JP2009072081A - Carrier for cell culture - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a carrier for cell culture which has superior cell growth properties, even in serum-free culture without the risk of contaminating infectious agents. <P>SOLUTION: The carrier for cell culture contains a water-absorbing resin (A) having a water holding capacity of 2-50 g/g, and polyvalent metal ions (B) of 0.2-10 mmol/g per 1 g of the water-absorbing resin (A) are used. Furthermore, an artificial peptide (P), having at least one cell adhesive minimum amino acid sequence (X) in one molecule, is preferably contained. The water-absorbing resin (A) is, preferably, a cross-linked poly(meth)acrylic acid. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a cell culture carrier. More specifically, the present invention relates to a cell culture carrier suitable for serum-free culture.

  As cell culture carriers, dextran beads having animal-derived collagen (Non-patent Document 1) and the like are known.

Microcarrier cell culture principles & methods (Pharmacia Biotech Co., Ltd., issued October 10, 1996) 27-31 etc.

  However, the above beads are insufficient in cell proliferation in serum-free culture. Moreover, since the above dextran beads have animal-derived components, there is a problem that there is a risk of contamination with infectious agents such as viruses. That is, an object of the present invention is to provide a cell culture carrier that has excellent cell growth properties even in serum-free culture and is free from the risk of contamination with infectious agents.

  The cell culture carrier of the present invention is characterized by containing a water-absorbing resin (A) having a water retention amount of 2 to 50 g / g and a polyvalent metal ion (B), and containing the polyvalent metal ion (B). The point is that the amount is 0.2 to 10 mmol / g per 1 g of the water-absorbent resin (A).

  The gist of the method for producing a useful substance of the present invention includes a step of culturing cells using the cell culture carrier.

  Further, the gist of the method for producing a tissue or organ of the present invention includes a step of culturing cells using the carrier for cell culture described above.

  The cell culture carrier of the present invention exhibits excellent cell growth properties. Moreover, since it does not contain animal-derived components, there is no risk of contamination with infectious agents such as viruses.

  According to the method for producing a useful substance of the present invention, a large amount of a useful substance can be obtained because it exhibits excellent cell growth properties. In addition, useful substances not contaminated with infectious agents such as viruses can be easily obtained.

  According to the method for producing a tissue or organ of the present invention, the target tissue or organ can be easily obtained because it exhibits excellent cell growth. In addition, it is possible to easily obtain tissues and organs not contaminated with infectious agents such as viruses.

  The water retention amount (g / g) of the water absorbent resin (A) is preferably from 2 to 50, more preferably from 5 to 40, particularly preferably from 10 to 30, and most preferably from 10 to 25, from the viewpoint of cell growth. It is.

The water retention amount is measured as follows. That is, the swollen resin that had been swollen in advance by adding 1.0 g of a measurement sample while stirring about 100 mL of physiological saline was transferred to a bag (20 cm long, 10 cm wide) made of a nylon net having a mesh size of 57 μm. Soak in normal saline for 60 minutes. Next, the swollen resin together with the bag is placed in a centrifugal dehydrator, and centrifugal dehydration is performed at 150 G for 90 seconds, excess water is removed, and the weight (g2) after centrifugal dehydration is measured. And In addition, about the bag and physiological saline produced with the nylon net | network, it conforms to JISK7223-1996.
However, the measurement sample used is one that has been dried for 1 hour in a vacuum dryer {120 ° C., 0.1 kPa or less}.

(Water retention amount) = (g2) −1.0

  The water-absorbing resin (A) is not particularly limited as long as it has the above-described water retention amount and can absorb water, but includes the polymers (1) to (19).

(1) A starch-acrylic acid (salt) graft-crosslinked copolymer described in JP-B-53-46199 or JP-B-53-46200.
(2) Crosslinked polyacrylic acid (salt) obtained by aqueous solution polymerization (adiabatic polymerization, thin film polymerization, spray polymerization, etc.) described in JP-A-55-133413.
(3) Cross-linked polyacrylic acid (salt) obtained by reverse phase suspension polymerization described in JP-B-54-30710, JP-A-56-26909, or JP-A-11-5808.
(4) A saponified product of a copolymer of a vinyl ester and an unsaturated carboxylic acid or a derivative thereof described in JP-A-52-14689 or JP-A-52-27455.
(5) A copolymer of acrylic acid (salt) and a sulfo (sulfonate) group-containing monomer described in JP-A-58-2312 or JP-A-61-36309.
(6) A saponified product of crosslinked isobutylene-maleic anhydride copolymer described in US Pat. No. 4,389,513.
(7) A starch-acrylonitrile copolymer hydrolyzate described in JP-A-46-43995.
(8) Crosslinked carboxymethylcellulose as described in US Pat. No. 4,650,716 and the like.
(9) A polyalkylene (ethylene, propylene, etc.) glycol cross-linked product as described in the latest trend of polymer gels (CMC Publishing Co., Ltd., published in 2004).
(10) A crosslinked polyvinyl alcohol as described in the latest trend of polymer gels (CMC Publishing, published in 2004).
(11) A starch radiation cross-linked product described in JP-A-2003-48997.
(12) A carboxyl group-containing crosslinked cellulose described in JP-A-9-85080.
(13) A polyamino acid radiation cross-linked product described in JP-A-10-251402.
(14) The crosslinked polyaspartic acid described in JP-A-2002-179770.
(15) A polyvalent metal ion cross-linked product of a polysaccharide described in JP-A No. 2001-120992.
(16) JP 2001-2935 A, JP 2003-054242 A, JP 2003-082250 A, JP 2003-165883 A, JP 2003-176421 A, JP 2003-183528 A. JP, 2003-192732, JP 2003-225565, JP 2003-238696, JP 2003-335970, JP 2004-009673, JP 2004-121400, JP 2004-123835, JP 2005-075982, JP 2005-095759, JP 2005-097569, JP 2005-186015, JP 2005-186016, JP 2005. Highness described in Japanese Patent No. 247931 The water-absorbing resin.
(17) Polyvinyl alcohol polymers described in JP-A-64-43188, JP-A-58-36630, JP-A-8-73691, JP-A-9-316271, and the like.
(18) A polyacrylamide polymer described in Japanese Patent No. 2812863.
(19) A hydrophilic urethane resin-based polymer described in JP-A-9-51794.

  Of these, from the viewpoint of adhesion of the cell to the carrier, (1), (2), (3), (12), (13), (14), (16) and (18) are preferable, More preferred are (2), (3), (16) and (18), and particularly preferred are (3) and (16). That is, crosslinked poly (meth) acrylic acid (salt) is preferred.

In addition, (meth) acrylic acid means acrylic acid and / or methacrylic acid, and an acid (salt) means an acid and / or an acid salt.
Examples of acrylates and methacrylates include alkali metal (such as lithium, potassium and sodium) and polyvalent metals (alkaline earth metals (such as magnesium and calcium)) and boron group metals (such as aluminum and gallium). And transition metals (such as titanium, vanadium, chromium, manganese, iron, nickel, cobalt, copper, zinc, zirconium, molybdenum, ruthenium, rhodium, silver, cadmium, osmium, and platinum)} 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, particularly preferably alkali metal salts and alkaline earth metals. Salts, most preferably sodium, potassium, magnesium and calcium salts.

  If the cross-linked poly (meth) acrylic acid (salt) has (meth) acrylic acid (salt) as the main constituent unit, the other vinyl monomer copolymerizable with (meth) acrylic acid (salt) also has the constituent unit. be able to. Other vinyl monomers that can be copolymerized include known copolymerizable monomers (hydrophilic vinyl monomers and hydrophobic vinyl monomers), known cross-linkable monomers, etc. {Japanese Patent Laid-Open No. 11-5808 JP-A-2001-2935, JP-A-2003-165883, JP-A-2005-247931, JP-A-2005-186015, etc.}.

  When a copolymerizable monomer is used, the content (% by weight) of the copolymerizable monomer unit is preferably 0.001 to 10, based on the weight of the (meth) acrylic acid (salt) unit, More preferably, it is 0.01-7, Most preferably, it is 0.03-5, Most preferably, it is 0.05-3. In addition, it is preferable not to contain a copolymerizable monomer unit from the viewpoint of cell proliferation.

  Among the crosslinkable monomers, from the viewpoint of cell proliferation and the like, a crosslinkable monomer having two or more ethylenically unsaturated groups and a crosslinkable monomer having two or more reactive functional groups are preferable. Preferably it is a crosslinkable monomer having two or more reactive functional groups, particularly preferably polyethyleneimine, ethylene glycol diglycidyl ether, diethylene glycol diglycidyl ether, propylene glycol diglycidyl ether, glycerin (di or tri) glycidyl ether. Most preferred is ethylene glycol diglycidyl ether.

  When the crosslinkable monomer is used, the content (% by weight) of the crosslinkable monomer unit is preferably 0.001 to 50, more preferably based on the weight of the (meth) acrylic acid (salt) unit. Is 0.005-15, particularly preferably 0.02-5, most preferably 0.1-2. Within this range, cell proliferation is further improved.

  Cross-linked poly (meth) acrylic acid is (1) (meth) acrylic acid (salt) with stirring in a hydrophobic organic solvent, and, if necessary, other vinyl monomers copolymerizable, polymerization initiator, chain transfer agent And / or a method in which a graft substrate is continuously supplied to carry out known reverse phase suspension polymerization (Japanese Patent Laid-Open Nos. 11-5808, 2001-2935, 2003-165883, and 2005-2005). (2) (meth) acrylic acid (salt), and if necessary, other vinyl monomers that can be copolymerized, a polymerization initiator, a chain transfer agent, and / or Methods for polymerizing a graft substrate in a known aqueous solution (JP 2005-075982 A, JP 2005-095759 A, JP 2005-097569 A). Patent may be produced by 2005-186015 and JP 2005-186016 JP etc.) and the like.

  The water-absorbent resin (A) can be subjected to surface cross-linking treatment with a surface cross-linking agent as necessary. When the surface vicinity of the water-absorbent resin (A) is subjected to a crosslinking treatment with a surface crosslinking agent, the cell growth property is further improved.

  Examples of the surface cross-linking agent include polyvalent glycidyl described in JP-A-59-189103 (ethylene glycol diglycidyl ether and glycerin diglycidyl ether are preferred); JP-A-58-180233 or special Polyhydric alcohols, polyhydric amines, polyhydric aziridines and polyhydric isocyanates described in Japanese Utility Model Laid-Open No. 61-16903, etc .; Silane cups described in Japanese Patent Application Laid-Open No. 61-212305 or Japanese Patent Application Laid-Open No. 61-252212 Examples of the ring agent include polyvalent metals described in JP-A No. 51-136588 or JP-A No. 61-257235. Of these surface cross-linking agents, polyvalent glycidyl, polyvalent amine and silane coupling agent are preferred, polyvalent glycidyl and silane coupling agent are more preferred, and polyvalent glycidyl is particularly preferred.

  When surface cross-linking treatment is performed, the amount (% by weight) of the surface cross-linking agent varies depending on the type of surface cross-linking agent, conditions for cross-linking, etc., but based on the weight of the water-absorbent resin (A) before surface cross-linking treatment 0.002 to 8 is preferable, 0.01 to 4 is more preferable, 0.05 to 2 is particularly preferable, and 0.05 to 1 is most preferable.

  As a method for performing the surface crosslinking treatment, conventionally known methods can be applied (for example, JP-A-11-5808, JP-A-2001-2935, JP-A-2003-165883, JP-A-2005-247931). And JP-A-2005-186015).

  The water absorbent resin (A) is subjected to drying treatment, pulverization treatment and / or classification treatment as necessary. The method of drying is not particularly limited, and is a method of drying with hot air at a temperature of 50 to 230 ° C., a thin film drying method using a drum dryer or the like heated to 50 to 230 ° C., a (heating) vacuum drying method, and freeze drying. And drying methods using infrared rays can be used. The method for pulverization is not particularly limited, and the pulverization can be performed using a normal apparatus (hammer-type pulverizer, impact-type pulverizer, roll-type pulverizer, jet airflow-type pulverizer, etc.). Further, the classification method is not particularly limited, and classification can be performed using a normal apparatus (a dry vibration sieve, a wet vibration sieve, a wind classifier, etc.).

  The outer shape of the water-absorbent resin (A) may be any of spherical, acicular, flat (elliptical) (spindle), flaky, grape tuft, irregularly crushed, and fibrous. From this viewpoint, a spherical shape and a flat (elliptical) shape (spindle shape) are preferable, and a spherical shape is more preferable.

The volume average particle diameter (μm) of the swollen particles obtained by swelling the water absorbent resin (A) with physiological saline is preferably 10 to 2000, more preferably 25 to 1000, particularly from the viewpoint of cell proliferation. Preferably it is 50-500, Most preferably, it is 100-300.
The volume average particle size of the swollen particles is obtained by immersing swollen particles prepared by immersing 1 part by weight of a measurement sample in 100 parts by weight of physiological saline (0.9 wt%) at 25 ° C. for 60 minutes. Based on JIS Z8825-1: 2001, it can be measured by a laser type particle size distribution measuring device (for example, LA-920 manufactured by Horiba, Ltd., dispersion medium: physiological saline, measurement temperature: 25 ° C.).

  The water-absorbing resin (A) can be easily obtained from the market. Aqua Pearl (registered trademark) series {Sundia Polymer Co., Ltd.}, Sun Fresh (registered trademark) series {Sanyo Chemical Industries Co., Ltd.}, Aqua Keep ( Registered trademark) series {Sumitomo Seika Co., Ltd.}, Aronzap (registered trademark) series (Toa Gosei Co., Ltd.) and Aquaric (registered trademark) series (manufactured by Nippon Shokubai Co., Ltd.). Of these, the Aqua Pearl series and Aqua Keep series are preferred, and the Aqua Pearl series is more preferred.

The polyvalent metal ion (B) means a metal ion having at least a divalent valence.
Examples of the polyvalent metal ion (B) include alkaline earth metals (magnesium, calcium, etc.), boron group metals (aluminum, gallium, indium, etc.) and transition metals (titanium, vanadium, chromium, manganese, iron, nickel, cobalt, Copper, zinc, zirconium, molybdenum, ruthenium, rhodium, silver, cadmium, osmium, platinum, etc.). Among these, from the viewpoint of cytotoxicity, magnesium, calcium, barium, manganese, iron, copper and zinc are preferable, magnesium, calcium and iron are more preferable, and magnesium and calcium are particularly preferable.
The polyvalent metal ion contained in the cell culture carrier of the present invention may be one type or two or more types.

  The content (mmol / g) of the polyvalent metal ion (B) is preferably 0.2 to 10 and more preferably 0.5 to 5 per 1 g of the water-absorbent resin (A) from the viewpoint of cell proliferation and the like. Especially preferably, it is 1-3.

The content of the polyvalent metal ion (B) was measured by placing 1 g of a measurement sample in a crucible (constant weight), then heating and ashing with an electric furnace {550 to 600 ° C., 6 hours}, and about 25 After cooling to 0 ° C., 3 ml of 0.1 mol / L hydrochloric acid aqueous solution is added and left for 60 minutes, and the supernatant is quantified by ICP emission spectroscopic analysis.
However, the measurement sample used is one that has been dried for 1 hour in a vacuum dryer {120 ° C., 0.1 kPa or less}.

The polyvalent metal ion only needs to exist in an ion state during cell culture, and may exist as a metal compound until it is used for cell culture.
The metal compound is not limited as long as it can be dissociated into metal ions when it comes into contact with water. For example, metal hydroxide, metal salt {metal halide salt, metal carbonate, hydrogen carbonate metal salt, phosphate metal salt , Metal nitrates, metal sulfates, fatty acid metal salts, metal organic sulfonates, metal organic phosphates, etc.} and simple metals.

  Therefore, the carrier for cell culture of the present invention may be obtained by mixing (1) the water absorbent resin (A) and the polyvalent metal ion (B), or (2) the water absorbent resin (A) and It may be obtained by mixing with a metal compound, or (3) when the water absorbent resin (A) is produced, it may be obtained as a metal salt of the water absorbent resin using a metal compound or the like.

(1) As a method of mixing the water absorbent resin (A) and the polyvalent metal ion (B), the water absorbent resin {or an artificial polypeptide-bonded water absorbent resin described later} is immersed in an aqueous solution containing the polyvalent metal ion. The method to do is applicable. If necessary, filtration, vacuum drying, etc. can be performed.
Examples of the aqueous solution containing polyvalent metal ions include an aqueous solution of a metal compound.
The concentration of the aqueous solution containing a polyvalent metal ion is not limited as long as the content of the polyvalent metal ion (B) is within the above range when mixed with the water absorbent resin (A). Preferably it is about 0.1-230} mmoml / L.
There is no restriction | limiting also in the temperature to immerse, It is about 5-50 {preferably 20-30} degreeC.

(2) As a method of mixing the water absorbent resin (A) and the metal compound, a known powder mixing method or the like of the water absorbent resin (A) and the metal compound can be applied.
In powder mixing, the water-absorbent resin (A) may be a water-swelling gel containing water or may be dried particles.
During powder mixing, the size of the metal compound is not limited as long as it can be dissociated into metal ions when it comes into contact with water, but it is 0.1 to 5000 {preferably 1 from the viewpoint of ease of mixing and uniformity. ˜200} μm.

(3) In the production of the water absorbent resin (A), as a method of using a metal compound or the like to form a metal salt of the water absorbent resin, a method widely applied in the field of the water absorbent resin can be applied, For example, a method of polymerizing using a metal compound as a constituent monomer or dispersant of the water absorbent resin; a method of neutralizing with a metal hydroxide after obtaining the water absorbent resin, and the like are included.

  The cell culture carrier of the present invention preferably further contains an artificial peptide (P) having at least one cell adhesion minimal amino acid sequence (X) in one molecule. When the artificial polypeptide (P) is contained, cell proliferation is further improved.

  “Cell adhesion” means a property in which a specific minimum amino acid sequence is recognized by a cell integrin receptor, and the cell easily adheres to a substrate (Osaka Prefectural Maternal and Child Medical Center, Vol. 8, No. 1, 58-66, 1992).

  Examples of the cell adhesion minimal amino acid sequence (X) include “Pathophysiology, Vol. 9, No. 7, pp. 527-535, 1990” and “Osaka Prefectural Maternal and Child Medical Center, Vol. 8, No. 1, 58-66”. Page, 1992 ", etc. are used.

  Among these minimum amino acid sequences (X), Arg Gly Asp sequence, Leu Asp Val sequence, Leu Arg Glu sequence, His Ala Val sequence, Arg Glu Asp Val sequence (1), Tyr Ile Gly Ser Arg sequence (2) Pro Asp Ser Gly Arg sequence (3), Arg Tyr Val Val Leu Pro Arg sequence (4), Leu Gly Thr Ile Pro Gly sequence (5), Arg Asn Ile Ala Glu Ile Ile Lys Asp Ile sequence (6), Ile Lys Val Ala Val sequence (7), Asp Gly Glu Ala sequence (8), Gly Val Lys Gly Asp Lys Gly Asn Pro Gly Trp Pro Gly Ala Pro sequence (9), Gly Glu Phe Tyr Phe Asp Leu Arg Leu Lys Gly Asp Lys sequence (10), Tyr Lys Leu Asn Val Asn Asp Ser sequence (11), Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys sequence (12), Asn Arg Trp His Ser Ile Tyr Ile Thr Arg Phe Gly sequence (13) , Thr Trp Tyr Lys Ile Ala Phe Gln Arg Asn Arg Lys sequence (14), Arg Lys Arg Leu Gln Val Gln Leu Ser Ile Arg Thr (15) and Pro His Ser Arg Asn (16) Species are preferable, and from the viewpoint of cell adhesion, Arg Gly Asp sequence, Tyr Ile Gly Ser Arg sequence (2), Ile Lys Val Ala Val sequence (7), Arg Lys Arg Leu Gln Val Gln Leu Ser Ile At least one selected from the group consisting of Arg Thr (15) and Pro His Ser Arg Asn (16), particularly preferably Arg Gly Asp sequence.

  At the both ends of these minimum amino acid sequences (X), other amino acids {alanine (Ala), glycine (Gly), serine (Ser), threonine (Thr), valine (Val), leucine (Leu), isoleucine (Ile) ), Cysteine (Cys), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), proline (Pro), tryptophan (Trp), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid ( Glu), arginine (Arg), lysine (Lys), histidine (His), etc.}.

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

  In addition to the minimum amino acid sequence (X), the artificial polypeptide (P) preferably has an auxiliary amino acid sequence (Y) from the viewpoint of improving the thermal stability of the artificial polypeptide (P).

  As the auxiliary amino acid sequence (Y), an amino acid sequence other than the minimum amino acid sequence (X) can be used. From the viewpoint of the thermal stability of the artificial polypeptide (P), a sequence having Gly and / or Ala is preferable.

  The auxiliary amino acid sequence (Y) includes (Gly Ala) a sequence, (Gly Ala Gly Ala Gly Ser) b sequence, (Gly Ala Gly Ala Gly Tyr) c sequence, (Gly Ala Gly Val Gly Tyr) d sequence, ( Gly Ala Gly Tyr Gly Val) e sequence, {Asp Gly Gly (Ala) f Gly Gly Ala} g sequence, (Gly Val Pro Gly Val) h sequence, (Gly) i sequence, (Ala) j sequence, (Gly Gly Ala) k sequence, (Gly Val Gly Val Pro) m sequence, (Gly Pro Pro) n sequence, (Gly Ala Gln Gly Pro Ala Gly Pro Gly) o sequence, (Gly Ala Pro Gly Ala Pro Gly Ser Gln Gly Ala Pro Gly Leu Gln) p sequence and / or (Gly Ala Pro Gly Thr Pro Gly Pro Gln Gly Leu Pro Gly Ser Pro) sequence having q sequence and the like are included. Among these, (Gly Ala) a sequence, (Gly Ala Gly Ala Gly Ser) b sequence, (Gly Ala Gly Ala Gly Tyr) c sequence, (Gly Ala Gly Val Gly Tyr) d sequence, (Gly Ala Gly Tyr Gly Val) e, {Asp Gly Gly (Ala) f Gly Gly Ala} g sequence, (Gly Val Pro Gly Val) h sequence, (Gly Val Gly Val Pro) m sequence and / or (Gly Pro Pro) n sequence Those having a (Gly Ala Gly Ala Gly Ser) b sequence, (Gly Val Pro Gly Val) h sequence, (Gly Val Gly Val Pro) m sequence and / or (Gly Pro Pro) n sequence are preferred. Particularly preferred are those having the (Gly Ala Gly Ala Gly Ser) b sequence.

  A is an integer of 5 to 100, b is an integer of 1 to 33, c, d and e are integers of 2 to 33, f is an integer of 1 to 194, and g is {1} to {200 / (6 + f). } An integer obtained by rounding down the decimal point, h is an integer of 2 to 40, i and j are integers of 10 to 200, k is an integer of 3 to 66, m is an integer of 2 to 40, and n is an integer of 3 to 66. , O is an integer from 1 to 22, and p and q are integers from 1 to 13.

  The auxiliary amino acid sequence (Y) preferably contains glycine (Gly) and / or alanine (Ala). When glycine (Gly) and alanine (Ala) are contained, the total content (%) thereof is preferably 10 to 100, more preferably 20 to 95, based on the total number of amino acids in the auxiliary amino acid sequence (Y). Especially preferably, it is 30-90, Most preferably, it is 40-85. Within this range, the thermal stability is further improved.

  When both glycine (Gly) and alanine (Ala) are included, the content ratio (Gly / Ala) is preferably 0.03 to 40, more preferably 0.08 to 13, particularly preferably 0.2. ~ 5. Within this range, the thermal stability is further improved.

  In addition to the above examples, the auxiliary amino acid sequence (Y) includes other amino acids {alanine (Ala), glycine (Gly), serine (Ser), threonine (Thr), valine (Val), leucine (Leu), Isoleucine (Ile), Cysteine (Cys), Methionine (Met), Phenylalanine (Phe), Tyrosine (Tyr), Proline (Pro), Tryptophan (Trp), Asparagine (Asn), Glutamine (Gln), Aspartic acid (Asp) , Glutamic acid (Glu), arginine (Arg), lysine (Lys), histidine (His), etc.}.

Examples of the auxiliary amino acid sequence having the (Gly Ala) a sequence include amino acid sequences represented by SEQ ID NOs: (17) to (19).
Examples of the auxiliary amino acid sequence having (Gly Ala Gly Ala Gly Ser) b sequence include amino acid sequences represented by SEQ ID NOs: (20) to (22).
Examples of the auxiliary amino acid sequence having (Gly Ala Gly Ala Gly Tyr) c sequence include amino acid sequences represented by SEQ ID NOs: (23) to (25).
Examples of the auxiliary amino acid sequence having (Gly Ala Gly Val Gly Tyr) d sequence include amino acid sequences represented by SEQ ID NOs: (26) to (28).
Examples of the auxiliary amino acid sequence having the (Gly Ala Gly Tyr Gly Val) e sequence include amino acid sequences represented by SEQ ID NOs: (29) to (31).
Examples of the auxiliary amino acid sequence having the {Asp Gly Gly (Ala) f Gly Gly Ala} g sequence include amino acid sequences represented by SEQ ID NOs: (32) to (34).
Examples of the auxiliary amino acid sequence having (Gly Val Pro Gly Val) h sequence include amino acid sequences represented by SEQ ID NOs: (35) to (38).
Examples of the auxiliary amino acid sequence having the (Gly) i sequence include amino acid sequences represented by SEQ ID NOs: (39) to (41).
Examples of the auxiliary amino acid sequence having the (Ala) j sequence include amino acid sequences represented by SEQ ID NOs: (42) to (44).
Examples of the auxiliary amino acid sequence having the (Gly Gly Ala) k sequence include amino acid sequences represented by SEQ ID NOs: (45) to (47).
Examples of the auxiliary amino acid sequence having (Gly Val Gly Val Pro) m sequence include amino acid sequences represented by SEQ ID NOs: (48) to (50).
Examples of the auxiliary amino acid sequence having (Gly Pro Pro) n sequence include amino acid sequences represented by SEQ ID NOs: (51) to (53).
Examples of the auxiliary amino acid sequence having the (Gly Ala Gln Gly Pro Ala Gly Pro Gly) o sequence include amino acid sequences represented by SEQ ID NOs: (54) to (56).
(Gly Ala Pro Gly Ala Pro Gly Ser Gln Gly Ala Pro Gly Leu Gln) Examples of the auxiliary amino acid sequence having the p sequence include amino acid sequences represented by SEQ ID NOs: (57) to (59).
(Gly Ala Pro Gly Thr Pro Gly Pro Gln Gly Leu Pro Gly Ser Pro) Examples of the auxiliary amino acid sequence having the q sequence include amino acid sequences represented by SEQ ID NOs: (60) to (62).

  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 preferred, More preferably, SEQ ID NOs: (18), (20), (21), (22), (24), (27), (30), (34), (35), (36), (37), ( 38), (40), (43), (46), (49), (52), (55), (58) or (61), particularly preferably Column number (20), the amino acid sequence represented by (21) or (38).

  When the auxiliary amino acid sequence (Y) is included, the content of (Y) is preferably 2 to 50, more preferably 3 to 30, in 1 molecule of the artificial polypeptide (P) from the viewpoint of thermal stability. Especially preferably, it is 4-20, Most preferably, it is 5-15. Further, the artificial polypeptide (P) may contain two or more auxiliary amino acid sequences (Y).

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

  The artificial polypeptide (P) preferably has a structure in which the minimum amino acid sequence (X) and the auxiliary amino acid sequence (Y) are alternately chemically bonded from the viewpoints of cell adhesion and thermal stability. In this case, the number (units) of repeating units (XY) of the minimum amino acid sequence (X) and the auxiliary amino acid sequence (Y) is preferably 1 to 50, more preferably 2 from the viewpoint of cell adhesiveness. -40, particularly preferably 3-30, most preferably 4-20.

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

  Further, other amino acids may be included in the terminal portion of the artificial polypeptide (P) (from the minimum amino acid sequence (X) or auxiliary amino acid sequence (Y) to the end of the peptide). When other amino acids are included, the number thereof is preferably 1 to 1000, more preferably 3 to 300, and particularly preferably 10 to 100 per artificial polypeptide (P).

  The weight average molecular weight (hereinafter referred to as Mw) of the artificial polypeptide (P) is preferably 1,000 to 1,000,000, more preferably 2,000 to 700,000, particularly preferably 3,000 to 400,000. Most preferably, it is 4,000 to 200,000.

  In addition, Mw of artificial polypeptide (P) is well-known, such as the method of isolate | separating a measurement sample {polypeptide etc.} by SDS-PAGE (SDS polyacrylamide gel electrophoresis), and comparing a migration distance with a reference material. It is determined by the method (hereinafter the same).

A part of preferable artificial polypeptide (P) is illustrated below.
(1) When the minimum amino acid sequence (X) is Arg Gly Asp sequence (x1)
It has 13 Arg Gly Asp sequences (x1) and 12 (Gly Ala Gly Ala Gly Ser) 9 sequences (21) (y1), and has an Mw of about 11 having a structure in which these are alternately chemically bonded. Ten thousand polypeptides {"Pronectin F", Pronectin is a registered trademark (Japan and the United States) of Sanyo Chemical Industries, Ltd. Sanyo Chemical Industries Co., Ltd. (hereinafter the same)}; (x1) and (Gly Ala Gly Ala Gly Ser) 3 sequence (20) (y2) 5 A polypeptide having a structure of about 20,000 Mw (“pronectin F2”); three of (x1) and (Gly Val Pro Gly Val) 2 Gly Gly (Gly Ala Gly Ala Gly Ser) 3 sequence (38) ( a polypeptide having a structure of about 10,000 Mw (“pronectin F3”) and the like having a structure in which three of y3) and these are alternately chemically bonded.

(2) When the minimum amino acid sequence (X) is the Ile Lys Val Ala Val sequence (x2) The Arg Gly Asp sequence (x1) of pronectin F, pronectin F2 or pronectin F3 is converted into the Ile Lys Val Ala Val sequence (7) (x2) “Pronectin L”, “pronectin L2”, “pronectin L3”, etc.

(3) When the minimum amino acid sequence (X) is the Tyr Ile Gly Ser Arg sequence (x3) The Arg Gly Asp sequence (x1) of pronectin F, pronectin F2 or pronectin F3 was changed to the Tyr Ile Gly Ser Arg sequence (x3) “Pronectin Y”, “Pronectin Y2”, “Pronectin Y3” and the like.

  In addition to the polypeptides (1) to (3), RetroNectin (recombinant human fibronectin CH-296) manufactured by Takara Shuzo Co., Ltd. {Arg Gly Asp sequence (x1) and Leu Asp Val sequence as the minimum amino acid sequence (X) Mw containing about 60,000 polypeptide}, RGDS-Protein A {Mw about 30,000 polypeptide containing Arg Gly Asp sequence (x1) as the minimum amino acid sequence (X)} can be preferably used {however, This polypeptide is derived from nature and does not contain the auxiliary amino acid sequence (Y). Therefore, thermal stability etc. are inferior to said (1)-(3). The amino acid sequences of these polypeptides are disclosed in JP-A-2-31498. }.

  Artificial polypeptide (P) is artificially synthesized, for example, organic synthesis methods (solid phase synthesis method, liquid phase synthesis method, etc.) and biochemical synthesis methods [genetically modified microorganisms (yeast, bacteria) , E. coli, etc.)] and the like. That is, the artificial polypeptide (P) does not contain cell-adhesive proteins such as animal-derived collagen and fibronectin.

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

  In the carrier for cell culture of the present invention, the water-absorbing resin (A) and the artificial polypeptide (P) are usually chemically bonded (ionic bond, hydrogen bond and / or covalent bond, etc.) and / or physical adsorption (Vandel). Bonded by adsorption by Waals force). A chemical bond is preferable and a covalent bond is more preferable in that the water absorbent resin (A) and the artificial polypeptide (P) are firmly bonded.

  The artificial polypeptide (P) includes a water absorbent resin (A) and a polyvalent metal ion (B) {metal compound and the like. } May be combined with the water absorbent resin (A), but the artificial polypeptide and the water absorbent resin (A) may be combined to obtain an artificial polypeptide-bound water absorbent resin, Polypeptide bond water-absorbing resin and polyvalent metal ion (B) {including metal compound and the like. } Is preferably mixed.

  Examples of the method for covalently bonding the artificial polypeptide (P) to the water-absorbent resin (A) include the method described in “Basics and Experiments of Peptide Synthesis, Issued by Maruzen Co., Ltd., October 5, 1997”. Specifically, it is as the following (1) to (3).

(1) When the artificial polypeptide (P) having a primary amino group or secondary amino group is reacted with the carboxyl group of the water-absorbent resin (A), the carboxyl group is reacted with a carbodiimide compound in advance. After obtaining urea {R′—N═C (OCOR) —NH—R ′ (portion where —OCOR is derived from the cell culture carrier)}, the primary amino group or secondary of the artificial polypeptide (P) By adding a compound having an amino group to the acylisourea, an amide bond can be formed between the water absorbent resin (A) and the artificial polypeptide (P).
Examples of the carbodiimide compound include N, N′-dicyclohexylcarbodiimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride.

(2) Artificial polypeptide (P) having primary amino group or secondary amino group and water-absorbent resin (A) having hydroxyl group {As copolymerization monomer, hydroxyl group-containing monomer When used, the hydroxyl group of the water absorbent resin (A) is reacted with a carbonyldiimidazole compound in advance, and the imidazole derivative {R-Im, Im is an imidazoline ring, and R is a water absorbent resin (A). After obtaining the origin}, by adding the artificial polypeptide (P) having a primary amino group or secondary amino group to the imidazole derivative, the water absorbent resin (A) and the artificial polypeptide (P) Can be N—C bonded.
Examples of the carbonyldiimidazole compound include N, N′-carbonyldiimidazole.

  (3) When the artificial polypeptide (P) having a hydroxyl group is reacted with the carboxyl group of the water absorbent resin (A), the carboxyl group of the water absorbent resin (A) is reacted with a carbodiimide compound in advance. After obtaining urea, by adding the artificial polypeptide (P) having a hydroxyl group to the acylisourea, the water-absorbing resin (A) and the polypeptide can be ester-bonded.

The artificial polypeptide (P) and the water-absorbing resin (A) are introduced into a solvent or the like as a method for physically adsorbing, ion-bonding and / or hydrogen-bonding the artificial polypeptide (P) to the water-absorbing resin (A). And a method of mixing them. Although there is no restriction | limiting in particular as a solvent, An inorganic salt, organic acid salt, an acid, and / or a base are 0.001 to 50 weight% (preferably 0.005 to 30 weight%, More preferably, 0.01 to 10 weight% ) Containing aqueous solution (described in JP-A No. 2003-189848, etc.) can be used.
Among these solvents, an aqueous solution containing an inorganic salt, an acid and / or a base, and water are preferable, and an aqueous solution containing an inorganic salt, an acid and / or a base, and ion-exchanged distilled water, particularly preferably. An aqueous solution containing an inorganic salt, acid and / or base.

  The content of the artificial polypeptide (P) is preferably 5 ng / g to 500 mg / g, more preferably 50 ng / g to 50 mg / g, in terms of cell adhesiveness, etc. Preferably 500 ng / g to 50 mg / g, then preferably 500 ng / g to 20 mg / g, particularly preferably 500 ng / g to 5 mg / g, most preferably 5 μg to 5 mg / g.

  In addition, the content of the artificial polypeptide (P) is, when the content of the artificial polypeptide (P) exceeds 500 μg / g, the Biuret method (for example, “Biochemistry Experimental Course 1, Protein Chemistry I, edited by Japanese Biochemical Society I”). Pp. 45-55 (December 11, 1979; issued by Tokyo Chemical Co., Ltd.).

  On the other hand, when the content of the artificial polypeptide (P) is 500 μg / g or less, the Kjeldahl method (for example, the Biochemical Experiment Course 1, Protein Chemistry I, pages 45-55 (December 1979) 11th; issued by Tokyo Chemical Co., Ltd.)

Moreover, it can also measure using the immunoassay (it describes in Unexamined-Japanese-Patent No. 2004-049921 etc.). Specifically, (1) Cell culture carrier with known artificial polypeptide (P) content {for cell culture with known artificial polypeptide (P) content by Biuret method, Kjeldahl method, etc. The carrier} is immersed in physiological saline, the antibody that binds to the artificial polypeptide (P) is reacted with an enzyme labeled (hereinafter referred to as an enzyme-labeled antibody), and the amount of the enzyme of the reacted enzyme-labeled antibody is measured for absorbance. A calibration curve (content of artificial polypeptide (P) and absorbance relative thereto) is prepared. (2) Similarly, the absorbance of the specimen (cell culture carrier whose content of artificial polypeptide (P) is unknown) is measured. The content of the artificial polypeptide (P) in the sample can be determined from the calibration curve obtained in (1) and the absorbance obtained in (2).
In addition, as a measurement sample, what was dried for 1 hour with the vacuum dryer {120 degreeC, 0.1 kPa or less} is used.

  The cell culture carrier of the present invention may contain a cell growth factor in order to enhance cell proliferation. Cell growth factors include substances that promote cell growth, such as fibroblast growth factor, transforming growth factor, epidermal growth factor, hepatocyte growth factor, platelet-derived growth factor, insulin-like growth factor, vascular endothelial cell Growth factor, nerve growth factor, stem cell factor, leukemia inhibitory factor, bone morphogenetic factor, heparin-binding epidermal growth factor, neurotrophic factor, connective tissue growth factor, angiopoietin, chondromodulin, tenomodulin, interferon, interleukin, tumor necrosis factor And bioactive polypeptides such as colony-stimulating factor, adrenamodulin and natriuretic peptide (for example, described in “Ueda Minoru Tissue Engineering” (1999) published by Nagoya University Press). Among these cell growth factors, the range of tissue cells that can be applied is wide, and from the viewpoint of higher cell proliferation, fibroblast growth factor, transforming growth factor, epidermal growth factor, hepatocyte growth factor Platelet-derived growth factor, insulin-like growth factor, vascular endothelial growth factor, bone morphogenetic factor, interleukin and tumor necrosis factor, more preferably fibroblast growth factor, epithelial cell growth factor, insulin-like growth factor, blood vessel Endothelial cell growth factor, interleukin and tumor necrosis factor.

  The cell growth factor is preferably bound to the water absorbent resin (A). For this bond, chemical bond and / or physical adsorption can be used as well as the bond between the water-absorbent resin (A) and the artificial polypeptide (P), and preferable chemical bond and / or physical adsorption are also the same.

  When a cell growth factor is contained, the content is preferably 10 pg / g to 1000 μg / g, more preferably 100 pg / g to 100 μg / g, per 1 g of the water-absorbent resin (A), from the viewpoint of cell growth properties and the like. Particularly preferably, it is 1000 pg / g to 10 μg / g.

The outer shape of the carrier for cell culture of the present invention matches the outer shape of the water absorbent resin (A).
The volume average particle diameter (μm) of the swollen particles obtained by swelling the cell culture carrier of the present invention with physiological saline is preferably 10 to 2000, more preferably 25 to 1000, from the viewpoint of cell proliferation. Especially preferably, it is 50-500, Most preferably, it is 100-300.

  The water retention amount (g / g) of the carrier for cell culture of the present invention is preferably 2 to 50, more preferably 5 to 40, particularly preferably 10 to 30, and most preferably 10 to 10 from the viewpoint of cell proliferation. 25.

  The cell culture carrier of the present invention may be sterilized as 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 only by one type of method, or two or more types may be combined.

  The cell (CE) that can adhere to the cell culture carrier of the present invention is not limited as long as it is a cell. However, when the cell culture carrier of the present invention is used, cell proliferation is high, so that useful substances such as pharmaceuticals can be produced and treated. For example, normal cells derived from mammals, established cell lines derived from mammals, and insect cells are suitable.

  Normal cells derived from mammals include cells involved in the skin (epithelial cells, fibroblasts, vascular endothelial cells, smooth muscle cells, etc.), cells involved in blood vessels (vascular endothelial cells, smooth muscle cells, fibroblasts, etc.) ), Cells involved in muscle (muscle cells, etc.), cells involved in fat (fat cells, etc.), cells involved in nerves (nerve cells, etc.), cells involved in liver (hepatocytes, etc.), involved in pancreas Cells (pancreatic islet cells, etc.), kidney-related cells (renal epithelial cells, proximal tubular epithelial cells, mesangial cells, etc.), lung-bronchial cells (epithelial cells, fibroblasts, vascular endothelial cells and Smooth muscle cells, etc.), cells involved in the eyes (visual cells, corneal epithelial cells and corneal endothelial cells, etc.), cells involved in prostate (epithelial cells, stromal cells, smooth muscle cells, etc.), cells involved in bone ( Osteoblasts, bone cells and osteoclasts) Cells involved in cartilage (such as chondroblasts and chondrocytes), cells involved in teeth (such as periodontal ligament cells and osteoblasts), cells involved in blood (such as leukocytes and erythrocytes), and stem cells {eg, bone marrow Differentiated mesenchymal stem cells, skeletal muscle stem cells, hematopoietic stem cells, neural stem cells, hepatic stem cells (oval cells, small hepatocytes, etc.), adipose tissue stem cells, embryonic stem (ES) cells, epidermal stem cells, intestinal stem cells, sperm stem cells, embryos Reproductive stem (EG) cells, pancreatic stem cells (pancreatic ductal epithelial stem cells, etc.), leukocyte stem cells, lymphocyte stem cells, corneal stem cells, progenitor cells (adipose precursor cells, vascular endothelial precursor cells, cartilage precursor cells, lymphoid precursor cells) , NK progenitor cells, etc.)} and the like.

  Cell lines derived from mammals include 3T3 cells, A549 cells, AH130 cells, B95-8 cells, BHK cells, BOSC23 cells, BS-C-1 cells, C3H10T1 / 2 cells, C-6 cells, CHO cells, COS cells, CV-1 cells, F9 cells, FL cells, FL5-1 cells, FM3A cells, G-361 cells, GP + E-86 cells, GP + envAm12 cells, H4-II-E cells, HEK293 cells, HeLa cells, HEp− 2 cells, HL-60 cells, HTC cells, HUVEC cells, IMR-32 cells, IMR-90 cells, K562 cells, KB cells, L cells, L5178Y cells, L-929 cells, MA104 cells, MDBK cells, MDCK cells, MIA PaCa-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 cell, PG13 cell, QGH cell, Raji cell, RPMI-1788 cell, SGE1 cell, Sp2 / O-Ag14 cell, ST2 cell, THP-1 cell, U-937 cell, V79 cell, VERO cell, WI-38 cell , Ψ2 cells, ψCRE cells, etc. {Cell culture technology (edited by the Japanese Society for Tissue Culture, published by Asakura Shoten Co., Ltd., 1999)}.

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

  Among these cells, normal cells derived from mammals and established cell lines derived from mammals are preferable from the viewpoint of production of useful substances such as pharmaceuticals and treatment. In terms of usefulness in treatment, smooth muscle cells, hepatocytes, osteoblasts, epithelial cells, fibroblasts, vascular endothelial cells and stem cells, particularly preferably epithelial cells are preferred. Moreover, 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 MDCK cells and VERO cells.

As a medium (ME) used in the cell culture method using the cell culture carrier of the present invention, a serum-free medium (Grace medium, IPL-41 medium, Schneider's medium, Opti-PRO ^™ SFM medium, Opti-MEM ^™ I Medium, VP-SFM medium, CD293 medium, 293SFMII medium, CD-CHO medium, CHO-S-SFMII medium, FreeStyle ^™ 293 medium, CD-CHO ATG ^TM medium, and mixed media thereof; General medium (RPMI medium) , MEM medium, Eagle's MEM medium, BME medium, DME medium, αMEM medium, IMEM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, ASF103 medium, ASF104 medium, ASF301 medium, TC-100 Medium, Sf-900I Medium, Ex-cell405 medium, Express-Five medium, Drosophila medium and mixed media); and include mixtures media.

Of these, a serum-free medium is preferable from the viewpoint of preventing contamination with substances that may infect humans (such as viruses derived from serum), and more preferably Opti-PRO ^™ SFM medium, Opti-MEM ^™. I medium, VP-SFM medium, CD293 medium, 293SFMII medium, CD-CHO medium, CHO-S-SFMII medium, FreeStyle ^™ 293 medium, CD-CHO ATG ^™ medium and mixed media thereof, particularly preferably Opti-PRO ^™ SFM medium, VP-SFM medium, CD293 medium, 293SFMII medium, FreeStyle ^™ 293 medium, and mixed media thereof.

  In addition, serum can be added to these media, but it is preferable not to add serum from the viewpoint of preventing contamination with substances (such as viruses derived from serum) that may infect humans. .

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

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

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

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

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

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

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

A known method can be used as a method for counting the number of cells, for example, it can be measured by a cell nucleus counting method using crystal violet {Cell culture technology (edited by the Japanese Society for Tissue Culture, published by Asakura Shoten, 1999) )}.
Further, the dry weight (g) of the cell culture carrier to be introduced into the medium can be appropriately determined depending on the type of cells to be cultured, etc., but is preferably 0.005 to 800, more preferably 0.02 to 200 per liter of the medium. Especially preferably, it is 0.1-40.

  The culture conditions are not particularly limited, and conditions such as carbon dioxide (CO2) concentration of 1 to 20% by volume, 5 to 45 ° C. for 1 hour to 100 days, and culture if the medium is changed every 1 to 10 days as necessary. Is applicable. Preferable conditions are conditions in which the culture is performed while changing the medium every 1 to 3 days, at a CO2 concentration of 3 to 10% by volume, 30 to 40 ° C., 1 to 20 days.

Known methods can be used to detach cells from the cell culture carrier, such as chelating agents (EDTA, etc.), non-animal derived proteolytic enzymes {plant-derived proteolytic enzymes (papain, etc.)}, genes A method of peeling with a recombinant synthase (trade name: TrypLE ^™ Select, manufactured by Invitrogen Co., Ltd.) and / or an animal-derived proteolytic enzyme (trypsin, collagenase, etc.) can be used.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples. Unless otherwise specified, “part” means “part by weight” and “%” means “% by weight”.
<Production Example 1>
<Preparation of water absorbent resin (A-1)>
A polymerization reaction vessel equipped with a stirrer, monomer supply pipe, nitrogen gas introduction pipe, thermometer, and reflux condenser was charged with 624 parts of cyclohexane and 3.1 parts of sorbitan monostearate as a polymerization dispersant, and nitrogen bubbling was performed for 30 minutes or more. Then, the dissolved air was driven out and the temperature was raised to 75 ° C.

  A separate reactor was charged with 173 parts of an 80% aqueous acrylic acid solution, and neutralized by adding 207 parts of a 28% aqueous sodium hydroxide solution while cooling. To this aqueous solution was added 5.72 parts of a crosslinkable monomer {ethylene glycol diglycidyl ether}, 0.278 parts of a polymerization initiator {potassium persulfate}, and 0.053 parts of a chain transfer agent {sodium hypophosphite}. Thereafter, nitrogen bubbling was performed to drive out dissolved air to obtain an aqueous monomer solution.

While supplying the monomer aqueous solution from the monomer supply pipe of the above polymerization reactor to the cyclohexane liquid continuously stirred at a rate of 6.5 ml / min (stirring speed is 500 rpm) in about 1 hour. Polymerization was carried out under reflux of cyclohexane.
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 dried crosslinked polyacrylic acid sodium salt.
The dried crosslinked polyacrylic acid sodium salt was classified with a sieve having an aperture of 63 μm and a sieve having a size of 53 μm (JIS Z8801-1: 2000) to obtain particles having a particle size of 53 to 63 μm.

  Next, the particles and 7.5 parts of a methanol / ion-exchanged water (volume ratio 70/30) solution containing 2% ethylene glycol diglycidyl ether as a solution concentration are uniformly mixed, and the methanol is air-dried. The mixture was kept at 80 ° C. for 1 hour for surface crosslinking. Then, it was made to dry at 120 degreeC for 30 minute (s) in a normal air dryer, and water absorbing resin (A-1) was obtained. The water retention amount of the water absorbent resin (A-1) was 10 g / g.

<Production Example 2>
<Preparation of water absorbent resin (A-2)>
A water-absorbent resin (A-2) was produced in the same manner as in Production Example 1 except that the amount of the crosslinkable monomer {ethylene glycol diglycidyl ether} was changed from “5.72 parts” to “0.014 parts”. ) The water retention amount of the water absorbent resin (A-2) was 30 g / g.

<Production Example 3>
<Preparation of water absorbent resin (A-3)>
A water-absorbent resin (A-3) was prepared in the same manner as in Production Example 1 except that the amount of the crosslinkable monomer {ethylene glycol diglycidyl ether} was changed from “5.72 parts” to “0.9 parts”. ) The water retention amount of the water absorbent resin (A-3) was 21 g / g.

<Production Example 4>
<Preparation of water absorbent resin (A-4)>
The amount of the crosslinkable monomer {ethylene glycol diglycidyl ether} used was changed from “5.72 parts” to “0.9 parts” and “a sieve having an opening of 63 μm and a 53 μm sieve” A water absorbent resin (A-4) was obtained in the same manner as in Production Example 1 except that the opening was changed to a 32 μm sieve and a 45 μm sieve ”. The water retention amount of the water absorbent resin (A-4) was 21 g / g.

<Production Example 5>
<Preparation of water absorbent resin (A-5)>
The amount of the crosslinkable monomer {ethylene glycol diglycidyl ether} used was changed from “5.72 parts” to “0.9 parts” and “a sieve having an opening of 63 μm and a 53 μm sieve” A water-absorbent resin (A-5) was obtained in the same manner as in Production Example 1 except that the opening was changed to a 90 μm sieve and a 125 μm sieve. The water retention amount of the water absorbent resin (A-5) was 21 g / g.

<Production Example 6>
<Preparation of water absorbent resin (A-6)>
A water-absorbent resin (A-6) was produced in the same manner as in Production Example 1 except that the amount of the crosslinkable monomer {ethylene glycol diglycidyl ether} was changed from “5.72 parts” to “0.0111 parts”. ) The water retention amount of the water absorbent resin (A-6) was 33 g / g.

<Production Example 7>
<Preparation of water absorbent resin (A-7)>
A water-absorbing resin (A-7) was prepared in the same manner as in Production Example 1 except that the amount of the crosslinkable monomer {ethylene glycol diglycidyl ether} was changed from “5.72 parts” to “4.52 parts”. ) The water retention amount of the water absorbent resin (A-7) was 13 g / g.

<Example 1>
<Preparation of artificial polypeptide (P1)>
According to the method described in the examples in JP-T-3-502935, it has 13 Arg Gly Asp sequences and 12 (Gly Ala Gly Ala Gly Ser) 9 sequences (21), which are alternately A peptide “pronectin F” having a Mw of about 110,000 having a structure formed by chemically bonding to was produced as an artificial polypeptide (P1).

<Preparation of water-absorbent resin to which artificial polypeptide (P1) is bound>
To 1 g of the water-absorbent resin (A-1), 50 mL of 0.02 M, pH 7.2 phosphate buffer (hereinafter referred to as PBS) containing 0.85% sodium chloride was added, and left for 30 minutes. After swelling the water-absorbent resin (A-1) and removing the excess PBS by suction with an aspirator, water-soluble carbodiimide (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, Dojin Chemical Research Co., Ltd.) 40 mL of a PBS solution containing 60 mM in a concentration was added, stirred with a stirring blade made of polyfluorinated ethylene resin, and reacted for 2 hours to obtain a modified water absorbent resin. After the reaction for 2 hours, the reaction solution was removed by suction, and 40 mL of PBS was added, followed by a washing operation of removing by suction five times.

  Next, 40 mL of a PBS solution containing the artificial polypeptide (P1) at a concentration of 600 μg / mL is added to the modified water-absorbent resin, stirred with a stirring blade made of polyfluorinated ethylene resin, and reacted for 2 hours. The solution is removed by suction, and 40 ml of PBS is added and removed by suction 5 times. Further, 40 mL of ion-exchanged water is added, and the washing operation is removed by suction 2 times. Subsequently, a vacuum dryer {120 ° C., 0.1 kPa or less} was dried under reduced pressure for 4 hours to obtain an artificial polypeptide-bound water-absorbing resin (D-1).

  1 g of the artificial polypeptide-bound water-absorbing resin (D-1) is immersed in 7 mL of an aqueous solution containing calcium chloride at a concentration of 2.5 g / L and allowed to stand at 25 ° C. for 1 hour. It was filtered and vacuum dried at 40 ° C. and 0.1 kPa or less to obtain a cell culture carrier (E-1) of the present invention. This carrier (E-1) contained 2 mmol / g calcium ions and 2 mg / g artificial polypeptide, and the water retention amount was 10 g / g. Further, swollen particles obtained by swelling the carrier (E-1) with physiological saline (hereinafter abbreviated as swollen particles). } Had a volume average particle diameter of 150 μm.

<Example 2>
An artificial polypeptide-bound water-absorbing resin (D-2) was prepared in the same manner as in Example 1 except that “water-absorbing resin (A-1)” was changed to “water-absorbing resin (A-2)”. The cell culture carrier (E-2) of the present invention was obtained. This carrier (E-2) contained 2 mmol / g calcium ion and 2 mg / g artificial polypeptide, and the water retention amount was 30 g / g. The volume average particle diameter of the swollen particles was 200 μm.

<Example 3>
“Water absorbent resin (A-1)” was changed to “Water absorbent resin (A-3)” and “Aqueous solution containing calcium chloride at a concentration of 2.5 g / L” was changed to “Magnesium sulfate 5 g / L”. An artificial polypeptide-bound water-absorbing resin (D-3) was prepared in the same manner as in Example 1 except that the aqueous solution was changed to “Aqueous solution containing at a concentration of” to obtain the cell culture carrier (E-3) of the present invention. It was. This carrier (E-3) contained 2 mmol / g magnesium ions and 2 mg / g artificial polypeptide, and the water retention amount was 20 g / g. The volume average particle diameter of the swollen particles was 180 μm.

<Example 4>
“Water absorbent resin (A-1)” was changed to “Water absorbent resin (A-3)” and “Water solution containing calcium chloride at a concentration of 2.5 g / L” was changed to “2.5 g of calcium chloride”. / L, an artificial polypeptide-bonded water-absorbing resin (D-3) was prepared in the same manner as in Example 1 except that it was changed to “an aqueous solution containing 0.8 g / L of magnesium sulfate”. A cell culture carrier (E-4) was obtained. This carrier (E-4) contained 2 mmol / g calcium ion, 1 mmol / g magnesium ion and 2 mg / g artificial polypeptide, and the water retention amount was 19 g / g. The volume average particle diameter of the swollen particles was 180 μm.

<Example 5>
“Water absorbent resin (A-1)” was changed to “Water absorbent resin (A-3)” and “Aqueous solution containing calcium chloride at a concentration of 2.5 g / L” was changed to “150 mg / L of calcium chloride”. An artificial polypeptide-bound water-absorbing resin (D-3) was prepared in the same manner as in Example 1 except that the aqueous solution was changed to “Aqueous solution containing at a concentration of” to obtain the cell culture carrier (E-5) of the present invention. It was. This carrier (E-5) contained 0.2 mmol / g calcium ion and 2 mmol / g artificial polypeptide, and the water retention amount was 21 g / g. Further, the volume average particle diameter of the swollen particles was 185 μm.

<Example 6>
“Water absorbent resin (A-1)” was changed to “Water absorbent resin (A-3)”, and “Aqueous solution containing calcium chloride at a concentration of 2.5 g / L” was changed to “400 mg / L calcium chloride”. An artificial polypeptide-bound water-absorbing resin (D-3) was prepared in the same manner as in Example 1 except that the aqueous solution was changed to “Aqueous solution containing at a concentration of” to obtain the cell culture carrier (E-6) of the present invention. It was. This carrier (E-6) contained 0.5 mmol / g calcium ions and 2 mg / g artificial polypeptide, and the water retention amount was 21 g / g. Further, the volume average particle diameter of the swollen particles was 185 μm.

<Example 7>
“Water absorbent resin (A-1)” was changed to “Water absorbent resin (A-3)” and “Aqueous solution containing calcium chloride at a concentration of 2.5 g / L” was changed to “10 g / L calcium chloride”. An artificial polypeptide-bound water-absorbing resin (D-3) was prepared in the same manner as in Example 1 except that the aqueous solution was changed to “Aqueous solution containing at a concentration of” to obtain the cell culture carrier (E-7) of the present invention. It was. This carrier (E-7) contained 5 mmol / g calcium ion and 2 mg / g artificial polypeptide, and the water retention amount was 19 g / g. The volume average particle diameter of the swollen particles was 180 μm.

<Example 8>
“Water absorbent resin (A-1)” was changed to “Water absorbent resin (A-3)” and “Aqueous solution containing calcium chloride at a concentration of 2.5 g / L” was changed to “25 g / L calcium chloride”. An artificial polypeptide-bound water-absorbing resin (D-3) was prepared in the same manner as in Example 1 except that the aqueous solution was changed to “Aqueous solution containing at a concentration of” to obtain the cell culture carrier (E-8) of the present invention. It was. This carrier (E-8) contained 10 mmol / g calcium ions and 2 mg / g artificial polypeptide, and the water retention amount was 15 g / g. The volume average particle diameter of the swollen particles was 165 μm.

<Example 9>
“Water absorbent resin (A-1)” was changed to “Water absorbent resin (A-4)” and “Aqueous solution containing calcium chloride at a concentration of 2.5 g / L” was changed to “2.5 g of calcium chloride”. / L, an artificial polypeptide-bound water-absorbing resin (D-4) was prepared in the same manner as in Example 1 except that the aqueous solution was changed to an aqueous solution containing magnesium sulfate at a concentration of 0.8 g / L. A cell culture carrier (E-9) was obtained. This carrier (E-9) contained 2 mmol / g calcium ion, 1 mmol / g magnesium ion and 2 mg / g artificial polypeptide, and the water retention amount was 19 g / g. The volume average particle diameter of the swollen particles was 100 μm.

<Example 10>
“Water absorbent resin (A-1)” was changed to “Water absorbent resin (A-5)” and “Water solution containing calcium chloride at a concentration of 2.5 g / L” was changed to “2.5 g of calcium chloride”. / L, an artificial polypeptide-bound water-absorbing resin (D-5) was prepared in the same manner as in Example 1 except that the solution was changed to “an aqueous solution containing magnesium sulfate at a concentration of 0.8 g / L”. A cell culture carrier (E-10) was obtained. This carrier (E-10) contained 2 mmol / g calcium ion, 1 mmol / g magnesium ion and 2 mg / g artificial polypeptide, and the water retention amount was 19 g / g. The volume average particle diameter of the swollen particles was 300 μm.

<Example 11>
“Water absorbent resin (A-1)” was changed to “Water absorbent resin (A-3)”, “PBS solution containing water-soluble carbodiimide at a concentration of 60 mM” “Water-soluble carbodiimide at a concentration of 2 mM” Changed to “PBS solution”, “PBS solution containing artificial polypeptide (P1) at a concentration of 600 μg / mL” to “PBS solution containing artificial polypeptide (P1) at a concentration of 20 μg / mL”, Example 1 except that “an aqueous solution containing calcium chloride at a concentration of 2.5 g / L” was changed to “an aqueous solution containing calcium chloride at a concentration of 2.5 g / L and magnesium sulfate at a concentration of 0.8 g / L”. In the same manner as described above, an artificial polypeptide-bound water-absorbing resin (D-6) was prepared to obtain the cell culture carrier (E-11) of the present invention. This carrier (E-11) contained 2 mmol / g calcium ion, 1 mmol / g magnesium ion, and 500 ng / g artificial polypeptide, and the water retention amount was 19 g / g. The volume average particle diameter of the swollen particles was 180 μm.

<Example 12>
“Water absorbent resin (A-1)” was changed to “Water absorbent resin (A-3)”, “PBS solution containing water-soluble carbodiimide at a concentration of 60 mM” and “Water-soluble carbodiimide at a concentration of 150 mM” Changing to “PBS solution”, changing “PBS solution containing artificial polypeptide (P1) at a concentration of 600 μg / mL” to “PBS solution containing artificial polypeptide (P1) at a concentration of 2000 μg / mL”, Example 1 except that “an aqueous solution containing calcium chloride at a concentration of 2.5 g / L” was changed to “an aqueous solution containing calcium chloride at a concentration of 2.5 g / L and magnesium sulfate at a concentration of 0.8 g / L”. In the same manner as described above, an artificial polypeptide-bonded water-absorbing resin (D-7) was prepared to obtain the cell culture carrier (E-12) of the present invention. This carrier (E-12) contained 2 mmol / g calcium ion, 1 mmol / g magnesium ion and 5 mg / g artificial polypeptide, and the water retention amount was 19 g / g. The volume average particle diameter of the swollen particles was 180 μm.

<Example 13>
“207 parts of 28% aqueous sodium hydroxide solution” was changed to “152 parts of 48% aqueous sodium hydroxide solution and 55 parts of 38% calcium hydroxide” and the amount of crosslinkable monomer {ethylene glycol diglycidyl ether} used Was changed from “6.32 parts” to “0.9 parts” in the same manner as in Production Example 1 to obtain a water absorbent resin (A-8).

  Artificial polypeptide-bound water-absorbing resin (D-8) {this invention], except that “water-absorbing resin (A-1)” is changed to “water-absorbing resin (A-8)”. A cell culture carrier (E-13)} was obtained. This carrier (E-13) contained 1 mmol / g calcium ion and 2 mg / g artificial polypeptide, and the water retention amount was 20 g / g. The volume average particle diameter of the swollen particles was 180 μm.

<Example 14>
“Water absorbent resin (A-1)” was changed to “Water absorbent resin (A-2)” and “Aqueous solution containing calcium chloride at a concentration of 2.5 g / L” was changed to “150 mg / L of calcium chloride”. An artificial polypeptide-bound water-absorbing resin (D-2) was prepared in the same manner as in Example 1 except that the aqueous solution was changed to “Aqueous solution containing at a concentration of” to obtain the cell culture carrier (E-14) of the present invention. It was. This carrier (E-14) contained 0.2 mmol / g calcium ion and 2 mmol / g artificial polypeptide, and the water retention amount was 30 g / g. The volume average particle diameter of the swollen particles was 200 μm.

<Example 15>
Artificial polypeptide-bound water-absorbent resin in the same manner as in Example 1 except that “aqueous solution containing calcium chloride at a concentration of 2.5 g / L” was changed to “aqueous solution containing calcium chloride at a concentration of 400 mg / L”. (D-1) was prepared to obtain the cell culture carrier (E-15) of the present invention. This carrier (E-15) contained 0.5 mmol / g calcium ion and 2 mg / g artificial polypeptide, and the water retention amount was 10 g / g. The volume average particle diameter of the swollen particles was 150 μm.

<Example 16>
“Water absorbent resin (A-1)” was changed to “Water absorbent resin (A-6)” and “Aqueous solution containing calcium chloride at a concentration of 15 mg / L” was changed to “Concentration of calcium chloride at 10 g / L”. An artificial polypeptide-bound water-absorbing resin (D-9) was prepared in the same manner as in Example 1 except that the aqueous solution contained in ”was changed to“ Aqueous solution contained in ”to obtain the cell culture carrier (E-16) of the present invention. This carrier (E-16) contained 5 mmol / g calcium ion and 2 mg / g artificial polypeptide, and the water retention amount was 30 g / g. The volume average particle diameter of the swollen particles was 200 μm.

<Example 17>
“Water absorbent resin (A-1)” was changed to “Water absorbent resin (A-7)” and “Aqueous solution containing calcium chloride at a concentration of 2.5 g / L” was changed to “25 g / L calcium chloride”. An artificial polypeptide-bound water-absorbing resin (D-10) was prepared in the same manner as in Example 1 except that the aqueous solution was changed to “Aqueous solution containing at a concentration of” to obtain the cell culture carrier (E-17) of the present invention. It was. This carrier (E-17) contained 10 mmol / g calcium ions and 2 mg / g artificial polypeptide, and the water retention amount was 10 g / g. The volume average particle diameter of the swollen particles was 150 μm.

<Comparative Example 1>
The artificial polypeptide-bonded water absorbent resin (D-1) prepared in Example 1 was used as a carrier for cell culture (HE-1) for comparison. This carrier (HE-1) contained 2 mg / g of artificial polypeptide, and the water retention amount was 10 g / g. The volume average particle diameter of the swollen particles was 150 μm.

<Comparative example 2>
The artificial polypeptide-bonded water-absorbing resin (D-2) prepared in Example 2 was used as a carrier for cell culture (HE-2) for comparison. This carrier (HE-2) contained 2 mg / g of artificial polypeptide, and the water retention amount was 30 g / g. The volume average particle diameter of the swollen particles was 200 μm.

<Comparative Example 3>
The artificial polypeptide-bound water-absorbing resin (D-4) prepared in Example 9 was used as a carrier for cell culture (HE-3) for comparison. This carrier (HE-3) contained 2 mg / g of artificial polypeptide, and the water retention amount was 20 g / g. The volume average particle diameter of the swollen particles was 100 μm.

<Comparative example 4>
The artificial polypeptide-bonded water-absorbing resin (D-5) prepared in Example 10 was used as a carrier for cell culture (HE-4) for comparison. This carrier (HE-4) contained 2 mg / g of artificial polypeptide, and the water retention amount was 20 g / g. The volume average particle diameter of the swollen particles was 300 μm.

<Comparative Example 5>
“Dextran beads having animal-derived collagen” described in Non-Patent Document 1 (trade name Cytodex-3, manufactured by Amersham Biosciences) was used as a carrier for cell culture (HE-5) for comparison. The water retention amount of this carrier (HE-5) was 15 g / g. The volume average particle diameter of the swollen particles was 175 μm.

<Evaluation of cell proliferation>
The evaluation sample {carrier for cell culture} was swollen with physiological saline, and then centrifuged at 150 G for 90 seconds to remove excess water to prepare a swollen carrier.
4 g of swelling carrier is put into a spinner flask, physiological saline is added at 100 mL / flask, and autoclaved (121 ° C., 20 minutes). -A medium containing 0.2% of an antibacterial agent (trade name: PSA, manufactured by Cascade Co., Ltd.) in SFM (manufactured by Invitrogen Corporation) was added at 50 mL / flask and left for 1 hour. Remove the medium from the spinner flask by suction, add the same medium again at 100 mL / flask, leave the spinner flask in a CO ₂ incubator with a carbon dioxide gas concentration of 5% by volume at 37 ° C. for 2 hours, and pre-culture in advance. VERO cells (manufactured by Dainippon Sumitomo Pharma Co., Ltd.) were seeded in the medium so that the cell concentration was 200,000 cells / mL. Subsequently, the cells were cultured for 7 days in a CO ₂ incubator with a carbon dioxide gas concentration of 5% by volume at 37 ° C. while stirring at 30 rpm. On the 4th, 5th and 6th days of culture, half of the medium was replaced with a new medium. Sampling was performed on the 7th day of culture, the number of cell nuclei per unit volume was counted by the cell nuclei counting method using crystal violet, the cell concentration (10,000 cells / mL) in the medium was measured, and the results are shown in Table 1.

  From the above results, the cell culture carriers (Examples 1 to 17) of the present invention have much higher proliferation ability of VERO cells than the cell culture carriers (Comparative Examples 1 to 5) not containing polyvalent metal ions. Turned out to be high.

The cell culture carrier of the present invention can exhibit excellent cell growth in serum-free culture and has no risk of contamination with infectious agents such as viruses. It is extremely useful for production and treatment.
For research and development, cell culture for cell function evaluation such as differentiation function, culture of substitute cells for animal experiments (toxicity test, stimulation test, metabolic function test, etc.), cell culture for gene and protein introduction, etc. Available.
For the production of useful substances, it can be used for culturing cells for production of cytokines, thrombolytic agents, blood coagulation factor preparations, vaccines, hormones, antibiotics, antibodies, growth factors and the like. Among these, it is suitable for culturing cells for vaccine production.
For treatment, skin, skull, muscle, skin tissue, bone, cartilage, blood vessel, nerve, tendon, ligament, follicular tissue, mucosal tissue, periodontal tissue, dentin, bone marrow, retina, serosa, gastrointestinal tract and fat It can be used for cell culture of tissues such as lung, liver, pancreas and kidney.

Claims (5)

A water-absorbing resin (A) having a water retention amount of 2 to 50 g / g and a polyvalent metal ion (B) are contained, and the content of the polyvalent metal ion (B) is 0 per 1 g of the water-absorbing resin (A). A carrier for cell culture, which is 2 to 10 mmol / g. The carrier for cell culture according to claim 1, further comprising an artificial peptide (P) having at least one cell adhesion minimal amino acid sequence (X) in one molecule. The cell culture carrier according to claim 1 or 2, wherein the water absorbent resin (A) is a crosslinked poly (meth) acrylic acid. A method for producing a useful substance, comprising a step of culturing cells using the cell culture carrier according to claim 1. A method for producing a tissue or organ, comprising a step of culturing cells using the cell culture carrier according to any one of claims 1 to 3.