JPS63196272A - Substrate material for cell culture - Google Patents

Abstract

PURPOSE:To obtain a substrate for cell culture having excellent adhesiveness to cell and distensibility and proliferativity of cell, capable of carrying out high-density and long-period cell culture, by supporting a saccharide, protein, etc., on the surface of a high polymer substrate irradiated with ultraviolet. CONSTITUTION:The whole surface of a substrate for cell culture which consists of an olefinic polymer, polyester resin, etc., and molded preferably into a porous or tubular shape, hollow yarn, etc., is irradiated with ultraviolet rays or the surface of the substrate is partially irradiated with the ultraviolet rays in a lattice pattern, striped pattern, polkadotted pattern, etc., to form a hydrophilic group on the surface of the base material. Then one or more selected from a saccharide, protein, lipid and complex compound thereof is supported on the surface of the substrate by a conventional procedure.

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.

BACKGROUND 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 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.

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 its denatured product gelatin are already commercially available, as well as cross-linked soluble fibroin. A cell culture bed (see Japanese Unexamined Patent Application Publication No. 61-52280) has been proposed.

<Problems to be solved by the invention> However, with the above-mentioned prior art, firstly, the immobilization of glycoproteins, etc. to the polymer base material is insufficient (they are easily detached), and secondly, the surface is Because glycoproteins, etc. are immobilized on a homogeneous polymer base, the glycoproteins, etc. become unstable, resulting in poor adhesion to cells, as well as insufficient spread and proliferation of adhered cells, leading to high density and long length. There is a problem that cell culture cannot be carried out for a long time.

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

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 sugar, sugar,
It is characterized by supporting one or more of 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 substrate is partially treated with ultraviolet rays, and it is particularly preferable that the substrate be treated with ultraviolet rays to form a checkered pattern, a striped pattern, a polka dot pattern, or the like.

According to the cell culture substrate having the above configuration, since glycoproteins and the like are immobilized on the surface of the substrate made of a polymeric material, adhesiveness with cells can be improved. 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. Furthermore, since the surface of the base material has been surface-treated with ultraviolet rays, the surface has hydrophilic groups such as carbonyl groups, carboxyl groups, hydroxyl groups, etc. Adhesiveness with glycoproteins etc. is increased, and in turn, adhesion between cells and substrates can be enhanced.

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.

On the surface of a substrate that has been microfabricated by partial treatment with ultraviolet rays, and areas with different degrees of hydrophilicity are arranged in a fine pattern, glycoproteins, etc. are suitable for cell expansion and proliferation. Since it is immobilized on the substrate with its arrangement and shape, it has excellent adhesion to cells and can be used as a cell culture substrate that is more effective for the spread and proliferation of adhered cells. In particular, those that have been treated with ultraviolet light to have a checkered pattern, striped pattern, polka dot pattern, etc. can further enhance the above effects.

The present invention will be described in 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 by ultraviolet irradiation.

As the material for the polymer base material, any material can be used as long as it has shapeability and mechanical strength. For example, polyethylene, polypropylene, chlorinated polyethylene, olefin polymers such as ionomers, polytetrafluorocarbon Fluorine resins such as ethylene and polyvinylidene fluoride, styrene resins such as polystyrene, acrylic resins such as polymethyl methacrylate, polyvinyl alcohol, polyvinyl acetate, polyvinyl acecool, polyacrylonitrile, polyvinyl chloride, polyvinylidene chloride, polycarbonate , polyester resins such as polyarylate, polyphenylene oxide, polyethylene terephthalate, and polybutylene terephthalate, various polymers or copolymers such as epoxy resins, polyamides, polyimides, polysulfones, cellulose resins, silicone resins, polyurethanes, or blends thereof. can be exemplified.

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 with pores that facilitate material metabolism are preferable, and for high-density culture, tubes and hollow fiber forms are preferable. is suitable. 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, such as those with an inner diameter of 5O-1000II.
In addition, when the cell culture substrate according to 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 ultraviolet rays used in the ultraviolet irradiation treatment of the base material made of the polymeric material can have a wavelength that causes a chemical reaction on the surface of the polymeric base material. 200nm of ultraviolet light
Far ultraviolet rays with less than 300 nm have more light energy and can be processed more efficiently.

Although a xenon arc, a metal halide lamp, or the like can be used as the light source for emitting the ultraviolet rays, a mercury lamp that can process a large area or a laser that can perform coherent and fine processing is preferably used. As for the above mercury lamp,
High-pressure mercury lamp, whose main wavelength is 368 nm, 253.7r
An example is a low-pressure mercury lamp that simultaneously emits light with wavelengths of +a+ and 184.9 nm.

Further, as the laser, in addition to lasers such as Ar and He-Cd5N2, an excimer laser that emits light with a short wavelength and high output can be used. An excimer laser is preferably used because it applies high energy to the base material in a short time and can significantly modify the base material chemically and physically. The above treatment with ultraviolet rays is carried out by irradiating the surface of a desired polymeric substrate with ultraviolet rays in various atmospheres. For example, when ultraviolet rays are irradiated in air, not only carbonyl groups are generated on the surface of the polymer base material, but also hydroperoxide groups and peracid groups are generated. Therefore, by reducing with a reducing agent such as potassium iodide, it is possible to introduce functional groups, such as hydroxyl groups and carboxy groups, which have excellent affinity and adhesion to glycoproteins into the polymeric substrate.

The above-mentioned ultraviolet irradiation may be performed on the entire surface of the base material made of a polymer base material, but the ultraviolet irradiation may be performed partially,
By microfabrication, glycoproteins and the like can be firmly and stably immobilized on the base material, and it is possible to have excellent adhesion with cells and to increase the spread and proliferation of cells. In the case of partial irradiation with ultraviolet rays, a useful surface that can further enhance the above effects can be formed by irradiating fine patterns such as grids, stripes, and polka dots.

In addition, excimer lasers can apply high energy to the base material in a short time, chemically modifying the base material significantly, and also physically modify the base material surface by micromachining it into minute irregularities. It has the advantage that it can also promote the metabolism of cellular substances.

Note that the above-mentioned fine pattern can be formed by any appropriate means. For example, by irradiating the above-mentioned ultraviolet rays using a photomask or operating a laser, the above-mentioned fine pattern can be formed with a desired accuracy on the order of microns. It can be drawn in a pattern with an interval of .

After performing these ultraviolet irradiation treatments, glycoproteins and the like are supported on the surface of the base material. Any protein can be used as long as it has an affinity for cells and promotes adhesion between the substrate and cells, such as oligosaccharides such as lactose and galactose, and proteins such as albumin. , lipids such as phospholipids, glycolipids that are complexes of sugars and lipids such as globosides and gangliosides, riboproteins that are complexes of lipids and proteins contained in cytoplasm and serum,
Examples include glycoproteins, which are complexes of sugar and protein, and glycoproteins such as oligosaccharides, collagen, gelatin, fibronectin, laminin, chondronectin, vitronectin, and fibrin are particularly preferably used. These are 2
Combinations of one or more species may also be useful. Any conventional technique can be applied to the method of supporting glycoprotein etc. on the surface of the base material. Generally, this is carried out by immersing a polymeric substrate that has been irradiated with ultraviolet irradiation in a solution containing one or more of the above-mentioned glycoproteins, or by applying the solution and then drying it. In particular, it is preferable to dry under conditions that do not denature glycoproteins and the like. Glycoproteins and the like may be supported in a monomolecular layer or in a multimolecular layer.

The cell culture substrate of the present invention can be used to culture 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 Liver% human lung-derived eudiploid fibroblasts IRC-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

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

Example 1 A circular low-density polyethylene film (45 Mφ) was irradiated with ultraviolet rays for 1 hour from a distance of 6 − using low-pressure mercury or the like. This film was set in a 45 mm diameter glass jar, and after sterilization with ethylene oxide gas, a sterile collagen solution (type 1, concentration 0.3%) was applied, excess liquid was removed, and the film was dried at room temperature. .

All these operations were performed in a sterile clean bench. Next, Chinese hamster lung-derived cells (V-79 cells) were added to this petri dish at IX per 1'11 of culture solution.
104 cells were seeded and cultured. Eagle's MEM medium containing 10% by weight tallow serum was used as the culture solution, and culture was carried out for 7 days in an environment of 5% carbon dioxide, 95% air, and a temperature of 37°C. After culturing, the average number of cells was 5.4 x 106 cells per culture solution, and good proliferation was observed.

Comparative Example 1 A low-density polyethylene film that was not subjected to ultraviolet treatment was sterilized with ethylene oxide gas, and then collagen was applied and cultured in the same manner as in Example 1.

The collagen solution was repelled on the film and rarely adhered. In addition, the number of cells after culture was 2.6 x 104
It remained small.

Example 2 Circular polyethylene terephthalate film (45 mm
φ) was irradiated with ultraviolet rays with a wavelength of 250 to 300 nm for 1 hour using a Deep UV exposure device through an image of a chrome mask drawn on a quartz plate, resulting in a lattice-like pattern of 1μ square (treated area width 1μff1). Got the film. This treated film was set in a glass petri dish with a diameter of 45 mm, and after high-pressure steam sterilization, a sterile collagen solution (type 1, concentration 0.8%) was applied, dried, and then sterile fibronectin obtained from rat plasma was applied. Solution (concentration 10
μg/'Ml) was applied and dried to form a collagen-fibronectin layer. Example 1 with the obtained sample
As a result of carrying out a culture test in the same manner as above, the number of cells was 11 in the culture solution.
Good growth was observed with an average of 6.4 x 106 cells per cell.

<Effects of the Invention> As described above, according to the cell culture substrate of the present invention, glycoproteins, etc. etc., it is possible to firmly immobilize glycoproteins etc. on the substrate. In addition, glycoproteins have excellent affinity with cells,
Since it is possible to improve adhesiveness and allow cells to adhere in a form and arrangement suitable for spreading and proliferation, high-density and long-term cell culture is possible. In particular, substrates with microfabricated surfaces formed by partial irradiation with ultraviolet rays can be immobilized while maintaining their higher-order structures, such as glycoproteins, which improves their adhesion with cells. It also has the unique effect of being excellent in cell spreadability and proliferation. 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. Polymer base material surface treated with ultraviolet rays On the surface, sugars, proteins, lipids and Supported by one or more of the complex compounds of A cell culture substrate characterized by Material. 2. Characteristic that the polymer base material is porous The details set forth in claim 1 above, which is Substrate for cell culture. 3. Features that the polymer base material is a hollow fiber Claim 1 or Substrate for cell culture according to item 2. 4. The surface of the polymer base material is partially exposed to ultraviolet light. Claim 1 above as processed Cell culture according to any one of paragraphs 3 to 3. Base material for use. 5. The polymer base material is UV treated in a grid pattern. The above patent claim is characterized in that: The cell culture substrate according to scope 4. 6. The polymer base material is treated with ultraviolet light in a striped pattern. The scope of the above patent claim is characterized in that The cell culture substrate according to item 4. 7. The polymer base material is treated with UV light to create a polka dot pattern. The above patent claim is characterized in that: The cell culture substrate according to scope 4.