JPS63196275A - 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 vitronectin 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 of form of hollow fiber, etc., is pretreated, preferably physically or chemically. Then vitronectin 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 <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.

<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 adhere and proliferate adherent animal cells on a cultured 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. 81-52280) 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 action for solving the above problems> The cell culture substrate of the present invention, which has been made to achieve the above object, has vitronectin 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 vitronectin is partially supported on the substrate, particularly preferably in a checkered pattern, striped pattern, polka dot pattern, etc.

In the above configuration, vitronectin is a glycoprotein with a molecular weight of 75-Da that exists in plasma, platelets, kidneys, muscles, etc., 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, and these particles freely move inside the lipid bilayer. It is involved in cell adhesion. The vitronectin has a site where it can bind to intramembrane particles through ionic bonds, hydrophobic bonds, etc., so it has a high affinity with cells and can maintain cells in a stable shape and arrangement. Therefore, in the cell culture substrate of the present invention, in which vitronectin is supported, vitronectin plays a role of connecting cells and the substrate, and it has excellent cell adhesion and maintains a stable form of cells 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. In particular, vitronectin functions in adhesion with fibroblasts.

It should be noted that the above-mentioned polymer material whose surface has been chemically or physically treated has improved wettability and excellent adhesion between vitronectin and the base material. Moreover, the above polymer material is
When the material is porous, 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 vitronectin is partially supported on the substrate, especially when vitronectin 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 Since adhesion occurs at predetermined intervals, adhesion with cells is further stabilized, and cell spread and proliferation can be further increased.

The present invention will be explained in detail below.

The cell culture substrate of the present invention consists of a substrate made of a polymeric material and vitronectin 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, Examples include various polymers or copolymers, or blends thereof, such as polyester resins such as polyphenylene oxide, polyethylene terephthalate, and polybutylene terephthalate, epoxy resins, polyamides, polyimides, polysulfones, cellulose resins, silicone resins, and polyurethanes.

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 μm.

The base material made of the above-mentioned polymeric material may be untreated, but in order to improve adhesion with vitronectin,
It may be physically or chemically treated. 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 vitronectin with the substrate and improve its adhesion. 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, 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 vitronectin on a substrate made of a polymeric material. It can be obtained by immersing the base material in a solution containing the base material or by applying the solution onto the base material and then drying the base material. At this time, it is preferable to dry under conditions that do not easily denature vitronectin.

The vitronectin may be supported in either a monomolecular layer or a multimolecular layer, and may be supported on the entire surface of the substrate, but it is preferably supported partially on the surface of the substrate, particularly in a patterned manner. By patterning and supporting the cells in this manner, it is possible to control the arrangement of cells adhering to the substrate, which in turn stabilizes cell adhesion and enhances cell spread, proliferation, and functional expression. Furthermore, when vitronectin is partially supported as described above, a useful surface that can further enhance the above effects can be formed, particularly by supporting it in a fine pattern such as a lattice pattern, a striped pattern, a polka dot pattern, etc. . To pattern and support vitronectin on the substrate, for example, a technique such as screen printing can be applied.

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.

Example 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 was 100 W,
Pressure is 0.3 Torr % ammonia gas flow rate is 7 C
C/min, and the treatment time was 10 minutes.

The treated film was placed in a 45 mmφ glass petri dish and sterilized using high-pressure steam, followed by a phosphate buffer solution (concentration 10 μg/111) of vitronectin (manufactured by Calbiochem).
was added and allowed to stand for 10 minutes, then excess liquid was removed and vitronectin was fixed on the surface by drying at room temperature.

All of the above operations were performed in a sterile box.

Next, a suspension of mouse fibroblasts L929 (4 x 10'
cells / 11. In Eagle's MEM medium supplemented with 10% tallow serum) 41! was added to a petri dish and cultured for 6 days in an environment of 5% carbon dioxide gas and 95% air at a temperature of 37°C. After culturing, the average number of cells is 7.2 x 104 cells.
Good growth was observed at s/111.

Comparative Example The porous film used in the above example was used as it was for 45 m.
It was set in an mφ glass petri dish, and after high-pressure steam sterilization, the same culture test as in the example was conducted. Number of cells after culture, 4.
The result was 4 x 10' cells/111.

<Effects of the Invention> As described above, according to the cell culture substrate of the present invention, vitronectin, which is a glycoprotein, is supported on the surface of the polymeric substrate, and vitronectin has a high affinity with cells. It has excellent properties and can increase adhesion, as well as allow cells to adhere in a form and arrangement suitable for expansion and proliferation.
High-density and long-term cell culture is possible. Furthermore, if the substrate is partially supported with vitronectin in a patterned manner, the adhesion position of the cells can be controlled.
It 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.

Patent applicant: Sumitomo Electric Industries, Ltd. Representative: Patent attorney Hirokatsu Kamei (and 3 others)

Claims (1)

[Claims] 1. On the surface of the base material made of polymer material, Confirm that vitronectin is supported. Characteristic cell culture substrate. 2. If the polymer material is chemically or physically The above claims are surface treated. The cell culture substrate according to item 1. 3. The above where the polymeric material is a porous material Claim 1 or 2 Substrate for cell culture. 4. The above patent claim wherein the base material is a hollow fiber. Recorded in any of the range items 1 to 3. Substrate for cell culture. 5. Vitronectin is distributed on the surface of the base material. The above partially claimed claims Stated in any of paragraphs 1 to 4 of the box. Substrate for cell culture. 6. Vitronectin is supported in a lattice pattern As stated in claim 5 above, Substrate for cell culture. 7. Vitronectin is carried in a striped pattern As stated in claim 5 above, Substrate for cell culture. 8. Vitronectin is supported in a polka dot pattern As stated in claim 5 above, Substrate for cell culture.