JP7039308B2 - Cell culture carrier - Google Patents

Description

The present invention relates to a carrier for cell culture.

As a carrier for cell culture, a carrier for cell culture using a water-absorbent resin having a positively charged functional group (Patent Document 1) and the like are known.

Japanese Unexamined Patent Publication No. 2010-148486

However, the cell culture carrier described in the above-mentioned prior literature deteriorates over time, the initial spherical shape is deformed into irregularities, the cell concentration is further lowered, and the cell adhesion rate to the medium is lowered, so that the cells are cells. There is a problem that it cannot be cultured.
An object of the present invention is to provide a cell culture carrier that does not easily deteriorate over time and can be used for cell culture even after long-term storage.

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 cell culture carrier (D) containing a water-absorbent resin particle (A) and a polypeptide modification inhibitor (C), wherein the water-absorbent resin particle (A) has a carboxyl group and a first. The water-absorbent resin particles (E) having a to tertiary amino group and the polypeptide (B1) having at least one cell-adhesive minimum amino acid sequence (X) in one molecule are amide-bonded, and the polypeptide is modified. A cell culture carrier in which the inhibitor (C) is a sugar and / or an amino acid.

The carrier for cell culture of the present invention does not easily change with time and can be cultured even after long-term storage. Further, the shape of the carrier is maintained without causing unevenness over time.

Photograph of the cell culture carrier obtained in Example 1 stored at 60 ° C. for 65 days and inflated with an electron microscope (magnification magnification 40 times). Photograph of the cell culture carrier obtained in Comparative Example 1 stored at 60 ° C. for 65 days and inflated with an electron microscope (magnification magnification 40 times).

The cell culture carrier of the present invention is a cell culture carrier (D) containing a water-absorbent resin particle (A) and a polypeptide modification inhibitor (C), and the water-absorbent resin particle (A) has a carboxyl group. The water-absorbent resin particles (E) having a primary to tertiary amino group and the polypeptide (B1) having at least one cell-adhesive minimum amino acid sequence (X) in one molecule are amide-bonded to form a polypeptide. A cell culture carrier in which the denaturation inhibitor (C) is a sugar and / or an amino acid.

The composition of the water-absorbent resin used for the carrier for cell culture of the present invention is a water-absorbent resin (A') having a general carboxyl group and further having a primary to tertiary amino group.

Examples of the water-absorbent resin (A') having a carboxyl group include the following water-absorbent resins (1) to (5).
(1) Crosslinked polyacrylic acid (salt) obtained by aqueous solution polymerization (adiabatic polymerization, thin film polymerization, spray polymerization, etc.) described in JP-A-55-133413.
(2) Cross-linked polyacrylic acid (salt) obtained by reverse phase suspension polymerization described in JP-A-54-30710, JP-A-56-26909, JP-A-11-5808, and the like.
(3) The polyamino acid radiation crosslinked product described in JP-A No. 10-251402.
(4) Cross-linked polyaspartic acid described in JP-A-2002-179770.
(5) JP-A-2001-2935, JP-A-2003-052742, JP-A-2003-082250, JP-A-2003-165883, JP-A-2003-176421, JP-A-2003-183528. , JP-A-2003-192732, JP-A-2003-225565, JP-A-2003-238696, JP-A-2003-335970, JP-A-2004-091673, JP-A-2004-121400, Special Publication. Kai 2004-123835, JP-A-2005-075982, JP-A-2005-095759, JP-A-2005-097569, JP-A-2005-186015, JP-A-2005-186016, JP-A-2005 -Cross-linked poly (meth) acrylic acid (salt) described in JP-A-247931 and the like.

Of these, (2) and (5) are preferable as the water-absorbent resin (A') having a carboxyl group from the viewpoint of adhesion to the carrier of cells and the like. That is, a crosslinked polymer obtained by polymerizing a monomer having a carboxyl group is preferable, and a crosslinked poly (meth) acrylic acid (salt) is more preferable.

In addition, (meth) acrylic acid means acrylic acid and / or methacrylic acid, and acid (salt) means acid and / or acid salt.
Examples of acrylates and methacrylic acid salts include alkali metal (lithium, potassium and sodium, etc.) salts of acrylic acid or methacrylic acid, polyvalent metals {alkaline earth metals (magnesium, calcium, etc.), and boron metal (aluminum, gallium, etc.). And indium, etc.), and transition metals (titanium, vanadium, chromium, manganese, iron, nickel, cobalt, copper, zinc, zirconium, molybdenum, ruthenium, rhodium, silver, cadmium, osmium, platinum, etc.)} Salts and ammonium salts Etc. are used.

Of these, alkali metal salts and polyvalent metal salts are preferable from the viewpoint of cytotoxicity and the like, more preferably alkali metal salts, alkaline earth metal salts and transition metal salts, and particularly preferably alkali metal salts and alkaline earth metals. Salts, most preferably sodium salts, potassium salts, magnesium salts and calcium salts.

The crosslinked poly (meth) acrylic acid (salt) has (meth) acrylic acid (salt) as the main constituent unit, and other vinyl monomers copolymerizable with (meth) acrylic acid (salt) as the constituent unit. be able to. Other copolymerizable vinyl monomers include known copolymerizable monomers (hydrophilic vinyl monomers and hydrophobic vinyl monomers), known crosslinkable monomers and the like {Japanese Patent Laid-Open No. 11-5808. , JP-A-2001-2935, JP-A-2003-165883, JP-A-2005-247931, JP-A-2005-186015, etc.}.

The crosslinked poly (meth) acrylic acid is (1) in a hydrophobic organic solvent under stirring, (meth) acrylic acid (salt), and, if necessary, other copolymerizable vinyl monomers, polymerization initiators, chain transfer agents. And / or a method of continuously supplying a graft substrate and performing known reverse phase suspension polymerization (Japanese Patent Laid-Open Nos. 11-5808, 2001-2935, 2003-165883, 2005- 247931 and 2005-186015, etc.); And (2) (meth) acrylic acid (salt) and, if necessary, other copolymerizable vinyl monomers, polymerization initiators, chain transfer agents and / or Methods for polymerizing a graft substrate with a known aqueous solution (Japanese Patent Laid-Open No. 2005-075982, JP-A-2005-095759, JP-A-2005-097569, JP-A-2005-186015 and JP-A-2005-1860116 It can be manufactured according to the publication etc.).

The water-absorbent resin particles (A) used in the carrier for cell culture of the present invention are water-absorbent resins having not only a carboxyl group but also a primary to tertiary amino group.
The water-absorbent resin (A') having no amino group has a poor adhesion rate at the initial stage of cell culture, low cell adhesion, and having a primary to tertiary amino group significantly improves cell adhesion.

A method of introducing a primary to tertiary amino group into a water-absorbent resin (A') having a carboxyl group to produce the water-absorbent resin particles (A) of the present invention having a carboxyl group and a primary to tertiary amino group. Examples thereof include a method of chemically modifying a water-absorbent resin (A') having a carboxyl group with a compound (S) having a primary to tertiary amino group.

Further, as a method for chemically modifying the water-absorbent resin (A') having a carboxyl group with the compound (S) having a primary to tertiary amino group, specifically, the following methods (1) and (2) are used. Can be mentioned.
(1) Functional groups (alkyl halide groups, acyl halide groups, glycidyl ether groups, hydroxyl groups, carbodiimide groups, carbonate groups, amino groups, oxazoline groups and aziridines) that can react with the carboxyl groups in the water-absorbent resin (A'). Group, etc.) and a method of reacting with a compound (S1) having a primary to tertiary amino group;
(2) The carboxyl group in the water-absorbent resin (A') includes a functional group (amino group, alkyl halide group, acyl halide group, glycidyl ether group, hydroxyl group, carbodiimide group, carbonate group, which can react with the carboxyl group. A functional group (alkyl halide group, acyl halide group, glycidyl ether group, carbodiimide group, carbonate) capable of reacting the hydroxyl group with the hydroxyl group by reacting the compound (S2) having an oxazoline group and an aziridine group) and a hydroxyl group. Method for reacting a compound (S3) having a group (group, azilydin group, etc.) and a primary to tertiary amino group Of these, (2) is preferable from the viewpoint of cell adhesion.

In the functional group capable of reacting with the carboxyl group and the compound having a hydroxyl group (S2), the functional group capable of reacting with the carboxyl group is preferably an amino group, a glycidyl ether group and an alkyl halide group from the viewpoint of bond strength. , More preferably an amino group.
The compound (S2) includes a compound having a primary alcohol, for example, a compound having an amino group and a hydroxyl group {alkanolamine (including a compound having an alkylene group having 1 to 4 carbon atoms, and specifically, 2-Aminoethanol, 4-amino-1-butanol, 2-amino-2-methyl-1-propanol, 3-amino-1-propanol, etc.)}, compounds having a glycidyl ether group and a hydroxyl group {2 , 3-Epoxy-1-propanol, glycerol-2,10-diglycidyl ether, etc.}, compounds having an alkyl halide group and a hydroxyl group {methanol halide, 2-ethanol halide, 3-propanol propanol, 4- Examples thereof include butanol halides. Examples of the halogen include fluorine, chlorine, bromine and the like, but chlorine is preferable from the viewpoint of reactivity, ease of handling and safety of by-products. } And so on.
As the compound (S2), a compound having a primary alcohol is preferable, and an alkanolamine is more preferable, and an ethanolamine is most preferable, from the viewpoint of reactivity.

As a method for reacting the water-absorbent resin (A') having a carboxyl group with the compound (S2), a known method for reacting the carboxyl group with each functional group can be used, and the compound (S2) is reactive. When it has an amino group as a group, for example, a method of amidating a water-absorbent resin (A') and a compound (S2) in the presence of a condensing agent such as carbodiimide can be mentioned.
Specifically, after the carboxyl group is reacted with the carbodiimide compound in advance to obtain acylisourea {R'-N = C (OCOR) -NH-R'(the portion where -OCOR is derived from (A'))}. , (A') and (S2) can be amide-bonded by adding an amino group to this acylisourea.

In the compounds (S1) and (S3) having a primary to tertiary amino group, the primary to tertiary amino groups include a primary amino group (-NH2), a secondary amino group (-NHR), and a tertiary amino group. A secondary amino group (-NHR2), is included.
In the tertiary amino group, R may be the same or different.
R is a monovalent hydrocarbon having 1 to 4 carbon atoms, and from the viewpoint of cell adhesion, a monovalent aliphatic hydrocarbon having 1 to 3 carbon atoms is preferable, and an alkyl group having 1 to 2 carbon atoms is more preferable. It is particularly preferably a methyl group and an ethyl group.

Examples of the primary amino group include aminoalkyl groups such as amino group, aminomethyl group, aminoethyl group and aminopropyl group, and aminoalkoxyalkyl such as 3-amino-1-ethoxypropyl group and 1-amino-ethoxymethyl group. The basis etc. can be mentioned.
Examples of the secondary amino group include an amino group substituted with one hydrocarbon group. For example, an N-alkylaminoalkyl group is included, and examples thereof include an N-alkylaminoalkyl group such as an N-methylaminoethyl group and an N-ethylaminoethyl group, and an imidazole group.
Examples of the tertiary amino group include an amino group substituted with two hydrocarbon groups. Examples of the functional group having a tertiary amino group include N-dimethylaminoethyl group, N-dimethylaminopropyl group, N-diethylaminoethyl group, N-dibutylaminoethyl group and the like.

The primary to tertiary amino groups may be salts with an acid. Examples of the acid include hydrochloric acid, odorous acid, iodide, acetic acid, sulfuric acid, nitric acid, phosphoric acid and the like.

In the compound (S1), the functional group capable of reacting with the carboxyl group is preferably an alkyl halide group from the viewpoint of reactivity.
Specific examples of the compound (S1) include a halogenated alkyl compound having a primary to tertiary amino group {a compound having a divalent alkylene group having 2 to 4 carbon atoms, and halogens include fluorine and chlorine. Etc., for example, 2-chloro-N, N-diethylethylamine hydroxychloride, (3-chloro-2-hydroxypropyl) trimethylammonium chloride, etc.} and the like.
As the compound (S1), a halogenated alkyl compound having a primary to tertiary amino group is preferable, and more preferably, a divalent alkylene group having 2 to 3 carbon atoms and a primary to tertiary amino. It is a halogenated alkyl compound having a group, and particularly preferably 2-chloro-N, N-diethylethylamine hydroxychloride.

In the compound (S3), the functional group capable of reacting with the hydroxyl group is preferably an alkyl halide group from the viewpoint of reactivity.
Specific examples and preferred compounds of compound (S3) are the same as those of compound (S1).

In the water-absorbent resin particles (A) of the cell culture carrier of the present invention, the water-absorbent resin particles (E) having the above-mentioned carboxyl group and primary to tertiary amino groups have the smallest cell-adhesive amino acid sequence (X). It is amide-bonded to a polypeptide (B1) having at least one in one molecule.

The water-absorbent resin particles (A) of the present invention have an amino group of a polypeptide (B1) having at least one cell-adhesive minimum amino acid sequence (X) in one molecule, and the above-mentioned carboxyl group and the first to third grades. The carboxyl group in the water-absorbent resin particles (E) having an amino group is bonded by an amide bond.

As a method of amide-bonding the water-absorbent resin particles (E) and the polypeptide (B1), a carboxyl group is reacted with a carbodiimide compound in advance, and acylisourea {R'-N = C (OCOR) -NH-R'(- After obtaining the moiety from which OCOR is derived from (E)}, the polypeptide (B1) having a primary amino group or a secondary amino group is added to the acylisourea to obtain water-absorbent resin particles (E). ) And the polypeptide (B1) can be amide-bonded.
Examples of the carbodiimide compound include N, N'-dicyclohexylcarbodiimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride.

The polypeptide (B1) having at least one cell-adhesive minimum amino acid sequence (X) used in the present invention in one molecule will be described below.

<Polypeptide (B1)>
The cell adhesion minimum amino acid sequence (X) includes "Pathological Physiology, Vol. 9, No. 7, pp. 527-535, 1990" and "Osaka Women's and Children's Medical Center Magazine, Vol. 8, No. 1, 58-66". The one described in "Page, 1992" is used.

Among these minimum amino acid sequences (X), RGD sequence, LDV sequence, LRE sequence, HAV sequence, REDV sequence (1), YIGSR sequence (2), PDSGR sequence (3), RYVVLPR sequence (4), LGTIPG sequence. (5), RNIAEIIKDI sequence (6), IKVAV sequence (7), DGEA sequence (8), GVKGDDKGNPPGWPGAP sequence (9), GEFYFDRLLKGDK sequence (10), YKLNVNDS sequence (11), AKPSYPPTYK sequence (12), NRW ), TWYKIAFQRNRK sequence (14), RKRLQVQLSIRT (15) and PHSRN (16), at least one selected from the group is preferable, and from the viewpoint of cell adhesion, the RGD sequence, YIGSR sequence (2), IKVAV sequence are more preferable. (7), at least one selected from the group consisting of RKRLQVQLSIRT (15) and PHSRN (16), particularly preferably an RGD sequence.

At both ends of these minimum amino acid sequences (X) are 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 (asparagin), Q (glutamine), D (aspartic acid), E ( Glutamic acid), R (arginine), K (lysine), H (histidine), etc.} may be contained.

The polypeptide (B1) may have at least one minimum amino acid sequence (X) in one molecule, but one having 1 to 50 in one molecule is preferable, and more preferably, from the viewpoint of cell adhesion and the like. The number is 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 polypeptide (B1) 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 polypeptide (B1).

As the auxiliary amino acid sequence (Y), an amino acid sequence other than the minimum amino acid sequence (X) can be used, and from the viewpoint of thermal stability of the polypeptide (B1), a sequence having G (glycine) and / or A (alanine). 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, (GVPPGV) _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 is preferable to have at least one selected from the group consisting of a sequence, a (GVPGV) _h sequence, a (GVGVP) _m sequence and a (GPP) _n sequence, and more preferably a (GAGAGS) _b sequence, a (GVPPGV) _h sequence, ( It has at least one selected from the group consisting of a GVGVP) _m sequence and a (GPP) _n sequence, and is particularly preferably one having a (GAGAGS) _b sequence.
Note that 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 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 is an integer of 1 to 22, and p and q are integers of 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 the auxiliary amino acid sequence (Y), in addition to the above examples, 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 (asparagin), Q (glutamine), 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 amino acid sequences represented by SEQ ID NOs: (17) to (19).
Examples of the auxiliary amino acid sequence having the (GAGAGS) b sequence include amino acid sequences represented by SEQ ID NOs: (20) to (22).
Examples of the auxiliary amino acid sequence having the (GAGAGY) c sequence include amino acid sequences represented by SEQ ID NOs: (23) to (25).
Examples of the auxiliary amino acid sequence having the (GAGVGY) d sequence include amino acid sequences represented by SEQ ID NOs: (26) to (28).
Examples of the auxiliary amino acid sequence having the (GAGYGV) e sequence include amino acid sequences represented by SEQ ID NOs: (29) to (31).
Examples of the auxiliary amino acid sequence having the {DGG (A) fGGA} g sequence include amino acid sequences represented by SEQ ID NOs: (32) to (34).
Examples of the auxiliary amino acid sequence having the (GVPVGV) h sequence include amino acid sequences represented by SEQ ID NOs: (35) to (38).
(G) Examples of the auxiliary amino acid sequence having the i sequence include amino acid sequences represented by SEQ ID NOs: (39) to (41).
(A) Examples of the auxiliary amino acid sequence having the j sequence include amino acid sequences represented by SEQ ID NOs: (42) to (44).
Examples of the auxiliary amino acid sequence having the (GGA) k sequence include amino acid sequences represented by SEQ ID NOs: (45) to (47).
Examples of the auxiliary amino acid sequence having the (GVGVP) m sequence include amino acid sequences represented by SEQ ID NOs: (48) to (50).
Examples of the auxiliary amino acid sequence having the (GPP) n sequence include amino acid sequences represented by SEQ ID NOs: (51) to (53).
Examples of the auxiliary amino acid sequence having the (GAQGPAGPG) o sequence include amino acid sequences represented by SEQ ID NOs: (54) to (56).
(GAPGAPGSQGAPGLQ) Examples of the auxiliary amino acid sequence having a p sequence include amino acid sequences represented by SEQ ID NOs: (57) to (59).
Examples of the auxiliary amino acid sequence having the (GAPGTPGPQGLPGSP) q sequence include the amino acid sequences represented by SEQ ID NOs: (60) to (62).
Note that 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 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 is an integer of 1 to 22, and p and q are integers of 1 to 13.

Among these auxiliary amino acid sequences, SEQ ID NOs: (17), (18), (20), (21), (22), (23), (24), (26), (27), (29), (30), (32), (33), (34), (35), (36), (38), (39), (40), (42), (43), (45), (46) ), (48), (49), (51), (52), (54), (55), (57), (58), (60) or (61) is preferable. More preferably, SEQ ID NOs: (18), (20), (21), (22), (24), (27), (30), (34), (35), (36), (37), ( The amino acid sequence represented by 38), (40), (43), (46), (49), (52), (55), (58) or (61), particularly preferably SEQ ID NO: (20), It is an amino acid sequence represented by (21) or (38).

When the auxiliary amino acid sequence (Y) is contained, the number of (Y) contained is preferably 2 to 50, more preferably 3 to 30, particularly preferably 3 to 30 in one molecule of the polypeptide (B1) from the viewpoint of thermal stability and the like. It is preferably 4 to 20, most preferably 5 to 15. Further, the polypeptide (B1) may contain two or more kinds of auxiliary amino acid sequences (Y).

The polypeptide (B1) may contain a branched chain, may be partially crosslinked, or may contain a cyclic structure.
However, the polypeptide (B1) 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 polypeptide (B1) has a minimum amino acid sequence (X) and an auxiliary amino acid sequence (Y), and if necessary, other amino acids between (X) and (Y). It is preferable to have a structure in which chemical bonds are alternately formed via a sequence, and a structure in which an amino acid sequence containing an intervening amino acid sequence (Z) at both ends of (X) and (Y) are alternately chemically bonded. It is even more preferable to have.
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. It is -40, particularly preferably 3-30, most preferably 4-20.

As the intervening amino acid sequence (Z), the GAAVTG sequence (65), GLPGPKGD sequence (66), GPAVTG sequence (67), and AGPKGADGSPGPAVTG sequence (68) are located at the N-terminal of the minimum amino acid sequence (X) from the viewpoint of cell adhesion. ), GAAVCEPG sequence (69), GAALCVSEPG sequence (70), SPASAALCVSEPG sequence (71), SPASAAVCEPG sequence (72), AGPKGADGSPGPAVCEPG sequence (73), AGPKGADGSPGPALCVSEPG sequence (74), GPAVCEPG sequence (75), GPAVCEPG sequence (75) It is preferable that at least one intervening amino acid sequence (Z) selected from the group consisting of the GAAPGAS sequence (77) is bound, and the GAAVTG sequence (65), GLPGPKGD sequence (66), GPAVTG sequence (67) and AGPKGADGSPGPAVTG sequence are bound. At least one selected from the group consisting of (68) is more preferable, and the GAAVTG sequence (65) is particularly preferable.

As the intervening amino acid sequence (Z), the SPASAAGY sequence (78), the SPASAALCVS sequence (79), the SPASAAVC sequence (80), and the AGPKGADGSPGP sequence (81) are located at the C-terminal of the minimum amino acid sequence (X) from the viewpoint of cell adhesion. ), AGPKGADGSPGPAVC sequence (82), AGPKGADGSPGPALCVS sequence (83), AGPKGADGSP sequence (84), SPASAAGPVGSP sequence (85), CDAGY sequence (86), CDAGPVGSP sequence (87) and SAGPSAGY sequence (88). It is preferable that one type of intervening amino acid sequence (Z) is bound, and the SPASAAGY sequence (78), SPASAALCCVS sequence (79), SPASAAVC sequence (80), AGPKGADGSPGPP sequence (81), AGPKGADGSPGPAVC sequence (82), AGPKGADGSPGPALCVS sequence. (83), at least one selected from the group consisting of the AGPKGADGSP sequence (84) and the SPASAAGPVGSP sequence (85) is more preferred, and the SPASAAGY sequence (78) is particularly preferred.

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 polypeptide (B1) is preferably 0.5 to 2, more preferably 0.9 to 1.4. , Particularly preferably 1 to 1.3.

Further, other amino acids may be contained in the terminal portion of the polypeptide (B1) (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 polypeptide (B1), more preferably 3 to 300, and particularly preferably 10 to 100.

The weight average molecular weight (hereinafter, Mw) of the polypeptide (B1) 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, it is 4,000 to 200,000.

The Mw of the polypeptide (B1) is a known method such as a method of separating a measurement sample {polypeptide or the like} by SDS-PAGE (SDS polyacrylamide gel electrophoresis) and comparing the migration distance with a standard substance. Demanded by (the same applies hereinafter).

The polypeptide (B1) may be further modified with 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), 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 compound containing a primary to tertiary amino group and its salt and the compound containing an ammonium salt (AM-1) include polyamines, aminoalcohols, halides having an amino group, amino group-containing monomers and amino group-containing monomers. A polymer having the above as a constituent monomer, a hydrohalide hydrogen salt thereof, a quaternary compound, and the like can be used.
As the polyamine, a polyamine having at least one primary amino group or a secondary amino group (carbon number 2 to 56) 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.) and polyalkylene polyamines having an alkylene group having 2 to 6 carbon atoms (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 alkanolamine 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 (1-18 carbon atoms) 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.

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. are used. , 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, 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 ) 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. Aminoethyl (meth) acrylamide, N-benzyl-N-methylaminoethyl (meth) acrylamide, morpholinoethyl (meth) acrylamide, N-methylpipetidinoethyl (meth) acrylamide and the like can be mentioned.

Examples of the amino group-containing aromatic vinyl hydrocarbon 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.

Amino acids include arginine, histidine, isoleucine, leucine, methionine, phenylalanine, threonine, tryptophan, tyrosine, valine, alanine, aspartic acid, aspartic acid, glutamine, glutamic acid, proline, cysteine, lysine, serine, glycine and 3-aminopropionic acid. , 8-Aminoactanic acid, 20-Aminoeicosanoic acid and the like.

Examples of the polymer of the amino group-containing monomer include vinyl polymers composed of amino group-containing vinyl compounds, polyethyleneimine, and polypeptides (excluding (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 substance: polystyrene standard (manufactured by Tosoh) 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, N, N-diethylaminoethyl chloride and the like.

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.).

Of these compounds containing primary to tertiary 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 is more preferable.

The compound containing a carboxyl group (AM-2) is a carboxylic acid having 2 to 30 carbon atoms, and specifically, formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, and apple. Examples thereof include 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 and phenylacetic acid. .. 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 toluenesulfonic acid. Acids, sulfanic 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 sulfonic acid-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 and butanol. 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 polypeptide (B1) before modification {covalent bond, ionic bond and / or hydrogen bond, etc.}, and (2) A method of physically adsorbing the compound (AM) to the polypeptide (B1) before modification (adsorption by van der Waals force) or the like can be applied.
Of these, the method of chemical bonding (1) is preferable, and more preferably a covalent bond, from the viewpoint of cell proliferation and the like.

(1) In a method of chemically bonding a compound (AM) and an unmodified polypeptide (B1) {covalent bond, ion bond and / or hydrogen bond, etc.}, the polypeptide (B1) is used to chemically bond the compound (B1). It is preferable to have 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, 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 polypeptide (B1) has at least one reactive group, but from the viewpoint of cell proliferation, those having 2 to 50 in one molecule are preferable, and one molecule is more preferable. It has 3 to 30 cells, 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. Examples include sulfoxide, dimethylacetamide and methanol.

As a specific example of covalently binding the compound (AM) and the polypeptide (B1) before modification, amino acids (for example, Ser and Tyr) containing a hydroxyl group in the side chain in the polypeptide (B1) are added to 1-3. A method for alkyl etherifying with a class amino group and / or an ammonium salt, a carboxyl group, a sulfone group, an alkyl halide having a hydroxyl group, and amino acids containing a carboxyl group in the side chain (for example, Asp and Glu). Examples thereof include a method of esterifying with an alcohol having a group and / or an ammonium salt.

Some of the preferred polypeptides (B1) 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 (21) (y1), and these are alternately chemical. A polypeptide having a Mw of about 110,000 having a bound structure (sequence (89)) {"Pronectin F", Pronectin is a registered trademark of Sanyo Kasei Kogyo Co., Ltd. (Japan and the United States). 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 (90)) having 5 of (x1) and 5 of (GAGAGS) 3 sequences (20) and (y2) and having a structure in which these are chemically bonded alternately. ("Pronectin F2"); Mw having a structure having three (x1) and three (GVPVGV) 2GG (GAGAGS) 3 sequences (38) (y3) and chemically bonding these alternately. 10,000 polypeptides (sequence (91)) ("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.

In addition to the polypeptides (1) to (3), RetroNectin (recombinant human fibronectin CH-296) manufactured by Takara Shuzo Co., Ltd. {Mw containing the RGD sequence (x1) and LDV sequence as the minimum amino acid sequence (X). 60,000 polypeptides} and RGDS-Protein A {polypeptides with Mw of about 30,000 containing RGD sequence (x1) as the minimum amino acid sequence (X)} can also be preferably used {however, these polypeptides are naturally occurring. It is derived and does not contain the auxiliary amino acid sequence (Y). Therefore, the thermal stability and the like are inferior to the above (1) to (3). The amino acid sequences of these polypeptides are disclosed in JP-A No. 2-311498. }.

Polypeptides (B1) include those that are artificially synthesized (artificial polypeptides), and are, for example, organic synthesis methods (solid phase synthesis method, liquid phase synthesis method, etc.) and biochemical synthesis methods [genetical recombination]. Manufactured by microorganisms (yeast, bacteria, Escherichia coli, etc.)]. That is, the polypeptide (B1) does not contain cell-adhesive proteins such as collagen and fibronectin derived from animals.

The organic synthesis method is described in, for example, "Continued Biochemistry Experiment Course 2, Protein Chemistry (2)", pp. 641-694 (May 20, 1987; published by Tokyo Chemical 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 Japanese Patent Publication No. 3-502935 is used. A biochemical synthesis method using a recombinant microorganism is preferable, and a method for synthesizing using recombinant Escherichia coli is particularly preferable because the polypeptide (B1) can be easily synthesized.

A polypeptide (B1) having at least one cell adhesion minimum amino acid sequence (X) in one molecule is amide-bonded to the water-absorbent resin particles (E) having a carboxyl group and a primary to tertiary amino group. The amount of the residue of the amide-bound polypeptide (B1) bound is preferably 100 ng / g to 50 mg / g, more preferably 100 ng / g to 50 mg / g, based on the dry weight of the cell culture carrier, from the viewpoint of improving cell adhesion. Is 200 ng / g to 40 mg / g, particularly preferably 300 ng / g to 30 mg / g, and most preferably 1 μg to 1 mg / g.
The dry weight of the cell culture carrier as a reference here means a state of being dried at 120 ° C. for 4 hours in a desiccator.

When the content of the polypeptide (B1) exceeds 500 μg / g, the binding amount of the polypeptide (B1) is the Biuret method (for example, "Biochemistry Experiment Course 1, Protein Chemistry I" edited by The Japanese Biochemical Society, "No. Requested from pages 45 to 55 (December 11, 1979; published by Tokyo Chemical Co., Ltd.).

On the other hand, when the binding amount of the polypeptide (B1) is 500 μg / g or less, the Kjeldahl method (for example, “Biochemistry Experiment Course 1, Protein Chemistry I” edited by The Japanese Biochemical Society, pp. 45-55 (December 11, 1979)) Sun; issued by Tokyo Chemical Co., Ltd.).

Further, it can also be measured by using an immunological measurement method (described in JP-A-2004-049921). In particular,
(1) A cell culture carrier whose content of the polypeptide (B1) is known {a carrier for cell culture whose content of the polypeptide (B1) is known by the Biuret method, the Kjeldahl method, etc.} is used as a physiological saline solution. Immerse and react the antibody that binds to the polypeptide (B1) with an enzyme-labeled antibody (hereinafter referred to as enzyme-labeled antibody), measure the enzyme amount of the reacted enzyme-labeled antibody, measure the absorbance, and perform a calibration curve (polypeptide (polypeptide). The content of B1) and its absorbance) are prepared.
(2) Similarly, the absorbance of the sample (cell culture carrier whose content of the polypeptide (B1) is unknown) is measured. The content of the polypeptide (B1) of the sample can be determined from the calibration curve obtained in (1) and the absorbance obtained in (2).
As the measurement sample, a sample dried in a vacuum dryer {120 ° C., 0.1 kPa or less} for 1 hour is used.

The cell culture carrier of the present invention has an amide bond not only to the polypeptide (B1) in which the water-absorbent resin particles (E) have at least one cell adhesion minimum amino acid sequence (X) in one molecule, but also to collagen (B2). It is preferable that it is.

The type of collagen (B2) is not particularly limited, and may be any of type I, type II, type III, type IV, type V, etc. Examples include those synthesized by engineering. Further, as commercially available products, Cellmartlix Type I-A (type I, manufactured by Nitta Gelatin Co., Ltd.) and collagen type III derived from bovine dermis (type III, manufactured by Nippi Co., Ltd.) are available.
As collagen (B2), type I is preferable from the viewpoint of cell proliferation.

From the viewpoint of cell proliferation, the amount of collagen (B2) bound is preferably 50 ng / g to 400 mg / g, more preferably 200 ng / g to 200 mg / g, and particularly preferably 200 ng / g to 200 mg / g, based on the dry weight of the cell culture carrier. Is 500 ng / g to 50 mg / g, most preferably 20 μg / g to 20 mg / g.
The method for measuring the collagen (B2) content per dry unit weight of the cell culture carrier is the same as that for the above-mentioned polypeptide (B1).

The total binding amount of (B1) and (B2) is preferably 100 ng / g to 50 mg / g, more preferably 200 ng / g or more, per dry weight of the cell culture carrier from the viewpoint of cell adhesion and cell proliferation. It is 40 mg / g, and particularly preferably 300 ng / g to 30 mg / g.

When the polypeptide (B1) and collagen (B2) are amide-bonded, the weight ratio [(B1) / (B2)] between (B1) and (B2) used for the amide-bonding is cell proliferation. From the viewpoint of the above, 0.1 to 0.4 is preferable.

The carrier for cell culture of the present invention contains a polypeptide denaturation inhibitor (C) for the purpose of suppressing denaturation and aggregation of polypeptides, and examples thereof include sugars and / or amino acids.

Sugars that can be used as the polypeptide denaturation inhibitor (C) include monosaccharides {triose (ketotriose, etc.), tetrose (ketotetrose, etc.), pentose (ketopentose, deoxysaccharide, etc.), hexose (glucose, fructose, mannose, allose, and galactose). Etc.), Heptose (sedhepturose, etc.), etc.}, Disaccharides (martose, lactose, sucrose, trehalose, etc.), Trisaccharides (raffinose, meregitos, maltotriose, etc.), Polysaccharides (glycogen, starch, dextran, etc.), Sugar alcohol (Glycerin, xylitol, sorbitol, etc.) and the like.
Of the sugars, disaccharides are preferable, and trehalose is more preferable, from the viewpoint of storage stability.

Examples of the amino acid that can be used as the polypeptide modification inhibitor (C) include glycine, alanine, arginine, aspartic acid, aspartic acid, phenylalanine, tryptophan, tyrosine, leucine, lysine, histidine and salts thereof.

As (C), sugar is preferable, disaccharide is more preferable, and trehalose is most preferable, from the viewpoint of storage stability.
In addition, one type of polypeptide denaturation inhibitor (C) may be used, or two or more types may be used in combination.

The content of the polypeptide denaturation inhibitor (C) is preferably 0.005 g / g to 10 g / g, more preferably 1 from the viewpoint of storage stability, based on the dry weight of the water-absorbent resin particles (A). It is 0.0 g / g to 5.7 g / g, and particularly preferably 1.5 g / g to 3.5 g / g.

In the cell culture carrier, the weight ratio ((B) / (C)) of the polypeptide (B) to the polypeptide denaturation inhibitor (C) is preferably 0.0005 to 0.05 from the viewpoint of stability. , More preferably 0.001 to 0.009, and particularly preferably 0.003 to 0.008.

As a method for containing (C) in the cell culture carrier, the cell culture carrier carrying the water-absorbent resin particles (E) or the polypeptide (B1) is freeze-dried in an aqueous solution containing (C). By doing so, it can be contained. As the freeze-drying method, a known method used in the technical field can be used.

The shape of the water-absorbent resin particles (A) is spherical or spindle-shaped from the viewpoint of cell proliferation, and is preferably spherical, that is, beads.

The volume average particle diameter of the swollen particles (GB) of the cell culture carrier of the present invention by the laser diffraction / scattering method is preferably 125 to 225 μm from the viewpoint of cell proliferation.
The swelling particles (GB) are swelling particles after mixing 100 parts by weight of 0.85 (w / v)% sodium chloride aqueous solution and 1 part by weight of a cell culture carrier until they are dispersed, and allowing them to stand at room temperature for 2 hours. It means that.

The volume average particle diameter of the swollen particles (GB) is dispersed in a 0.85% (w / v) sodium chloride aqueous solution and measured by a laser diffraction type particle size distribution measuring device [Microtrac (manufactured by Nikkiso Co., Ltd.)]. can.

In the cell culture carrier of the present invention, the zeta potential of the water-absorbent resin particles (A) is preferably +5 to +35 mV. When the zeta potential of the water-absorbent resin particles (A) is in the above range, the cell adhesion is further improved.
The zeta potential of the water-absorbent resin particles (A) is measured by the following method.

<Measuring method of zeta potential of water-absorbent resin particles (A)>
Put 500 mL of deionized water in a 500 mL beaker, add about 0.5 g of water-absorbent resin particles (A), and let stand (completely swell) for 1 hour or more. Stir well before measurement, set the beaker on the "SZN 06 Zeta Potential Meter" (manufactured by MUTEK), and start suction. At this time, in order to prevent bubbles from entering as much as possible, slowly turn the valve ON to start suction. The mesh (53 μm) is clogged with the water-absorbent resin particles (A), and suction is continued until the cell is full. When it is full, wait for about 30 seconds until the suction of the water-absorbent resin particles (A) settles down, and then start the measurement.

The zeta potential of the water-absorbent resin particles (A) is preferably +5 to +35 mV, more preferably +10 to +25 mV, and particularly preferably +12 to +20 mV from the viewpoint of cell adhesion.

The content of the primary to tertiary amino groups in the water-absorbent resin particles (A) is 0.5 to 5 mmol / g from the viewpoint of cell adhesion and cytotoxicity based on the dry weight of the cell culture carrier. It is preferable, more preferably 1 to 2 mmol / g.
The content of the primary to tertiary amino groups in the water-absorbent resin particles (A) is measured by the following method.

<Contents of primary to tertiary amino groups in the water-absorbent resin particles (A)>
Weigh 10.0 mg of the water-absorbent resin particles (A) and shake with 4 mL of 0.1 M-HCl for 3 hours. Then, centrifuge at 3,000 g for 5 minutes to remove the supernatant.
The precipitated water-absorbent resin particles (A) are shaken with 4 mL of deionized water for 15 minutes or more, and centrifuged at 3,000 g for 5 minutes to remove the supernatant. This washing operation is repeated a total of 4 times.
After the fourth centrifugation, the supernatant is removed, 4 mL of 10% (w / v) sodium sulfate is added to the precipitated water-absorbent resin particles (A), and the mixture is shaken for 3 hours or more. Then, centrifuge at 3,000 g for 5 minutes to collect the supernatant, being careful not to inhale the particles.
The supernatant is diluted with ultrapure water, and the chloride ion content is quantified by an ion chromatogram (DIONEX, column used: DIONEX IonPac AS12A 4 × 200 mm). The content of the primary to tertiary amino groups is calculated assuming that the quantified amount of chlorine ions is equal to the amount of the primary to tertiary amino groups in the sample.

The carrier for cell culture of the present invention may contain a cell growth factor in order to enhance cell proliferation.
Examples of cell growth factors include substances that promote cell growth, such as fibroblast growth factor, transforming growth factor, epithelial cell growth factor, hepatocellular growth factor, platelet-derived growth factor, insulin-like growth factor, and vascular endothelial cells. Growth Factors, Nerve Growth Factors, Stem Cell Factors, Leukemia Inhibitors, Bone Formation Factors, Heparin-Binding Epithelial Cell Growth Factors, Neuronutrient Factors, Bonded Tissue Growth Factors, Angiopoetin, Chondromodulin, Tenomodulin, Interferon, Interleukin, Tumor Necrosis Factor , Colony stimulating factors, physiologically active polypeptides such as adrenamodulin and sodium diuretic peptide, etc. (for example, described in "Ueda Minoru Tissue Engineering" (1999) published by Nagoya University Press). Among these cell growth factors, fibroblast growth factor, transforming growth factor, epithelial cell growth factor, hepatocellular growth factor, etc., from the viewpoint of wide range of applicable tissue cells and higher cell growth. , Platelet-derived growth factor, insulin-like growth factor, vascular endothelial cell growth factor, bone formation factor, interleukin and tumor necrosis factor, more preferably fibroblast growth factor, epithelial cell growth factor, insulin-like growth factor, vascular. It is an endothelial cell growth factor, interleukin and tumor necrosis factor.

The cell growth factor is preferably bound to the water-absorbent resin particles (A).

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

The cell culture carrier of the present invention may be sterilized if necessary. As a sterilization method, a sterilization method using radiation, ethylene oxide gas, plasma, γ-rays, alcohol, autoclave, dry heat or the like can be applied. These may be performed by only one method, or may be a combination of two or more.

The cells (CE) that can adhere to the cell culture carrier of the present invention are not limited as long as they are cells, but since the cell culture carrier of the present invention has high cell proliferation, useful substances such as pharmaceuticals can be produced and treated. Such as normal cells derived from mammals, established cells 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 endothelial 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 involved (osteoblasts, bone cells, osteoblasts, etc.), cells involved in cartilage (chondroblasts, chondrocytes, etc.), cells involved in teeth (root membrane cells, osteoblasts, etc.), blood involved Cells (leukocytes and erythrocytes, etc.) and stem cells {eg, bone marrow undifferentiated mesenchymal stem cells, skeletal muscle stem cells, hematopoietic stem cells, nerve stem cells, hepatic stem cells (oval cell, small hepatocyte, etc.), adipose tissue stem cells, embryonic Stem (ES) cells, epidermal stem cells, intestinal stem cells, sperm stem cells, germ reproductive stem (EG) cells, pancreatic stem cells (pancreatic duct epithelial stem cells, etc.), leukocyte stem cells, lymphoid stem cells, corneal stem cells, precursor cells (adipose precursor) Cells, vascular endothelial precursor cells, cartilage precursor cells, lymphoid precursor cells, NK precursor cells, etc.)} and the like.

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 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, honeybee cells, ginger cells, sentinel flies. Examples include cells, human stag beetle cells, swallowtail cells, stag beetle cells, Irakusakikinuwaba cells (Tn-5 cells, HIGH FIVE cells, MG1 cells, etc.) {Insect Bio Factory (edited by Shigeru Kimura, published by Industrial Research Council, 2000) Year).

Among these cells, 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, smooth muscle cells, hepatocytes, osteoblasts, epithelial cells, fibroblasts, vascular endothelial cells and stem cells, and particularly preferably kidney cells and epithelial cells. Most preferably kidney 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, most preferably CRFK cells.

The medium (ME) used in the cell culture method using the cell culture carrier of the present invention is a serum-free medium (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 media thereof, etc.); 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 a mixed medium thereof); and a mixed medium thereof.

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 adding serum, human serum, bovine serum, and horse serum are preferable. In addition, the origin of animal serum includes serum derived from adults, serum derived from offspring, serum derived from newborns, serum derived from fetus and the like. When serum is added, among these, serum derived from pups, serum derived from newborns, and serum derived from fetuses are preferable, and serums derived from newborns and serums derived from fetuses, particularly preferably serums derived from fetuses are used. be. When the serum is added, the serum may be further deactivated, an antibody 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.

If necessary, cell growth factors can be contained in the medium. By containing a cell growth factor, the cell growth rate can be increased or 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, 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 fibroblasts, 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 a cell growth factor is used, the content (% by weight) of the cell growth factor varies depending on the type of the cell growth factor, but is preferably 10-16 to 10-3, more preferably 10-, based on the weight of the medium. 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 the antibacterial agent, but is preferably 10-6 to 10, more preferably 10-5 to 1, and particularly preferably 10-5 to 1 based on the weight of the medium. It is 10-4 to 0.1.

The concentration (cells / mL) of the 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 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 carrier 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 200, per 1 L of the medium. , 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, a temperature of 30 to 40 ° C., and a condition of culturing while exchanging the medium every 1 to 3 days for 1 to 20 days.

As a method for exfoliating cells from a cell culture carrier, a known method can be used, for example, a chelating agent (EDTA etc.), a non-animal-derived proteolytic enzyme {plant-derived proteolytic enzyme (papain etc.)}, a gene. A method of exfoliating with a recombinant synthetic enzyme (trade name: TrypLE ^TM Select, manufactured by Invitrogen Co., Ltd.) and / or an animal-derived proteolytic enzyme (trypsin, collagenase, etc.) can be used.

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.
In the following, Examples 6 to 8 are Reference Examples 1 to 3, respectively.

<Manufacturing example 1>
<Preparation of water-absorbent resin (A'-1)>
A reaction vessel equipped with a stirrer, a monomer supply pipe, a nitrogen gas introduction pipe, a thermometer, and a reflux condenser was charged with 624 parts of decan and 3.1 parts of sorbitan monostearate as a polymerization dispersant, and nitrogen bubbling was performed for 30 minutes or more. , The dissolved air was expelled and the temperature was raised to 75 ° C.
173 parts of an 80% acrylic acid aqueous solution was charged in another reactor, and 207 parts of a 28% sodium hydroxide aqueous solution was added while cooling to neutralize. 4.52 parts of a crosslinkable monomer {ethylene glycol diglycidyl ether}, 0.278 parts of a polymerization initiator {potassium persulfate}, and 0.053 parts of a chain transfer agent {sodium hypophosphate} were added to this aqueous solution. After that, nitrogen bubbling was performed to expel the dissolved air to obtain an aqueous monomer solution.
The obtained monomer aqueous solution was continuously put into a decan solution in the polymerization reactor at a rate of 6.5 ml / min from the monomer supply pipe of the above polymerization reactor (stirring rate is 500 rpm) over about 1 hour. It was fed and polymerized under decan reflux.
Next, 160 parts of water was extracted by azeotropic dehydration, and then the hydrogel polymer was taken out and further dried at 120 ° C. for 2 hours to obtain a dry crosslinked sodium polyacrylate salt.
The dry crosslinked sodium polyacrylate particles are classified by a sieve having a mesh size of 63 μm and a sieve having a mesh size of 53 μm (JIS Z8801-1: 2000) to obtain particles {crosslinked sodium polyacrylate particles} having a particle diameter of 53 to 63 μm. rice field.
Next, 7.5 parts of a methanol / ion-exchanged water (volume ratio 70/30) solution containing 2% of ethylene glycol diglycidyl ether as a solution concentration was added to the obtained particles and uniformly mixed. Then, after air-drying the methanol, the mixture was placed in a closed container and held at 80 ° C. for 1 hour for surface cross-linking. Then, it was dried in a dryer at 120 ° C. for 30 minutes to obtain a water-absorbent resin (A'-1).

<Manufacturing example 2>
<Manufacturing of water-absorbent resin particles (E) into which a tertiary amino group has been introduced>
3.83 g of water-soluble carbodiimide (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Dojin Chemical Laboratory Co., Ltd.)) in 25 mL of 0.03 M phosphate buffer (pH 5.2). In addition, it was completely dissolved. Next, 3.9 g of 2-aminoethanolamine hydrochloride (Wako Junyaku) was added to this solution and completely dissolved. Next, the water-absorbent resin (A'-1) was added to this solution. ) 1 g was added, and the mixture was stirred at room temperature with a stirring blade made of polyvinyl fluoride resin at a stirring speed of 300 rpm for about 20 minutes. After complete dispersion, the temperature of the solution was adjusted to 40 ° C. and the reaction was carried out for 4 hours. After completion, the reaction solution was filtered through a tea bag (length 20 cm, width 10 cm) made of a 95 μm mesh nylon net to recover the water-absorbent resin (A'-1-1) into which a hydroxyl group was introduced.
The recovered particles were taken out, suspended in 200 mL of ion-exchanged water, and adjusted to pH 7.2 with 1N NaOH. Then, the cells were filtered again, the tea bags were closed, and the cells were washed with stirring in 500 mL of ion-exchanged water. The ion-exchanged water was exchanged every 3 hours, and this operation was performed 3 times. After the washing was completed, it was dried in a dryer at 40 ° C. for 24 hours or more.
To 10 mL of ion-exchanged water, 8.56 g of 2-chloro-N, N-diethylethylamine hydroxychloride (manufactured by Sigma-Aldrich Co., Ltd.) was added and completely dissolved. Next, 3.92 mL of 48% (w / v) NaOH was added. Stir vigorously until it becomes uniform, and when it becomes uniform, add 1.0 g of a water-absorbent resin (A'-1-1) having a hydroxyl group introduced, and add it at room temperature with a stirring blade made of polyvinyl fluoride resin at a stirring speed of 300 rpm. The mixture was stirred for about 20 minutes until dispersed. After complete dispersion, the temperature of the solution was set to 60 ° C. and the reaction was carried out for 5 hours. After completion of the reaction, the reaction solution was filtered through a tea bag (length 20 cm, width 10 cm) made of a nylon net having an opening of 95 μm, and water-absorbent resin particles (E-1) into which a tertiary amino group was introduced were recovered.
The recovered water-absorbent resin particles (E-1) were taken out, suspended in 200 mL of ion-exchanged water, and adjusted to pH 7.2 with 1N NaOH. Then, the cells were filtered again, the tea bags were closed, and the cells were washed with stirring in 500 mL of ion-exchanged water. The ion-exchanged water was exchanged every 3 hours, and this operation was performed 3 times. After washing is completed, it is dried in a dryer at 40 ° C. for 24 hours or more. After drying, the content of the tertiary amino group was measured by an ion chromatogram and found to be 2.4 mmol / g. Moreover, when the zeta potential of the water-absorbent resin particle (E-1) was measured, it was 13.9 mV.

<Measuring method of zeta potential of water-absorbent resin particles (E-1)>
500 mL of deionized water was placed in a 500 mL beaker, about 0.5 g of water-absorbent resin particles (E-1) was added, and the mixture was allowed to stand (completely swell) for 1 hour or longer. After stirring well before the measurement, the beaker was set in the "SZN 06 zeta potential meter" (manufactured by MUTEK), and suction was started. At this time, in order to prevent bubbles from entering as much as possible, the valve was slowly turned ON to start suction. The mesh (53 μm) was clogged with the water-absorbent resin particles (E-1), and suction was continued until the cell was full. After it was full, it waited for about 30 seconds until the suction of the water-absorbent resin particles (E-1) settled down, and then the measurement was started.

<Total content of tertiary amino groups in the water-absorbent resin particles (E-1)>
10.0 mg of water-absorbent resin particles (E-1) was weighed and shaken with 4 mL of 0.1 M-HCl for 3 hours. Then, the supernatant was removed by centrifugation at 3,000 g for 5 minutes. The precipitated water-absorbent resin particles (E-1) were shaken with 4 mL of deionized water for 15 minutes or more, and centrifuged at 3,000 g for 5 minutes to remove the supernatant liquid. This washing operation was repeated a total of 4 times. After the fourth centrifugation, the supernatant was removed, 4 mL of 10% (w / v) sodium sulfate was added to the precipitated water-absorbent resin particles (E-1), and the mixture was shaken for 3 hours or more. Then, the supernatant was collected by centrifuging at 3,000 g for 5 minutes, being careful not to inhale the particles. The supernatant was diluted with ultrapure water, and the chloride ion content was quantified by an ion chromatogram (DIONEX, column used: DIONEX IonPac AS12A 4 × 200 mm). Assuming that the quantified amount of chloride ion is equal to the amount of the tertiary amino group in the sample, the total content of the tertiary amino group was calculated.

<Manufacturing example 3>
<Preparation of polypeptide (B1-1)>
A structure having 13 RGD sequences and 12 (GAGAGS) ₉ sequences (21), which are chemically bonded alternately, according to the method described in Examples in JP-A No. 3-502935. "Pronectin F", which is a polypeptide (sequence (89)) having Mw of about 110,000, was produced.
After dissolving 50 mg of the polypeptide and 150 mg of N, N-dimethylaminoethyl chloride (special grade reagent) in 1.5 mL of a 4.5 M aqueous solution of lithium perchlorate at 20-40 ° C, the solution is stirred at 20-40 ° C. While doing so, 1.325 mL of a 4.5 M lithium perchlorate aqueous solution in which 100 mg of sodium hydroxide (special grade reagent) was dissolved was added dropwise at a constant rate over 45 to 50 seconds. After stirring at room temperature (25 ° C.) for 1 hour, the reaction solution was dialyzed against 10 L of deionized water for 48 hours using a dialysis membrane having a fractional molecular weight of 12,000 to 14,000. For the first 12 hours, the deionized water was replaced every 4 hours. The obtained aqueous solution was freeze-dried at −20 ° C. and 0.1 kPa or less for 24 hours to obtain a water-soluble polypeptide (B1-1). The number of N, N-dimethylaminoethyl chlorides introduced was 12 in one molecule as a result of measurement according to the method described in Examples in JP-A No. 10-500701.

<Manufacturing example 4>
<Manufacturing of water-absorbent resin particles (A-1) containing a polypeptide (B1-1)>
Water-soluble carbodiimide (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydro) in 12 mL of 0.03 M phosphate buffer (pH 5.2) (hereinafter, PBS) containing 0.85% sodium chloride. 0.21 g of chloride (manufactured by Dojin Chemical Laboratory Co., Ltd.) was added and completely dissolved. Next, 1 g of water-absorbent resin particles (E-1) was added, and the stirring speed was increased with a stirring blade made of polyvinyl fluoride resin at room temperature. The mixture was stirred for about 20 minutes until dispersed at 300 rpm. After complete dispersion, the temperature of the solution was set to 40 ° C. and the reaction was carried out for 1 hour. The water-absorbent resin particles in which a part of the carboxyl group in the water-absorbent resin particles (E-1) was modified with carbodiimide were recovered by filtering with a length of 20 cm and a width of 10 cm. 2) Suspended in 12 mL. Next, 1 mL of PBS solution (pH 7.2) containing the polypeptide (B1-1) at a concentration of 2.48 mg / mL was added, and the temperature of the solution was raised to 40 ° C. for 2 hours. After the reaction was completed, the reaction solution was filtered through a tea bag (length 20 cm, width 10 cm) made of a 95 μm nylon mesh, the water-absorbent resin particles were collected, the tea bag was closed, and 500 mL of ion exchange was performed. The mixture was washed with stirring in water. Ion-exchanged water was exchanged every 3 hours, and this operation was performed 3 times to obtain water-absorbent resin particles (A-1) containing the polypeptide (B1-1).

<Manufacturing example 5>
<Production of water-absorbent resin particles (A-2) containing polypeptide (B1-1) and collagen (B2-1)>
Water-soluble carbodiimide (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydro) in 12 mL of 0.03 M phosphate buffer (pH 5.2) (hereinafter, PBS) containing 0.85% sodium chloride. 0.21 g of chloride (manufactured by Dojin Chemical Laboratory Co., Ltd.) was added and completely dissolved. Next, 1 g of water-absorbent resin particles (E) was added, and the stirring blade was made of polyvinyl fluoride resin at a stirring speed of 300 rpm at room temperature. The mixture was stirred for about 20 minutes until it was completely dispersed. After complete dispersion, the temperature of the solution was set to 40 ° C. and the reaction was carried out for 1 hour. , 10 cm wide) to recover water-absorbent resin particles in which some of the carboxyl groups in the water-absorbent resin particles (E) were modified with carbodiimide. The recovered particles were taken out and used in 12 mL of a PBS solution (pH 7.2). Suspended.
Next, 1 mL of a PBS solution (pH 7.2) containing the polypeptide (B1-1) at a concentration of 2.48 mg / mL and a 3 mg / mL collagen (B2-1) solution (trade name: Cellmartlix Type I-A,). Nitta Gelatin Co., Ltd.) was added in an amount of 3.9 mL, and the solution was brought to a temperature of 40 ° C. and reacted for 2 hours. After completion of the reaction, the reaction solution is filtered through a tea bag (length 20 cm, width 10 cm) made of a nylon net having an opening of 95 μm, water-absorbent resin particles are collected, the tea bag is closed, and the mixture is stirred in 500 mL of ion-exchanged water. I washed it while cleaning. Ion-exchanged water was exchanged every 3 hours, and this operation was performed 3 times to obtain water-absorbent resin particles (A-2) containing the polypeptide (B1-1) and collagen (B-2).
The weight ratio [(B1-1) / (B-2)] of the polypeptide (B1-1) to the collagen (B-2) was 0.26.

<Example 1>
1 g of the water-absorbent resin particles (A-1) was suspended in 10 mL of a 0.3 M trehalose aqueous solution and freeze-dried to obtain the cell culture carrier (D-1) of the present invention. The content of trehalose in the cell culture carrier (D-1) was 1.05 g / g by the following method.

<Contents of polypeptide denaturation inhibitor (C) in cell culture carrier>
The content of the polypeptide denaturation inhibitor (C) in the cell culture carrier was calculated assuming that all of the (C) used for freeze-drying was adsorbed on the water-absorbent resin particles (A-1).

<Example 2>
In Example 1, a carrier for cell culture (D-2) was obtained in the same manner except that "suspended in 10 mL of 1 M trehalose aqueous solution" was used instead of "suspended in 10 mL of 1 M trehalose aqueous solution". ..

<Example 3>
In Example 1, a carrier for cell culture (D-3) was obtained in the same manner except that "water-absorbent resin particles (A-2)" were used instead of "water-absorbent resin particles (A-1)". ..

<Example 4>
In Example 1, the cell culture carrier (D-4) was prepared in the same manner except that "suspended in 10 mL of 0.3 M sucrose aqueous solution" was used instead of "suspended in 10 mL of 0.3 M trehalose aqueous solution". Obtained.

<Example 5>
In Example 1, a carrier for cell culture (D-5) was obtained in the same manner except that "suspended in 10 mL of 1 M sucrose aqueous solution" was used instead of "suspended in 10 mL of 0.3 M trehalose aqueous solution". ..

<Example 6>
In Example 1, the carrier for cell culture (D-6) was prepared in the same manner except that "suspended in 10 mL of 0.5 M arginine aqueous solution" was used instead of "suspended in 10 mL of 0.3 M trehalose aqueous solution". Obtained.

<Example 7>
In Example 1, the same applies except that "suspended in 10 mL of 100 mg / mL dextran (molecular weight: 15,000 to 20,000) aqueous solution" is used instead of "suspended in 10 mL of 0.3 M trehalose aqueous solution". , Cell culture carrier (D-7) was obtained.

<Example 8>
In Example 1, the same applies except that "suspended in 10 mL of 100 mg / mL dextran (molecular weight: 60,000 to 90,000) aqueous solution" is used instead of "suspended in 10 mL of 0.3 M trehalose aqueous solution". , Cell culture carrier (D-8) was obtained.

<Comparative Example 1>
In Example 1, "suspension in 10 mL of ion-exchanged water" was used instead of "suspension in 10 mL of 0.3 M trehalose aqueous solution", and the same was applied except that the polypeptide modification inhibitor (C) was not used. , Cell culture carrier (D'-1) was obtained.

<Comparative Example 2>
In Example 1, cells are similarly used except that "water-absorbent resin particles (A'-1)" having no primary to tertiary amino groups are used instead of "water-absorbent resin particles (A-1)". A culture carrier (D'-2) was obtained.

The following performance evaluation was performed.
<Evaluation of shape retention after storage at 60 ° C for 65 days>
After freeze-drying, put 0.1 g of each cell culture carrier (D-1) to (D-8) and (D'-1), (D'-2) into the centrifuge tube, and put each centrifuge tube into the centrifuge tube. Stored at 25 ° C. After 65 days, 10 mg of each cell culture carrier was weighed, placed in an Eppen tube, 1 mL of 0.85% sodium chloride aqueous solution was added, and the cells were inflated for 1 hour.
After that, photographs (magnification of 40 times) were taken of 100 arbitrarily selected carriers for cell culture with an electron microscope (manufactured by Olympus Corporation, "IX71"), and the photographed surface of each carrier had irregularities. The carrier in which the above was not confirmed was counted as a normal carrier, and the carrier in which the unevenness was confirmed was counted as a deteriorated carrier, and the shape retention rate was calculated from the following formula.
Shape retention rate (%) = (number of normal carriers) / (number of normal carriers + number of deteriorated carriers) x 100
The results are shown in Table 1.

<Cell culture evaluation>
The following evaluations were performed using the cell culture carriers (D-1) to (D-8) and (D'-1) and (D'-2) after preparation and after storage at 60 ° C. for 65 days, respectively. ..
Add 0.3 g of each cell culture carrier (D-1) to (D-8), (D'-1), and (D'-2) to each Spinner flask, and prepare from powdered DMEM (Gibco). 100 mL of DMEM solution (pH unadjusted) was added to each container and sterilized by autoclave (121 ° C., 20 minutes). After autoclave sterilization, the DMEM solution was aspirated and removed with an aspirator, and then 1 volume% fetal bovine serum (manufactured by Invitrogen Co., Ltd.) was added to MEM Dalbecco liquid medium (manufactured by Dainippon Sumitomo Pharmaceutical Co., Ltd.). 50 mL each was added and left for 1 minute. The medium was removed by suction and 90 mL each of the same serum medium was added again.
After leaving the Spinner flask in a CO ₂ incubator at 37 ° C and a carbon dioxide gas concentration of 5% by volume for 1 hour, 200,000 CRFK cells (manufactured by Dainippon Pharmaceutical Co., Ltd.) that had been pre-cultured in advance were placed. The seeds were seeded in the medium so as to be / mL.
Incubation was carried out for 7 days in a CO ₂ incubator at 37 ° C. and a carbon dioxide gas concentration of 5% by volume with stirring at 30 rpm.
Half of the medium was replaced on the 3rd, 4th, 5th and 6th days of the culture. Sampling was performed 3 hours after the start of culture, and the cell adhesion rate at the initial stage of cell culture to each cell culture carrier was measured by the following method. In addition, sampling was performed on the 7th day of culture, the number of cell nuclei per unit volume was counted by a cell nucleus counting method using crystal violet, and the cell concentration (cells / mL) in the medium was measured. The results are shown in Table 1.

<Cell adhesion rate at the initial stage of cell culture>
Photographs (magnification of 40 times) were taken of 100 arbitrarily selected carriers for cell culture with an electron microscope (manufactured by Olympus Co., Ltd., "IX71"), and 50 of the area of the photographed surface of each carrier was taken. A carrier having cells attached to% or more was counted as a carrier to which cells were attached, and a carrier having less than 50% of cells attached was counted as a carrier to which cells were not attached, and the cell adhesion rate was calculated from the following formula. The results are shown in Table 1.
Cell adhesion rate (%) = (number of adhered carriers) / (number of adhered carriers + number of non-attached carriers) × 100

<Storage stability as a carrier for cell culture>
As an evaluation of storage stability as a carrier for cell culture, cell adhesion stability and cell proliferation stability were calculated by the following formulas.
Cell adhesion stability (%) = (adhesion rate of cultured cells when using a cell culture carrier after production) / (adhesion of cultured cells when using a cell culture carrier after storage at 60 ° C for 65 days) rate)
Cell proliferation stability (%) = (cell concentration when using the cell culture carrier after production) / (cell concentration when using the cell culture carrier after storage at 60 ° C. for 65 days)
The results are shown in Table 1.

From the results in Table 1, the cell culture carriers of Examples 1 to 8 have storage stability (cell adhesion stability) as cell culture carriers as compared with Comparative Example 1 which does not contain the polypeptide denaturation inhibitor (C). It can be seen that the sex and cell proliferation stability) are high, the cells do not easily deteriorate over time, and the cells can be cultured even after long-term storage.
Further, from the results of the shape retention rate after storage at 60 ° C. for 65 days, it can be seen that the appearance of the cell culture carrier does not easily change in Examples 1 to 5 using trehalose and sucrose.

Since the carrier for cell culture of the present invention can exhibit excellent cell adhesion and cell proliferation, it is extremely useful for research and development, production of useful substances and the like related to cells.
For research and development, for culturing cells for cell function evaluation 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.

Claims (6)

A cell culture carrier containing the water-absorbent resin particles (A) and the polypeptide modification inhibitor (C), wherein the water-absorbent resin particles (A) have a carboxyl group and a primary to tertiary amino group. The water-absorbent resin particles are amide-bonded with the resin particles (E) and the polypeptide (B1) having at least one cell-adhesive minimum amino acid sequence (X) in one molecule, and are a polypeptide modification inhibitor (C). ) Is one or more sugars selected from trehalose and sucrose ,
The polypeptide (B1) has 13 RGD sequences and 12 (GAGAGS) ₉ sequences (21), and is a polypeptide having a structure in which these are chemically bonded alternately.
The cell culture carrier (D) in which the content of the polypeptide denaturation inhibitor (C) is 1.0 g / g to 3.78 g / g based on the dry weight of the water-absorbent resin particles (A ). The cell culture carrier according to claim 1 , wherein the polypeptide denaturation inhibitor (C) is trehalose. The cell culture carrier according to claim 1 or 2 , wherein the binding amount of the polypeptide (B1) is 100 ng / g to 50 mg / g based on the dry weight of the water-absorbent resin particles (A). The content of the primary to tertiary amino groups in the water-absorbent resin particles (A) is 0.5 to 5 mmol / g based on the dry weight of the water-absorbent resin particles (A), according to claims 1 to 3 . The cell culture carrier according to any one of the following items. The item according to any one of claims 1 to 4 , wherein the weight ratio ((B) / (C)) of the polypeptide (B1) to the polypeptide denaturation inhibitor (C) is 0.001 to 0.009. Carrier for cell culture. The water-absorbent resin particles (A) are water-absorbent resin particles in which the water-absorbent resin particles (E), the polypeptide (B1) and the collagen (B2) are amide-bonded, and the polypeptide (B1) and the collagen (B2) are bonded. The cell culture carrier according to any one of claims 1 to 5 , wherein the weight ratio [(B1) / (B2)] is 0.1 to 0.4.

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