JPS63196277A - 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> In recent years, research has been actively conducted 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 adhere and proliferate adherent animal cells on a cultured substrate, it is necessary to ensure that the adhesion between the substrate surface and the cells is good, and that the morphology and arrangement of the adhered cells are effective for cell expansion and proliferation. It is necessary to be in the form.

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.

On the other hand, sugars, proteins, lipids, and their complex compounds are
It is known to be involved in cell adhesion. Attempts have been made to coat polymeric materials with these substances as culture substrates, and culture dishes coated with collagen or gelatin, a modified product of collagen, are already commercially available, as well as cross-linked soluble fibroin. A stacked cell culture bed (see JP-A-61-52280) has been proposed.

<Problems to be Solved by the Invention> However, the above-mentioned conventional technology has a problem in that glycoproteins and the like are not sufficiently immobilized on the polymeric substrate and are easily detached. Furthermore, all of the cell culture substrates produced by these methods can only be obtained with the entire surface of the material treated, and the arrangement and morphology of adhered cells can be controlled to be favorable for cell expansion and proliferation. The problem is that I can't. Therefore, the above-mentioned cell culture substrate has insufficient adhesion with cells, cell spreadability and proliferation, and there is a problem in that cell culture cannot be carried out at high density and over a long period of time.

Purpose This invention was made in view of the above-mentioned problems, and it has excellent adhesion with cells, can proliferate cells and maintain their functions, and enables high-density, long-term cell culture. The purpose is to provide a cell culture substrate that can be conditioned.

Means and action for solving the above problems> The cell culture substrate of the present invention, which was made to solve the above problems, has a polymer substrate surface-treated with ozone, and on the surface of the polymer substrate,
It is characterized in that it supports one or more of sugars, proteins, lipids, and complex compounds thereof (hereinafter referred to as glycoproteins, etc.).

Note that the polymer base material is preferably a porous material. Further, it is preferable that the base material is a hollow fiber. Furthermore, it is preferable that the glycoprotein or the like is partially supported on the base material, particularly preferably in a checkered pattern, striped pattern, polka dot pattern, etc.

The cell culture substrate of the present invention has the above-mentioned structure, and since glycoproteins, etc. are supported on the surface of the polymer substrate oxidized by ozone, the adhesion between the substrate and glycoproteins, etc. is strong. can do. Furthermore, glycoproteins and the like have excellent adhesion to cells, allowing cells to adhere in a stable form and arrangement.

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 above-mentioned glycoproteins and the like have sites that can bind to intramembrane particles through ionic bonds, hydrophobic bonds, etc., they can increase adhesion to cells and maintain cells in a stable shape and arrangement. Therefore, the cell culture substrate of the present invention can promote stable adhesion of cells and enable good spread and proliferation of adhered cells.

Furthermore, when the polymer base 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 polymer base 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.

In addition, when glycoproteins, etc. are partially supported on the surface of the substrate, especially when glycoproteins, etc. are supported in a checkered pattern, striped pattern, polka dot pattern, etc., the position of the adhering cells can be adjusted. Since the cells adhere at predetermined intervals, the adhesion with the cells is further stabilized, and the spread and proliferation of the cells can be further increased.

This invention will be explained in more detail below.

The cell culture substrate of the present invention has a structure in which one or more types of glycoproteins or the like are supported on the surface of a polymer substrate that has been surface-treated with ozone.

As the material for the polymer base material, any material can be used as long as it has shapeability and mechanical strength, such as polyethylene, polypropylene, chlorinated polyethylene, olefin polymers such as ionomers, polytetra Fluorine resins such as fluoroethylene and polyvinylidene fluoride, styrene resins such as polystyrene, acrylic resins such as polymethyl methacrylate, polyvinyl alcohol, polyvinyl acetate, polyvinyl acecool, polyacrylonitrile, polyvinyl chloride, polychloride Various polymers or copolymers such as vinylidene, polycarbonate, polyarylate, polyphenylene oxide, polyethylene terephthalate, polyester resin such as polybutylene terephthalate, epoxy resin, polyamide, polyimide, polysulfone, cellulose resin, silicone resin, polyurethane, or the like. An example is a blend of the following.

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 polymer substrates having pores that facilitate material metabolism are preferable, and for high-density culture,
A hollow fiber 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, the culture solution is perfused into the hollow part or outside of the hollow fiber,
Cells can be grown and proliferated on the hollow fibers by sending carbon dioxide gas, air, etc. to the hollow portions of the hollow fibers as needed. Note that the hollow fibers can be of various sizes, for example, those with an inner diameter of 50 to 1000 μm.
- 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 substrate used has a particle size of about 100 to 300 mm.

A conventional method is used to treat the surface of the polymer base material with ozone, for example, ozone generated by an ozone generator (if necessary) is placed in a reaction vessel in which the polymer base material is placed. , as a mixed gas with an inert gas such as argon). If desired, the reaction may be carried out under pressurized conditions, or an organic solvent or the like may be used. The concentration of ozone in the reaction container is usually 0.2 to 1.
The amount is 0% by volume, preferably 0.3-3% by volume, and the reaction is usually carried out at room temperature. After the ozone treatment, it is washed with water and dried according to the usual method.

After performing the above ozone treatment, glycoproteins and the like are supported on the base material. The glycoprotein used here can be any substance that has an affinity for cells and promotes adhesion between the substrate and cells, such as oligosaccharides such as lactose and galactose, albumin, etc. proteins, lipids such as phospholipids, glycolipids which are complexes of sugars and lipids such as globosides and gangliosides, riboproteins which are complexes of lipids and proteins contained in cytoplasm and serum, and complexes of sugars and proteins. Examples include complex glycoproteins, especially oligosaccharides, collagen, gelatin, fibronectin, laminin,
Glycoproteins such as chondronectin, vitronectin, and fibrin are preferably used, and it is also useful to use two or more of these in combination. Any conventional technique can be applied to the method of supporting glycoprotein etc. on the substrate. In general, a polymer substrate surface-treated with ozone is immersed in a solution containing one or more of the above-mentioned glycoproteins, etc., or after the solution is applied onto the substrate,
This is done by drying. At this time, it is preferable to dry under conditions that do not easily denature glycoproteins and the like.

The above-mentioned glycoproteins, etc. may be supported in either a monomolecular layer or a multimolecular layer, or may be laminated over the entire surface of the base material, but it is preferable to support them partially, especially in a pattern, on the surface of the base material. Preferably, by supporting the cells in a patterned manner, it is possible to control the arrangement of cells adhering to the substrate, which in turn stabilizes the adhesion of the cells, making it advantageous for cell spreading, proliferation, and functional expression. can. Furthermore, when glycoproteins, etc. are partially supported as described above, in particular, by supporting them 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. be able to. Glycoproteins and the like can be patterned and supported on the base material by, for example, applying techniques 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, and examples include living body-derived cells, hybridomas, etc. For example, Chinese hamster lung-derived cells V- 79, human uterine cancer-derived cells HeLa, human fetal lung-derived cells MRC-5, human liver-derived cells Chang Livers, human lung-derived eudiploid fibroblasts IRe-90, human lymphoma-derived Namalva cells, and the like.

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

The cell culture substrate of the present invention can be applied to the proliferation of animal cells in various conventionally known modules.

An example of a module using a film-like substrate as a cell culture substrate of the present invention will be described below based on FIG.

The cell culture vessel shown in Figure 1 has a support screen (2).
Both ends of the film-like base material (1) placed thereon are held by spacers (6) provided on both sides within a housing (3) made of polycarbonate or the like.

Further, the housing (3) is provided with a hole (4) for filling the housing (3) with a cell suspension to be proliferated, and a tube 6) for perfusing the culture solution is attached. It is being The hole (4) is covered with a lid (7) equipped with a filter to prevent bacteria from entering.

In order to proliferate cells using the cell culture device described above, a cell suspension is injected through the hole (4) and the cells are grown in the substrate (1).
), the hole (4) is covered with the lid (7) with a filter, and the culture solution is allowed to flow through the tube (5) for a predetermined period of time under predetermined culture conditions. It is done.

<Examples> Hereinafter, the present invention will be described in more detail based on Examples.

Example 1 and Comparative Examples Polysulfone (manufactured by UCC, Udel P17
00) with a thickness of 50μ and a diameter of 45m + nφ was set in a desiccator equipped with a gas inlet and an outlet, and air containing 1.5% by volume of ozone was flowed through it at room temperature. Treated for 2 hours. After treatment, wash thoroughly with water.
Set in a 45 mmφ glass petri dish, and after high-pressure steam sterilization, add a phosphate buffer solution (concentration 0.1 m+; +/if) of sterile fibronectin (manufactured by Sigma, collected from bovine serum).
was applied and dried at room temperature. All these operations were performed aseptically. Human cervical cancer cells (Hela 5-3) were cultured in this petri dish. Culture solution is 10%
Culture medium 111 in Eagle MEM medium containing beef tallow serum.
When 2 x 104 cultured cells were seeded per cell and cultured for 3 days in an environment of 5% carbon dioxide and 95% air at a temperature of 37°C, an average of 3°4
The number of cells was 105, and good proliferation was observed.

On the other hand, in a comparative example in which the test was conducted in the same manner as in the above example except that the polysulfone film was used as it was, the culture solution 1
The average number of cells per '11 was 1.2 x 105.

Example 2 Polyetherimide (trade name, ULTEM, manufactured by Engineering Plastics Co., Ltd.) was dissolved in N-methyl-2-pyrrolidone to prepare a 18% by weight solution. This solution was cast onto a glass plate with a doctor knife to a thickness of 300 μm, and after being left for a certain period of time, the N-
It was immersed in a 5% aqueous solution of methyl-2-pyrrolidone and coagulated to obtain a porous film. This membrane was treated in the same manner as in Example 1 with air containing 1.5% by volume of ozone for 3 hours. The treated film was placed in running water for 1 hour, and after drying,
It was punched out into a 0 mm diameter circle and sterilized with high pressure steam. Then, using a metal screen, sterile collagen solution (
Type 11 concentration 0.3%) treated width 40, untreated width 4
After forming a striped pattern of 0 μm, it was dried to support the striped collagen.

The above composite film as a cell culture substrate (1) was previously sterilized with ethylene oxide gas in a cell culture vessel (with an inner diameter of 4
7 mmφ) together with the support screen (2), and human fetal foreskin-derived cells (F 1 ow
7000) was filled with Eagle MEM (10% tallow serum supplemented) suspension (2 x 104 cells/animal). Hole (4
) was covered with a lid (7) equipped with a filter to cut out bacteria, fresh Eagle MEM medium was poured into the tank through the tube (5), and cultured at 37°C for one week. After culturing, the number of cells attached to the film was measured and found to be 5.2X.
It was found that the number of cells had grown to 104/11.

<Effects of the Invention> As described above, according to the cell culture substrate of the present invention, glycoproteins, etc. are supported on the surface of the polymer substrate surface-treated with ozone. can be firmly immobilized on the substrate. Furthermore, glycoproteins etc.
It has excellent affinity with cells and can increase adhesion, as well as allow cells and cells to adhere in a form and arrangement suitable for expansion and proliferation, making it possible to culture cells at high density and over long periods of time. It has a unique effect. 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 the drawing]

The figure is a sectional view showing an example of a cell culture device using the cell culture substrate of the present invention. (1)...Substrate for cell culture, (2)...Support screen, (3).../1 Uzing, (4)...Hole, (5)...Tube.

Claims (1)

[Claims] 1. Polymer base material surface treated with ozone sugars, proteins, lipids and One or more of these complex compounds are A cell culture characterized by supporting Nutrient substrate. 2. The above patent claimant in which the polymer base material is porous The cell culture substrate according to item 1. 3. The above patent application in which the polymer base material is a hollow fiber Cells according to claim 1 or 2 Substrate for culture. 4. Sugar, protein, Both lipids and their complex compounds or one or more species are partially supported Claims 1 to 3 above The cell culture substrate according to any one of the above. 5. Sugars, proteins, lipids and their complexes One or more of the compounds has a lattice pattern. The above claims are carried as follows: 4. The cell culture substrate according to item 4. 6. Sugars, proteins, lipids and their complexes One or more of the compounds has a striped pattern Claim No. Substrate for cell culture according to item 4. 7. Sugars, proteins, lipids and their complexes One or more of the compounds has a polka dot pattern. The above claims are carried as follows: 4. The cell culture substrate according to item 4.