JPH04173086A - Carrier for culturing animal cell - Google Patents

Abstract

PURPOSE:To improve cell proliferation by covalently bonding a synthetic polypeptide containing amino acid residues such as cell adhesive factor arginine to specific particles through a polyethyleneimine placed therebetween. CONSTITUTION:A continuous foam, made of cellulose and having a threedimensional network structure with 0.3-2.0mm average pore diameter, 1.0-10.0m<2>/g specific surface area, >=97% porosity and 1.4-1.7g/cm<3> specific gravity is cut to provide a foam having 0.5-5.0mm particle diameter. An amino acid in which arginine (Arg), glycine (Gly) and aspartic acid (Asp) are linked from the N-terminal through peptide bonds according to the sequence expressed by formula I is subsequently contained to afford a synthetic peptide such as formula II (R1 and R2 are peptide chain composed of amino acid residue). A polyethyleneimine, selected as a spacer, having >=3000 molecular weight and expressed by formula III is then covalently bonded to a hydrophilic polymer which is the aforementioned foam. The above-mentioned synthetic peptide is further covalently bonded to the spacer terminal to prepare a carrier for culturing animal cells.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a carrier used for culturing animal cells. Generally, cell adhesion proteins contained in animal serum are required to adhere and spread animal cells on a solid surface for the purpose of culturing animal cells. The present invention relates to a carrier for culturing animal cells that enables immobilization.

[Conventional technology]

When culturing animal cells in large quantities for purposes such as producing physiologically active substances, most cells, with the exception of some cells that can grow in suspension, cannot grow unless they adhere to some kind of solid surface. For this reason, efforts are being made to find ways to increase the culture area on which cells can adhere and increase productivity. Polymers such as polysaccharides and polystyrene are used as culture carriers that have been devised in this way.
The microcarrier culture method using this carrier is one of the methods that allows the largest culture area per volume. However, since these polymers do not have sufficient ability to adhere and spread cells, improvements have been made such as coating the polymer with collagen or imparting a positive charge through chemical synthesis. Collagen has an amino acid sequence that allows cells to adhere to each other, and since the carrier is positively charged and the cell surface is negatively charged, an electrical adsorption phenomenon occurs.

[Problem to be solved by the invention]

To attach animal cells to a solid surface and to place them in a stretched state where they can proliferate.

- Serum is added in the injection method, but this method requires cell adhesion proteins such as fibronectin and vitronectin in serum that activate cell adhesion and spreading functions.

Polymers that have been given a positive charge through chemical synthesis cannot reach a stretched state in which cells can proliferate simply by attracting cells with electrostatic force, so cell adhesion proteins in serum are added to the polymer to attract cells using electrostatic force. It is necessary to create a state of chemical cell adhesion.

On the other hand, collagen-coated polymers provide adhesive properties to cells due to collagen's biological affinity for cells and the positive charge of collagen as a protein, but collagen may peel off from the carrier or dissolve in the culture medium. The problem remains that the process is completed. Cross-linking collagen by chemical methods can avoid detachment of collagen from polymers and dissolution in culture medium, but this has the drawback of inferior biocompatibility compared to untreated collagen. In addition, Ar, which is the active site of cell adhesion protein,
Although collagen has the amino acid sequence g-Gly-Asp, the three-dimensional structure resulting from the molecular structure of collagen makes it difficult for this active site to directly reach the binding site within the cell receptor, resulting in its adhesion. At present, the sexual activation effect is low, and as a result, it is necessary to add serum.

The use of serum for cell culture has the following problems.

(2) Serum for culture is expensive, and in the direct culture method, about 90% of the cost of the culture solution is serum-based.

■There are unexplained lot differences in serum, which may make it difficult to obtain reproducible results.

■Since serum is composed of various components such as proteins,
It is difficult to purify useful substances produced by cells from culture fluid.

As a method that does not use serum, cell adhesion and spreading can proceed to a certain extent by adding only cell adhesion proteins such as fibronectin and vitronectin, but cell adhesion proteins obtained by purification from serum are more effective than serum. It is far more expensive and its application to mass culture is impractical.

[Means to solve the problem]

In order to solve the above-mentioned problems, the present inventors have completed the present invention by proceeding with the practical application in view of the following knowledge.

Arg, which is common to proteins such as fibronectin, vitronectin, fibrinogen, laminin, and collagen,
The amino acid sequence Arg-Gly-Asp, in which three amino acids Gly and Asp are peptide-bonded in this order from the N-terminus, is the active site of a cell adhesion protein and has the function of activating cell adhesion and spreading functions. In other words, cells have receptors that control adhesion and spreading, and Arg
It is only when the amino acid sequence of -Gly-Asp acts specifically on the binding site within its receptor that cells begin to adhere to a solid surface and then spread. Therefore, if a synthetic peptide containing exactly the same amino acid sequence is present near a solid surface, cell receptors will bind to the synthetic peptide even in a culture medium without cell adhesion proteins, and the activated cells will be activated by this. , it can be directly attached to and stretched onto adjacent polymer surfaces.

The present invention applies this phenomenon to carriers for animal cell culture. The content is a spacer with a molecular weight of 3.0
00 or more is covalently bonded to a hydrophilic polymer, and a synthetic peptide containing the amino acid sequence of Arg-Gly-Asp is covalently bonded to the end of the spacer, allowing cells to adhere to the carrier and spread even in serum-free medium. It is used as a carrier for cell culture.

The spacer that is inserted when covalently bonding a synthetic peptide to a hydrophilic polymer is used to make it easier for the active peptide of Arg-Gly-Asp to bind to the binding site within the receptor, and to make it easier for cells to bind to the polymer after binding. This is to make it possible to take a position where it is easy to extend.

This is because the binding site within the receptor is located deep on the cell membrane, away from the adhesive area, and the distance from the adhesive area to the binding site is longer than the length of the synthetic peptide. In other words, the polymer itself cannot get close to the binding site, so if you just covalently bond a synthetic peptide directly to the polymer, the active site of the synthetic peptide will not reach the binding site, and even if it were able to bind to the receptor, cells would not be able to reach the binding site. Since the steric degree of freedom that allows for sufficient stretching is taken away, the adhesion and spreading functions of cells cannot be enhanced as a result. For this reason, it becomes necessary to insert a spacer between the synthetic peptide and the polymer.

Polyethyleneimine (1) is used for the spacer.

(-C2H,-N(-C,H,-NH,)-C,H,-
NH-)n(1) Polyethyleneimine is obtained by polymerization in the presence of an acid catalyst, and its structure has a highly branched dendritic structure, and its properties are clear and viscous water-soluble with cationic groups. Polymer. Polyethyleneimine is used as the spacer of the present invention because it improves cell adsorption due to the following characteristics and can prevent the spacer itself from being eluted into the culture medium. In order for peptides to approach cell receptors, it is necessary to first adsorb cells to a carrier, but polyethyleneimine has the highest cation density among existing polymer materials, so it has the ability to adsorb cells through electrostatic attraction. Moreover, since it is a water-soluble polymer, there is no repulsion with cells due to hydrophobic interaction, and it can easily come into contact with cells in the culture medium.

If the spacer polymer is a straight chain, if the molecular chain is broken due to damage, the end that is not bound to the carrier will elute into the culture medium, but polyethyleneimine molecules with a branched structure has a structure that makes it difficult to release because each of its multiple terminal groups is bonded to a carrier.

The molecular weight of this polyethyleneimine is 3 for the following reasons.
,000 or more. That is.

This is because if it is less than 3.000, the length of the molecule will be short, and a sufficient effect will not be obtained, similar to when a synthetic peptide is directly bonded to a polymer.

An open cellulose foam is used as the wood-philic polymer used as the support of the present invention. This foam is made by mixing viscose, which is made by chemically treating high-purity pulp made from wood, with a small amount of reinforcing material and crystals that form pores, injecting it into a mold, heating it, and then cooling it to solidify. It can be obtained by This material was selected as a carrier because the surface of cellulose exhibits strong hydrophilicity, so as long as it is used in an aqueous system, the surface of the support can behave in the same way as an aqueous solution.

The synthetic peptides used in the present invention include Arg, Gly, A
The three amino acids of sp are Arg-Gly from the N-terminus.
- Contains a peptide-bonded amino acid sequence in the order Asp.

It is an oligopeptide or a polypeptide. The general formulas of this synthetic peptide are (2), (3), and (4) below.

It can be expressed as either (5).

H-R1-Arg-Gly-Asp-R2-OH(2
) H-R, -Arg-Gly-Asp-OH (3) H-
Arg-Gly-Asp-R2-OH(4)H-Ar
g -Gly -Asp -OH (5) R and R2 are peptide chains consisting of one amino acid residue or multiple amino acid residues. The type of amino acid is not particularly limited, but the configuration of the amino acid is preferably the L configuration, which is the same as the configuration of natural amino acids, Arg-Gly-A
The bonding order of amino acids other than sp is not particularly limited. The total number of amino acids constituting the synthetic peptide shall be 3 or more.

〔Example〕

Some examples are shown below as examples of the active peptide-imparted carrier for culturing animal cells according to the present invention, but the present invention is not limited to these examples.

Example 1 1. Peptide synthesis The peptide was synthesized by the solid phase synthesis method proposed by Merrifield. This synthetic method involves binding amino acids to a support that is insoluble in organic solvents, and extending the peptide chain from there. In the synthesis, a Boa-amino acid with an α-amino group protected by a Boa group was used as the amino acid, and a 1-2% divinylbenzene crosslinked polystyrene having a chloromethyl group was used as a support insoluble in an organic solvent.

The synthesized peptide was roughly purified by gel filtration and then purified using high performance liquid chromatography to obtain peptide (6).

H-Gly-Arg-GLy-Asp-5ar-Guy
-DH(6)2, Preparation of carrier material The open cellulose foam was cut into a cubic shape with a side length of 1 inch to a size suitable for cell culture. (manufactured by Sakai Engineering) 3. Preparation of Spacer Polyethyleneimine with a molecular weight of 10,000 was used for the spacer.

4. Synthesis of active peptide-attached carrier The active peptide-attached carrier is produced by grafting polyethyleneimine onto cellulose foam using a crosslinking agent, and then linking the α-carboxyl group of the peptide and the amino group of polyethyleneimine with an acid amide bond using a coupling reagent. I got it by doing that. All reactions were carried out in polar organic solvents such as DMF and DMS○.

Example 2 The peptide (7) was synthesized, and the synthesis was carried out using the same spacer and carrier material as in Example 1 to obtain an active peptide-attached carrier.

H-Arg-Giy-Asp-OH (7) Example 3 A cellulose open cell foam cut into cubic shapes with a side length of 5 cm was used as a carrier material, and the same spacer and peptide were used according to Example 1. Synthesis was carried out to obtain an active peptide-attached carrier.

Example 4 Synthesis was carried out using polyethyleneimine having a molecular weight of 3,000 as a spacer and the same carrier material and peptide as in Example 1 to obtain an active peptide-attached carrier.

Comparative Example 1 An open cellulose foam made alkaline was reacted with 2-aminoethyl sulfate to obtain an open foam of aminoethyl cellulose, and a synthetic peptide similar to that in Example 1 (6
) was directly covalently bonded with a coupling reagent and used as a carrier for animal cell culture.

Comparative Example 2 Cytodsx3 manufactured by Pharmacia, in which the surface of cross-linked dextran beads was coated with collagen, was used as a carrier for animal cell culture.

Comparative Example 3 Pfeifer in which the surface of cross-linked dextran beads was substituted with N,N-diethylaminoethyl chloride hydrochloride
Ilormacall manufactured by & Langen was used as a carrier for animal cell culture.

Specific Examples Cell culture was carried out in a 5% fetal bovine serum medium and a serum-free medium using Examples 1 to 4 and Comparative Examples 1 to 3 as carriers for animal cell culture. Chinese hamster ovary cells were used as the culture medium, and a 1:1 mixture of Dulbecco's MEM medium and Ham's F-12 medium was used as the culture medium. Detailed procedures for the serum culture method and serum-free culture method are shown below.

1. Culture using serum (1) Add 25 μl of carrier to a 250-liter Bellco spinner flask, and add (, a34, N g
2+-free phosphate buffer (hereinafter referred to as PBS (-
) 1001111, 121℃, 3
Carry out autoclaving for 0 minutes.

(2) Discard PBS(-) and add serum medium 200-
After repeating this operation twice, the mixture was stirred at 37°C for 2 hours.

(3) Discard the medium and add 2 x 10'cells/l1
The carrier is seeded in 30 ml of serum medium containing cells at a concentration of llI in such a manner that the carrier is absorbed.

(4) After leaving at 37°C for 2 hours or more, add serum medium up to 250 d and culture with stirring at 37°C at a speed of 50 rpm.

2. Serum-free culture The serum medium from item 1 above does not contain serum and contains insulin,
Ethanolamine, transferrin.

Culture is performed in the same manner by replacing the cells with a serum-free medium containing substances that promote cell proliferation, such as selenium.

Example Results Table 1 shows the maximum cell density determined from the culture results.

As is clear from the results in Table 1, cells proliferated in the medium supplemented with serum, regardless of the carrier used.
Proliferation was observed only in Examples 1 to 4 in serum-free medium. In the case of the carriers of Comparative Examples 1 to 3, cell proliferation could not be confirmed in any serum-free medium.

Table 1 Cell density (number of cells/amount of culture solution cells/ml) [Effects of the invention] As explained above, the animal cell culture carrier of the present invention has a sufficiently long spacer extending to reach the binding site of the receptor. Furthermore, since the spacer has an active peptide that controls receptors involved in adhesion and spreading at the end of the spacer, cells can be attached and spread on the carrier not only in serum-containing medium but also in serum-free medium. Cells can be sufficiently proliferated by providing only the required amount of nutrients. Therefore, by using the carrier for animal cell culture of the present invention, cell culture without using serum becomes possible, and various problems caused by the addition of serum can be overcome.

Patent applicant: Sakai Engineering Co., Ltd. Dairin Beer Co., Ltd.

Claims (1)

[Claims] 1: Average pore diameter is 0.3 to 2.0 mm, specific surface area is 1.0
~10.0m^2/g, porosity is 97% or more, specific gravity is 1
．． A cellulose open foam with a three-dimensional network structure of 4 to 1.7 g/cm^3 was cut into particles with a particle diameter of 0.5 mm to 5.0 mm, and arginine (hereinafter referred to as Arg) and glycine (hereinafter referred to as Gly) were cut into pieces with a particle size of 0.5 mm to 5.0 mm. ), aspartic acid (hereinafter referred to as A
Arg-Gly-A, a cell adhesion factor consisting of
A synthetic peptide containing sp amino acid residues with a molecular weight of 3,
A carrier for animal cell culture, in which polyethyleneimine of 000 or more is interposed and covalently bonded to the above-mentioned foam.