JPS63279785A - Substrate for cell culture - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a substrate for cell culture. More specifically, the present invention relates to a cell culture substrate used for culturing animal cells.

<Prior art and problems to be solved by the invention> In recent years,
BACKGROUND OF THE INVENTION Research is actively being carried out to cultivate biological cells and produce useful physiologically active substances, such as vaccines, hormones, and interferons, through the metabolic activities of the cells.

In such methods, adherent animal cells have conventionally been cultured using glass or plastic petri dishes, test tubes, culture bottles, and the like.

Recently, attempts have been made to use microcarriers and hollow fibers as culture substrates to achieve higher-density culture and longer-term culture. In order to allow adherent animal cells to adhere and proliferate on a culture substrate, it is necessary to have good adhesion between the cells and the surface of the substrate, as well as the morphology and arrangement of the adhered cells.
It is necessary to have a form that is effective for cell expansion and proliferation.

However, although the polymer materials conventionally used as substrates for cell culture have excellent shapeability and durability, they are inadequate in terms of adhesive properties, etc., and are difficult to perform high-density and long-term cell culture. However, none of them have been able to achieve sufficient results.

In order to improve this point, we have developed methods that coat collagen, which is a biopolymer, and gelatin, which is a modified product of collagen, on a polymer material (see Japanese Patent Application Laid-open No. 71884/1984), and coated soluble fibroin on a polymer material. A cell culture bed in which a crosslinked body is formed (see Japanese Patent Laid-Open No. 61'-52280) has been proposed.

However, when collagen is used as the natural polymer material in the cell culture substrate made by combining the above-mentioned polymer material with a natural polymer, cell adhesion and proliferation can be improved; There are problems in that it is difficult to handle and lacks reproducibility because it has very low solubility under acidic conditions where it has good stability and is susceptible to denaturation at room temperature. Furthermore, when other natural polymer materials are used, there is a problem that the adhesion of cells and the spreadability, proliferation and activity maintenance of adhered cells are still insufficient.

Purpose This invention was made in view of the above problems, and it is easy to handle, has good adhesion to cells, cell spreading,
The purpose of the present invention is to provide a cell culture substrate that exhibits excellent growth and activity maintenance and enables high-density, long-term culture.

Means and Effects for Solving the Problems> The cell culture substrate of the present invention, which has been made to achieve the above object, has a structure in which the side chain amino groups are replaced by guanidyl groups on the surface of the polymer substrate. It is characterized by supporting converted gelatin (hereinafter referred to as guanidylated gelatin).

This invention consists of the above-mentioned structure. Gelatin is a type of derived protein made by boiling collagen with water to irreversibly make it water-soluble, and guanidylated gelatin is produced by converting the amino group in the side chain of gelatin to a guanidyl group. It has the characteristics of high solubility in water, good stability, and excellent affinity with cells. In other words, the structure of the cell membrane on the cell surface is such that various glycoproteins, glycolipids, etc., called intramembrane particles, are embedded in a lipid bilayer in a distributed manner, and these particles freely move inside the lipid bilayer. It is involved in cell adhesion. The above-mentioned guanidylated gelatin has a site capable of bonding to intramembrane particles through ionic bonding, hydrophobic bonding, etc., so it has high affinity with cells and can maintain cells in a stable shape and arrangement. Therefore,
The cell culture substrate of the present invention, which supports guanidylated gelatin, has excellent cell adhesion as the guanidylated gelatin plays a role in connecting cells and the substrate, and the cells are stable without damaging their higher order structure. Since the cells can be attached in a certain form and arrangement, the spread and proliferation of the cells will not be harmed.

Note that the polymer base material is preferably one that has been surface-treated chemically or physically, or a porous material. Further, it is preferable that the base material is a hollow fiber. If the surface of the polymer base material is chemically or physically treated, the wettability will be improved, and the adhesion between the guanidylated gelatin and the base material will be excellent. When the material is a porous material, substance metabolism becomes easy through the pores of the porous material, and cells can be cultured for a long period of time. In particular, when the polymer base material is a hollow fiber, it is possible to grow and proliferate cells on the hollow fiber at high density by perfusing a culture solution, etc. inside the hollow part or outside of the hollow fiber. Can be done.

Furthermore, it is preferable that the guanidylated gelatin is partially supported on the base material, and in particular, it is preferable that the guanidylated gelatin is partially supported on the base material.
Those supported in a polka dot pattern or the like are preferred. Guanidylated gelatin is partially supported on the base material,
In particular, when guanidylated gelatin is supported in a checkered pattern, striped pattern, polka dot pattern, etc., the position of the adhering cells can be adjusted, and the cells adhere at predetermined intervals, making the adhesion with the cells even more stable. , cell spreading and proliferation can be further increased.

The present invention will be explained in detail below.

The cell culture substrate of this invention consists of a polymeric substrate and guanidylated gelatin supported on this substrate.

The above-mentioned guanidylated gelatin can be easily obtained by reacting commercially available gelatin with a guanidylating agent such as O-methylisourea or 1-guanyl-3,5-dimethylpyrazole in the presence of a basic compound. This reaction is usually carried out in a solvent, and water, hydrous alcohol, etc. are used as the solvent. As the basic compound, alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates or hydrogen carbonates such as sodium carbonate, potassium carbonate, and sodium hydrogen carbonate are used. The reaction is carried out at room temperature to heating, and after the reaction, reprecipitation,
Guanidylated gelatin can be separated and purified by a conventional method such as dialysis. The amount of guanidyl groups introduced into the guanidylated gelatin used in this invention is not particularly limited, and gelatins with various degrees of substitution can be used. Further, the amount of guanidyl group introduced can be adjusted by appropriately selecting the amount of guanylating agent used, reaction time, etc.

The guanidylated gelatin thus obtained has high solubility in water, excellent stability, and is easy to handle.

Furthermore, as the material for the polymer base material, any material can be used as long as it has formability and mechanical strength.
For example, olefin polymers such as polyethylene, polypropylene, chlorinated polyethylene, and ionomers, fluorine resins such as polytetrafluoroethylene and polyvinylidene fluoride, styrene resins such as polystyrene, acrylic resins such as polymethyl methacrylate, and polyvinyl alcohol. , polyester resins such as polyvinyl acetate, polyvinyl acetal, polyacrylonitrile, polyvinyl chloride, polyvinylidene chloride, polycarbonate, polyarylate, polyphenylene oxide, polyethylene terephthalate, polybutylene terephthalate, epoxy resins, polyamides, polyimides, polysulfones, cellulose resins Examples include various polymers or copolymers such as silicone resin, polyurethane, or blends thereof.

The above-mentioned polymeric base material can be formed into various forms, such as molded products such as petri dishes and flasks, as well as forms such as films, tubes, hollow fibers, fibers, and fine particles. Among these forms, -9= For long-term cell culture, porous polymer substrates with pores that facilitate material metabolism are preferred, and for high-density culture, tubes, hollow A thread shape is preferred. Particularly preferred is a hollow fiber made of a porous polymer base material, which is easy to metabolize and can be cultured at high density for a long period of time. When using this hollow fiber, cells are grown on the hollow fiber by perfusing the culture solution into the hollow part or outside of the hollow fiber, and by sending carbon dioxide gas, air, etc. into the hollow part of the hollow fiber as necessary. It can be cultivated and multiplied. Note that the hollow fibers can be of various sizes; for example, the hollow fibers may have an inner diameter of 50 mm.
A thickness of about 1000 μm is used.

Further, when the cell culture substrate of the present invention is used as a bead carrier in a microcarrier method, the polymer substrate used has a particle size of about 100 to 300 μm.

The polymer base material may be untreated, but may be physically or chemically treated to improve adhesion to guanidylated gelatin. The above treatments include plasma treatment, ultraviolet light (including laser), electron beam,
Amino acids that undergo radiation treatment by irradiation with alpha, beta, or gamma rays, ion treatment by ion sputtering, ozone treatment in an ozone atmosphere, or oxidation, nitration, and reduction with hydrogen peroxide, potassium persulfate, chromate, etc. Chemical treatments such as introduction of groups and sulfonation with chlorosulfonic acid improve the wettability of the guanidylated gelatin with the substrate and improve adhesion.

In addition, among the above surface treatments, laser treatment can not only chemically modify the base material, but also physically modify the base material surface by processing it into minute irregularities, which can improve cell metabolism. The advantage is that it can be promoted.

The cell culture substrate of the present invention is obtained by supporting guanidylated gelatin on a polymer substrate. For example, if necessary, a physically or chemically pretreated polymer substrate is It can be obtained by immersing the base material in a solution containing gelatin or by applying the solution onto the base material and then drying it.

The above-mentioned guanidylated gelatin may be supported in either a monomolecular layer or a multimolecular layer, and may be supported on the entire surface of the base material, but it is preferable to support it partially on the surface of the base material, especially in a patterned manner. Preferably, by supporting the cells in a patterned manner, the arrangement of the cells adhering to the substrate can be controlled, and the adhesion of the cells can be stabilized, which can be advantageous for cell spreading, proliferation, and functional expression. . Furthermore, when guanylated gelatin is partially supported as described above, in particular, by supporting it in a fine pattern such as a lattice pattern, a striped pattern, a polka dot pattern, etc., a useful surface that can further enhance the above effects is formed. Can be done. Guanidylated gelatin can be patterned and supported on the base material by, for example, applying a technique such as screen printing.

The cell culture substrate of the present invention can be used for culturing various cells, and the type of cells is not particularly limited, but epithelial cells are particularly exemplified.

Furthermore, when culturing animal cells using the cell culture substrate of the present invention, various culture solutions are used depending on the type of cells to be cultured, and conditions such as optimal temperature and pH suitable for cell proliferation are used. Culture is carried out in

<Examples> The present invention will be described in more detail below based on examples. In addition, guanidylated gelatin was prepared by the method of the following reference example.

Reference example Distilled water 1 adjusted to pH 11 using sodium hydroxide
0.2 g of gelatin and 0.2 g of O-methylisourea are dissolved in 5 ml at 40°C. Thereafter, the gelatin was guanidylated by reacting at room temperature for 3 days. Next, the reaction solution was charged into a dialysis membrane and dialyzed in distilled water to obtain a guanidylated gelatin solution.

Example 1 and Comparative Examples 1 to 3 The guanidylated gelatin solution obtained in the above reference example was poured into a polystyrene Petri dish so as to cover the entire surface of the Petri dish, and was dried overnight under an ultraviolet lamp on a clean bench at a distance of about 300 I11. , a Petri dish coated with guanidylated gelatin was obtained.

On the other hand 1. As a comparative example, gelatin was mixed with the above guanidine = 1
3- Petri dish obtained by processing in the same manner as above using a solution adjusted to the same concentration as the gelatin solution (Comparative Example 1), a commercially available surface-treated polystyrene petri dish for cell culture (Comparative Example 2), and untreated Polystyrene petri dish (comparative example 3)
was used.

In each of these petri dishes, Ch of human normal hepatocytes was added to a culture solution containing Eagle MEM solution and 10% beef tallow serum.
A suspension of ang Liver cells adjusted to a concentration of 5 x 104 cells/ml was injected, and then incubated at 37°C for 1 hour in a carbon dioxide incubator with an atmosphere of 5% carbon dioxide and 95% air. Next, the culture solution in the Petri dish was discarded, and Ca++ and Mg
After thoroughly washing with a phosphate buffer that does not contain ++ to remove non-adherent cells, the adherent cells are detached with a solution containing trypsin-EDTA, and the number of adherent cells is determined using a hemocytometer to determine the cell adhesion rate. The results obtained are shown in the table below.

= 14- Example 2 A plurality of each of the petri dishes used in Example 1 and Comparative Examples 1 to 3 above were prepared, and changed in the same manner as in Example 1.
Liver cells were cultured, and the number of cells that adhered to the culture vessel and proliferated was measured over time using a hemocytometer to examine cell proliferation. The results are shown in the attached drawings.

As is clear from the above table and the accompanying drawings, the Petri dish coated with guanidylated gelatin showed excellent cell adhesion and proliferation.

Example 3 A porous tetrafluoroethylene resin film (Fluoropore FP-022, manufactured by Sumitomo Electric Industries, Ltd.) was subjected to plasma treatment in an ammonia gas atmosphere. The discharge output for this 15-hour period is 100W, and the pressure is 0.3 Torr.

The ammonia gas flow rate was 7 cc/min, and the treatment time was 10 minutes. The treated film was set in a 45InIllφ glass shear dish and sterilized with high pressure steam, followed by adding a guanidylated gelatin solution and drying to coat the film with guanidylated gelatin. Next, a suspension of Chang Liver cells derived from human normal hepatocytes (5×104 cells/m in Eagle MEM culture medium supplemented with 10% tallow serum)
1) was added to a Petri dish and cultured for 6 days in an air atmosphere containing 5% carbon dioxide at a temperature of 37°C. After culturing,
The average number of cells is 6.5. Good proliferation was observed at 105 cells/ml.

<Effects of the Invention> As described above, according to the cell culture substrate of the present invention, guanidylated gelatin is supported on the surface of the polymer substrate, and the guanidylated gelatin has high solubility and stability. Because of its good properties, it is easy to handle, and because it has excellent affinity with cells and can increase adhesiveness, it has the unique effect of allowing high-density and long-term cell culture. Therefore, the cell culture substrate of the present invention can be used in a system for producing useful products such as hormones by culturing animal cells, and can also form an artificial pancreas by, for example, attaching and culturing insulin-producing cells to the surface of the substrate. It can be used to construct artificial organs.

[Brief explanation of drawings]

The attached drawings are diagrams showing cell proliferation properties of cell culture substrates of the present invention and comparative examples.

Claims (1)

[Claims] 1. On the surface of the polymer base material, side chain amino Gelatin with groups converted to guanidyl groups A cell characterized by carrying Substrate for culture. 2. The polymer base material is chemically or physically exposed. Claim No. 1, which is surface-treated The cell culture substrate according to item 1. 3. The above feature where the polymer base material is a porous material. Claims 1 or 2 Substrate for cell culture. 4. The above patent application in which the polymer base material is a hollow fiber Any of the first to third terms of the scope of the request. The cell culture substrate described in . 5. On the surface of the polymer base material, side chain amino Gelatin with groups converted to guanidyl groups The above patent claim is partially claimed by In any of the ranges 1 to 4 of The described cell culture substrate. 6. Side chain amino group converted to guanidyl group The gelatin is supported in a checkered pattern. As stated in claim 5 above, Substrate for cell culture. 7. Amino group in side chain converted to guanidyl group gelatin is supported in a striped pattern. The details set forth in claim 5 above Substrate for cell culture. 8. Side chain amino group converted to guanidyl group The gelatin is supported in a polka dot pattern. As stated in claim 5 above, Substrate for cell culture.