JP6912903B2 - Cell production method and cell culture kit - Google Patents

Description

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

Traditionally, cell culture has been performed on the surface of glass or on the surface of polystyrene that has been subjected to various treatments. As an example, containers of various shapes composed of polystyrene surface-treated by plasma treatment, corona treatment, etc. coated with cell adhesion proteins such as collagen, fibronectin, or polylysine are used as cell culture groups. It is 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 also decomposes extracellular matrix which is beneficial for subsequent use. There is a problem that it ends up.

In order to solve these problems, a culture dish (Patent Document 1 and Non-Patent Document 1) in which poly (N-isopropylacrylamide) (PNIPAM) is surface-grafted and a culture dish consisting of polysaccharides (Patent Documents 2 to 4) have been reported. Has been done. In a culture dish on which PNIPAM is surface-grafted, cells are treated at 20 ° C. for about 30 minutes in order to collect the cells, but there is a problem that the cells cannot be applied to cells having high sensitivity to temperature.
Further, as a culture dish made of a polysaccharide, there are a method using a hydrophilic polysaccharide (alginic acid and the like) and a method using a poorly water-soluble polysaccharide (cellulose and the like). In the method using hydrophilic polysaccharides, cells are cultured on a gel of hydrophilic polysaccharides gelled by metal ions or the like, but unlike petri dishes such as polystyrene, the hardness of the gel is insufficient. There is a problem of causing poor cell adhesion and poor shape of adherent cells. Further, although it is known that a gel is hardened by a chelating agent, the chelating agent used to harden the gel causes cytotoxicity. In the method using a poorly water-soluble polysaccharide, there are a method of exfoliating cells with a glycolytic enzyme and a method of decomposing modified cellulose with an amino group-containing compound. Since the method using a glycolytic enzyme is poorly water-soluble, the base material is too hard, and there is a problem that polysaccharides are not easily decomposed in a water-based system in which cells are cultured. Further, in the method of decomposing with an amino group-containing compound, there is a problem that an amino acid (amino group-containing compound) essential for cell culture cannot be added to the medium.

Special Fair 6-104061 Gazette JP-A-2007-259735 JP-A-2015-48467 Japanese Unexamined Patent Publication No. 2005-110537

Tatsuya Shimizu, Teruo 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 can detach cells in a short time. The purpose.

The present inventors have arrived at the present invention as a result of diligent studies to solve these problems.
That is, the present invention is a method for producing cells in which cells (X) are cultured using a cell culture substrate, and the cells contain a cross-linked product (C) of the polysaccharide (A) by a cross-linking agent (B). A culture substrate, which is an organic compound having at least two functional groups (b) in which the cross-linking agent (B) can react with an active hydrogen group, and has a storage elasticity G'at 37 ° C. of the cell culture substrate. includes but 1000-1 × Ri ¹⁰ 10 Pa der method of producing cells, after culturing the cells (X) on the cell culture substrate, the step of decomposing the cell culture substrate by decomposing agent (E) , A method for producing cells in which the degrading agent (E) is a polysaccharide-degrading enzyme (E1) ; a cell culture kit containing a cell culture base material and a degrading agent (E), and the cell culture base material is numerous. An organic compound containing a cross-linked product (C) of a saccharide (A) by a cross-linking agent (B) and having at least two functional groups (b) in which the cross-linking agent (B) can react with an active hydrogen group, and is a cell culture. A cell culture kit in which the storage elasticity G'at 37 ° C. of the base material is 1000 to 1 × 10 ¹⁰ Pa, and the degrading agent (E) is a polysaccharide-degrading enzyme (E1) .

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

The base material for cell culture of the present invention is a base material for cell culture containing a crosslinked product (C) of the polysaccharide (A) by the cross-linking agent (B), and the cross-linking agent (B) reacts with the active hydrogen group. It is an organic compound having at least two possible functional groups (b), and is a cell culture base material having a storage elasticity G'at 37 ° C. of the cell culture base material of 1000 to 1 × 10 ¹⁰ Pa.

Examples of the polysaccharide (A) include those in which 10 or more monosaccharides (a) are bound by glycosidic bonds and derivatives thereof.
As the monosaccharide (a), the hydroxyl group and / or the hydroxymethyl group of the monosaccharide (a1) having 3 to 7 carbon atoms or the above (a1) is a carboxyl group, an amino group, an N-acetylamino group, a sulfooxy group, and the like. 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 the triose include dihydroxyacetone and glyceraldehyde.
Examples of tetrose include erythrose, threose, erythrulose and the like.
Examples of the pentose include arabinose, xylose, ribose, xylulose, ribulose and deoxyribose.
Examples of the hexose include glucose, mannose, galactose, fructose, sorbose, tagatose, fucose, fuculose and rhamnose.
Examples of sedoheptulose include sedoheptulose and the like.

As the monosaccharide (a2), _{a monosaccharide (a2-1) in which one or more hydroxymethyl groups (-CH 2} OH) are replaced with a carboxyl group (-COOH), or 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 an N-acetylamino group (-NHCOCH 3} ), 1 A monosaccharide (a2-4) in which one or more hydroxyl groups are _{substituted with a sulfooxy group (-OSO 3} H), and a monosaccharide (a2) in which one or more hydroxymethyl groups are substituted with a _{methoxycarbonyl group (-COOCH 3).} -5) A monosaccharide (a2-6) in which one or more hydroxymethyl groups are _{substituted with a carboxymethyl group (-CH 2} COOH) and a plurality of types of substitutions in which two or more hydroxyl groups and / or hydroxymethyl groups are substituted. Examples thereof include a monosaccharide (a2-7) substituted with a group (E).

Examples of the (a2-1) include uronic acids such as glucuronic acid, iduronic acid, mannuronic acid and galacturonic acid.
Examples of the above (a2-2) include amino sugars such as glucosamine, galactosamine, mannosamine and muramic acid.
Examples of (a2-3) include N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, N-acetylmuramic acid and the like.
Examples of the (a2-4) include galactose-3-sulfuric acid and the like.
Examples of the (a2-5) include glucose methyl esters and methyl esters of the carboxylic acid in the above (a2-1).
Examples of (a2-7) include N-acetylglucosamine-4-sulfate, iduronic acid-2-sulfate, glucuronic acid-2-sulfate, N-acetylgalactosamine-4-sulfate, neuraminic acid and N-acetylneuraminic acid. And so on.

Specific examples of the polysaccharide (A) include cellulose, amylopectin, amylopectin, glycosaminoglycan, alginic acid, carrageenan, hyaluronic acid, glycogen, dextrin, glucan, fructan, chitin, tamarind seed gum, guar gum, locust bean gum, and arabic. Examples thereof include gum, carrageenan, pectin, xanthan gum, julan gum, agrobacterium succinoglycan, Daiyutan gum, mannan, glucomannan, chitosan, tara gum, psyllium seed gum and derivatives thereof (carboxymethyl cellulose and the like).
Of the polysaccharides (A), from the viewpoint of industrial utility and degradability, cellulose, amylose, amylopectin, glycosaminoglycan, alginic acid, carrageenan, hyaluronic acid, glycogen, dextrin, glucan, fructan, chitin, tamarind seed gum, Selected from the group consisting of guar gum, locust bean gum, arabic gum, karaya gum, pectin, xanthan gum, julan gum, agrobacterium succinoglycan, daiyutan gum, mannan, glucomannan, chitosan, tara gum, psyllium seed gum and derivatives thereof. At least one is preferable, and at least one selected from the group consisting of cellulose, alginic acid, carrageenan, hyaluronic acid, xanthan, amylose, dextran and derivatives thereof, and particularly preferably carboxymethyl cellulose.

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

In the present invention, the cross-linking agent (B) is an organic compound having at least two functional groups (b) capable of reacting with an active hydrogen group.
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.
Of 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 reactivity and strength of the crosslinked product (C).

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

The cross-linking agent (B) having a carboxyl group includes a polycarboxylic acid.
Specific examples of the polycarboxylic acid include those having two carboxyl groups {alcandicarboxylic acids having 2 to 50 carbon atoms (oxalic acid, malonic acid, succinic acid, adipic acid, lepargic acid, sebacic acid, etc.) ) And aromatic dicarboxylic acids with 8 to 36 carbon atoms (phthalic acid, isophthalic acid, terephthalic acid, naphthalenedicarboxylic acid, etc.), unsaturated dicarboxylic acids with 4 to 50 carbon atoms (maleic acid, fumaric acid and 12 to 46 carbon atoms). (Elkenyl succinic acid having an alkenyl group, etc.)}, etc.}, having 3 or more carboxyl groups {aromatic polycarboxylic acid having 9 to 20 carbon atoms (trimellitic acid, pyromellitic acid, etc.), carbon number Examples thereof include 6 to 36 aliphatic tricarboxylic acids (hexanetricarboxylic acid and the like).

Anhydrides of these carboxylic acids, lower alkyl (1 to 4 carbon atoms) esters (methyl esters, ethyl esters, isopropyl esters, etc.) may be used as those having a carboxyl group, and the anhydrides and / Alternatively, a lower alkyl esterified product and a product having the above-mentioned carboxyl group may be used in combination.

Further, even if an unsaturated monocarboxylic acid is used as a cross-linking agent, the carboxyl group in the unsaturated monocarboxylic acid is reacted with the active hydrogen of the polysaccharide (A), and the double bond is further reacted to carry out cross-linking. good.
The unsaturated monocarboxylic acid includes an unsaturated monocarboxylic acid having 2 to 30 carbon atoms, specifically, acrylic acid, methacrylic acid, propiolic acid, 2-butyric acid, crotonic acid, isocrotonic acid, and 3-butene. Acids, angelic acid, tigric acid, 4-pentenoic acid, 2-ethyl-2-butenoic acid, 10-undecenoic acid, 2,4-hexadienoic acid, myristoleic acid, palmitoleic acid, sapienoic acid, oleic acid, ellaidic acid, Examples thereof include baxenoic acid, gadrain acid, eicosenoic acid, erucic acid and nervonic acid.

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

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

Those having an isocyanate group include aliphatic diisocyanate compounds, alicyclic diisocyanates and aromatic diisocyanate compounds.

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-butylenediocyanate, and dodecamethylene diisocyanate. And 2,4,4-trimethylhexamethylene diisocyanate and the like.

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

Examples of the aromatic diisocyanate compound include phenylenediocyanate, tolylene diisocyanate (2,4-toluene diisocyanate and 2,6-tolylene diisocyanate, hereinafter abbreviated as TDI), 2,2'-diphenylmethane diisocyanate, 4, 4'-diphenylmethane diisocyanate (hereinafter abbreviated as MDI), 4,4'-toluene diisocyanate, 4,4'-diphenyl ether diisocyanate, 4,4'-diphenyl diisocyanate, 1,5-naphthalenediocyanate, xylylene diisocyanate, etc. Can be mentioned.

As those having an isocyanate group, from the viewpoint of reactivity and strength of the crosslinked product (C), preferred ones are aromatic diisocyanates having 6 to 15 carbon atoms, aliphatic diisocyanates having 4 to 12 carbon atoms and 4 carbon atoms. It is at least one selected from the group consisting of ~ 15 alicyclic diisocyanates, and particularly preferable is at least one selected from the group consisting of TDI, MDI, HDI, hydrogenated MDI and IPDI.

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

The cell culture substrate of the present invention is not particularly limited as long as it contains a crosslinked product (C), but has fibronectin, collagen, laminin and the following minimum amino acid sequence (X) from the viewpoint of cell adhesion. It preferably contains at least one cell adhesion molecule 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), RNIAEI IKDI sequence (6), IKEVAV At least one selected from the group consisting of sequence (7), LRE sequence, DGEA sequence (8), GVKGDKGNPGWPGAP sequence (9), GEFYFDLRLKGDK sequence (10), HAV sequence and YKLNVNDS sequence (11).
In the present invention, "sequence (1)" means "SEQ ID NO: (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 the property that a specific minimum amino acid sequence is recognized by the integrin receptor of the cell and the cell easily adheres to the substrate (Osaka Prefectural Maternal and Child Medical Center Magazine, No. 1). Volume 8, No. 1, pp. 58-66, 1992).

The minimum amino acid sequence (X) is "Pathological Physiology, Vol. 9, No. 7, pp. 527-535, 1990" and "Osaka Prefectural Maternal and Child Medical Center Magazine, Vol. 8, No. 1, pp. 58-66, 1992". The ones described in "Year" and the like are known, and the above sequences (1) to (11) are preferable, and more preferably RGD sequences, from the viewpoint of cell adhesion.

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 and the like, it is preferable that the cell adhesion peptide (D) has 1 to 50 in one molecule. It is more preferably 2 to 50, then preferably 3 to 30, particularly preferably 4 to 20, and most preferably 5 to 15. In addition, two or more kinds of minimum amino acid sequences (X) may be contained in one molecule.

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

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

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, (GVPVGV) _h sequence, (G) _i sequence, (A) _j sequence, (GGA) _k sequence, (GVGVP) _m sequence, (GPP) _n sequence, (GAQGPAGPG) _o sequence, (GAPGAPGSQGAPGLQ) _p sequence and / or (GAPGTPGPQGLPGSP) A _{sequence having a q} sequence or the like is included.
Of 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, a (GVPVGV) _h sequence, a (GVGVP) _m sequence and a (GPP) _{n sequence, and more preferably a (GAGAGS) b} sequence, a (GVPVGV) _h sequence, ( It has at least one selected from the group consisting of the GVGVP) _m sequence and the (GPP) _{n sequence, and particularly preferably has the (GAGAGS) b} sequence.
Note that a is an integer of 5 to 100, b is an integer of 1-33, c, d and e are integers of 2-33, f is an integer of 1 to 194, and g is {1} to {200 / (6 + f). } Is an integer rounded down to the nearest whole number, 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 are integers from 1 to 22, and p and q are integers from 1 to 13.

The auxiliary amino acid sequence (Y) preferably comprises G (glycine) and / or A (alanine). When G (glycine) and A (alanine) are contained, the total content ratio (%) of these 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). It is particularly preferably 30 to 90, and most preferably 40 to 85. Within this range, the thermal stability is further improved.
When both G (glycine) and A (alanine) are contained, the content ratio (G / A) of these is preferably 0.03 to 40, more preferably 0.08 to 13, and 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 (aspartic acid), Q (glutamic acid), D (aspartic acid) , E (glutamic acid), R (arginine), K (lysine), H (histidine), etc.} may be contained.

Examples of the _{auxiliary amino acid sequence having the (GA) a} sequence include the amino acid sequences represented by SEQ ID NOs: (12) to (14).
Examples of the _{auxiliary amino acid sequence having the (GAGAGS) b} sequence include 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).
Examples of the _{auxiliary amino acid sequence having the (GAGVGY) d} sequence include amino acid sequences represented by SEQ ID NOs: (21) to (23).
Examples of the _{auxiliary amino acid sequence having the (GAGYGV) e} sequence include amino acid sequences represented by SEQ ID NOs: (24) to (26).
{DGG (A) _f GGA} Examples of the _{auxiliary amino acid sequence having a g} sequence include amino acid sequences represented by SEQ ID NOs: (27) to (29).
Examples of the _{auxiliary amino acid sequence having the (GVPVGV) h} sequence include amino acid sequences represented by SEQ ID NOs: (30) to (33).
(G) _Examples of the auxiliary amino acid sequence having the i sequence include the amino acid sequences represented by SEQ ID NOs: (34) to (36).
(A) _Examples of the auxiliary amino acid sequence having the j sequence include the amino acid sequences represented by SEQ ID NOs: (37) to (39).
Examples of the _{auxiliary amino acid sequence having the (GGA) k} sequence include amino acid sequences represented by SEQ ID NOs: (40) to (42).
Examples of the _{auxiliary amino acid sequence having the (GVGVP) m} sequence include amino acid sequences represented by SEQ ID NOs: (43) to (45).
Examples of the _{auxiliary amino acid sequence having the (GPP) n} sequence include amino acid sequences represented by SEQ ID NOs: (46) to (48).
Examples of the _{auxiliary amino acid sequence having the (GAQGPAGPG) 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 the 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 preferable. More preferably, SEQ ID NOs: (13), (15), (16), (17), (19), (22), (25), (29), (30), (31), (32), ( The amino acid sequence represented by 33), (35), (38), (41), (44), (47), (50), (53) or (56), particularly preferably SEQ ID NO: (15), It is an amino acid sequence represented by (16) or (33).

When the auxiliary amino acid sequence (Y) is contained, the number of (Y) contained is preferably 2 to 50, more preferably 3 to 30 in one molecule of the cell adhesion peptide (D) from the viewpoint of thermal stability and the like. , Particularly preferably 4 to 20, most preferably 5 to 15. Moreover, the cell adhesion peptide (D) may contain two or more kinds of auxiliary amino acid sequences (Y).

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

It may also have an intervening amino acid sequence (Z).
As the intervening amino acid sequence (Z), the GAAVTG sequence (58), GLPGPKGD sequence (59), GPAVTG sequence (60), and AGPKGADGSPGPAVTG sequence (61) are located at the N-terminal of the smallest amino acid sequence (X) from the viewpoint of cell adhesion. ), GAAVCEPG sequence (62), GAALCCVSEPG sequence (63), SPASAALCEPG sequence (64), SPASAAVCEPG sequence (65), AGPKGADGSPGPAVCEPG sequence (66), AGPKGADGSPGPALCVSEPG sequence (67), GPAVCEPG sequence (68), GPAVCEPG sequence (68) It is preferable that at least one intervening amino acid sequence (Z) selected from the group consisting of GAAPGAS sequence (70) is bound, and GAAVTG sequence (58), GLPGPKGD sequence (59), GPAVTG sequence (60) and AGPKGADGSPGPAVTG sequence. At least one selected from the group consisting of (61) is more preferable, and the GAAVTG sequence (58) is particularly preferable.

As the intervening amino acid sequence (Z), the SPASAAGY sequence (71), the SPASAALCS sequence (72), the SPASAAVC sequence (73), and the AGPKGADGSPGP sequence (74) are located at the C-terminal of the smallest 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). It is preferable that one type of intervening amino acid sequence (Z) is bound, and it is preferable that the SPASAAGY sequence (71), the SPASAALCCVS sequence (72), the SPASAAVC sequence (73), the AGPKGADGSPGGP sequence (74), the AGPKGADGSPGPAVC sequence (75), and the AGPKGADGSPGPALCVS sequence are linked. (76), at least one selected from the group consisting of the AGPKGADGSP sequence (77) and the SPASAAGPVGSP sequence (78) is more preferred, and the SPASAAGY sequence (71) is particularly preferred.

The cell adhesion peptide (D) may contain a branched chain, may be partially crosslinked, or may contain a cyclic structure. However, the cell adhesion peptide (D) is preferably uncrosslinked, more preferably an uncrosslinked linear structure, and particularly preferably a non-crosslinked linear structure without a cyclic structure. The linear structure also includes a β structure (a secondary structure in which linear peptides are bent and these portions are arranged in parallel, and hydrogen bonds are formed between them).

From the viewpoint of cell adhesion and thermal stability, the cell-adhesive peptide (D) has the minimum amino acid sequence (X) and the auxiliary amino acid sequence (Y), if necessary, between (X) and (Y). It is preferable that the structure is formed by alternately chemically bonding via the amino acid sequence of (X), and the amino acid sequence containing the intervening amino acid sequence (Z) at both ends of (X) and (Y) are alternately chemically bonded. The structure is more preferable. In this case, the number (pieces) of the repeating unit (XY) of the minimum amino acid sequence (X) and the auxiliary amino acid sequence (Y) is preferably 1 to 50, more preferably 2 from the viewpoint of cell adhesion and the like. ~ 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. If they are different, it is preferable that the content of either one is one less than the number of the other {in this case, the auxiliary amino acid sequence (Y) is preferably less}. The content ratio (X / Y) of 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. 0.4, particularly preferably 1-1.3.

Further, other amino acids may be contained in the terminal portion of the cell adhesion peptide (D) (from the minimum amino acid sequence (X) or the auxiliary amino acid sequence (Y) to the peptide terminal). When other amino acids are contained, the content thereof is preferably 1 to 1000 per cell adhesion peptide (D), more preferably 3 to 300, and particularly preferably 10 to 100.

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

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

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

Examples of the compounds containing 1st to 3rd grade amino groups and their salts and the compounds containing ammonium salts (AM-1) include polyamines, aminoalcohols, halides having amino groups, amino group-containing monomers and amino group-containing monomers. A polymer containing the above as a constituent monomer, and hydrogen halides and quaternized compounds thereof can be used.
As the polyamine, a polyamine having at least one primary amino group or a secondary amino group (2-56 carbon atoms) or the like is used, and an aliphatic polyamine, an alicyclic polyamine, a heterocyclic polyamine, an aromatic polyamine and the like are used. Is used.

Examples of the aliphatic polyamine include alkylenediamine (ethylenediamine, propylenediamine, trimethylenediamine, tetramethylenediamine, hexamethylenediamine, etc.), polyalkylene polyamine having 2 to 6 carbon atoms in the alkylene group (diethylenetriamine, iminobispropylamine, etc.). Triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, etc.) and their alkyl (1-18 carbon atoms) substitutions (dimethylaminopropylamine, diethylaminopropylamine, dipropylaminopropylamine, methylethylaminopropylamine, etc.) Trimethylhexamethylenediamine, N, N-dioctadecylethylenediamine, trioctadecylethylenediamine, methyliminobispropylamine, etc.) and the like.

Alicyclic polyamines include 1,3-diaminocyclohexane, 1,3-bis (methylamino) cyclohexane, 1,3-bis (dihydroxyamino) cyclohexane, isophoronediamine, mentandiamine and 4,4'-methylenedicyclohexane. Examples include diamine.

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

Examples of aromatic polyamines include phenylenediamine, N, N'-dimethylphenylenediamine, N, N, N'-trimethylphenylenediamine, diphenylmethanediamine and 2,6-diaminopyridine, tolylene diamine, diethyl tolylene diamine, 4, Examples thereof 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 or the like is used, and an alkanol amine having 2 to 10 carbon atoms [monoethanolamine, diethanolamine, triethanolamine, monoisopropanolamine, monobutanolamine, triethanolamine, tri. Propanolamine, tributanolamine, N, N-bis (hydroxyethyl) ethylenediamine and N, N, N', N'-tetrakis (hydroxyethyl) ethylenediamine, etc.], alkyl (carbon number 1-18) substitutions thereof [ N, N-Dimethylethanolamine, N, N-diethylethanolamine, N-ethyldiethanolamine, N-octadecyldiethanolamine, N, N-diethyl-N', N'-bis (hydroxyethyl) ethylenediamine, N, N-di Octadecyl-N', N'-bis (hydroxyethyl) ethylenediamine and N, N, N'-trioctadecyl-N'-hydroxyethylethylenediamine, etc.] and the like can be mentioned.

As the halide having an amino group, a halogen (chlorine, bromine, etc.) compound of an alkylamine having 2 to 17 carbon atoms is used, and aminoethyl chloride, N-methylaminopropyl chloride, N, N-dimethylaminoethyl chloride, etc. , N, N-dibenzylaminoethyl 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, and the like are used.
As the amino group-containing vinyl compound, an amino group-containing (meth) acrylate, an amino group-containing (meth) acrylamide, an amino group-containing aromatic vinyl hydrocarbon, an amino group-containing allyl ether and the like are used.

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

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

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

Amino acids include 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-Aminoactanic acid, 20-Aminoeicosanoic acid and the like.

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 the amino group-containing monomer is preferably 5 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 material: polystyrene standard (manufactured by Toso) having a molecular weight of 420 to 20,600,000}.

Examples of the hydrohalide salt include salts of the above amino group-containing compound and halides (hydrogen fluoride, hydrogen chloride, hydrogen bromide, hydrogen iodide, etc.).
Specific examples thereof 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 (methyl chloride, ethyl chloride, benzyl chloride, dimethyl carbonate, dimethyl sulfate, ethylene oxide, etc.).

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

The carboxyl group-containing compound (AM-2) is a carboxylic acid having 2 to 30 carbon atoms, and specifically, formic acid, acetic acid, propionic acid, benzoic acid, glycolic acid, gluconic acid, lactic acid, and apple. Acids, tartaric acid, sucrose, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, pyruvate, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, mesylic acid, salicylic acid, 4-hydroxybenzoic acid, phenylacetic acid and the like. .. Further, a halide having these carboxyl groups and the like can also be used. Specific examples thereof include chloroacetic acid and chloroformic acid. Among these carboxyl group-containing compounds (AM-2), chloroacetic acid is preferable from the viewpoint of cell proliferation.

The sulfo group-containing compound (AM-3) is a sulfonic acid having 2 to 30 carbon atoms, and specifically, ethanesulfonic acid, benzenesulfonic acid, pantothenic acid, 2-hydroxyethanesulfonic acid, and toluenesulfon. Acids, sulfanilic acid, cyclohexylaminosulfonic acid and the like can be mentioned. Further, a halide having these sulfo groups and the like can also be used. Specific examples thereof include chlorosulfonic acid and chloroethanesulfonic acid. Among these sulfone group-containing compounds (AM-3), chloroethanesulfonic acid is preferable from the viewpoint of cell proliferation.

Examples of the compound containing a hydroxyl group (AM-4) include methanol, ethanol, propanol, butanol and the like. Further, a halide having these hydroxyl groups and the like can also be used. Specific examples thereof include chloroethanol and chloropropanol. Among these hydroxyl group-containing compounds (AM-4), chloroethanol is preferable from the viewpoint of cell proliferation.

As a method of modifying with the compound (AM), (1) a method of chemically bonding the compound (AM) and the cell adhesion peptide (D) before modification {covalent bond, ionic bond and / or hydrogen bond, etc.} is used. Applicable.
Of these, from the viewpoint of cell proliferation and the like, the method of chemical bonding (1) is preferable, and covalent bonding is more preferable.

(1) In a method of chemically bonding a compound (AM) and an unmodified cell-adhesive peptide (D) {covalent bond, ionic bond and / or hydrogen bond, etc.}, a cell-adhesive peptide is used to chemically bond the compound (AM). It is preferable that (D) 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. Among the reactive groups, a hydroxyl group, a carboxyl group and a primary amino group are preferable, and a hydroxyl group and a carboxyl group are preferable, and a hydroxyl group is particularly preferable, from the viewpoint of cell proliferation. Further, it is sufficient that one molecule of the cell adhesion peptide (D) has at least one reactive group, but from the viewpoint of cell proliferation, it is preferable that one molecule has 2 to 50 reactive groups, and more preferably. It has 3 to 30 cells in one molecule, particularly preferably 5 to 20 cells in one molecule.

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

Specific examples of covalently binding the compound (AM) to the unmodified cell-adhesive peptide (D) include amino acids (eg, Ser and Tyr) containing a hydroxyl group in the side chain of the cell-adhesive peptide (D). Is alkyl etherified with an alkyl halide having a primary amino group and / or an ammonium salt, a carboxyl group, a sulfone group, or a hydroxyl group, and an amino acid containing a carboxyl group in the side chain (for example, Asp and Glu) can be esterified with an alcohol having an amino group and / or an ammonium salt.

Some of the preferred cell adhesion peptides (D) are illustrated below.
(1) When the minimum amino acid sequence (X) is the RGD sequence (x1) It has 13 RGD sequences (x1) and 12 (GAGAGS) ₉ sequences (16) (y1), and these are alternately chemically chemically. A polypeptide having a Mw of about 110,000 having a bound structure (sequence (82)) {"Pronectin F", Pronectin is a registered trademark of Sanyo Kasei Kogyo Co., Ltd. (Japan and the United States). Made by Sanyo Chemical Industries, Ltd. <Same below>};
A protein obtained by modifying Pronectin F with a compound (AM), specifically, a polypeptide modified with N, N-dimethylaminoethyl chloride as the compound (AM) (“Pronectin F Plus”);
A polypeptide having about 20,000 Mw (sequence (83)) having 5 (x1) and 5 (GAGAGS) ₃ sequences (15) and (y2) and having a structure formed by alternately chemically bonding these. ("Pronectin F2"); Mw having a structure consisting of three (x1) and three (GVPVGV) ₂ GG (GAGAGS) ₃ sequences (33) and (y3), which are chemically bonded alternately. Approximately 10,000 polypeptides (sequence (84)) ("Pronectin F3") and the like.

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

(3) When the minimum amino acid sequence (X) is the YIGSR sequence (x3) “Pronectin Y”, “Pronectin Y2”, in which 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.

The cell-adhesive peptide (D) includes an artificially synthesized peptide (artificial polypeptide), for example, an organic synthesis method (solid phase synthesis method, liquid phase synthesis method, etc.) and a biochemical synthesis method [gene]. Recombinant microorganisms (yeast, bacteria, Escherichia coli, etc.)], etc. That is, the cell adhesion peptide (D) does not contain cell adhesion proteins such as collagen and fibronectin derived from animals.

Regarding the organic synthesis method, for example, it is described in "Sequel Biochemistry Experiment Course 2, Protein Chemistry (2)", pp. 641-694 (May 20, 1987; published by Tokyo Kagaku Dojin Co., Ltd.), edited by The Japanese Biochemical Society. The method used is used. As for the biochemical synthesis method, for example, the method described in JP-A-3-502935 is used. A biochemical synthesis method using a genetically modified microorganism is preferable, and a method for synthesizing using genetically modified Escherichia coli is particularly preferable because the cell adhesion peptide (D) can be easily synthesized.

As the cell adhesion factor, at least one selected from the group consisting of fibronectin, collagen, laminin, pronectin F and pronectin F plus is preferable 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 sheet-like, fibrous, rod-like, and spherical (bead-like, etc.) shapes. Of these, sheet-shaped and spherical are preferable from the viewpoint of being easy to use for cell culture.
In the case of a sheet, the thickness of the sheet is not particularly limited, but from the viewpoint of ease of cell culture and degradability, a sheet having a thickness of 0.5 to 500 nm is preferable.

Cell culture substrate of the present invention, the storage modulus G 37 ° C. 'is 1000-1 × ¹⁰ 10 Pa, preferably a ^{1 × 10 4 ~1 × 10 8} . If the storage elastic modulus G'is less than 1000 Pa, there is a problem that the adhesion of cells is lowered and the shape of adherent cells is changed. Further, if ^{it exceeds 1 × 10 10} Pa, it is difficult to prepare a base material for cell culture, and there is a problem that it is difficult to be decomposed by the decomposing agent (E) described later.
The storage elastic modulus G'is a value measured by a rheometer.

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

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

Examples of the method for producing a base material for cell culture of the present invention include those in which the polysaccharide (A) is carboxymethyl cellulose and includes the following steps (1) to (5).
(1) A step of dissolving carboxymethyl cellulose (hereinafter abbreviated as CMC) in water.
(2) A step of adding a cross-linking agent (B) to the base material obtained in (1) above to allow the cross-linking reaction to proceed.
(3) A step of processing the base material obtained in (2) above into a sheet or beads.
(4) A step of drying the base material obtained in (3) above to remove water.
(5) A step of modifying the cell adhesive peptide (D) on the substrate obtained in (4) above.

Examples of the method of 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 the stirrer with a stirrer, a method of stirring with a stirring blade, and a method of further heating the above 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 cross-linking agent (B) include a method of adding the cross-linking agent (B) while stirring the CMC solution with a stirrer. In this case, the cross-linking agent (B) may be added in its entirety at a time or gradually added using a pump or the like, but from the viewpoint of uniformly proceeding the cross-linking reaction, it is gradually added using a pump or the like. The method of

Examples of the method for uniformly processing the CMC solution after the addition of the cross-linking agent (B) into a petri dish into a sheet form include dip coating, spraying, roller coating and spin coating.
From the viewpoint of productivity and handleability, dip coating is not preferable, and spray coating, roller coating and spin coating are preferable. From the viewpoint of coating film uniformity, 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 for processing the CMC solution after adding the cross-linking agent (B) into beads, for example, a pulverization method in which the CMC solution is physically crushed into particles after drying and a CMC solution after adding the cross-linking agent are used. Examples thereof include a dispersion method in which the mixture is dispersed in a hydrophobic organic solvent.

As a pulverization method, it is preferable that the dried product is first roughly pulverized and then finely pulverized. At this time, a method of colliding with a collision plate in a jet stream to pulverize, colliding particles with each other in a jet stream to pulverize, 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 by removing fine particle portions by, for example, cyclone, decanter, centrifugation or the like.

In the dispersion method, examples of the method of dispersing the CMC solution after the addition of the cross-linking agent (B) in the organic solvent include a reverse phase suspension method, and the uniformity of the particle size, the circularity of the particles, and the surface shape of the particles. From the viewpoint of smoothness, dispersion by the reverse phase suspension method is preferable.
As the hydrophobic organic solvent, known ones (Patent No. 3363000, Japanese Patent Application Laid-Open No. 2003-147005) can be used, and basically any hydrophobic solvent can be used as long as it is difficult to dissolve in water. For example, aliphatic hydrocarbons such as n-pentane, n-hexane, n-heptane and n-octane, alicyclic hydrocarbons such as cyclohexane and methylcyclohexane, and aromatic hydrocarbons such as benzene, toluene and xylene. Examples include hydrogen.
As the dispersant, known ones as a reverse phase suspension polymerization dispersant can be used. Japanese Patent Application Laid-Open No. 61-209201, etc.), etc.), nonionic dispersants (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, a method of drying using a dryer such as a disc dryer, a turbo dryer and a drum dryer can be 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, it does not require a long time for drying and 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 dry atmosphere may be under normal pressure or reduced pressure (for example, 500 mmHg or less).

The moisture content after drying is defined by the amount of water at which the storage elastic modulus G'is 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 to each other when the cell culture substrate is bead-shaped. Further, when the cell culture substrate is in the form of a sheet, there is a problem that it is deformed or adhered when it comes into contact with another material.

The amount of water 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. It is less than% by weight. On the other hand, if the amount of water decreases too much, the surface roughness Ra increases due to volume shrinkage, so 10% by weight or more is more preferable.

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

As a method (1) for chemical bonding, the reactive group (-OH, -COOH, -NH-, -NH ₂ , -SH, etc.) of the cell-adhesive peptide (D) and the polysaccharide (A) are contained. Examples thereof include a method performed by cross-linking reactive groups (-OH, -COOH, -CHO, -C (= O)-, -NH-, -NH _{2 and -SH, etc.).}
Examples of the method (2) of physical adsorption include a method of physically adsorbing a solution containing a cell adhesion peptide (D) having a predetermined concentration and a cell culture substrate in coexistence for a predetermined time.
Examples of the enzyme modification method (3) include a method of binding the polysaccharide (A) to the cell adhesion peptide (D) using an enzyme such as glycosyltransferase.

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, but since the cell culture substrate of the present invention has high cell proliferation, it is a normal mammalian-derived cell used for the production of useful substances such as pharmaceuticals and treatment. Cells, cell lines derived from mammals and insect cells are suitable.

Examples of normal mammalian-derived cells include cells involved in skin (epithelial cells, fibroblasts, vascular endothelial cells, smooth muscle cells, etc.), cells involved in blood vessels (vascular endothelial cells, smooth muscle cells, fibroblasts, etc.). ), Muscle-related cells (muscle cells, etc.), fat-related cells (fat cells, etc.), nerve-related cells (nerve cells, etc.), liver-related cells (hepatic cells, etc.), pancreas Cells (pancreatic La Island cells, etc.), cells involved in the kidney (kidney cells, renal epithelial cells, proximal tubule epithelial cells, mesangium cells, etc.), cells involved in the lungs and bronchi (epithelial cells, fibroblasts, blood vessels, etc.) Involvement in endothelium cells and smooth muscle cells, etc.), cells involved in eyes (photoreceptor cells, corneal epithelial cells, corneal endothelial cells, etc.), cells involved in prostate (epithelial cells, stromal cells, smooth muscle cells, etc.), bone Cells (osteoblasts, bone cells, osteoblasts, etc.), cells involved in cartilage (chondrocytes, chondrocytes, etc.), cells involved in teeth (root membrane cells, osteoblasts, etc.), blood involved Cells (leukocytes, erythrocytes, etc.) and stem cells {for example, undifferentiated mesenchymal stem cells, skeletal muscle stem cells, hematopoietic stem cells, nerve stem cells, hepatic stem cells (oval cell, small hepatocite, etc.), 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 epithelial stem cells, etc.), leukocyte stem cells, lymphoid stem cells, corneal stem cells, etc.}, Precursor cells (adipose progenitor cells, vascular endothelial progenitor cells, cartilage progenitor cells, lymphoid progenitor cells, NK progenitor cells, etc.)} and the like can be mentioned.

Examples of mammalian-derived cell lines include CRFK cells, 3T3 cells, A549 cells, AH130 cells, B95-8 cells, BHK cells, BOSC23 cells, BS-C-1 cells, C3H10T1 / 2 cells, and 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, NG108-15 cells, NRK cells, OC10 cells, OTT6050 cells, P388 cells, PA12 cells, PA317 cells, PC-12 cells, PER. C6 cells, PG13 cells, QGH cells, Razi 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, ψCRE cells, etc. {Cell culture technology (edited by the Japan Society for Tissue Culture, published by Asakura Shoten Co., Ltd., 1999)}.

Insect cells include silkworm cells (BmN cells, BoMo cells, etc.), quail cells, sakusan cells, Shinjusan cells, yotoga cells (Sf9 cells, Sf21 cells, etc.), mulberry cells, beetle cells, ginger cells, sentinel flies. Examples include cells, human staghorn cells, swallowtail cells, swallowtail cells and Irakusakin uwaba cells (Tn-5 cells, HIGH FIVE cells, MG1 cells, etc.) {Insect Bio Factory (edited by Shigeru Kimura, published by Industrial Research Association, 2000) Year).

Among these cells (X), normal cells derived from mammals and cell lines derived from mammals are preferable from the viewpoint of production of useful substances such as pharmaceuticals and treatment. And, in terms of usefulness for treatment, more preferably, kidney cells, muscle cells, hepatocytes, osteoblasts, epithelial cells, fibroblasts, vascular endothelial cells, stem cells and progenitor cells. Further, in terms of being useful for the production of useful substances such as pharmaceuticals, more preferably, 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.

The medium (ME) used in the method for producing cells of the present invention includes serum-free medium (STEMPRO hESC SFM17, TeSR 2, E8 medium, hESF9 medium, hESF-FX medium, hESF92ai, StemFit AK02N, S-mediaum, mTeSR ^TM. , Grace medium, IPL-41 medium, Schneider's medium, Opti-PRO ^TM SFM medium, Opti-MEM ^TM I medium, VP-SFM medium, CD293 medium, 293SFMII medium, CD-CHO medium, CHO-S-SFMII medium. , FreeStyle ^TM 293 medium, CD-CHO ATG ^TM medium and mixed medium thereof, etc.); General medium (RPMI medium, MEM medium, Eagle's MEM medium, BME medium, DME medium, αMEM medium, IMEM medium, ES medium, etc. 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 a mixture of these. ); And a mixed medium thereof and the like.

In addition, serum can be added to these 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.).
When serum is added, human serum, bovine serum, and horse serum are preferable. In addition, the origin of animal serum includes adult-derived serum, offspring-derived serum, neonatal-derived serum, fetal-derived serum and the like. When sera are added, among these, pup-derived serum, newborn-derived serum, and fetal-derived serum are preferable, and more preferably newborn-derived serum and fetal-derived serum, particularly preferably fetal-derived serum. be. When the serum is added, the serum may be further deactivated, an antibody may be removed, or the like.

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

Cell growth factors can be contained in the medium, if 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 cell growth factor, hepatocellular growth factor, platelet-derived growth factor, insulin-like growth factor, vascular endothelial growth factor, nerve growth factor, stem cell factor, and leukemia. Physiologically active peptides such as inhibitors, bone formation factors, heparin-binding epithelial cell growth factors, neurotrophic factors, binding tissue growth factors, angiopoetin, cytokines, interleukins, adrenamodulin and sodium diuretic peptides are included. Of these, fibroblast growth factor, transforming growth factor, insulin-like growth factor and bone formation factor are preferable, and more preferably fibroblast, from the viewpoint that the range of applicable cells is wide and the healing period can be shortened. Growth factors, transforming growth factors and insulin-like growth factors.

When using the cell growth factor, but the content of the cell growth factor (wt%) depends on the type of cell growth factor, based on the weight of the medium, preferably 10 ^-16 to 10 ^-3, more preferably 10 ^{- It is 14} to 10 ^-5 , particularly preferably 10 ^-12 to 10 ^-7 .

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

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

A known method can be used for counting the number of cells, and for example, it can be measured by a cell nucleus counting method using trypan blue or crystal violet {Cell culture technology (edited by the Japan Tissue Culture Society, published by Asakura Shoten Co., Ltd.). , 1999)}.
The dry weight (g) of the cell culture substrate to be added to 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 0.02 to 1 L of the medium. 200, particularly preferably 0.1 to 40.

The culture conditions are not particularly limited, and the culture conditions are such that the carbon dioxide (CO ₂ ) concentration is 1 to 20% by volume, the culture medium is changed at 5 to 45 ° C. for 1 hour to 100 days, and if necessary, the medium is changed every 1 to 10 days. Etc. can be applied. Preferred conditions are a CO ₂ concentration of 3 to 10% by volume, 30 to 40 ° C., 1 to 20 days, and a condition of culturing while exchanging the medium every 1 to 3 days.

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

The preferred polysaccharide-degrading enzyme (E1) is appropriately selected from the polysaccharide (A) used in the crosslinked 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, carboxymethyl cellulose and other cellulose derivatives} and a degrading agent (E) {cellulase}.
A combination of a polysaccharide (A) {at least one selected from the group consisting of dextrins and dextrin derivatives} and a degrading agent (E) {dextranase}.
A combination of a polysaccharide (A) {at least one selected from the group consisting of carrageenan and carrageenan derivatives} and a degrading 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 degrading agent (E) {alginate lyase}.
A combination of a polysaccharide (A) {at least one selected from the group consisting of hyaluronic acid and hyaluronic acid derivatives} and a degrading agent (E) {hyaluronidase}.
A combination of a polysaccharide (A) {at least one selected from the group consisting of chitin and chitin derivatives} and a degrading agent (E) {chitinase}.
A combination of a polysaccharide (A) {at least one selected from the group consisting of chitosan and chitosan derivatives} and a degrading agent (E) {chitosanase}.

The decomposing agent (E) can be used as a decomposing agent (E) solution.
The concentration of the decomposing agent (E) in the degrading agent (E) solution is the type of the decomposing agent (E) used and the polysaccharide (A) used in the crosslinked product (C) constituting the cell culture substrate. It is appropriately selected depending on the type, but from the viewpoint of cytotoxicity and degradability of the cell culture substrate, 0.1 to 5% by weight is preferable, and more preferably 0. 1 to 1% by weight.

Specific preferred 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 above-mentioned cell culture substrate. After culturing, cells are recovered from the cell culture substrate using the degrading agent (E).
(2) Culture of cell sheet Adhesive cells (X-1) are cultured for a predetermined period using the above-mentioned cell culture substrate. After the cells become confluent, the cell sheet is recovered from the cell culture substrate using the degrading agent (E).
(3) Culturing of cell constituents Adhesive cells (X-1) are cultured for a predetermined period of time using the above-mentioned cell culture substrate molded into a desired structure. After culturing, the cell culture substrate is removed with the degrading agent (E) to obtain a cell constituent.

The cell culture kit of the present invention contains the cell culture substrate and the degrading agent (E).
The preferred base material for cell culture and the decomposing agent (E) are the same as described above.
Further, the decomposing agent (E) may be included in the kit as the above-mentioned decomposing agent (E) solution.

The present invention will be described in more detail below as examples, but the present invention is not limited to these examples. In the following, unless otherwise specified, parts mean parts by weight and% means% by weight.

<Example 1>
Put 800 parts of water in a container equipped with a stirrer, add 100 parts of CMC (Daiichi Kogyo Seiyaku Co., Ltd., "Cerogen F-SC") gradually while stirring (50 rpm) so as not to get lumpy. A CMC solution was obtained. Then, 100 parts of ethylene glycol diglycidyl ether (Nagase ChemteX Corporation, "Denacol EX-810"), which is a compound having an epoxy group, was added as the cross-linking 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 advancing the crosslinking reaction in a dryer at 100 ° C. A sheet-shaped (diameter 70 mm, thickness 0.1 mm) cell culture substrate (1) having a water content of 20% by weight was obtained. Storage modulus G of the resulting substrate (1) 'was 10 ⁸ Pa.

<Example 2>
Sheet-shaped (diameter 70 mm, thickness 0.1 mm) having a water content of 20% by weight in the same manner as in Example 1 except that the amount of the cross-linking agent (B) added is "67 parts" instead of "100 parts". The cell culture substrate (2) was obtained. Storage modulus G of the resulting substrate (2) 'was 10 ⁶ Pa.

<Example 3>
Sheet-shaped (diameter 70 mm, thickness 0.1 mm) having a water content of 20% by weight in the same manner as in Example 1 except that the amount of the cross-linking agent (B) added is "50 parts" instead of "100 parts". The cell culture substrate (3) was obtained. Storage modulus G of the resulting substrate (3) 'was 10 ⁴ Pa.

<Example 4>
The cell culture substrate (2) obtained in Example 2 was used as a cell-adhesive peptide (D), Pronectin F (a polypeptide having a sequence of SEQ ID NO: (82), Mw of about 110,000, Sanyo Kasei Kogyo (Sanyo Kasei Kogyo). The cell culture substrate (4) was obtained by immersing it in a PeptideF solution (37 ° C.) containing 0.001% by weight of (manufactured by Co., Ltd.) for 2 hours and modifying the surface of PeptideF by physical adsorption.

<Example 5>
The cell culture substrate (2) obtained in Example 2 is a fibronectin solution (37 ° C.) containing 0.001% by weight of fibronectin (“Fibronectin Solution, from Human Plasma” manufactured by Wako Pure Chemical Industries, Ltd.). ) Was immersed for 2 hours to obtain a cell culture substrate (5) whose surface was modified with fibronectin by physical adsorption.

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

<Comparative example 1>
A sheet-like (diameter 70 mm, thickness 0.1 mm) cell culture substrate (1') 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. rice field. The storage elastic modulus G'of the obtained base material (1') was 800 Pa.

<Comparative example 2>
Comparative cell culture substrate (2') having a water content of 20% by weight in the same manner as in Example 1 except that the amount of the cross-linking agent (B) added was "200 parts" instead of "100 parts". Got The storage elastic modulus G'of the obtained substrate (2') was 1 × 10 ¹¹ Pa. In addition, it became a lump shape before being molded by the spin coater, and could not be molded into a sheet shape.

<Comparative example 3>
A 96-well plate for cell culture (manufactured by Nunc) was used as a base material for cell culture (3') for comparison.

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

<Comparative example 5>
Fibronectin (manufactured by Wako Pure Chemical Industries, Ltd., "Fibronectin Solution, from Human Plasma") is modified on a cellulose film (manufactured by Whatman, "pure cellulose paper") according to the description of JP-A-2001-321157 for comparison. Cell culture substrate (5') was obtained.

<Evaluation of physical properties of cell culture substrate>
Storage elastic modulus G': According to JIS K7244-10 "Plastic-Test method for dynamic mechanical properties-Part 10: Complex shear viscosity by parallel plate vibration rheometer", Viscoelasticity measuring device [Equipment name "ARES-24A" , Rheometric Co., Ltd.] was used to measure the storage elastic modulus at 37 ° C.
Contact angle with water (37 ° C.): Measured according to JIS R3257.
Surface Roughness (Ra): Surface roughness was measured using a commercially available cantilever and an atomic force microscope [equipment name "E-sweep", manufactured by SII Nanotechnology Co., Ltd.]. The height image (three-dimensional image) was measured by scanning at a constant speed (0.3 Hz to 1.2 Hz) with the measurement range set to 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>
^{VERO cells were seeded at 3 × 10 5} cells / cm ² on cell culture substrates (1) to (6) and (1') to (5') in DMEM medium (containing 10 wt% FBS). The cells were cultured at 37 ° C., 5% by volume CO ₂ , and under wet conditions (humidity 100% RH). After 24 hours, the DMEM medium was removed, 0.1 ml of a 100-fold diluted solution of cellulase (Endolase 5000 L, manufactured by Novozymes) was added as a degrading agent (E) for 2 hours at 37 ° C., 5% by volume CO ₂ , and wet conditions (wet conditions). It was cultured in 100% humidity RH). After 2 hours, 10 μl / 100 μl of the viable cell count reagent SF (manufactured by Nacalai Tesque, Inc.) and 100 μl of DMEM medium (containing 10 wt% FBS) were added to the culture medium, and after 1 hour, 450 nm (reference wavelength 600 nm). Was measured. The cell viability was calculated assuming that the cell viability was 100% when the degrading agent (E) was not added (blank).
Further, in the cell culture substrate (3) of Comparative Example 3, the same procedure was applied except that "0.1 ml of trypsin / EDTA Solution (manufactured by Thermo)" was used instead of "0.1 ml of cellulase". Cell viability was evaluated.
Cell viability (%) = [sample absorbance-blank absorbance] / [unadded absorbance-blank absorbance] x 100

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

<Evaluation of cell culture: peeling time>
^{VERO cells were seeded at 3 × 10 5} cells / cm ² on cell culture substrates (1) to (6) and (1') to (5'), and were seeded in DMEM (10% FBS) at 37 ° C., 5 The cells were cultured under volume% CO ₂ and wet conditions (humidity 100% RH).
After 24 hours, a 100-fold diluted solution of cellulase (Endolase 5000L, manufactured by Novozymes) was added to the cell culture medium as a degrading agent (E), and the degree of cell detachment over time was confirmed, and the time during which the cells were completely detached ( Minutes) was measured.

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.
Further, the cell culture substrates of Examples 1 to 6 are extremely suitable for cell culture because they have extremely high cell viability and cell adhesion rate, are easily decomposed by a degrading agent, and have a short cell detachment time. I understand. In particular, from the results of the cell culture substrates of Examples 4 to 6, it can be seen that the cell adhesion rate can be improved by containing the cell adhesion factor. On the other hand, the cell culture substrates of Comparative Examples 1 (non-crosslinked) and 4 (metal crosslinked) have high cell viability and degradability, but low cell adhesion and are suitable for cell culture. It turns out that there is no such thing. Further, it can be seen that the cell culture substrate, which 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 a decomposing agent, and it takes a long time to detach 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, so that it can be used for research and development, production of useful substances, treatment, etc. related to cells. Extremely useful.
For research and development, for culturing cells for evaluating cell function such as differentiation function, culturing alternative cells for animal experiments (toxicity test, stimulus test, metabolic function test, etc.), culturing cells for introducing genes and proteins, etc. Available.
For 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. Of 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 (9)

A cell process for the production of culturing cells (X) using the cell culture substrate for
The cell culture substrate contains the crosslinked product (C) of the polysaccharide (A) with the crosslinking agent (B).
The cross-linking agent (B) is an organic compound having at least two functional groups (b) capable of reacting with an active hydrogen group, and the storage elastic modulus G'at 37 ° C. of the cell culture substrate is 1000 to 1 × 10 ¹⁰ Pa. And
The method for producing cells includes a step of culturing cells (X) on a cell culture substrate and then degrading the cell culture substrate with a degrading agent (E).
A method for producing cells in which the degrading agent (E) is a polysaccharide-degrading enzyme (E1). 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, arabic gum, karaya gum, pectin The cell according to claim 1, which is at least one selected from the group consisting of xanthan gum, julan gum, agrobacterium succinoglycan, dieutan gum, mannan, glucomannan, chitosan, tara gum, psyllium seed gum and derivatives thereof. Production method. The method for producing a cell 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 method for producing cells according to any one of claims 1 to 3, wherein the contact angle of the cell culture substrate with respect to water (37 ° C.) on the cell culture surface is 20 to 60 °. The method for producing cells according to any one of claims 1 to 4, wherein the surface roughness (Ra) on the cell culture surface of the cell culture substrate is 1 μm or less. Claims 1 to 5 in which the cell culture substrate contains at least one cell adhesion factor selected from the group consisting of fibronectin, collagen, laminin and the cell adhesion peptide (D) having the following minimum amino acid sequence (X). The method for producing cells according to any one of the above.
Minimum amino acid sequence (X): RGD sequence, LDV sequence, REDV sequence (1), YIGSR sequence (2), PDSGR sequence (3), RYVVLPR sequence (4), LGTIPG sequence (5), RNIAEI IKDI sequence (6), IKEVAV At least one selected from the group consisting of sequence (7), LRE sequence, DGEA sequence (8), GVKGDKGNPGWPGAP sequence (9), GEFYFDLRLKGDK sequence (10), HAV sequence and YKLNVNDS sequence (11). The cell-adhesive peptide (D) is chemically modified with N, N-dimethylaminoethyl chloride hydrochloride for the polypeptide represented by SEQ ID NO: (82) and / or the peptide represented by SEQ ID NO: (82) in the sequence listing. The method for producing a cell according to claim 6, which is a polypeptide obtained . The method for producing cells according to any one of claims 1 to 7, wherein the cell culture substrate has a sheet shape. A kit for cell culture comprising cell culture base material and decomposition agent (E),
The cell culture substrate contains the crosslinked product (C) of the polysaccharide (A) with the crosslinking agent (B).
The cross-linking agent (B) is an organic compound having at least two functional groups (b) capable of reacting with an active hydrogen group, and the storage elastic modulus G'at 37 ° C. of the cell culture substrate is 1000 to 1 × 10 ¹⁰ Pa. And
A cell culture kit in which the degrading agent (E) is a polysaccharide-degrading enzyme (E1).