auxiliary amino acid sequence (Y) is preferably 1 to 50, more preferably 2 from the viewpoint of cell adhesiveness. -40, particularly preferably 3-30, most preferably 4-20.

Moreover, you may have an intervening amino acid sequence (Z).
As an intervening amino acid sequence (Z), from the viewpoint of cell adhesion, a GAAVTG sequence (58), a GLPGPKGD sequence (59), a GPAVTG sequence (60), an AGPKGADGSGPPAVTG sequence (61) ), GAAVCEPG sequence (62), GAALCVSEPG sequence (63), SPASAALC VSEPG sequence (64), SPASAAVCEPG sequence (65), AGPKGADGSGPPAVCCEPG sequence (66), AGPKGADGSPGPALCVSEPG sequence (67), GPAVCEPG sequence (68), GPG Preferably, at least one intervening amino acid sequence (Z) selected from the group consisting of the GAAPGAS sequence (70) is bound, and the GAAVTG sequence (58 , GLPGPKGD sequence (59), at least one more preferably selected from the group consisting of GPAVTG sequence (60) and AGPKGADGSPGPAVTG sequence (61), GAAVTG sequence (58) is particularly preferred.

  As the intervening amino acid sequence (Z), the SPASAAGY sequence (71), SPASAALCVS sequence (72), SPASAAVC sequence (73), AGPKGADGSGPP sequence (74) are present at the C-terminus of the minimal amino acid sequence (X) from the viewpoint of cell adhesion. ), AGPKGADGSPGPAVC sequence (75), AGPKGADGSPGPALCVS sequence (76), AGPKGADGSP sequence (77), SPASAAGPVGSP sequence (78), CDAGY sequence (79), CDAGPVGSP sequence (80) and SAGPSAGY sequence (81) Preferably, one intervening amino acid sequence (Z) is linked, and SPASAAGY sequence (71), SPASAALCVS sequence (72), SPASAAVC sequence (73), AGPKG At least one selected from the group consisting of DGSGPP sequence (74), AGPKGADGSGPPAVC sequence (75), AGPKGADGSGPPALCVS sequence (76), AGPKGADGSP sequence (77) and SPASAAGPVGSP sequence (78) is more preferred, and SPASAAGY sequence (71) is particularly preferred. .

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

  The cell adhesion peptide (D) has a minimum amino acid sequence (X) and an auxiliary amino acid sequence (Y), if necessary, between (X) and (Y) from the viewpoint of cell adhesion and thermal stability. It is preferable that the structure is formed by alternately chemical bonding via the amino acid sequence of (X), and the amino acid sequence including the intervening amino acid sequence (Z) at both ends of (X) and (Y) are alternately chemically bonded. More preferably, it is a structure. In this case, the number (units) of repeating units (XY) of the minimum amino acid sequence (X) and the auxiliary amino acid sequence (Y) is preferably 1 to 50, more preferably 2 from the viewpoint of cell adhesiveness. -40, particularly preferably 3-30, most preferably 4-20.

  Further, the number of the minimum amino acid sequence (X) and the auxiliary amino acid sequence (Y) may be the same or different. When they are different from each other, it is preferable that the content of one of them is one less than the content of the other {in this case, the auxiliary amino acid sequence (Y) is preferably small}. The content ratio (X / Y) between the minimum amino acid sequence (X) and the auxiliary amino acid sequence (Y) in the cell adhesion peptide (D) is preferably 0.5 to 2, more preferably 0.9 to 1. .4, particularly preferably 1-1.3.

  Moreover, you may include another amino acid in the terminal part (Minimum amino acid sequence (X) or auxiliary amino acid sequence (Y) to the terminal of a peptide) of a cell adhesive peptide (D). When other amino acids are contained, the content is preferably 1 to 1000, more preferably 3 to 300, and particularly preferably 10 to 100 per cell adhesive peptide (D).

  The weight average molecular weight (hereinafter referred to as Mw) of the cell adhesion peptide (D) is preferably 1,000 to 1,000,000, more preferably 2,000 to 700,000, particularly preferably 3,000 to 400,000. 000, most preferably 4,000 to 200,000.

  The Mw of the cell adhesion peptide (D) is a well-known method such as separating a measurement sample {polypeptide etc.} by SDS-PAGE (SDS polyacrylamide gel electrophoresis) and comparing the migration distance with a standard substance. (Hereinafter the same).

  The cell adhesion peptide (D) may be further modified with a compound (AM). As the compound (AM), a compound containing a primary to tertiary amino group and a salt thereof, an ammonium salt (AM-1), a compound containing a carboxyl group (AM-2), a compound containing a sulfo group (AM -3) as well as compounds containing a hydroxyl group (AM-4). When it is modified with (AM), the cell proliferation is further improved.

Examples of the compound containing a primary to tertiary amino group and a salt thereof and an ammonium salt-containing compound (AM-1) include polyamines, amino alcohols, halides having an amino group, amino group-containing monomers, and amino group-containing monomers. Can be used, as well as hydrogen halide salts and quaternized compounds thereof.
As the polyamine, a polyamine having at least one primary amino group or secondary amino group (having 2 to 56 carbon atoms) is used, such as an aliphatic polyamine, an alicyclic polyamine, a heterocyclic polyamine, and an aromatic polyamine. Is used.

  Aliphatic polyamines include alkylene diamines (ethylene diamine, propylene diamine, trimethylene diamine, tetramethylene diamine, hexamethylene diamine, etc.), polyalkylene polyamines having 2 to 6 carbon atoms in the alkylene group (diethylene triamine, iminobispropylamine, Triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, etc.), and alkyl (carbon number 1-18) substitution products thereof (dimethylaminopropylamine, diethylaminopropylamine, dipropylaminopropylamine, methylethylaminopropylamine, Trimethylhexamethylenediamine, N, N-dioctadecylethylenediamine, trioctadecylethylenediamine, methyliminobispropylamine, etc. Etc. The.

  Examples of alicyclic polyamines include 1,3-diaminocyclohexane, 1,3-bis (methylamino) cyclohexane, 1,3-bis (dihydroxyamino) cyclohexane, isophorone diamine, menthane diamine, and 4,4′-methylene dicyclohexane. Examples include diamines.

  Examples of the heterocyclic polyamine include piperazine, N-methylpiperazine, N-aminoethylpiperazine and 1,4-diaminoethylpiperazine.

  Aromatic polyamines include phenylenediamine, N, N′-dimethylphenylenediamine, N, N, N′-trimethylphenylenediamine, diphenylmethanediamine and 2,6-diaminopyridine, tolylenediamine, diethyltolylenediamine, 4, Examples include 4′-bis (methylamino) diphenylmethane and 1-methyl-2-methylamino-4-aminobenzene.

  As the amino alcohol, an amino alcohol having 2 to 58 carbon atoms is used, and an alkanolamine having 2 to 10 carbon atoms [monoethanolamine, diethanolamine, triethanolamine, monoisopropanolamine, monobutanolamine, triethanolamine, triethanolamine, Propanolamine, tributanolamine, N, N-bis (hydroxyethyl) ethylenediamine and N, N, N ′, N′-tetrakis (hydroxyethyl) ethylenediamine, etc.], alkyl (C 1-18) substituted products thereof [ N, N-dimethylethanolamine, N, N-diethylethanolamine, N-ethyldiethanolamine, N-octadecyldiethanolamine, N, N-diethyl-N ′, N′-bis (hydroxyethyl) ethylenediamine, N, - dioctadecyl -N ', N'-bis (hydroxyethyl) ethylenediamine and N, N, N'trioctadecyl -N'- hydroxyethyl ethylenediamine] and the like.

  Examples of the halide having an amino group include halogenated (such as chlorine and bromine) of alkylamines having 2 to 17 carbon atoms, such as aminoethyl chloride, N-methylaminopropyl chloride, N, N-dimethylaminoethyl chloride. N, N-diethylaminoethyl chloride, N, N-dibenzylaminoethyl bromide, N, N-dimethylaminopropyl bromide, N, N-diethylaminopropyl chloride, N, N-dibenzylaminopropyl chloride and the like.

As the amino group-containing monomer, an amino group-containing vinyl compound having 5 to 21 carbon atoms, ethyleneimine, an amino acid having 2 to 20 carbon atoms, or the like is used.
Examples of the amino group-containing vinyl compound include amino group-containing (meth) acrylates, amino group-containing (meth) acrylamides, amino group-containing aromatic vinyl hydrocarbons, amino group-containing allyl ethers, and the like.

  As the amino group-containing (meth) acrylate, aminoethyl (meth) acrylate, N-methylaminoethyl (meth) acrylate, N, N-dimethylaminoethyl (meth) acrylate, N, N-diethylaminopropyl (meth) acrylate, N, N-dipropylaminoethyl (meth) acrylate, N-benzyl-N-methylaminoethyl (meth) acrylate, N, N-dibenzylaminoethyl (meth) acrylate, N, N-dibenzylaminopropyl (meth) ) Acrylate, morpholinoethyl (meth) acrylate, N-methylpipetidinoethyl (meth) acrylate, and the like.

  Examples of amino group-containing (meth) acrylamides include aminoethyl acrylamide, N-methylaminopropyl acrylamide, N, N-dimethylaminoethyl (meth) acrylamide, N, N-diethylaminopropyl (meth) acrylamide, and N, N-dipropyl. Examples include aminoethyl (meth) acrylamide, N-benzyl-N-methylaminoethyl (meth) acrylamide, morpholinoethyl (meth) acrylamide, and N-methylpipetidinoethyl (meth) acrylamide.

  Examples of amino group-containing aromatic vinyl hydrocarbons include aminoethylstyrene, N-methylaminoethylstyrene, N, N-dimethylaminostyrene, N, N-dipropylaminostyrene, and N-benzyl-N-methylaminostyrene. Can be mentioned.

  Examples of the amino group-containing allyl ether include aminoethyl allyl ether, N-methylaminoethyl allyl ether, N, N-dimethylaminoethyl allyl ether, and N, N-diethylaminoethyl allyl ether.

  As amino acids, arginine, histidine, isoleucine, leucine, methionine, phenylalanine, threonine, tryptophan, tyrosine, valine, alanine, asparagine, aspartic acid, glutamine, glutamic acid, proline, cysteine, lysine, serine, glycine, 3-aminopropionic acid , 8-aminoactanoic acid and 20-aminoeicosanoic acid.

Examples of the polymer of the amino group-containing monomer include a vinyl polymer composed of an amino group-containing vinyl compound, polyethyleneimine, and a polypeptide (not including (B1)).
The weight average molecular weight of the polymer of amino group-containing monomers is preferably 500 to 1,000,000, more preferably 1,000 to 800,000, and particularly preferably 2,000 to 500,000. The weight average molecular weight can be measured by gel permeation chromatography (GPC) {reference substance: polystyrene standard having a molecular weight of 420 to 20,600,000 (manufactured by Tosoh Corp.)}.

Examples of the hydrogen halide salt include salts of the amino group-containing compound and halides (hydrogen fluoride, hydrogen chloride, hydrogen bromide, hydrogen iodide, etc.).
Specific examples include N, N-dimethylaminoethyl chloride hydrochloride and N, N-diethylaminoethyl chloride hydrochloride.

  Examples of these quaternized products include those obtained by quaternizing these amino groups with a quaternizing agent (such as methyl chloride, ethyl chloride, benzyl chloride, dimethyl carbonate, dimethyl sulfate and ethylene oxide).

  Among these compounds containing 1 to 3 amino groups and salts thereof and ammonium salts (AM-1), N, N-dimethylaminoethyl chloride hydrochloride and N, N-diethylamino hydrochloride are preferable from the viewpoint of cell proliferation. Ethyl chloride is preferable, and N, N-dimethylaminoethyl chloride hydrochloride is more preferable.

  The compound (AM-2) containing a carboxyl group is a carboxylic acid having 2 to 30 carbon atoms, specifically, formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, apple Acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, mesylic acid, salicylic acid, 4-hydroxybenzoic acid and phenylacetic acid . Moreover, the halide etc. which have these carboxyl groups can also be used. Specific examples include chloroacetic acid and chloroformic acid. Of these carboxyl group-containing compounds (AM-2), chloroacetic acid is preferred from the viewpoint of cell proliferation.

  The compound (AM-3) containing a sulfo group is a sulfonic acid having 2 to 30 carbon atoms, specifically, ethanesulfonic acid, benzenesulfonic acid, pantothenic acid, 2-hydroxyethanesulfonic acid, toluenesulfone. Examples include acid, sulfanilic acid and cyclohexylaminosulfonic acid. Moreover, the halide etc. which have these sulfo groups can also be used. Specific examples include chlorosulfonic acid and chloroethanesulfonic acid. Of these sulfone group-containing compounds (AM-3), chloroethanesulfonic acid is preferred from the viewpoint of cell proliferation.

  Examples of the compound (AM-4) containing a hydroxyl group include methanol, ethanol, propanol, butanol and the like. Moreover, the halide etc. which have these hydroxyl groups can also be used. Specific examples include chloroethanol and chloropropanol. Of these hydroxyl group-containing compounds (AM-4), chloroethanol is preferred from the viewpoint of cell proliferation.

Examples of the method of modifying with compound (AM) include (1) a method of chemically bonding {covalent bond, ionic bond and / or hydrogen bond, etc.} between compound (AM) and cell adhesive peptide (D) before modification. Applicable.
Among these, from the viewpoint of cell proliferation, the chemical bonding method (1) is preferable, and covalent bonding is more preferable.

  (1) In order to chemically bond the compound (AM) and the cell adhesive peptide (D) before modification in a chemical bond {covalent bond, ionic bond and / or hydrogen bond, etc.} (D) preferably has an amino acid having a reactive group {hydroxyl group, carboxyl group, mercapto group, primary or secondary amino group, etc.} as an amino acid residue. Of the reactive groups, from the viewpoint of cell proliferation, a hydroxyl group, a carboxyl group and a primary amino group are preferable, a hydroxyl group and a carboxyl group are more preferable, and a hydroxyl group is particularly preferable. Further, at least one reactive group may be contained in one molecule of the cell adhesion peptide (D), but from the viewpoint of cell proliferation, those having 2-50 in one molecule are preferable, and more preferably One having 3 to 30 and particularly preferably 5 to 20 in one molecule.

  As a method for chemical bonding, a known method can be applied, and examples thereof include a method described in JP-A-2007-51127. A reaction solvent may be used for the chemical bond formation reaction, and a known solvent can be used, for example, water, lithium bromide aqueous solution, lithium perchlorate aqueous solution, methanol, ethanol, isopropanol, acetone, dimethyl Examples include sulfoxide, dimethylacetamide, and tetrahydrofuran.

  Specific examples of covalently bonding the compound (AM) and the cell-adhesive peptide (D) before modification include amino acids containing a hydroxyl group in the side chain in the cell-adhesive peptide (D) (for example, Ser and Tyr) And alkyl ethers with alkyl halides having 1 to 3 amino groups and / or ammonium salts, carboxyl groups, sulfone groups, hydroxyl groups, and amino acids containing carboxyl groups in the side chain (for example, Asp and Glu) may be esterified with an alcohol having an amino group and / or an ammonium salt.

A part of preferable cell adhesion peptide (D) is illustrated below.
(1) When minimum amino acid sequence (X) is RGD sequence (x1) It has 13 RGD sequences (x1) and 12 (GAGAGS) ₉ sequences (16) (y1). Polypeptide with a molecular weight of about 110,000 (sequence (82)) {"pronectin F", pronectin is a registered trademark (Japan and USA) of Sanyo Chemical Industries. Manufactured by Sanyo Chemical Industries Co., Ltd.
A protein obtained by modifying pronectin F with compound (AM), specifically, a polypeptide ("pronectin F plus") modified with N, N-dimethylaminoethyl chloride hydrochloride as compound (AM);
A polypeptide having an Mw of about 20,000 (5) (x1) and 5 (GAGAGS) ₃ sequences (15) and (y2) and having a structure in which these are alternately chemically bonded (sequence (83)) (“Pronectin F2”); Mw having three (x1) and three (GVPVG) ₂ GG (GAGAGS) ₃ sequences (33) (y3), and these are alternately chemically bonded About 10,000 polypeptides (sequence (84)) ("pronectin F3") and the like.

(2) When the minimum amino acid sequence (X) is an IKVAV sequence (x2) “Pronectin L”, “Pronectin” in which the RGD sequence (x1) of pronectin F, pronectin F2 or pronectin F3 is changed to the IKVAV sequence (7) (x2) L2 "or" Pronectin L3 ".

(3) When the minimum amino acid sequence (X) is a YIGSR sequence (x3) “Pronectin Y”, “Pronectin Y2”, wherein the RGD sequence (x1) of pronectin F, pronectin F2 or pronectin F3 is changed to the YIGSR sequence (x3), Or “pronectin Y3” or the like.

  Cell adhesion peptides (D) include those artificially synthesized (artificial polypeptides). For example, organic synthesis methods (solid phase synthesis methods, liquid phase synthesis methods, etc.) and biochemical synthesis methods [genes Recombinant microorganisms (yeast, bacteria, E. coli, etc.)] and the like. That is, the cell adhesion peptide (D) does not include cell adhesion proteins such as animal-derived collagen and fibronectin.

  The organic synthesis method is described in, for example, “Sequel Biochemistry Experiment Course 2, Protein Chemistry (Part 2)” pages 641-694 (May 20, 1987; published by Tokyo Chemical Co., Ltd.). The method etc. currently used are used. As for the biochemical synthesis method, for example, the method described in JP-T-3-502935 is used. A biochemical synthesis method using a genetically modified microorganism is preferable in that the cell adhesive peptide (D) can be easily synthesized, and a method using a genetically modified Escherichia coli is particularly preferable.

  The cell adhesion factor is preferably at least one selected from the group consisting of fibronectin, collagen, laminin, pronectin F and pronectin F plus from the viewpoint of cell adhesion.

  In the cell culture substrate of the present invention, the content of the cell adhesion factor is preferably 0 to 10% by weight, more preferably 0 to 5%, based on the weight of the cell culture substrate, from the viewpoint of cell adhesion. % By weight.

The shape of the cell culture substrate of the present invention is not particularly limited, and examples thereof include a sheet shape, a fiber shape, a rod shape, and a spherical shape (such as a bead shape). Among these, from the viewpoint of easy use in cell culture, a sheet shape and a spherical shape are preferable.
In the case of a sheet, the thickness of the sheet is not particularly limited, but preferably 0.5 to 500 nm from the viewpoint of ease of cell culture and degradability.

The substrate for cell culture of the present invention has a storage elastic modulus G ′ at 37 ° C. of 1000 to 1 × 10 ¹⁰ Pa, preferably 1 × 10 ⁴ to 1 × 10 ⁸ . When the storage elastic modulus G ′ is less than 1000 Pa, there are problems that the adhesiveness of the cells is lowered and the shape of the adherent cells is changed. Moreover, when it exceeds 1 × 10 ¹⁰ Pa, it is difficult to produce a cell culture substrate, and there is a problem that it is difficult to be decomposed by the decomposition agent (E) described later.
The storage elastic modulus G ′ is a value measured by a rheometer.

  The contact angle with respect to water (37 ° C.) on the cell culture surface of the cell culture substrate of the present invention is preferably 20 to 60 °, more preferably 30 to 50 ° from the viewpoint of cell adhesion.

  From the viewpoint of cell adhesion, the surface roughness (Ra) of the cell culture substrate of the present invention is preferably 1 μm or less, more preferably 0.5 μm or less.

As a manufacturing method of the base material for cell cultures of this invention, a polysaccharide (A) is carboxymethylcellulose and the thing including the process of following (1)-(5) is mentioned, for example.
(1) A step of dissolving carboxymethyl cellulose (hereinafter abbreviated as CMC) in water.
(2) A step of adding a crosslinking agent (B) to the substrate obtained in (1) and causing the crosslinking reaction to proceed.
(3) The process of processing the base material obtained by said (2) into a sheet form or bead form.
(4) The process of drying the base material obtained by said (3), and removing a water | moisture content.
(5) A step of modifying the cell adhesion peptide (D) on the substrate obtained in (4).

  Examples of the method for dissolving CMC in water include a method of adding CMC while stirring water with a stirrer. In order to ensure the uniformity of the solution, a method of gradually adding CMC is preferably included.

Examples of the stirring method for dissolving CMC in water include a method of rotating a stirring bar with a stirrer, a method of stirring with a stirring blade, and a method of further heating the two methods.
Since the viscosity of the CMC solution is high, a method of stirring with a stirring blade is preferable, and a method of stirring with a stirring blade while heating is more preferable.

  Examples of the method of adding the crosslinking agent (B) include a method of adding the crosslinking agent (B) while stirring the CMC solution with a stirrer. In this case, the cross-linking agent (B) may be added all at once, or may be gradually added using a pump or the like, but from the viewpoint of allowing the crosslinking reaction to proceed uniformly, it is gradually added using a pump or the like. Is preferred.

Examples of the method for uniformly processing the CMC solution after addition of the crosslinking agent (B) on a petri dish into a sheet form include dip coating, spraying, roller coating, and spin coating.
From the viewpoint of productivity and handling properties, dip coating is not preferred, and spraying, roller coating and spin coating are preferred. From the viewpoint of the uniformity of the coating film, roller coating and spin coating are more preferable. From the viewpoint of efficiency from application to drying, spin coating is most preferable.

  As a method of processing the CMC solution after addition of the cross-linking agent (B) into beads, for example, a pulverizing method in which the CMC solution is physically pulverized after drying and a CMC solution after addition of the cross-linking agent is used. Examples of the dispersion method include dispersion in a hydrophobic organic solvent.

  As a pulverization method, it is preferable to first coarsely pulverize the dried product and then finely pulverize. At this time, a method of pulverizing by colliding with a collision plate in a jet stream, pulverizing particles by colliding with each other in a jet stream, or pulverizing with a narrow gap between a mechanically rotating rotor and a stator is preferably used.

  In the classification step, the pulverized product obtained in the pulverization step is classified and adjusted to particles having a predetermined particle size. Classification can be performed, for example, by removing fine particle portions by a cyclone, a decanter, centrifugation, or the like.

In the dispersion method, as a method of dispersing the CMC solution after addition of the crosslinking agent (B) in the organic solvent, there may be mentioned a reverse phase suspension method, etc., including uniformity of particle diameter, particle circularity and particle surface shape. From the viewpoint of smoothness, dispersion by a reverse phase suspension method is preferred.
As the hydrophobic organic solvent, known ones (Patent No. 3363000, JP-A No. 2003-147005) can be used, and basically any solvent can be used as long as it is hardly soluble in water. For example, aliphatic hydrocarbons such as n-pentane, n-hexane, n-heptane and n-octane, alicyclic hydrocarbons such as cyclohexane and methylcyclohexane, aromatic carbons such as benzene, toluene and xylene. Hydrogen etc. are mentioned.
As the dispersant, those known as reverse phase suspension polymerization dispersants can be used. For example, anionic dispersants (polyoxyethylene alkyl ether sulfate (described in JP-A-6-93008), monoalkyl phosphates ( Nonionic dispersant (sucrose fatty acid ester, sorbitol fatty acid ester, aliphatic alcohol alkylene oxide adduct, alkylphenol alkylene oxide adduct, etc.) and the like.

The method for removing excess water is not particularly limited, and examples thereof include a drying method using a dryer. For example, the method of drying using dryers, such as a disk dryer, a turbo dryer, and a drum dryer, is mentioned.
The drying temperature is preferably 60 to 230 ° C, more preferably 100 to 200 ° C. When the drying temperature is 60 ° C. or higher, drying does not require a long time and it is economical in terms of energy consumption. On the other hand, when the drying temperature is 230 ° C. or lower, side reactions and resin decomposition do not occur. Therefore, it is preferable. The drying atmosphere may be normal pressure or reduced pressure (for example, 500 mmHg or less).

The moisture content after drying is defined by the amount of water that gives a storage elastic modulus G ′ of 1000 to 1 × 10 ¹⁰ Pa. When the storage elastic modulus G ′ is less than 1000 Pa, there is a problem that the particles are coalesced when the cell culture substrate has a bead shape. Further, when the cell culture substrate is in the form of a sheet, there is a problem that it deforms or adheres when it is in contact with other materials.

The water content at which the storage elastic modulus G ′ is 1000 to 1 × 10 ¹⁰ Pa is preferably 40% by weight or less based on the weight of the cell culture substrate, and more preferably 30 from the viewpoint of improving the storage elastic modulus. % By weight or less. On the other hand, if the amount of water is excessively reduced, the surface roughness Ra increases due to volume shrinkage, and thus it is more preferably 10% weight or more.

  As a method for modifying the cell adhesion peptide (D) to the cell culture substrate, (1) a chemical bond {covalent bond, ionic bond and / or hydrogen bond, etc.}, (2) a cell adhesion peptide (D) Examples include physical adsorption (adsorption by van der Waals force) and (3) enzyme modification.

As the chemical bonding method (1), the reactive group (—OH, —COOH, —NH—, —NH ₂ , —SH, etc.) possessed by the cell adhesive peptide (D) and the polysaccharide (A) are possessed. Examples thereof include a method performed by crosslinking a reactive group (—OH, —COOH, —CHO, —C (═O) —, —NH—, —NH _2, —SH, etc.).
Examples of the physical adsorption method (2) include a physical adsorption method in which a solution containing a cell-adhesive peptide (D) having a predetermined concentration and a cell culture substrate coexist for a predetermined time.
Examples of the enzyme modification method (3) include a method of binding the polysaccharide (A) to the cell adhesive peptide (D) using an enzyme such as glycoside transferase.

  The cell production method of the present invention is a cell production method for culturing cells (X) using the cell culture substrate.

  The cell (X) is not limited as long as it is a cell. However, when the cell culture substrate of the present invention is used, cell proliferation is high, and normal derived from mammals used for production of useful substances such as pharmaceuticals and treatment. Cells, mammalian cell lines and insect cells are suitable.

  Normal cells derived from mammals include cells involved in the skin (epithelial cells, fibroblasts, vascular endothelial cells, smooth muscle cells, etc.), cells involved in blood vessels (vascular endothelial cells, smooth muscle cells, fibroblasts, etc.) ), Cells involved in muscle (muscle cells, etc.), cells involved in fat (fat cells, etc.), cells involved in nerves (nerve cells, etc.), cells involved in liver (hepatocytes, etc.), involved in pancreas Cells (pancreatic islet cells, etc.), kidney-related cells (kidney cells, renal epithelial cells, proximal tubular epithelial cells, mesangial cells, etc.), lung-bronchial cells (epithelial cells, fibroblasts, blood vessels) Endothelial cells and smooth muscle cells, etc.), cells involved in the eyes (visual cells, corneal epithelial cells, corneal endothelial cells, etc.), cells involved in the prostate (epithelial cells, stromal cells, smooth muscle cells, etc.), and bones Cells (osteoblasts, bone cells and fractures) Cells), cells involved in cartilage (such as chondroblasts and chondrocytes), cells involved in teeth (such as periodontal ligament cells and osteoblasts), cells involved in blood (such as leukocytes and erythrocytes), and stem cells { For example, bone marrow undifferentiated mesenchymal stem cells, skeletal muscle stem cells, hematopoietic stem cells, neural stem cells, hepatic stem cells (such as oval cells, small hepatocytes), adipose tissue stem cells, iPS cells, embryonic stem (ES) cells, epidermal stem cells, Intestinal stem cells, sperm stem cells, embryonic germ stem (EG) cells, pancreatic stem cells (pancreatic duct epithelial stem cells, etc.), leukocyte stem cells, lymphocyte stem cells, corneal stem cells, etc.}, precursor cells (adipose precursor cells, vascular endothelial precursor cells, Cartilage progenitor cells, lymphocyte progenitor cells, NK progenitor cells, etc.)} and the like.

  Cell lines derived from mammals 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 PaCG-2 cells, N18 cells, Namalwa cells, NG108- 5 cells, NRK cells, OC10 cells, OTT6050 cells, P388 cells, PA12 cells, PA317 cells, PC-12 cells, PER. C6 cell, PG13 cell, QGH cell, Raji cell, RPMI-1788 cell, SGE1 cell, Sp2 / O-Ag14 cell, ST2 cell, THP-1 cell, U-937 cell, V79 cell, VERO cell, WI-38 cell , Ψ2 cells, ψCRE cells, etc. {Cell culture technology (edited by the Japanese Society for Tissue Culture, published by Asakura Shoten Co., Ltd., 1999)}.

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

  Of these cells (X), normal cells derived from mammals and established cell lines derived from mammals are preferred from the viewpoint of production of useful substances such as pharmaceuticals and treatment. In terms of usefulness for treatment, kidney cells, muscle cells, hepatocytes, osteoblasts, epithelial cells, fibroblasts, vascular endothelial cells, stem cells and progenitor cells are more preferable. Further, 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, particularly preferably CRFK cells, MDCK cells and VERO cells.

As a medium (ME) used in the method for producing cells of the present invention, a serum-free medium (STEMPRO hESC SFM17, TeSR 2, E8 medium, hESF9 medium, hESF-FX medium, hESF92ai, StemFit AK02N, S-medium, mTeSR ^™ Grace medium, IPL-41 medium, Schneider's medium, Opti-PRO ^™ SFM medium, Opti-MEM ^™ I 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 medium (RPMI medium, MEM medium, Eagle's 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 medium thereof ); And mixed media thereof.

Moreover, serum can be added to these culture media.
Serum includes human serum and animal serum (bovine serum, horse serum, goat serum, sheep serum, pig serum, rabbit serum, chicken serum, rat serum, mouse serum, etc.).
Of these, human serum, bovine serum, and horse serum are preferred when serum is added. The origin of animal serum includes adult-derived serum, offspring-derived serum, newborn-derived serum, fetal-derived serum, and the like. When adding serum, among these, serum derived from offspring, serum derived from newborn, and serum derived from fetus are more preferable, serum derived from newborn, and serum derived from fetus, particularly preferably serum derived from fetus. is there. When serum is added, the serum may be further subjected to inactivation treatment, antibody removal treatment, or the like.

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

  In the culture medium, a cell growth factor can be contained as necessary. By containing a cell growth factor, the cell growth rate can be increased and the cell activity can be increased.

  Cell growth factors include fibroblast growth factor, transforming growth factor, epithelial growth factor, hepatocyte growth factor, platelet-derived growth factor, insulin-like growth factor, vascular endothelial growth factor, nerve growth factor, stem cell factor, leukemia Inhibitory factors, osteogenic factors, heparin-binding epithelial cell growth factor, neurotrophic factor, connective tissue growth factor, angiopoietin, cytokines, interleukins, adrenamodulin and natriuretic peptides are included. Of these, fibroblast growth factor, transforming growth factor, insulin-like growth factor, and osteogenic factor are preferable, and more preferably fibroblast, from the viewpoint that the range of applicable cells is wide and the healing period can be further shortened. Growth factors, transforming growth factors and insulin-like growth factors.

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

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

  Although there is no restriction | limiting in particular as a density | concentration (cell / mL) of the cell disperse | distributed to a culture medium, 100-100 million is preferable per 1 mL of culture medium, More preferably, it is 1000-10 million, Most preferably, it is 10,000-1 million. .

A known method can be used for counting the number of cells. For example, it can be measured by a cell nucleus counting method using trypan blue or crystal violet. {Cell culture technology (edited by the Japanese Society for Tissue Culture, published by Asakura Shoten Co., Ltd.) 1999)}.
The dry weight (g) of the cell culture substrate to be introduced into the medium can be appropriately determined depending on the type of cells to be cultured, etc., but is preferably 0.005 to 800, more preferably 0.02 to 1 L of the medium. 200, particularly preferably 0.1 to 40.

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

The method for producing cells of the present invention is not particularly limited as long as the cells (X) are cultured using the cell culture substrate, but from the viewpoint of industrial applicability, the cells are produced on the cell culture substrate. It is preferable to include a step of decomposing the cell culture substrate with the decomposing agent (E) after culturing (X).
Examples of the decomposing agent (E) include polysaccharide degrading enzymes (E1), organic acids (E2), and inorganic acids (E3).
Of these, polysaccharide degrading enzyme (E1) is preferred.
As the polysaccharide degrading enzyme (E1), those known as enzymes degrading polysaccharides can be used without limitation. Specifically, cellulase, amylase, dextranase, carrageenase, alginate lyase, hyaluronidase, chitinase and chitosanase Etc.
By decomposing the cell culture substrate with the decomposing agent (E), the cells can be recovered without destroying the shape of the proliferated cells. For example, sheet-like, spherical and tissue-like cells can be obtained. it can.

A preferred polysaccharide-degrading enzyme (E1) is appropriately selected depending on the polysaccharide (A) used in the cross-linked product (C) constituting the cell culture substrate from the viewpoint of degradability of the cell culture substrate. However, specifically, the following combinations are preferable.
A combination of a polysaccharide (A) {at least one selected from the group consisting of cellulose, carboxymethylcellulose and other cellulose derivatives} and a degradation agent (E) {cellulase}.
Combination of polysaccharide (A) {at least one selected from the group consisting of dextrin and dextrin derivatives} and degradation agent (E) {dextranase}.
A combination of a polysaccharide (A) {at least one selected from the group consisting of carrageenan and carrageenan derivatives} and a degradation agent (E) {carrageenase}.
A combination of a polysaccharide (A) {at least one selected from the group consisting of alginic acid and alginic acid derivatives} and a degradation agent (E) {alginate lyase}.
Combination of polysaccharide (A) {at least one selected from the group consisting of hyaluronic acid and hyaluronic acid derivatives} and a decomposing agent (E) {hyaluronidase}.
A combination of a polysaccharide (A) {at least one selected from the group consisting of chitin and chitin derivatives} and a degradation agent (E) {chitinase}.
A combination of a polysaccharide (A) {at least one selected from the group consisting of chitosan and chitosan derivatives} and a degradation agent (E) {chitosanase}.

The decomposer (E) can be used as a decomposer (E) solution.
The concentration of the decomposing agent (E) in the decomposing agent (E) solution depends on the type of the decomposing agent (E) used and the polysaccharide (A) used in the cross-linked product (C) constituting the substrate for cell culture. Although it is appropriately selected depending on the kind, from the viewpoint of cytotoxicity and degradability of the substrate for cell culture, 0.1 to 5% by weight is preferable based on the weight of the decomposition agent (E) solution, and more preferably 0.8. 1-1% by weight.

Preferred specific examples of the cell production method are shown below.
(1) Cell production method by general cell culture Adhesive cells (X-1) are cultured for a predetermined period using the cell culture substrate. After culturing, the cells are recovered from the cell culture substrate using the degradation agent (E).
(2) Culture of cell sheet Adhesive cells (X-1) are cultured for a predetermined period using the cell culture substrate. After the cells become confluent, the cell sheet is recovered from the cell culture substrate using the degradation agent (E).
(3) Culture of cell components Adhesive cells (X-1) are cultured for a predetermined period using the cell culture substrate formed into a desired structure. After culturing, the cell culture substrate is removed with a degrading agent (E) to obtain a cell composition.

The cell culture kit of the present invention comprises the cell culture substrate and the degradation agent (E).
Preferable examples of the cell culture substrate and the degradation agent (E) are the same as described above.
Moreover, the decomposition agent (E) may be contained in the kit as the decomposition agent (E) solution.

  Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples. In the following, unless otherwise specified, “part” means “part by weight” and “%” means “% by weight”.

<Example 1>
Add 800 parts of water to a container equipped with a stirrer, and gradually add 100 parts of CMC (Daiichi Kogyo Seiyaku Co., Ltd., “Serogen F-SC”) while stirring (50 rpm) so as not to become lumps. A CMC solution was obtained. Thereafter, 100 parts of ethylene glycol diglycidyl ether (Nagase ChemteX Co., Ltd., “Denacol EX-810”), which is a compound having an epoxy group, was added as a crosslinking agent (B) with stirring.
The obtained solution was applied using a spin coater (“Spin Coater 7094” manufactured by Imoto Seisakusho Co., Ltd.) on a circular glass plate having a diameter of 70 mm and dried for 3 hours while proceeding with a crosslinking reaction with a dryer at 100 ° C. A cell culture substrate (1) in a sheet form (diameter 70 mm, thickness 0.1 mm) having a water content of 20% by weight was obtained. The obtained substrate (1) had a storage elastic modulus G ′ of 10 ⁸ Pa.

<Example 2>
Except for changing the addition amount of the crosslinking agent (B) to “67 parts” instead of “100 parts”, a sheet shape having a water content of 20% by weight (diameter: 70 mm, thickness: 0.1 mm), except that the amount is “67 parts”. The cell culture substrate (2) was obtained. The storage modulus G ′ of the obtained substrate (2) was 10 ⁶ Pa.

<Example 3>
Except for changing the addition amount of the crosslinking agent (B) to “50 parts” instead of “100 parts”, a sheet shape (diameter 70 mm, thickness 0.1 mm) having a water content of 20% by weight is the same as in Example 1. A cell culture substrate (3) was obtained. The storage modulus G ′ of the obtained substrate (3) was 10 ⁴ Pa.

<Example 4>
The cell culture substrate (2) obtained in Example 2 was used as a cell adhesion peptide (D), Pronectin F (polypeptide having a sequence of Mw of about 110,000 having the sequence of SEQ ID NO: (82), Sanyo Chemical Industries ( The cell culture substrate (4) whose surface was modified by physical adsorption was obtained by immersing in a ProctinF solution (37 ° C.) containing 0.001% by weight).

<Example 5>
The cell culture substrate (2) obtained in Example 2 was used as a fibronectin solution (37 ° C.) containing 0.001% by weight of fibronectin (manufactured by Wako Pure Chemical Industries, Ltd., “Fibroctin Solution, from Human Plasma”). ) To obtain a cell culture substrate (5) having a surface modified with fibronectin by physical adsorption.

<Example 6>
The cell culture substrate (2) obtained in Example 2 is a collagen solution (37 ° C, containing 0.3% by weight of collagen (manufactured by Nitta Gelatin Co., Ltd., “Cell matrix Type I-A”)). It was immersed in pH 3) for 2 hours to obtain a cell culture substrate (6) in which collagen was modified on the surface by physical adsorption.

<Comparative Example 1>
A cell culture substrate (1 ′) for comparison in the form of a sheet (diameter 70 mm, thickness 0.1 mm) having a water content of 20% by weight was obtained in the same manner as in Example 1 except that the cross-linking agent (B) was not added. It was. The obtained base material (1 ′) had a storage elastic modulus G ′ of 800 Pa.

<Comparative Example 2>
Comparative cell culture substrate (2 ′) for comparison having a water content of 20% by weight in the same manner as in Example 1 except that the amount of the crosslinking agent (B) added was changed to “200 parts” instead of “100 parts”. Got. The storage modulus G ′ of the obtained substrate (2 ′) was 1 × 10 ¹¹ Pa. In addition, before forming with a spin coater, it became lumpy and could not be formed into a sheet.

<Comparative Example 3>
A 96-well plate for cell culture (manufactured by Nunc) was used as a cell culture substrate (3 ′) for comparison.

<Comparative example 4>
Put 800 parts of water in a container equipped with a stirrer, and 2.4 parts of alginic acid (Wako Pure Chemical Industries, Ltd., “Sodium Alginate 300-400”), so as not to become lumpy while stirring (800 rpm). The solution was gradually added to obtain an alginic acid solution. The obtained solution was applied using a spin coater (“Spin Coater 7094” manufactured by Imoto Seisakusho Co., Ltd.) on a circular glass plate having a diameter of 70 mm, and immersed in 1M calcium chloride solution for 3 hours to crosslink alginic acid. The sheet was taken out from the 1M calcium chloride solution and dried with a dryer at 100 ° C. for 1 hour to obtain a cell culture substrate (4 ′) having a water content of 20% by weight (diameter 70 mm, thickness 0.1 mm). The obtained base material (4 ′) had a storage elastic modulus G ′ of 900 Pa.

<Comparative Example 5>
According to the description in JP-A No. 2001-321157, fibronectin (manufactured by Wako Pure Chemical Industries, Ltd., “Fibroticin Solution, from Human Plasma”) is modified on a cellulose film (manufactured by Whatman, “pure cellulose paper”) for comparison. A cell culture substrate (5 ′) was obtained.

<Evaluation of physical properties of cell culture substrate>
Storage elastic modulus G ′: JIS K7244-10 “Plastics—Testing method of dynamic mechanical properties—Part 10: Complex shear viscosity by parallel plate vibration rheometer” Viscoelasticity measuring device [equipment name “ARES-24A” , Manufactured by Rheometric Co., Ltd.], and the storage elastic modulus at 37 ° C. was measured.
Contact angle to water (37 ° C.): Measured according to JIS R3257.
Surface roughness (Ra): The surface roughness was measured using a commercially available cantilever and an atomic force microscope [device name “E-sweep”, manufactured by SII Nanotechnology, Inc.]. The height range (stereoscopic image) was measured by scanning at a constant speed (0.3 Hz to 1.2 Hz) with a measurement range of 4 μm. The surface roughness (Ra) was calculated from the measurement results according to the method defined in JIS B0601.

<Evaluation of cytotoxicity: cell viability>
Cell culture substrates (1) to (6) and (1 ′) to (5 ′) were seeded with VERO cells at 3 × 10 ⁵ cells / cm ² and in DMEM medium (containing 10 wt% FBS). The cells were cultured at 37 ° C., 5% by volume CO ₂ , and wet conditions (humidity 100% RH). After 24 hours, the DMEM medium was removed, 0.1 ml of a 100-fold dilution of cellulase (Endolase 5000L, Novozymes) was added as a degrading agent (E), and 2 hours at 37 ° C., 5 vol% CO ₂ , wet conditions ( (Humidity 100% RH). After 2 hours, 10 μl / 100 μl of live cell count reagent SF (manufactured by Nacalai Tesque) and 100 μl of DMEM medium (containing 10 wt% FBS) were added to the culture solution, and 450 nm (reference wavelength 600 nm) after 1 hour. Was measured. The cell viability was calculated assuming that the cell viability was 100% when no degradation agent (E) was added (blank).
Further, in the cell culture substrate (3) of Comparative Example 3, the procedure was the same except that “trypsin / EDTA (trypsin / EDTA Solution, manufactured by Thermo) 0.1 ml” was used instead of “cellulase 0.1 ml”. Cell viability was assessed.
Cell viability (%) = [absorbance of sample−absorbance of blank] / [absorbance without addition−absorbance of blank] × 100

<Evaluation of cell culture: adhesion rate>
Cell culture substrates (1) to (6) and (1 ′) to (5 ′) were seeded with VERO cells at 3 × 10 ⁵ cells / cm ² and in DMEM medium (containing 10 wt% FBS). The cells were cultured at 37 ° C., 5% by volume CO ₂ , and wet conditions (humidity 100% RH).
After culturing for 2 hours, non-adherent cells were removed by washing with phosphate buffer (PBS). After removing PBS, 10 μl / 100 μl of live cell count reagent SF (manufactured by Nacalai Tesque) and 100 μl of DMEM medium (containing 10 wt% FBS) were added, and 450 nm (reference wavelength 600 nm) was added after 1 hour. It was measured. The cell adhesion rate was calculated assuming that the non-adherent cell removal treatment was not performed as 100%.

<Evaluation of cell culture: peeling time>
Cell culture substrates (1) to (6) and (1 ′) to (5 ′) are seeded with VERO cells at 3 × 10 ⁵ cells / cm ² , in DMEM (10% FBS) at 37 ° C., 5 ° The culture was performed under the conditions of volume% CO ₂ and humid conditions (humidity 100% RH).
After 24 hours, a 100-fold dilution of cellulase (Endolase 5000L, Novozymes) was added to the cell culture medium as a decomposing agent (E), the degree of cell detachment over time was confirmed, and the time when the cells were completely detached ( Minute).

From Table 1, it can be seen that the cell culture substrates of Examples 1 to 6 having a storage elastic modulus G ′ in the range of 1000 to 1 × 10 ¹⁰ Pa are easily formed into a shape suitable for cell culture. On the other hand, the cell culture substrate of Comparative Example 2 having a storage elastic modulus G ′ of 10 ¹¹ could not be formed into a shape suitable for cell culture.
In addition, the cell culture substrates of Examples 1 to 6 have extremely high cell viability and cell adhesion rate, are easily decomposed by a degrading agent, and have a short cell detachment time, so that they are extremely suitable for cell culture. I understand. In particular, it can be seen from the results of the cell culture substrates of Examples 4 to 6 that the cell adhesion rate can be improved by containing a cell adhesion factor. On the other hand, the cell culture substrates of Comparative Examples 1 (not cross-linked) and 4 (metal cross-linked) have high cell viability and degradability, but have low cell adhesion and are suitable for cell culture. I understand that there is no. It can also be seen that the cell culture substrate that requires the use of a proteolytic enzyme for cell detachment in Comparative Example 3 has high cytotoxicity and low cell viability. Further, it can be seen that the cell culture substrate of Comparative Example 5 (cellulose) has a storage elastic modulus of 5 × 10 ^{10 which} is too high, so that it is difficult to be decomposed by the decomposing agent and it takes a long time to peel off the cells.

The cell culture substrate of the present invention exhibits excellent cell viability and cell adhesion, and can detach cells in a short time. Therefore, for cell-related research and development, production of useful substances, treatment, etc. Very useful.
For research and development, cell culture for cell function evaluation such as differentiation function, culture of substitute cells for animal experiments (toxicity test, stimulation test, metabolic function test, etc.), cell culture for gene and protein introduction, etc. Available.
For the production of useful substances, it can be used for culturing cells for production of cytokines, thrombolytic agents, blood coagulation factor preparations, vaccines, hormones, antibiotics, antibodies, growth factors and the like. Among these, it is suitable for culturing cells for vaccine production.
For treatment, skin, skull, muscle, skin tissue, bone, cartilage, blood vessel, nerve, tendon, ligament, follicular tissue, mucosal tissue, periodontal tissue, dentin, bone marrow, retina, serosa, gastrointestinal tract and fat It can be used for cell culture of tissues such as lung, liver, pancreas and kidney.

Claims (12)

A cell culture substrate containing a cross-linked product (C) of a polysaccharide (A) cross-linking agent (B), wherein the cross-linking agent (B) has at least 2 functional groups (b) capable of reacting with active hydrogen groups. A cell culture substrate, which is an organic compound and has a storage elastic modulus G ′ at 37 ° C. of 1000 to 1 × 10 ¹⁰ Pa of the cell culture substrate.   Polysaccharide (A) is cellulose, amylose, amylopectin, glycosaminoglycan, alginic acid, carrageenan, hyaluronic acid, glycogen, dextrin, glucan, fructan, chitin, tamarind seed gum, guar gum, locust bean gum, gum arabic, caraya gum, pectin 2. The cell according to claim 1, which is at least one selected from the group consisting of xanthan gum, duran gum, Agrobacterium succinoglycan, diyutan gum, mannan, glucomannan, chitosan, tara gum, psyllium seed gum and derivatives thereof. Culture substrate.   The cell culture substrate according to claim 1 or 2, wherein the functional group (b) is at least one selected from the group consisting of a carboxyl group, an epoxy group and an isocyanate group.   The cell culture substrate according to any one of claims 1 to 3, wherein a contact angle with respect to water (37 ° C) on the cell culture surface of the cell culture substrate is 20 to 60 °.   The cell culture substrate according to any one of claims 1 to 4, wherein the cell culture substrate has a surface roughness (Ra) of 1 µm or less on the cell culture surface. 6. The cell according to claim 1, comprising at least one cell adhesion factor selected from the group consisting of fibronectin, collagen, laminin and a cell adhesion peptide (D) having the following minimum amino acid sequence (X). Cell culture substrate.
Minimum amino acid sequence (X): RGD sequence, LDV sequence, REDV sequence (1), YIGSR sequence (2), PDSGR sequence (3), RYVVLPR sequence (4), LGTIPG sequence (5), RNIAEIIKDI sequence (6), IKVAV At least one selected from the group consisting of a sequence (7), an LRE sequence, a DGEA sequence (8), a GVGKGDKGNPGPGAP sequence (9), a GEFYFDLRLKGGDK sequence (10), a HAV sequence and a YKLNVNDS sequence (11).   The cell adhesion peptide (D) is chemically modified with the polypeptide represented by SEQ ID NO: (82) and / or the peptide represented by SEQ ID NO: (82) in the sequence listing using N, N-dimethylaminoethyl chloride hydrochloride. The substrate for cell culture according to any one of claims 1 to 6, which is a polypeptide obtained.   The cell culture substrate according to any one of claims 1 to 7, wherein the shape is a sheet.   The cell production method which culture | cultivates a cell (X) using the base material for cell cultures of any one of Claims 1-8.   The method for producing cells according to claim 9, comprising a step of decomposing the cell culture substrate with a degrading agent (E) after culturing the cells (X) on the cell culture substrate.   The method for producing cells according to claim 9 or 10, wherein the decomposing agent (E) is a polysaccharide degrading enzyme (E1).   A cell culture kit comprising the cell culture substrate according to any one of claims 1 to 8 and a degradation agent (E).