JPH01309682A - Substrate for animal cell culture - Google Patents

Abstract

PURPOSE:To obtain the subject substrate having excellent adhesion in culture of an animal cell, safe sterilization operation and high safeness and free from contamination of antigen ingredient by covalently bonding an adherent peptide containing a specific amino acid as an essential ingredient to a polymer substrate. CONSTITUTION:(A) An adherent peptide synthesized by using arginine, glycine and aspartic acid, preferably of L-configuration as essential ingredients and preferably having <=3000 molecular weight is covalently bonded to (B) a polymer substrate such as cellulose, nylon. etc., utilizing, e. g., a crosslinking agent to provide the aimed substrate.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a substrate for culturing animal cells. - Animal cells used in boat fishing are adhesion-dependent cells and need to be attached or adhered to some kind of substrate when being cultured in large quantities, and the present invention relates to a cell culture substrate that has improved animal cell adhesion to the substrate. It is something.

[Prior Art] Conventionally, in order to obtain useful components such as physiologically active substances produced by animals, methods of producing them by mass culturing animal cells have been studied. However, in animal cell culture, except for special cells, adhesion-dependent cells that cannot proliferate unless they are attached to a substrate are commonly used. When these cells are made of polysaccharides or synthetic polymers as a polymer substrate, the actual situation is that the adhesion to the substrate is usually insufficient and the cells do not progress or proliferate smoothly. Therefore, in order to improve the adhesion of animal cells, a method of positively charging the surface of the substrate (Cytod
ex 1, Pharmacia microcarrier) and collagen coating method (Cyt.

dex3. Microcarriers manufactured by Pharmacia) have been used.

[Problems to be solved by the invention] However, among these conventionally used animal cell culture substrates, the substrates whose surfaces are positively charged do not have sufficient adhesive properties, and Substrates coated with collagen often suffer from peeling or denaturation during sterilization operations or mass culture operations.

In addition, the surface treatment material of the substrate uses components obtained from in vivo or their derivatives, and the condition for using the above animal cell culture substrate is that the medium contains fetal bovine serum (hereinafter referred to as FC5).
・After mass culture,
It was extremely difficult to purify only useful components from the culture solution.

[Means for Solving the Problems] In view of the above-mentioned problems, the present inventors have worked diligently to find a substrate for animal cell culture that provides good adhesion and allows easy purification of useful components when culturing animal cells. As a result of study, we have arrived at the present invention. That is, the present invention provides a substrate for animal cell culture that has improved adhesion of animal cells by covalently bonding an adhesive peptide having arginine-glycine-aspartic acid as an essential constituent unit to a polymer substrate for animal cell culture. be.

The following three adhesive peptides are used in the present invention.
one amino acid, namely arginine (hereinafter referred to as Arg)
, glycine (hereinafter referred to as G1y), and aspartic acid (hereinafter referred to as Asp) bonded as essential components. The type and the order of bonding are not particularly limited.

Examples include peptides having a molecular weight of 3,000 or less and having as a constituent component. Preferably, a peptide chain of the general formula % formula % (2) Y,,'/2:O or 1 to 30 amino acid residues in which serine (hereinafter referred to as Ser) is bonded to Ar5-Gly-Asp. The types of amino acids and the order of bonding are not particularly limited.

A peptide having a molecular weight of 3,000 or less and particularly preferably containing the hydrophobic amino acid proline (hereinafter referred to as Pro). (3) Z1$Z2: One or more Pro A peptide chain of 1 to 30 amino acid residues containing. The types of amino acids and the order of bonding are not particularly limited.

It is a peptide with a molecular size of less than 3,000 m. X4 in general formulas (1), (2), and (3), x
2, Y4, Y2.2, . The amino acids constituting the 22 amino acid residues are not particularly limited, and include the amino acids listed in Biochemical Data Book I P29 to P59 (Nippon Biochemical Kinsen, published by Tokyo Kagaku Dojin).

The amino acid used in the adhesive peptide according to the present invention may be either L-form or 0-form, but preferably L-form. In addition, since the constituent amino acid Pro imparts hydrophobic properties to the adhesive peptide, organic solvents such as DMF (dimethylformamide) can be used as a reaction solvent when bonding to a polymer substrate. The reaction efficiency can be improved compared to the reaction in an aqueous solution.

The molecular weight of the adhesive peptide according to the present invention is approximately 100%.

Usually less than 3,000. If it exceeds 3,000,
The molecular weight of the peptide used in the present invention was set to 3,000 or less since it has the potential to become an antigen and a molecular weight of 10,000 or more acts as a complete antigen.

Methods for synthesizing peptides include, but are not particularly limited to, liquid phase methods, solid phase methods, and synthesis methods using automatic synthesizers applying solid phase methods. For details on these synthesis methods, please refer to "Biochemistry Experiment Course/Protein Chemistry IV P.
2O7~P495 (Published by Nippon Biochemical Gold Line/Tokyo Kagaku Dojin), Continued Biochemistry Experiment Course/Chemistry of Proteins (Part 2) P
641-P694 (Nippon Seikagaku Kinsen, published by Tokyo Kagaku Dojin), etc.

Polymer substrates for creating animal culture substrates include conventionally used polysaccharides such as cellulose, dextran, and chitin: nylon, glass fiber, polyvinyl alcohol, polyester, polypropylene, polycarbonate, urethane resin, fluororesin, and silicone. Examples include synthetic polymers such as resins. These polymer substrates may be processed into molded products such as beads, foams, elastomers, films, porous membranes, hollow tubes, hollow fibers, and fibers. Among these, bead-shaped polymer substrates are preferred from the standpoint of efficiency in mass culture.

In order to use the adhesive peptide used in the present invention on a substrate for animal culture, it is necessary to covalently bond the peptide to the substrate. The bonding method is not particularly limited, but includes a synthetic method in which a hydroxyl group, an amino group, a carboxylic acid group, etc. on the surface of the substrate is bonded to an adhesive peptide using a crosslinking agent, and a reaction when there is no reactive functional group on the surface of the substrate. Examples include a synthetic method in which a sexual functional group is bonded to an introduced L-. For example, peptide bond synthesis method using cyanogen bromide, acid azide, water-soluble carbodiimide, etc.; diazo synthesis method using diazonium compound obtained by reacting an aromatic amino group introduced into a substrate with sodium nitrite; halogen Algylation method that utilizes acetyl derivatives and triazinyl derivatives; Ship base formation synthesis method that utilizes the reaction between aldehyde groups such as glutaraldehyde and amino groups of the substrate; carbosyl group, amine 7 group,
Ugi reaction synthesis method in which condensation is carried out in the coexistence of aldehyde groups and isonitrile groups; tresyl chloride synthesis method using tresyl ester; Synthetic method using active ester group; dimethylsuberoimidate dibasic acid, methyl-4
- Synthesis method using imide ester groups such as mercaptobutyrimidate hydrochloride and methyl-4-azidobenzimidate hydrochloride; Synthesis method using maleimide groups such as p-phenylene bismaleimide; -Synthetic method activated with carbonyldiimidazole. The above synthetic method can be carried out in an aqueous solution or in a polar organic solvent such as DMF or pyridine. Preferred solvents are polar organic solvents.

Methods of bonding a crosslinking agent to a polymer substrate using a crosslinking agent include a method of directly bonding a crosslinking agent to a polymer substrate, and a method of grafting polyethylene glycol, polypropylene glycol, etc. to a polymer substrate and bonding the above crosslinking agent to the terminal thereof. There are several methods.

The method for using the cell culture substrate in culturing animal cells is not particularly limited and may be carried out in a conventional manner. for example,
A method in which animal cells are cultured by adhering to the bead surface by suspending beads treated with covalently bonded adhesive peptides in a culture medium and stirring at low speed; A method of culturing animal cells on a bottle, roller bottle, etc.; A method of culturing animal cells by circulating the culture solution through a hollow fiber that has been treated with an adhesive peptide to covalently bond it to the inner surface of the hollow fiber; Examples include a method using a column filled with treated beads; a method using a multi-disc or multi-tray treated with covalently bonded adhesive peptides, and the like.

Animal cells related to the present invention are not particularly limited, but include:
Examples include cells that produce useful components such as physiologically active substances. Examples include cells from various animal tissues, such as epithelial cells derived from amnion or kidney that produce interferon; renal cells that produce urokinase; and cells of pancreatic origin that produce insulin. It is also possible to inoculate and culture the varicella virus or hepatitis virus using the cultured cells as hosts for the production of vaccines and the like.

[Examples] Hereinafter, the present invention will be further explained with reference to Examples, but the present invention is not limited thereto.

Production Example 1 Synthesis of adhesive polypeptide Automated peptide synthesis by Merrifield method 14
Synthesis was performed using i. Boa for protection of α-amino group
Adhesive synthetic peptides showing a single peak on HPLC (high performance liquid chromatography) after purification by Sephadex gel, side-cellulose ion exchange chromatography, and partition chromatography.
I got it.

Table 1 Adhesive Synthetic Peptide Example 1 After reacting crosslinked dextran beads with hexamethylene diisocyanate in a DMF solvent, the isocyanate groups in the side chains were hydrolyzed and aminated.

Next, the amino groups were activated with di-N-hydroxysuccinimidyl perate (DSS) and reacted with the above adhesive synthetic peptide-1 in a buffer solution to obtain a substrate for animal cell culture.

Example 2.3 Adhesive synthetic peptide-2,3 to cross-linked dextran beads
were reacted according to Example 1 to obtain a substrate for animal cell culture.

Example 4 The surface hydroxyl groups of cross-linked dextran beads were activated with N,N'-carbonyldiimidazole and reacted with the above adhesive synthetic peptide-3 in a DMF solvent to obtain a substrate for animal cell culture.

Example 5 Cross-linked dextran beads were reacted with adhesive synthetic peptide-4 according to Example 4 to obtain a substrate for animal cell culture.

Comparative Example 1 The surface of cross-linked dextran beads was
) Cytodex 2 (manufactured by 7 Almasia) substituted with a charged group
was used as a substrate for animal cell culture.

Comparative Example 2 Cytodex 3 (manufactured by Pharmacia), in which the surface of cross-linked dextran beads was treated with denatured collagen (MW 60,000 to 200,000) obtained from pig epidermis, was used as a substrate for animal cell culture.

Test Example 1 (1) Immobilization Density of Adhesive Synthetic Peptide The immobilization density of Example 3.4 in which adhesive synthetic peptide-3 was immobilized was measured, and differences due to reaction conditions were investigated. When the ratio of carbon atoms and nitrogen atoms on the surface of the polymer substrate was measured, it was found that DM
Example 4, in which the reaction was carried out in the solvent F, had a higher immobilization density of 70
% increase.

(2) Evaluation of adhesion and proliferation of animal cells Cell culture was performed using the animal cell culture substrates prepared in Examples 1 to 5 and Comparative Example 1.2. The cells used were vascular endothelial cells, and the culture medium was Dulbecco's Modified Eagle.
e's Medium (hereinafter referred to as DMEM),
A culture solution prepared by adding 1Ox of Fe2 to DMEM was used.

After culturing, adhesion and proliferation were observed using a phase contrast microscope and a scanning electron microscope, and the results are shown in Table 2.

Table 2 Adhesion and proliferation of animal cells ○: Good, △: Slightly poor, ×: Poor C used for comparison
The cell adhesion and proliferation of ytodex-2 and 3 in Fe2-free DMEM medium were inferior to those of the substrates of Examples. Although it is improved by adding Fe2,
Removing the FC5 component from the medium required repeated gel filtration, and it was difficult to remove it completely.

[Effects of the Invention] The animal cell culture substrate of the present invention has the following effects because the adhesive peptide having Arg-Gly-Aspe as an essential constituent unit is covalently bonded to the polymer substrate. (2) Good adhesion of living cells to the polymer substrate is achieved even without the addition of fetal calf serum (hereinafter referred to as FC5) containing cell adhesive peptides such as fibronectin. ■Culture with cell adhesion can be performed well even without the addition of FCS, so when obtaining physiologically active pharmacological components by mass culturing cells using the animal cell culture substrate of the present invention, antigens contained in FC5 can be used. This eliminates the possibility of contamination with potentially dangerous ingredients, increasing safety for patients. Furthermore, since the adhesive peptide used in the present invention has a low molecular weight, the possibility that it itself becomes an antigen is reduced, further increasing safety. (2) Since the adhesive peptide of the present invention is covalently bonded to the substrate, it does not fall off from the substrate and can be safely sterilized. (2) Adhesive peptides containing Pro can react in organic solvents, and the reaction efficiency is significantly improved.

Claims (1)

[Claims] 1. An animal cell culture substrate with improved animal cell adhesion, which is obtained by covalently bonding an adhesive peptide having arginine-glycine-aspartic acid as an essential constituent unit to a polymer substrate.