JPS63196279A - 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.

<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. 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 laminin 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 laminin is partially supported on the substrate, particularly preferably in a checkered pattern, striped pattern, polka dot pattern, etc.

In the above configuration, laminin is a glycoprotein with a molecular weight of 800 Da that exists in the basement membrane of living organisms, 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. Since the laminin has a site capable of binding to intramembrane particles through ionic bonds, hydrophobic bonds, etc., 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 laminin is supported, laminin plays a role of connecting cells and the substrate, and the cell culture has excellent adhesion properties and the cells can 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.

It should be noted that the polymer material whose surface has been chemically or physically treated has excellent adhesion between laminin 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 laminin is partially supported on the substrate, especially when laminin is supported in a checkered pattern, striped pattern, polka dot pattern, etc., the position of the adhering cells can be adjusted.
Since cells adhere at predetermined intervals, adhesion with cells is further stabilized, and cell spreading and proliferation 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 laminin 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, those with an inner diameter of 50 to too μm.
It is used for various purposes.

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 soo I#.

The base material made of the above-mentioned polymeric material may be untreated, but may be physically or chemically treated to improve adhesion to laminin. 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 the introduction of groups and sulfonation with chlorosulfonic acid can improve the wettability of laminin 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 laminin 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 laminin.

The laminin 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 that it is supported partially on the surface of the base material, particularly in a patterned manner. Butter like this.

By supporting the cell in the form of a cell, it is possible to control the arrangement of the cells that adhere to the substrate, which in turn stabilizes the adhesion of the cells, making it possible to advantageously spread, proliferate, and express functions of the cells. Furthermore, when laminin is partially supported as described above, a useful surface that can further enhance the above effects can be formed by supporting laminin in a fine pattern such as a lattice pattern, a striped pattern, a polka dot pattern, etc. . Laminin 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 to culture 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.

Example 1 and Comparative Examples A circular nylon-6,6 film substrate (45 mmφ) was set in a 45 mmφ glass petri dish, and after high-pressure steam sterilization, laminin (manufactured by Bethesda Research) 5
0μg/l! The film surface was wetted with a phosphate buffer solution (sterilized) at a concentration of . After 10 minutes, excess solution was removed and the film was dried at room temperature to obtain a film on which laminin was laminated. Note that all of the above operations were performed in a sterile box.

Next, 2×10 4 isolated rat hepatocytes prepared by perfusion with 0.05% collagenase were seeded and cultured for 6 hours. The culture medium is serum-free Williams medium E containing 10μg/lI! of insulin! , Glucagon 10μg/xl, Evidermal Gross Factor 50ng/111, Prolactin 20μU/
1! , growth hormone 10μU/dai, copper 0.1μM, selenium 3μM, zinc 50pM were added. In addition, the culture conditions are
The cells were incubated at 37° C. in an atmosphere of 5% carbon dioxide and 95% air.

After culturing, the culture medium was removed from the Petri dish, washed with phosphate buffer, adhered cells were released with 0.1% collagenase solution, and the number of cells was measured using a hemocytometer. % were glued together.

On the other hand, as a comparative example, culture was carried out in the same manner as in the above example except that a film not laminated with laminin was used, and the adhesion rate of the seeded cells was 4%.

Example 2 A polypropylene film (thickness 100 μm) was coated with 18.
Plasma treatment was performed using a device having a 56 MHz high frequency power source. The processing conditions were a discharge output of 120W.

The pressure is 0.3 Torr, and the ammonia gas is 10cc.
The discharge was carried out for 10 minutes while flowing at a rate of 1/min. The treated film was punched out into a 45 mm diameter circle, and after being autoclaved, sterile laminin was supported in a striped pattern. That is, using a metal screen, the processing width of laminin was 40 μm,
After forming a striped pattern with an untreated width of 40 μm, it was dried.
Laminin was supported in a striped pattern. This composite film was set in a sterilized glass petri dish, and rat liver-derived cells were cultured for one week under the same culture conditions as in Example 1 above. When the state after culturing was observed using a phase contrast microscope, clusters of cells were formed, and good adhesion and spreading of the cells was confirmed.

<Effects of the Invention> As described above, according to the cell culture substrate of the present invention, laminin, which is a glycoprotein, is supported on the surface of the polymer substrate, and laminin has an affinity for cells. This method has excellent adhesion properties and allows cells to adhere in a form and arrangement suitable for spreading and proliferation, making it possible to culture cells at high density and over a long period of time. Furthermore, in the case of a substrate partially supported with laminin, particularly in a patterned manner,
Since the adhesion position of 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.

Claims (1)

[Scope of Claims] 1. A substrate for cell culture, characterized in that laminin 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 has been surface-treated chemically or physically. 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 laminin is partially supported on the surface of the substrate. 6. The cell culture substrate according to claim 5, wherein laminin is supported in a lattice pattern. 7. The cell culture substrate according to claim 5, wherein laminin is supported in a striped pattern. 8. The cell culture substrate according to claim 5, wherein laminin is supported in a polka dot pattern.