JPS63196278A - Substrate for cell culture - Google Patents

Abstract

PURPOSE:To obtain a substrate for cell culture excellent in adhesion with a cell and distension and proliferation of the cell and capable of cultivating the cell in high density for long term, by carrying a fibrin on the surface of a high polymer based substrate. CONSTITUTION:The surface of a substrate for cell culture consisting of a high polymer material such as olefinic polymer, polyester resin, etc., and preferably shaped into porous or tubular form or form of hollow fiber, etc., is pretreated, preferably physically or chemically. Then a fibrin is carried wholly or partially, e.g. in checked pattern, striped pattern, polka dots, etc., on the surface of the substrate.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application 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.

<Problems to be solved by conventional techniques and inventions> In recent years, there has been active research into culturing biological cells and producing useful physiologically active substances, such as vaccines, hormones, and interferons, through the metabolic activities of the cells. It is being done.

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 unsuitable in terms of adhesive properties, etc., and cannot be used for 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, cell culture substrates made by combining natural polymers with the above-mentioned polymeric materials have the problem that cell adhesion and adhesion, proliferation, and activity maintenance of adhered cells are still insufficient. .

Purpose This invention was made in view of the above-mentioned problems, and is a cell culture product that has excellent adhesion with cells, as well as cell spreading, proliferation, and maintenance of activity, and enables high-density, long-term culture. The purpose is to provide a base material.

Means and operation for solving the above problems> The cell culture substrate of the present invention, which has been made to achieve the above object, has fibrin supported on the surface of the substrate made of a polymeric material. It is characterized by:

Note that the above-mentioned polymeric 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. Furthermore,
It is preferable that fibrin be partially supported on the base material, particularly preferably in a checkered pattern, striped pattern, polka dot pattern, etc.

In the above structure, fibrin is a fibrous glycoprotein produced by the action of the enzyme thrombin on fibrinogen present in the blood plasma of an animal, and has the property of having 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.
These can move freely within the lipid bilayer and are involved in cell adhesion. The above-mentioned fibrin has a site that can bind to intramembrane particles through ionic bonds, hydrophobic bonds, etc., so it has a high affinity with cells and maintains a stable form of cells.
Can be held in place.

Therefore, the fibrin-supported cell culture substrate of the present invention has excellent cell adhesion as the fibrin plays a role in connecting the cells and the substrate, and the cells maintain a stable morphology without damaging their higher order structure. Since the cells can be attached to each other in the same manner, the spread and proliferation of the cells will not be impaired. Furthermore, fibrin has the effect of promoting cell proliferation, resulting in good results.

It should be noted that the polymer material whose surface has been chemically or physically treated has excellent adhesion between fibrin and the base material. Further, when the polymeric material is a porous material, substance metabolism is facilitated through the pores of the porous material, and cells can be cultured for a long period of time. In particular, when the base material made of the polymeric material is a hollow fiber, cells can be grown and multiplied at high density on the hollow fiber by perfusing a culture solution or the like into the hollow portion or outside of the hollow fiber. can.

In addition, when fibrin is partially supported on the base material, especially when fibrin is supported in a checkered pattern, striped pattern, polka dot pattern, etc., the position of the adhering cells can be adjusted, and the cells can be placed in a predetermined position. Since the cells adhere to each other with a spacing of , the adhesion with the cells is further stabilized, and the spreading and proliferation of the cells can be further increased.

The present invention will be described in detail below.

The cell culture substrate of the present invention consists of a substrate made of a polymeric material and fibrin supported on this substrate.

As the above-mentioned polymeric material, any material can be used as long as it has formability and mechanical strength. For example, olefinic polymers such as polyethylene, polypropylene, chlorinated polyethylene, and ionomers, polytetrafluoroethylene, and Fluorine resins such as vinylidene chloride, styrene resins such as polystyrene, acrylic resins such as polymethyl methacrylate, polyvinyl alcohol, polyvinyl acetate, polyvinyl acetal, polyacrylonitrile, polyvinyl chloride, polyvinylidene chloride, polycarbonate, polyarylate, Polyester resins such as polyphenylene oxide, polyethylene terephthalate, polybutylene terephthalate, epoxy resins, polyamides, polyimides, polysulfones, cellulose resins,
Examples include various polymers or copolymers such as silicone resins and polyurethanes, or blends thereof.

The base material made of the above-mentioned polymeric material can be formed into various shapes, including molded products such as petri dishes and flasks, as well as films, tubes, hollow fibers, fibers, and fine particles. Among these forms, for long-term cell culture, porous polymeric materials with pores that facilitate material metabolism are preferable, and for high-density culture, tubes and hollow fibers are preferable. suitable. Particularly preferred are hollow fibers made of porous polymeric materials that are 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, an inner diameter of 50 to 1000 μm.
A certain degree is used.

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

The base material made of the above-mentioned polymeric material may be untreated, but may be physically or chemically treated to improve adhesion to fibrin. The above treatments include plasma treatment, radiation treatment by ultraviolet rays (including lasers), electron beams, alpha rays, beta rays, gamma rays, etc., ion treatment by ion sputtering, ozone treatment in an ozone atmosphere, or peroxidation. Chemical treatments such as oxidation with hydrogen, potassium persulfate, chromate, etc., introduction of amino groups that nitrate and reduce, and sulfonation with chlorosulfonic acid improve the wettability of fibrin and the base material and improve adhesive properties. will improve. 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.
It has the advantage that it can also promote cellular metabolism.

The cell culture substrate of the present invention is obtained by supporting fibrin on a substrate made of a polymeric material. Since fibrin is insoluble in water and saline solutions, the method for supporting it is, for example, adsorbing fibrinogen onto a physically or chemically pretreated polymeric material as required, and then adding it to thrombin or blood coagulation factors. Examples include a method of converting into fibrin using χm. At this time, if a highly concentrated fibrinogen solution is used, a gel will be formed. Then, it is obtained by drying.

The fibrin 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 may be supported partially on the surface of the base material, particularly 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 fibrin is partially supported as described above, especially in the form of a lattice,
By supporting 1 in a fine pattern such as a striped pattern or a polka dot pattern, a useful surface that can further enhance the above effects can be formed. Fibrin can be patterned and supported on the base material by applying a technique such as screen printing, for example.

The cell culture substrate of the present invention can be used to culture various cells, and the type of cells is not particularly limited, but fibroblasts 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.

Examples and Comparative Examples 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 at this time is 50 W, the pressure is 0.2 Torr, and the ammonia gas flow rate is 5 cc/
The discharge time was 15 minutes. The treated film was set in a 45m5φ glass petri dish, and after high-pressure steam sterilization, human plasma fibrinogen (manufactured by Sigma, type l
) was added to the phosphate buffer solution (4 mg/ml), and thrombin (manufactured by Sigma) was added to the solution at 37°C.
Fibrin gel was formed on the film surface by allowing it to act for 0 hours. All these operations were performed aseptically.

Next, a suspension of mouse fibroblasts L929 (4X 10
' cells/ml in Eagle's MEM medium supplemented with 10% tallow baby serum) was added to a petri dish, and cultured for 60 hours in an environment of 5% carbon dioxide gas, 95% air atmosphere, and 37°C. After culturing, the average number of cells is 5.8 x 104 cells.
/ml, and good proliferation was observed.

On the other hand, as a comparative example, the porous film used in the above example was set in a 45 mmφ glass petri dish, sterilized with high pressure steam, and then cultured in the same manner as in the example. The average number of cells after culturing was 4.4X10'col Is/
It became ml.

<Effects of the Invention> As described above, according to the cell culture substrate of the present invention, fibrin, which is a glycoprotein, is supported on the surface of the polymer substrate, and fibrin has the effect of promoting cell proliferation. It has excellent affinity with cells and can enhance adhesiveness. Furthermore, since cells can be adhered in a form and arrangement suitable for expansion and proliferation, high-density and long-term cell culture is possible. Furthermore, in the case of substrates in which fibrin is partially supported in a patterned manner, the adhesion position of cells can be controlled, which has the unique effect of further increasing the spread and proliferation of adhered cells. . 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.

Claims (1)

[Scope of Claims] 1. A substrate for cell culture, characterized in that fibrin is supported on the surface of the substrate made of a polymeric material. 2. The cell culture substrate according to claim 1, wherein the polymer material is chemically or physically surface-treated. 3. The cell culture substrate according to claim 1 or 2, wherein the polymeric material is a porous material. 4. The cell culture substrate according to any one of claims 1 to 3, wherein the substrate is a hollow fiber. 5. The cell culture substrate according to any one of claims 1 to 4, wherein fibrin is partially supported on the surface of the substrate. 6. The cell culture substrate according to claim 5, wherein fibrin is supported in a lattice pattern. 7. The cell culture substrate according to claim 5, wherein fibrin is supported in a striped pattern. 8. The cell culture substrate according to claim 5, wherein fibrin is supported in a polka dot pattern.