JP2008263955A - Serum-free medium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a serum-free medium providing improved cell-proliferative properties in a serum-free medium, and hardly providing risk of mixing of an infectious factor. <P>SOLUTION: The serum-free medium contains an artificial polypeptide (P) having at least one minimum amino acid sequence (X) with cellular adhesiveness in one molecule. Preferably, (X) is Arg Gly Asp sequence. Preferably, (P) has at least one assistant amino acid sequence (Y) in one molecule of the (P). Preferably, (P) has a structure obtained by alternately chemically bonding an amino acid sequence containing an interstitial amino acid sequences (Z) and (Y) at both the terminals of (X). <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a serum-free medium. More particularly, the present invention relates to a serum-free medium that is particularly effective for cell growth in serum-free culture.

  As a serum-free medium, a medium containing animal-derived fibronectin (Patent Document 1) is known.

JP 2005-515777 A

  However, the above medium has insufficient cell growth in serum-free culture. In addition, since the above-mentioned medium has 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 culture medium that improves cell growth in serum-free culture and does not have a risk of contamination with infectious agents.

  A feature of the serum-free medium of the present invention is that it contains an artificial polypeptide (P) having at least one cell adhesion minimal amino acid sequence (X) in one molecule.

  The serum-free medium 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.

“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) and the like} are preferably linked to the intervening amino acid sequence (Z).

  From the viewpoint of cell adhesion, the N-terminal of the minimum amino acid sequence (X) is Gly Ala Ala Val Thr Gly sequence (65), Gly Leu Pro Gly Pro Lys Gly Asp sequence (66), Gly Pro Ala Val Thr Gly sequence. (67), Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Pro Ala Val Thr Gly sequence (68), Gly Ala Ala Val Cys Glu Pro Gly sequence (69), Gly Ala Ala Leu Cys Val Ser Glu Pro Gly sequence ( 70), Ser Pro Ala Ser Ala Ala Leu Cys Val Ser Glu Pro Gly sequence (71), Ser Pro Ala Ser Ala Ala Val Cys Glu Pro Gly sequence (72), Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Pro Ala Val Cys Glu Pro Gly Sequence (73), Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Pro Ala Leu Cys Val Ser Glu Pro Gly Sequence (74), Gly Pro Ala Val Cys Glu Pro Gly Sequence (75), Gly Pro At least one intervening amino acid sequence (Z) selected from the group consisting of Ala Leu Cys Val Ser Glu Pro Gly sequence (76) and Gly Ala Ala Pro Gly Ala Ser sequence (77) Gly Ala Ala Val Thr Gly sequence (65), Gly Leu Pro Gly Pro Lys Gly Asp sequence (66), Gly Pro Ala Val Thr Gly sequence (67) and Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Pro Ala Val Thr Gly sequence (68) is more preferred, and Gly Ala Ala Val Thr Gly sequence (65) is particularly preferred.

  From the viewpoint of cell adhesiveness, Ser Pro Ala Ser Ala Ala Gly Tyr sequence (78), Ser Pro Ala Ser Ala Ala Leu Cys Val Ser sequence (79), Ser Pro Ala Ser Ala Ala Val Cys sequence (80), Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Pro sequence (81), Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Pro Ala Val Cys sequence (82), Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Pro Ala Leu Cys Val Ser sequence (83), Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro sequence (84), Ser Pro Ala Ser Ala Ala Gly Pro Val Gly Ser Pro sequence (85) Cys Asp Ala Gly Tyr sequence (86), Cys Asp Ala Gly Pro Val Gly Ser Pro sequence (87) and Ser Ala Gly Pro Ser Ala Gly Tyr sequence (88) (Z) is preferably bound, Ser Pro Ala Ser Ala Ala Gly Tyr sequence (78), Ser Pro Ala Ser Ala Ala Leu Cys Val Ser sequence ( 9), Ser Pro Ala Ser Ala Ala Val Cys sequence (80), Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Pro sequence (81), Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Pro Ala Val Cys sequence ( 82), Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Pro Ala Leu Cys Val Ser sequence (83), Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro sequence (84) and Ser Pro Ala Ser Ala Ala Gly Pro Val Gly At least one selected from the group consisting of Ser Pro sequence (85) is more preferred, and Ser Pro Ala Ser Ala Ala Gly Tyr sequence (78) is particularly preferred.

  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 those having 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, from the viewpoint of thermal stability and the like, 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) The amino acid sequence represented is preferred, more preferably SEQ ID NO: (18), (20), (21), (22), (24), (27), (30), (34), (35), ( 36), (37), (38), (40), (43), (46), (49), (52), (55), (58) or amino represented by (61) Sequences, particularly preferably SEQ ID NO: 20 is 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) has a minimum amino acid sequence (X) and an auxiliary amino acid sequence (Y), if necessary, between (X) and (Y), from the viewpoint of cell adhesion and thermal stability. A structure formed by alternately chemically bonding via an amino acid sequence is preferable, and a structure formed by alternately chemically bonding (Y) an amino acid sequence containing an intervening amino acid sequence (Z) at both ends of (X) More preferably. 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 Gly Ala Ala Val Thr Gly sequence (N1 terminus of (x1) ( 65), a polypeptide having Ser W Ala Ser Ala Ala Gly Tyr sequence (78) at the C-terminus of (x1) (78) and having a structure in which these are alternately chemically bonded to each other with a molecular weight of about 110,000 {“pronectin F” Pronectin is a registered trademark (Japan and USA) 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) The Gly Ala Ala Val Thr Gly sequence (65) that binds to the N-terminus of the minimal amino acid sequence (X) is bound to the Gly Ala Ala Pro Gly Ala Ser sequence (77) and the C-terminus of the minimal amino acid sequence (X). Serpro Ala Ser Ala Ala Gly Tyr sequence (78) is changed to Ser Ala Gly Pro Ser Ala Gly Tyr sequence (88) "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 is reduced to the Tyr Ile Gly Ser Arg sequence (x3). The Gly Ala Ala Val Thr Gly sequence (65) that binds to the N terminus of the amino acid sequence (X) is linked to the Gly Ala Ala Val Cys Glu Pro Gly sequence (69), and the Ser Pro that binds to the C terminus of the minimal amino acid sequence (X). A “pronectin Y”, “pronectin Y2”, “pronectin Y3” or the like obtained by changing the Ala Ser Ala Ala Gly Tyr sequence (78) to the Cys Asp Ala Gly Tyr sequence (86).

  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. Regarding 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.

As serum-free medium (ME), 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 Medium, CHO-S-SFMII medium, FreeStyle ^™ 293 medium, CD-CHO AGT ^™ medium, RPMI medium, DMEM 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 Beauty mixtures media, and the like.
Among these, Opti-PRO ^™ SFM medium, Opti-MEM ^™ I medium, VP-SFM medium, CD293 medium, 293SFMII medium, CD-CHO medium, CHO-S-SFMII medium, FreeStyle ^™ 293 from the viewpoint of cell proliferation. Medium, CD-CHO AGT ^™ medium, DMEM medium, and mixed media thereof are preferable, and more preferably, Opti-PRO ^™ SFM medium, VP-SFM medium, CD293 medium, 293SFMII medium, FreeStyle ^™ 293 medium, DMEM medium, and these Mixed medium.

By containing the artificial polypeptide (P) in a serum-free medium (ME), the serum-free medium (A) of the present invention is obtained.
The content (mol / L, hereinafter abbreviated as M) of the minimum amino acid sequence (X) is preferably 1 nM to 1 mM with respect to the serum-free medium (A) from the viewpoint of improving cell proliferation. More preferably, it is 10 nM-100 micromol, Most preferably, it is 100 nM-10 micromol.

The serum-free medium (A) can contain a cell growth factor (Z) as necessary. By containing the cell growth factor (Z), the cell proliferation property can be further enhanced.
Cell growth factor (Z) includes fibroblast growth factor, transforming growth factor, epithelial cell growth factor, hepatocyte growth factor, platelet derived growth factor, insulin-like growth factor, vascular endothelial growth factor, nerve growth factor, stem cell Factors, leukemia inhibitory factors, bone morphogenetic factors, heparin-binding epithelial cell growth factor, neurotrophic factor, connective tissue growth factor, angiopoietin, cytokines, interleukins, adrenamodulin and natriuretic peptides are included. Among these, epithelial cell growth factor, fibroblast growth factor, transforming growth factor, insulin-like growth factor and bone morphogenetic factor are preferable from the viewpoint of wide range of applicable cells and high cell proliferation. Epidermal growth factor, fibroblast growth factor, transforming growth factor and insulin-like growth factor.
When the cell growth factor (Z) is used, the content (% by weight) of the cell growth factor varies depending on the type of the cell growth factor, but is 10 ⁻¹⁶ to 10 ⁻³ based on the weight of the serum-free medium (A). Is more preferably 10 ⁻¹⁴ to 10 ⁻⁵ , and particularly preferably 10 ⁻¹² to 10 ⁻⁷ .

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

As a method for producing the serum-free medium (A) of the present invention, an artificial polypeptide (P) can be introduced into a serum-free medium (ME), mixed and contained, and examples thereof include the following methods.
Using a glass container, a magnetic stirrer and a rotator, the artificial polypeptide (P) is added while stirring the serum-free medium (ME) at room temperature (25 ° C. ± 10) and normal pressure (1 atm). Although not particularly limited, the rotational speed of the magnetic stirrer is preferably 10 to 200 rpm. Stirring is continued for an additional hour after adding the artificial polypeptide (P). Next, it is sterilized by filtration using a disposable membrane filter unit (trade name: DISMIC-25cs, pore diameter: 0.45 μm, manufactured by Advantech Toyo Co., Ltd.) in a clean bench.

As a cell culture method using the serum-free medium (A) of the present invention, for example, the serum-free medium (A) is put into a tissue culture flask, and the cell solution has a cell concentration of 10,000 to 1,000,000 cells / ml. If the medium is changed every day for 5 days under the condition of carbon dioxide (CO ₂ ) concentration of 5% by volume and 37 ° C., the culture is performed.

The cell (CE) that can be cultured in the serum-free medium of the present invention is not limited as long as it is a cell. The mammal-derived normal cells, mammalian-derived cell lines and insect cells used 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, HT-1080 cell WI-38 cells, ψ2 cells, ψCRE cells, etc. {Cell culture technology (edited by the Japanese Tissue Culture Association, 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 are more preferred, and epithelial cells are particularly preferred. Moreover, 3T3 cells, BHK cells, CHO cells, HEK293 cells, HeLa cells, L-929 cells, MDCK cells, PER. C6 cell VERO cell, HT-1080 cell and WI-38 cell, particularly preferably MDCK cell, VERO cell and HT-1080 cell.

The concentration (cells / mL) of the cells dispersed in the serum-free medium is not particularly limited, but is preferably 100 to 100 million, more preferably 1000 to 10 million, and particularly preferably 10,000 to 100 ml per 1 ml of the serum-free medium. One 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) )}.

  The cell culture carrier in the case of culturing the cells can be used without particular limitation, for example, multi-well plate, petri dish, tissue culture flask, roller bottle, microcarrier (bead), film, non-woven fabric, Porous particles and holo-fibers are used. Of these, preferred are microcarriers (beads), films, multi-well plates, petri dishes, tissue culture flasks and roller bottles, and particularly preferred are tissue culture flasks, microcarriers (beads) and multi-well plates. Is.

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

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to this. In the following, parts and% indicate parts by weight and% by weight, respectively.

<Example 1>
<Preparation of Artificial Polypeptide (P1) “Pronectin F”>
According to the method described in the examples of JP-T-3-502935, it has 13 Arg Gly Asp sequences (x1) and 12 (Gly Ala Gly Ala Gly Ser) 9 sequences (21). An artificial polypeptide (P1) “Pronectin F” having a structure of Mw of about 110,000 having a structure formed by alternately bonding chemical bonds was produced.
<Preparation of serum-free medium (A1)>
Under normal temperature (25 ° C. ± 10) and normal pressure (1 atm), 1000 parts of a serum-free medium (ME1) (trade name: VP-SFM, manufactured by Invitrogen Corporation) was placed in a glass container. 2 parts of antibacterial agent {trade name: antibacterial agent GA solution (gentamicin, anteforin B), Kurashiki Boseki Co., Ltd.} in this serum-free medium (ME1) while stirring at 120 rpm using a magnetic stirrer and rotor And Pronectin F 8.46 × 10 ⁻⁶ parts. After the preparation, the mixture was further stirred for 1 hour. Next, it was sterilized by filtration using a disposable membrane filter unit in a clean bench to obtain a serum-free medium (A1).

<Example 2 to Example 7>
In Example 1, the amount of pronectin F charged was changed from 8.46 × 10 ⁻⁶ parts to 8.46 × 10 ⁻⁵ parts, 8.46 × 10 ⁻⁴ parts, 8.46 × 10 ⁻³ parts, respectively. Serum-free media (A2) to (A7) were obtained in the same manner as in Example 1 except for changing to 8.46 × 10 ⁻² parts, 8.46 × 10 ⁻¹ parts, and 8.46 parts.

<Examples 8 to 14>
In Example 1, 8.84 × 10 ⁻⁶ parts of Pronectin F were each GRGDS {artificial polypeptide (P2)} (trade name: Fibronectin Active Fragment, Gw Arg of Mw490 having one Arg Gly Asp sequence (x1). Artificial polypeptide of Gly Asp Ser sequence (63), manufactured by Peptide Institute, Inc.) 4.90 × 10 ⁻⁷ parts, 4.90 × 10 ⁻⁶ parts, 4.90 × 10 ⁻⁵ parts, 4.90 × ^10-4 parts, 4.90 × 10 ⁻³ parts, 4.90 × 10 ⁻² parts, 4.90 × 10 ⁻¹ parts ” A8) to (A14) were obtained.

<Example 15>
<Preparation of artificial polypeptide (P3)>
Preparation of artificial polypeptide (P3A): According to the DCC method described in “Basics and Experiments of Peptide Synthesis” Nos. 114 to 117 (published on Mar. 20, 1985), etc. A solution containing a cross-linked product reacted with the peptides (P2) at a concentration of about 5% by weight was prepared. After 10 mL of this solution was filtered through a 0.45 μm membrane filter, using a gel filtration column (trade name: Superdex 30 pg, manufactured by Amersham Biosciences), flow rate: 1 mL / min, eluent: phosphate buffer Liquid (pH 7.2 containing 0.515 g / l Na ₂ HPO ₄ · 12H ₂ O, 0.764 g / l KH ₂ PO ₄ and 8.16 g / l NaCl in deionized water) The solution was fractionated under the following conditions: Using the elution time of a molecular weight marker that had been gel-filtered in advance under the same conditions as an index, a fraction with an elution time of about 1000 Mw was collected. The fraction with a molecular weight of about 1,000 was desalted by dialysis in ion-exchanged water. After desalting, freeze-drying was performed for 24 hours under the conditions of −20 ° C. and 0.1 kPa or less. Next, it is freeze-dried and has about 2 artificial polypeptides (P3A) {Gly Arg Gly Asp Ser sequences (63). }.
Preparation of artificial polypeptide (P3B): “Instead of reacting with artificial polypeptide (P2), instead of reacting with artificial polypeptide (P3A)” and “Mw instead of a fraction of about 1,000 Mw Prepared in the same manner as in the preparation of artificial polypeptide (P3A) except that fractions of about 2,000 are collected, and about 4 artificial polypeptides (P3B) {Gly Arg Gly Asp Ser sequence (63) Have. }.
Preparation of artificial polypeptide (P3C): “Instead of reacting with artificial polypeptide (P2), instead of reacting with artificial polypeptide (P3B)” and “Mw instead of a fraction of about 1,000 Mw Prepared in the same manner as the above-mentioned artificial polypeptide (P3A) except that a fraction of about 4,000 is collected, and the artificial polypeptide (P3C) {Gly Arg Gly Asp Ser sequence (63) Eight. }.
Preparation of artificial polypeptide (P3): “Instead of reacting artificial polypeptides (P2) with each other, artificial polypeptide (P3A) and artificial polypeptide (P3C) are reacted at a molar ratio of 1: 1” and “ Prepared in the same manner as the above-described artificial polypeptide (P3A) except that a fraction having a Mw of about 5,000 is collected instead of a fraction having a Mw of about 1,000, and the artificial polypeptide (P3 ) {Gly Arg Gly Asp Ser sequence (63). }.
<Preparation of serum-free medium (A15)>
In Example 1, a serum-free medium (in the same manner as in Example 1 except that “8.46 × 10 ⁻⁶ parts of Pronectin F is changed to 4.90 × 10 ⁻⁴ parts of artificial polypeptide (P3)”) A15) was obtained.

<Example 16>
<Preparation of artificial polypeptide (P4)>
“Instead of reacting artificial polypeptides (P2) with each other, reacting artificial polypeptides (P3) with each other”, “Instead of using Superdex 30 pg as a gel filtration column, trade name: Superdex 75 pg is used. In the same manner as in the preparation of the artificial polypeptide (P3A) described in Example 15, except that “fractionation of about 10,000 Mw instead of fractionation of about 1,000 Mw”. Prepared and has about 20 artificial polypeptides (P4) {Gly Arg Gly Asp Ser sequence (63). }.
<Preparation of serum-free medium (A16)>
In Example 1, a serum-free medium (in the same manner as in Example 1 except that “8.46 × 10 ⁻⁶ parts of Pronectin F is changed to 4.90 × 10 ⁻⁴ parts of artificial polypeptide (P4)”) A16) was obtained.

<Example 17>
<Preparation of artificial polypeptide (P5)>
“Instead of reacting the artificial polypeptide (P2) with each other, the artificial polypeptide (P3) and the artificial polypeptide (P4) are reacted at a molar ratio of 1: 1”, “As a gel filtration column, trade name: Superdex 30 pg Except for using “Product name: Superdex 75 pg instead of using“ and “Making a fraction of about 15,000 Mw instead of a fraction of about 1,000 Mw”. 15 is prepared in the same manner as the artificial polypeptide (P3A) described in No. 15, and has about 30 artificial polypeptides (P5) {Gly Arg Gly Asp Ser sequence (63). }.
<Preparation of serum-free medium (A17)>
In Example 1, a serum-free medium (A17) was used in the same manner as in Example 1 except that “8.46 × 10 ⁻⁶ parts of Pronectin F was changed to 4.90 × 10 ⁻⁴ parts of artificial polypeptide (P5)”. )

<Example 18>
<Preparation of artificial polypeptide (P6)>
“Instead of reacting artificial polypeptides (P2) with each other, reacting artificial polypeptides (P4) with each other”, “Instead of using Superdex 30 pg as a gel filtration column, trade name: Superdex 75 pg is used. In the same manner as in the preparation of the artificial polypeptide (P3A) described in Example 15, except that a fraction with a Mw of about 20,000 is collected instead of a fraction with a Mw of about 1,000. Prepared and has about 40 artificial polypeptides (P6) {Gly Arg Gly Asp Ser sequences (63). }.
<Preparation of serum-free medium (A18)>
In Example 1, a serum-free medium (A18) was used in the same manner as in Example 1 except that “8.46 × 10 ⁻⁶ parts of Pronectin F was changed to 4.90 × 10 ⁻⁴ parts of artificial polypeptide (P6)”. )

<Example 19>
<Preparation of artificial polypeptide (P7)>
Except for “reacting artificial polypeptide (P2) with each other instead of reacting artificial polypeptide (P2) with Gly Ala Gly Ala Gly Ser sequence (64) at a molar ratio of 1: 1”, described in Example 15 The artificial polypeptide (P3A) was prepared in the same manner as that of the artificial polypeptide (P3A). About 1 of the artificial polypeptide (P7) {Gly Arg Gly Asp Ser sequence (63) and Gly Ala Gly Ala Gly Ser sequence (64) And have. }.
<Preparation of serum-free medium (A19)>
In Example 1, a serum-free medium (A19) was used in the same manner as in Example 1 except that “8.46 × 10 ⁻⁶ parts of Pronectin F was changed to 9.08 × 10 ⁻⁴ parts of artificial polypeptide (P7)”. )

<Example 20>
<Preparation of artificial polypeptide (P8)>
Preparation of artificial polypeptide (P8A): Artificial polypeptide described in Example 15 except that, instead of reacting between artificial polypeptides (P2), Gly Ala Gly Ala Gly Ser sequences (64) are reacted with each other It is prepared in the same manner as the preparation of (P3A), and has about 2 artificial polypeptides (P8A) {Gly Ala Gly Ala Gly Ser sequence (64). }.
Preparation of artificial polypeptide (P8B): “Instead of reacting with artificial polypeptide (P2), instead of reacting with artificial polypeptide (P8A)” and “Mw instead of a fraction of about 1,000 Mw, The artificial polypeptide (P8B) {Gly Ala Gly Ala Gly Ser sequence was prepared in the same manner as the artificial polypeptide (P3A) described in Example 15 except that a fraction of about 2,000 was collected. There are about 4 (64). }.
Preparation of artificial polypeptide (P8C): “Instead of reacting with artificial polypeptide (P2), instead of reacting with artificial polypeptide (P8B)” and “Mw instead of a fraction of about 1,000 Mw The artificial polypeptide (P8C) {Gly Ala Gly Ala Gly Ser sequence was prepared in the same manner as the artificial polypeptide (P3A) described in Example 15 except that a fraction of about 3,000 was collected. There are about 8 (64). }.
Preparation of artificial polypeptide (P8D): “Instead of reacting artificial polypeptides (P2) with each other, artificial polypeptide (P8A) and artificial polypeptide (P8C) are reacted at a molar ratio of 1: 1” and “ Prepared in the same manner as in the preparation of the artificial polypeptide (P3A) described in Example 15 except that a fraction having a Mw of about 4,000 is collected instead of a fraction having a Mw of about 1,000. Polypeptide (P8D) {has about 10 Gly Ala Gly Ala Gly Ser sequences (64). }.
Preparation of artificial polypeptide (P8): “Instead of reacting artificial polypeptides (P2) with each other, artificial polypeptide (P2) and artificial polypeptide (P8D) are reacted at a molar ratio of 1: 1” and “ Prepared in the same manner as in the preparation of the artificial polypeptide (P3A) described in Example 15 except that a fraction having a Mw of about 5,000 is collected instead of a fraction having a Mw of about 1,000. Polypeptide (P8) {has about 1 Gly Arg Gly Asp Ser sequence (63) and about 10 Gly Ala Gly Ala Gly Ser sequences (64). }.
<Preparation of serum-free medium (A20)>
In Example 1, a serum-free medium (A20) was used in the same manner as in Example 1 except that “8.46 × 10 ⁻⁶ parts of Pronectin F was changed to 4.67 × 10 ⁻³ parts of artificial polypeptide (P8)”. )

<Example 21>
<Preparation of artificial polypeptide (P9)>
Preparation of artificial polypeptide (P9A): “Instead of reacting with artificial polypeptide (P2), instead of reacting with artificial polypeptide (P8D)” and “Mw instead of a fraction of about 1,000 Mw The artificial polypeptide (P9A) {Gly Ala Gly Ala Gly Ser sequence was prepared in the same manner as the artificial polypeptide (P3A) described in Example 15, except that a fraction of about 8,000 was collected. There are about 20 (64). }.
Preparation of artificial polypeptide (P9): “Instead of reacting artificial polypeptides (P2) with each other, artificial polypeptide (P2) and artificial polypeptide (P9A) are reacted at a molar ratio of 1: 1” and “ Prepared in the same manner as in the preparation of the artificial polypeptide (P3A) described in Example 15 except that a fraction having a Mw of approximately 9,000 is collected instead of a fraction having a Mw of approximately 1,000. Polypeptide (P9) {has about 1 Gly Arg Gly Asp Ser sequence (63) and about 20 Gly Ala Gly Ala Gly Ser sequences (64). }.
<Preparation of serum-free medium (A21)>
In Example 1, a serum-free medium (A21) was used in the same manner as in Example 1 except that “8.46 × 10 ⁻⁶ parts of Pronectin F was changed to 8.85 × 10 ⁻³ parts of artificial polypeptide (P9)”. )

<Example 22>
<Preparation of artificial polypeptide (P10)>
Preparation of artificial polypeptide (P10A): “Instead of reacting artificial polypeptides (P2) with each other, artificial polypeptide (P8D) and artificial polypeptide (P9A) are reacted at a molar ratio of 1: 1”, “ As a gel filtration column, instead of using the product name: Superdex 30 pg, use the product name: Superdex 75 pg ”and“ fractionation of about 13,000 Mw instead of the fraction of about 1000 Mw. Except for “taking”, it is prepared in the same manner as the artificial polypeptide (P3A) described in Example 15, and has about 30 artificial polypeptides (P10A) {Gly Ala Gly Ala Gly Ser sequences (64). }.
Preparation of artificial polypeptide (P10): “Instead of reacting artificial polypeptides (P2) with each other, artificial polypeptide (P2) and artificial polypeptide (P10A) are reacted at a molar ratio of 1: 1”, “ As a gel filtration column, instead of using the product name: Superdex 30 pg, use the product name: Superdex 75 pg ”and“ fractionation of about 13,000 Mw instead of the fraction of about 1000 Mw. Except that the artificial polypeptide (P3A) described in Example 15 is prepared in the same manner as in Example 15, and about 1 of the artificial polypeptide (P10) {Gly Arg Gly Asp Ser sequence (63) and Gly Ala About 30 Gly Ala Gly Ser sequences (64). }.
<Preparation of serum-free medium (A22)>
In Example 1, a serum-free medium (A22) was used in the same manner as in Example 1 except that “8.46 × 10 ⁻⁶ parts of Pronectin F was changed to 1.30 × 10 ⁻² parts of artificial polypeptide (P10)”. )

<Example 23>
<Preparation of artificial polypeptide (P11)>
Preparation of artificial polypeptide (P11A): “Instead of reacting artificial polypeptides (P2) with each other, artificial polypeptide (P9A) and artificial polypeptide (P10A) are reacted at a molar ratio of 1: 1”, “ As a gel filtration column, instead of using the product name: Superdex 30 pg, use the product name: Superdex 75 pg. ”And“ Instead of fractioning around Mw about 1,000, fractions around Mw about 21,000 are separated. Except for “taking”, it is prepared in the same manner as the artificial polypeptide (P3A) described in Example 15, and has about 50 artificial polypeptides (P11A) {Gly Ala Gly Ala Gly Ser sequences (64). }.
Preparation of artificial polypeptide (P11): “Instead of reacting artificial polypeptides (P2) with each other, artificial polypeptide (P2) and artificial polypeptide (P11A) are reacted at a molar ratio of 1: 1”, “ As a gel filtration column, instead of using the product name: Superdex 30 pg, use the product name: Superdex 75 pg. ”And“ Instead of fractioning around Mw about 1,000, fractions around Mw about 21,000 are separated. Except that the artificial polypeptide (P3A) described in Example 15 was prepared in the same manner as in Example 15, and about 1 of the artificial polypeptide (P11) {Gly Arg Gly Asp Ser sequence (63) and Gly Ala About 50 of the Gly Ala Gly Ser sequences (64). }.
<Preparation of serum-free medium (A23)>
In Example 1, a serum-free medium (A23) was used in the same manner as in Example 1 except that “8.46 × 10 ⁻⁶ parts of Pronectin F was changed to 2.14 × 10 ⁻² parts of artificial polypeptide (P11)”. )

<Example 24>
<Preparation of artificial polypeptide (P12)>
According to the method described in the examples in JP-T-3-502935, five Arg Gly Asp sequences (x1) and five (Gly Ala Gly Ala Gly Ser) 3 sequences (20) (y2) And an artificial polypeptide (P12) “pronectin F2” having an Mw of about 20,000 having a structure in which these are alternately chemically bonded.
<Preparation of serum-free medium (A24)>
In Example 1, a serum-free medium (A24) was used in the same manner as in Example 1 except that “8.46 × 10 ⁻⁶ parts of Pronectin F was changed to 4.00 × 10 ⁻³ parts of artificial polypeptide (P12)”. )

<Example 25>
<Preparation of artificial polypeptide (P13)>
Three Arg Gly Asp sequences (x1) and (Gly Val Pro Gly Val) 2 Gly Gly (Gly Ala Gly Ala Gly Ser) 3 sequences in accordance with the method described in the Examples in JP-T-3-502935 (38) An artificial polypeptide (P13) “pronectin F3” having an Mw of about 10,000 having a structure in which three of (y3) and these are alternately chemically bonded was produced.
<Preparation of serum-free medium (A25)>
In Example 1, a serum-free medium (A25) was used in the same manner as in Example 1 except that “8.46 × 10 ⁻⁶ parts of Pronectin F was changed to 3.33 × 10 ⁻³ parts of artificial polypeptide (P13)”. )

<Example 26>
<Preparation of artificial polypeptide (P15)>
The artificial polypeptide described in Example 15 except that “the artificial polypeptide (P14) (Ile Lys Val Ala Val sequence (7) of Mw529, manufactured by NEOSYSTEM) is used instead of the artificial polypeptide (P2)”. Prepared in the same manner as the preparation of (P3), and has about 10 artificial polypeptides (P15) {Ile Lys Val Ala Val sequence (7). }.
<Preparation of serum-free medium (A26)>
In Example 1, “Change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME2) (trade name: MEM Dulbecco liquid medium, manufactured by Dainippon Sumitomo Pharma Co., Ltd.)” and “Pronectin F8 Serum-free medium (A25) was obtained in the same manner as in Example 1 except that “.46 × 10 ⁻⁶ parts were changed to 5.29 × 10 ⁻⁴ parts of artificial polypeptide (P15)”.

<Example 27>
<Preparation of artificial polypeptide (P16)>
In Example 16, it was prepared in the same manner as the preparation of the artificial polypeptide (P4) described in Example 16 except that “the artificial polypeptide (P15) is used instead of the artificial polypeptide (P3)”. Artificial polypeptide (P16) {has about 20 Ile Lys Val Ala Val sequences (7) }.
<Preparation of serum-free medium (A27)>
In Example 1, “Change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME2)” and “Pronectin F 8.46 × 10 ⁻⁶ parts with artificial polypeptide (P16) 5.29 × A serum-free medium (A27) was obtained in the same manner as in Example 1, except that “change to 10 ⁻⁴ parts”.

<Example 28>
<Preparation of artificial polypeptide (P17)>
In Example 17, except for using “artificial polypeptide (P15) and artificial polypeptide (P16) instead of using artificial polypeptide (P3) and artificial polypeptide (P4)”, Example 17 It is prepared in the same manner as the artificial polypeptide (P5) described in 1. and has about 30 artificial polypeptides (P17) {Ile Lys Val Ala Val sequence (7). }.
<Preparation of serum-free medium (A28)>
In Example 1, “Change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME2)” and “Pronectin F8.46 × 10 ⁻⁶ parts with artificial polypeptide (P17) 5.29 × A serum-free medium (A28) was obtained in the same manner as in Example 1 except that “change to 10 ⁻⁴ parts”.

<Example 29>
<Preparation of artificial polypeptide (P18)>
In Example 18, prepared in the same manner as in the preparation of the artificial polypeptide (P6) described in Example 18 except that “the artificial polypeptide (P16) is used instead of the artificial polypeptide (P4)”, Artificial polypeptide (P18) {has about 40 Ile Lys Val Ala Val sequences (7) }.
<Preparation of serum-free medium (A29)>
In Example 1, “Change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME2)” and “Pronectin F 8.46 × 10 ⁻⁶ parts with artificial polypeptide (P18) 5.29 × A serum-free medium (A29) was obtained in the same manner as in Example 1 except that “change to 10 ⁻⁴ parts”.

<Example 30>
<Preparation of artificial polypeptide (P19)>
In Example 19, “instead of using the artificial polypeptide (P2) and the Gly Ala Gly Ala Gly Ser sequence (64), the artificial polypeptide (P14) and the Gly Ala Gly Ala Gly Ser sequence (64) are used. Except that the artificial polypeptide (P7) described in Example 19 was prepared in the same manner as the artificial polypeptide (P19) {about 1 of the Ile Lys Val Ala Val sequence (7) and Gly Ala Gly About one of the Ala Gly Ser sequences (64). }.
<Preparation of serum-free medium (A30)>
In Example 1, “Change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME2)” and “Pronectin F 8.46 × 10 ⁻⁶ parts with artificial polypeptide (P19) 9.47 × A serum-free medium (A30) was obtained in the same manner as in Example 1 except that “change to 10 ⁻⁴ parts”.

<Example 31>
<Preparation of serum-free medium (A31)>
In Example 1, “Change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME2)” and “Pronectin F 8.46 × 10 ⁻⁶ parts with artificial polypeptide (P14) 5.29 × A serum-free medium (A31) was obtained in the same manner as in Example 1 except that “change to 10 ⁻⁴ parts”.

<Example 32>
<Preparation of artificial polypeptide (P20)>
In Example 20, except for “use artificial polypeptide (P14) and artificial polypeptide (P8D) instead of using artificial polypeptide (P2) and artificial polypeptide (P8D)”, Example 20 Prepared in the same manner as the artificial polypeptide (P8) described in the above, and the artificial polypeptide (P20) {about 1 of the Ile Lys Val Ala Val sequence (7) and the Gly Ala Gly Ala Gly Ser sequence (64) There are about 10 pieces. }.
<Preparation of serum-free medium (A32)>
In Example 1, "Change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME2)" and "Pronectin F8.46 × 10 ⁻⁶ parts are artificial polypeptide (P20) 4.71 × A serum-free medium (A32) was obtained in the same manner as in Example 1 except that the content was changed to 10 ⁻³ parts.

<Example 33>
<Preparation of artificial polypeptide (P21)>
In Example 21, except for using “artificial polypeptide (P14) and artificial polypeptide (P9A) instead of using artificial polypeptide (P2) and artificial polypeptide (P9A)”, Example 21 Prepared in the same manner as the artificial polypeptide (P9) described in the above, and the artificial polypeptide (P21) {about 1 of the Ile Lys Val Ala Val sequence (7) and the Gly Ala Gly Ala Gly Ser sequence (64) There are about 20 pieces. }.
<Preparation of serum-free medium (A33)>
In Example 1, "Change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME2)" and "Pronectin F 8.46 × 10 ⁻⁶ parts are artificial polypeptide (P21) 8.89 × A serum-free medium (A33) was obtained in the same manner as in Example 1 except that the content was changed to 10 ⁻³ parts.

<Example 34>
<Preparation of artificial polypeptide (P22)>
In Example 22, except for using “artificial polypeptide (P14) and artificial polypeptide (P10A) instead of using artificial polypeptide (P2) and artificial polypeptide (P10A)” in Example 22 Prepared in the same manner as the artificial polypeptide (P10) described in the above, and the artificial polypeptide (P22) {about 1 of the Ile Lys Val Ala Val sequence (7) and the Gly Ala Gly Ala Gly Ser sequence (64) There are about 30 pieces. }.
<Preparation of serum-free medium (A34)>
In Example 1, “change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME2)” and “pronectin F 8.46 × 10 ⁻⁶ parts of artificial polypeptide (P22) 1.31 × A serum-free medium (A34) was obtained in the same manner as in Example 1, except that “change to 10 ⁻² parts”.

<Example 35>
<Preparation of artificial polypeptide (P23)>
Except for “instead of using an artificial polypeptide (P2) and an artificial polypeptide (P11A), an artificial polypeptide (P14) and an artificial polypeptide (P11A)”, the artificial polyp Prepared in the same manner as the preparation of peptide (P11), artificial polypeptide (P23) {about 1 of Ile Lys Val Ala Val sequence (7) and about 50 of Gly Ala Gly Ala Gly Ser sequence (64) Have. }.
<Preparation of serum-free medium (A35)>
In Example 1, “Change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME2)” and “Pronectin F8.46 × 10 ⁻⁶ parts of artificial polypeptide (P23) 2.14 × A serum-free medium (A35) was obtained in the same manner as in Example 1 except that “change to 10 ⁻² parts”.

<Example 36>
<Preparation of artificial polypeptide (P24)>
In accordance with the method described in the examples in JP-T-3-502935, 13 Ile Lys Val Ala Val sequences (7) (x2) and (Gly Ala Gly Ala Gly Ser) 9 sequences (21) (y1 And an artificial polypeptide (P24) “pronectin L” having an Mw of about 110,000 having a structure in which these are alternately chemically bonded.
<Preparation of serum-free medium (A36)>
In Example 1, “Change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME2)” and “Pronectin F 8.46 × 10 ⁻⁶ parts with artificial polypeptide (P24) 8.46 × A serum-free medium (A36) was obtained in the same manner as in Example 1 except that “change to 10 ⁻³ parts”.

<Example 37>
<Preparation of artificial polypeptide (P25)>
According to the method described in the examples in JP-T-3-502935, five Ile Lys Val Ala Val sequences (7) (x2) and (Gly Ala Gly Ala Gly Ser) 9 sequences (21) (y1 ) And an artificial polypeptide (P25) “pronectin L2” having an Mw of about 20,000 having a structure in which these are alternately chemically bonded.
<Preparation of serum-free medium (A37)>
In Example 1, “Change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME2)” and “Pronectin F 8.46 × 10 ⁻⁶ parts of artificial polypeptide (P25) 4.00 × A serum-free medium (A37) was obtained in the same manner as in Example 1 except that “change to 10 ⁻³ parts”.

<Example 38>
<Preparation of artificial polypeptide (P26)>
According to the method described in the examples in JP-T-3-502935, three Ile Lys Val Ala Val sequences (7) (x2) and (Gly Ala Gly Ala Gly Ser) 9 sequence (21) (y1 ) And an artificial polypeptide (P26) (“pronectin L3”) having an Mw of about 10,000 having a structure in which these are alternately chemically bonded.
<Preparation of serum-free medium (A38)>
In Example 1, “Change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME2)” and “Pronectin F8.46 × 10 ⁻⁶ parts by artificial polypeptide (P26) 3.33 × A serum-free medium (A38) was obtained in the same manner as in Example 1 except that the content was changed to 10 ⁻³ parts.

<Example 39>
<Preparation of serum-free medium (A39)>
In Example 1, "Changing 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME3) (trade name: Opti-PRO ^™ SFM medium, manufactured by Invitrogen Corporation)" and "Pronectin F8. A serum-free medium (A39) was obtained in the same manner as in Example 1 except that “46 × 10 ⁻⁶ parts were changed to 8.46 × 10 ⁻³ parts”.

<Example 40>
<Preparation of serum-free medium (A40)>
In Example 1, “Change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME3)” and “Pronectin F 8.46 × 10 ⁻⁶ parts are 4.90 × artificial polypeptide (P2). A serum-free medium (A40) was obtained in the same manner as in Example 1 except that “change to 10 ⁻⁴ parts”.

<Example 41>
<Preparation of serum-free medium (A41)>
In Example 1, “Change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME3)” and “Pronectin F 8.46 × 10 ⁻⁶ parts are 4.90 × artificial polypeptide (P3). A serum-free medium (A41) was obtained in the same manner as in Example 1 except that “change to 10 ⁻⁴ parts”.

<Example 42>
<Preparation of serum-free medium (A42)>
In Example 1, “Change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME3)” and “Pronectin F 8.46 × 10 ⁻⁶ parts are 4.90 × artificial polypeptide (P4)”. A serum-free medium (A42) was obtained in the same manner as in Example 1, except that “change to 10 ⁻⁴ parts”.

<Example 43>
<Preparation of serum-free medium (A43)>
In Example 1, “Change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME3)” and “Pronectin F8.46 × 10 ⁻⁶ parts with 4.90 × artificial polypeptide (P5)”. A serum-free medium (A43) was obtained in the same manner as in Example 1 except that “change to 10 ⁻⁴ parts”.

<Example 44>
<Preparation of serum-free medium (A44)>
In Example 1, “Change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME3)” and “Pronectin F 8.46 × 10 ⁻⁶ parts with 4.90 × artificial polypeptide (P6)”. A serum-free medium (A44) was obtained in the same manner as in Example 1 except that “change to 10 ⁻⁴ parts”.

<Example 45>
<Preparation of serum-free medium (A45)>
In Example 1, “Change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME3)” and “Pronectin F 8.46 × 10 ⁻⁶ parts with artificial polypeptide (P7) 9.08 × A serum-free medium (A45) was obtained in the same manner as in Example 1, except that “change to 10 ⁻⁴ parts”.

<Example 46>
<Preparation of serum-free medium (A46)>
In Example 1, “Change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME3)” and “Pronectin F 8.46 × 10 ⁻⁶ parts of artificial polypeptide (P8) 4.67 × A serum-free medium (A46) was obtained in the same manner as in Example 1 except that “change to 10 ⁻³ parts”.

<Example 47>
<Preparation of serum-free medium (A47)>
In Example 1, “Change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME3)” and “Pronectin F 8.46 × 10 ⁻⁶ parts of artificial polypeptide (P9) 8.85 × A serum-free medium (A47) was obtained in the same manner as in Example 1 except that “change to 10 ⁻³ parts”.

<Example 48>
<Preparation of serum-free medium (A48)>
In Example 1, “Change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME3)” and “Pronectin F8.46 × 10 ⁻⁶ parts of artificial polypeptide (P10) 1.30 × A serum-free medium (A48) was obtained in the same manner as in Example 1 except that the content was changed to ^10-2 parts.

<Example 49>
<Preparation of serum-free medium (A49)>
In Example 1, “Change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME3)” and “Pronectin F8.46 × 10 ⁻⁶ parts of artificial polypeptide (P11) 2.14 × A serum-free medium (A49) was obtained in the same manner as in Example 1 except that the change was made to ^10-2 parts.

<Example 50>
<Preparation of serum-free medium (A50)>
In Example 1, “Change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME3)” and “Pronectin F 8.46 × 10 ⁻⁶ parts with artificial polypeptide (P12) 4.00 × A serum-free medium (A50) was obtained in the same manner as in Example 1 except that “change to 10 ⁻³ parts”.

<Example 51>
<Preparation of serum-free medium (A51)>
In Example 1, "Change 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME3)" and "Pronectin F 8.46 × 10 ⁻⁶ parts are artificial polypeptide (P13) 3.33 × A serum-free medium (A51) was obtained in the same manner as in Example 1 except that the content was changed to 10 ⁻³ parts.

<Comparative Example 1>
<Preparation of serum-free medium (RA1)>
In Example 1, a comparative serum-free medium (RA1) was obtained in the same manner as in Example 1 except that “the amount of pronectin F charged was changed from 8.46 × 10 ⁻⁶ parts to 0 parts”. .

<Comparative example 2>
<Preparation of serum-free medium (RA2)>
In Example 1, “Pronectin F8.46 × 10 ⁻⁶ parts were changed to 1.10 × 10 ⁻⁵ parts of fibronectin (polypeptide derived from an animal having two Arg Gly Asp sequences (x1), manufactured by BD Bioscience)”. A comparative serum-free medium (RA2) was obtained in the same manner as in Example 1 except that.

<Comparative Example 3>
<Preparation of serum-free medium (RA3)>
In Example 1, except for changing “pronectin F8.46 × 10 ⁻⁶ parts to fibronectin 1.10 × 10 ⁻⁴ parts”, a serum-free medium for comparison (RA3) was prepared in the same manner as in Example 1. Obtained.

<Comparative Example 4>
<Preparation of serum-free medium (RA4)>
In Example 1, a comparative serum-free medium (RA4) was prepared in the same manner as in Example 1 except that “8.46 × 10 ⁻⁶ parts of pronectin F was changed to 1.10 × 10 ⁻³ parts of fibronectin”. Obtained.

<Comparative Example 5>
<Preparation of serum-free medium (RA5)>
In Example 1, a comparative serum-free medium (RA5) was prepared in the same manner as in Example 1, except that “8.46 × 10 ⁻⁶ parts of pronectin F was changed to 1.10 × 10 ⁻² parts of fibronectin”. Obtained.

<Comparative Example 6>
<Preparation of serum-free medium (RA6)>
In Example 1, except for changing “pronectin F8.46 × 10 ⁻⁶ parts to fibronectin 1.10 × 10 ⁻¹ parts”, a comparative serum-free medium (RA6) was prepared in the same manner as in Example 1. Obtained.

<Comparative Example 7>
<Preparation of serum-free medium (RA7)>
In Example 1, a serum-free medium for comparison (RA7) was obtained in the same manner as in Example 1 except that “8.46 × 10 ⁻⁶ parts of pronectin F was changed to 1.10 parts of fibronectin”.

<Comparative Example 8>
<Preparation of serum-free medium (RA8)>
In Example 1, a serum-free medium for comparison (RA8) was obtained in the same manner as in Example 1 except that “8.46 × 10 ⁻⁶ parts of pronectin F was changed to 11.0 parts of fibronectin”.

<Comparative Example 9>
<Preparation of serum-free medium (RA9)>
In Example 1, a serum-free medium for comparison (RA9) was obtained in the same manner as in Example 1 except that “Pronectin F8.46 × 10 ⁻⁶ parts were changed to 110 parts of fibronectin”.

<Comparative Example 10>
<Preparation of serum-free medium (RA10)>
In Example 1, except for “changing 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME2)” and “changing pronectin F8.46 × 10 ⁻⁶ parts to 0 part” In the same manner as in Example 1, a serum-free medium (RA10) was obtained.

<Comparative Example 11>
<Preparation of serum-free medium (RA11)>
In Example 1, except for “changing 1000 parts of serum-free medium (ME1) to 1000 parts of serum-free medium (ME3)” and “changing pronectin F8.46 × 10 ⁻⁶ parts to 0 part” In the same manner as in Example 1, a serum-free medium (RA11) was obtained.

<Cell proliferation evaluation 1>
For the serum-free media (A1) to (A14) and (RA1 to RA9) of Examples 1 to 14 and Comparative Examples 1 to 9, the multi-well plate ( (Product name: MICROTEST ^TM Flat Bottom, 96 holes, non-treatment, polystyrene, BECTON DICKINSON). Next, pre-cultured VERO cells (manufactured by Dainippon Sumitomo Pharma Co., Ltd.) were seeded in each serum-free medium so that the cell concentration was 100,000 cells / ml. Culturing was carried out for 5 days in a CO ₂ incubator at 37 ° C. and a carbon dioxide gas concentration of 5% by volume. On the fifth day of culture, each serum-free medium was removed by aspiration, 50 μl / well of PBS and 10 μl / well of TetraColor ONE (Seikagaku Corporation) were introduced, and CO _{2 with} a carbon dioxide gas concentration of 5% by volume at 37 ° C. It was allowed to stand for 4 hours in an incubator. After 4 hours, the absorbance at 492 nm (reference wavelength: 620 nm) was measured using a plate reader (manufactured by Wako Pure Chemical Industries, Ltd.). Table 1 shows values obtained by dividing the measured absorbance of (RA1) by absorbance (blank) and dividing the absorbance measured by each by the absorbance (blank).

  From the results in Table 1, it was found that cell growth was reduced when a serum-free medium for comparison was used, whereas cell growth was improved when the serum-free medium of the present invention was used.

<Cell proliferation evaluation 2>
For each of the serum-free media (A4), (A11), (A15) to (A25) and (RA1) of Examples 4, 11, 15 to 25 and Comparative Example 1, 0.1 ml / well of each serum-free medium was used. As shown in FIG. Next, pre-cultured VERO cells were seeded in each serum-free medium so that the cell concentration was 100,000 cells / ml. Culturing was carried out for 5 days in a CO ₂ incubator at 37 ° C. and a carbon dioxide gas concentration of 5% by volume. On day 5 of culture, each serum-free medium was removed by aspiration, PBS (50 μl / well) and TetraColor ONE (10 μl / well) were added, and the plate was allowed to stand still in a CO ₂ incubator at 37 ° C. and 5% by volume of carbon dioxide gas for 4 hours. I put it. After 4 hours, the absorbance at 492 nm (reference wavelength: 620 nm) was measured using a plate reader. Table 2 shows values obtained by dividing the measured absorbance of (RA1) by absorbance (blank) and dividing the absorbance measured by each by the absorbance (blank).

  From the results in Table 2, it was found that the serum-free medium of the present invention has higher cell proliferation of VERO cells than the serum-free medium for comparison.

<Cell proliferation evaluation 3>
For the serum-free media (A26) to (A38) and (RA10) of Examples 26 to 38 and Comparative Example 10, each serum-free media was added to a multiwell plate so as to be 0.1 ml / well. Next, pre-cultured HT-1080 cells (Dainippon Sumitomo Pharma Co., Ltd.) were seeded in each serum-free medium so that the cell concentration was 100,000 cells / ml. Culturing was carried out for 5 days in a CO ₂ incubator at 37 ° C. and a carbon dioxide gas concentration of 5% by volume. On day 5 of culture, each serum-free medium was removed by aspiration, PBS (50 μl / well) and TetraColor ONE (10 μl / well) were added, and the plate was allowed to stand still in a CO ₂ incubator at 37 ° C. and 5% by volume of carbon dioxide gas for 4 hours. I put it. After 4 hours, the absorbance at 492 nm (reference wavelength: 620 nm) was measured using a plate reader. The measured value of the absorbance of (RA10) was taken as the absorbance (blank), and the values obtained by dividing the absorbance measured by the absorbance (blank) are shown in Table 3.

  From the results in Table 3, it was found that the serum-free medium of the present invention has higher cell growth of HT-1080 cells than the serum-free medium for comparison.

<Cell proliferation evaluation 4>
For the serum-free media (A39) to (A51) and (RA11) of Examples 39 to 51 and Comparative Example 11, each serum-free medium was added to a multiwell plate at 0.1 ml / well. Next, pre-cultured MDCK cells (Dainippon Sumitomo Pharma Co., Ltd.) were seeded in each serum-free medium so that the cell concentration was 100,000 cells / ml. Culturing was carried out for 5 days in a CO ₂ incubator at 37 ° C. and a carbon dioxide gas concentration of 5% by volume. On day 5 of culture, each serum-free medium was removed by aspiration, PBS (50 μl / well) and TetraColor ONE (10 μl / well) were added, and the plate was allowed to stand still in a CO ₂ incubator at 37 ° C. and 5% by volume of carbon dioxide gas for 4 hours. I put it. After 4 hours, the absorbance at 492 nm (reference wavelength: 620 nm) was measured using a plate reader. Table 4 shows values obtained by dividing the measured absorbance of (RA11) by the absorbance (blank) and dividing the absorbance measured by each by the absorbance (blank).

  From the results shown in Table 4, it was found that the serum-free medium of the present invention has higher cell proliferation of MDCK cells than the serum-free medium for comparison.

The serum-free medium of the present invention can exhibit excellent cell growth and there is no risk of contamination with infectious agents such as viruses. Very useful.
For research and development, cell culture for cell function evaluation such as differentiation function, culture of substitute cells for animal experiments (toxicity test, stimulation test, metabolic function test, etc.), cell culture for gene and protein introduction, etc. Available.
For the production of useful substances, it can be used for culturing cells for production of cytokines, thrombolytic agents, blood coagulation factor preparations, vaccines, hormones, antibiotics, antibodies and growth factors. 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 (6)

A serum-free medium (A) comprising an artificial polypeptide (P) having at least one cell adhesion minimal amino acid sequence (X) in one molecule. The serum-free medium according to claim 1, wherein the cell adhesion minimal amino acid sequence (X) is an Arg Gly Asp sequence. The serum-free medium according to claim 1 or 2, wherein the artificial polypeptide (P) is an artificial polypeptide further comprising at least one auxiliary amino acid sequence (Y) in one molecule. The artificial polypeptide (P) has a structure in which an amino acid sequence containing an intervening amino acid sequence (Z) and an auxiliary amino acid sequence (Y) are alternately chemically bonded to both ends of a cell adhesion minimal amino acid sequence (X). Item 4. A serum-free medium according to item 3. The serum-free medium according to any one of claims 1 to 4, wherein the content of the cell adhesion minimal amino acid sequence (X) is 1 nM to 1 mM with respect to the serum-free medium (A). A method for culturing cells, comprising using the serum-free medium according to any one of claims 1 to 5.