JPS63196271A - Substrate 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 irradiating the surface of high polymer substrate with ultraviolet rays. 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 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.

<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 for adherent animal cells to adhere and proliferate on cultured cells, it is necessary to have good adhesion between the substrate surface and the cells, as well as the morphology and arrangement of the adhered cells to be effective for their expansion and proliferation after adhesion. 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.

In order to improve this problem, cell culture substrates have been proposed in which the above-mentioned polymeric materials are surface-treated to make them hydrophilic. For example, tissue culture materials in which polymeric materials are treated with ozone (
JP-A-52-41291), tissue culture carrier particles treated with low-temperature plasma (JP-A-57-2289)
1) and a method for manufacturing a cell culture bed that forms a thin film on the surface of a substrate by plasma polymerization (Japanese Unexamined Patent Publication No. 61-522).
(see Publication No. 81) has been proposed.

<Problems to be solved by the invention> -However, the cell culture substrate obtained by the above ozone treatment only has a surface where the polymer material is oxidized by ozone, and it is not possible to improve adhesiveness. It is enough. In addition, when using low-temperature plasma, electrons, ions, radicals,
Due to the action of light, it is not only difficult to obtain a homogeneously treated substrate, but also difficult to control the processing conditions. Furthermore, with cell culture substrates produced by these methods, only the substrates in which the entire surface of the material is treated, including the depressions, can be obtained, and the arrangement and morphology of adhered cells can be made into a form that is advantageous for cell expansion and proliferation. The problem is that it cannot be controlled. Therefore,
The above-mentioned cell culture substrates have insufficient adhesion with cells, cell spreadability and proliferation, and have a problem in that cell culture cannot be carried out at high density and over a long period of time.

Purpose This invention has been made in view of the above problems, and it is possible to easily process base materials made of various polymers, and has excellent cell adhesion, spreading and proliferation, and allows high-density and long-term cell growth. The purpose of the present invention is to provide a cell culture substrate that enables cell culture.

Means and operation for solving the above problems> In order to achieve the above object, the cell culture substrate of the present invention is characterized in that a substrate made of a polymeric material is surface-treated with ultraviolet rays. It is something to do.

Note that the polymer 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, the substrate made of a polymeric material is surface-treated with ultraviolet rays, so that the surface of the polymeric material has hydrophilic groups with excellent affinity for cells, such as , a carbonyl group, a carboxy group, a hydroxyl group, etc., and the site having the above-mentioned hydrophilic group can enhance cell adhesion and effectively promote cell spreading and proliferation.

In addition, unlike the plasma treatment described above, there is no ion or electron bombardment and only UV irradiation is required, making it easy to control the reaction. Microfabrication can be easily performed by this method.

Furthermore, when the polymer material is a porous material,
Metabolism becomes easy 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.

The structure of the cell membrane on the cell surface consists of a lipid bilayer containing
Various glycoproteins and glycolipids, called intramembrane particles, are embedded in a distributed distribution, and these are involved in cell adhesion. On the surface of a substrate that has been microfabricated by partial treatment with ultraviolet rays and has portions with different degrees of hydrophilicity arranged in a fine pattern, the above-mentioned structure of the cell membrane is caused by ionic bonds, Hydrophobic bonds and the like allow cells to adhere to the substrate in a stable form and arrangement, which in turn can promote cell expansion and proliferation. In particular, checkered patterns,
The above-mentioned effects can be further enhanced by UV treatment with a certain pattern such as a striped pattern or a polka dot pattern.

The present invention will be described in detail below.

The cell culture substrate of the present invention is made of a polymeric material whose surface has been treated by ultraviolet irradiation.

As the polymer 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, polytetrafluoroethylene, Fluorine resins such as polyvinylidene fluoride, 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, and polybutylene terephthalate, various polymers or copolymers such as epoxy resins, polyamides, polyimides, polysulfones, cellulose resins, 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, and for example, those having an inner diameter of about 50 to 1000 μm are used.

Further, when the cell culture substrate according to 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 cell culture substrate of the present invention is produced by subjecting the surface of the substrate made of the above polymeric material to ultraviolet irradiation treatment.

The above-mentioned ultraviolet rays are of a wavelength that causes a chemical reaction on the surface of the polymer material, and among the ultraviolet rays, 200
Far ultraviolet rays having a wavelength of less than nm have a large optical energy and can therefore 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 388 nIIl, 253.
An example is a low-pressure mercury lamp that simultaneously emits light with wavelengths of 7 nm and 184.9 nm.

In addition, as a laser, Ar% He-Cd,
, N2, etc., excimer lasers that emit light of short wavelength and high power are available. The excimer laser is
It is preferably used because it can apply high energy to the base material in a short time and significantly modify the base material chemically and physically. The above treatment with ultraviolet rays is carried out by irradiating the surface of a base material made of a desired polymeric material with ultraviolet rays in various atmospheres.

For example, when ultraviolet rays are irradiated in the air, not only carbonyl groups are generated on the surface of the base material made of the polymer 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 with excellent affinity and adhesion to cells, such as hydroxyl groups and carboxy groups, into a base material made of a polymeric material. .

The above-mentioned ultraviolet irradiation may be performed on the entire surface of the base material made of a polymer material, but the ultraviolet irradiation may be performed partially,
Through microfabrication, cell adhesion is stabilized,
Cell spreading and proliferation can be increased. 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 apply high energy to the base material in a short period of time,
In addition to being able to significantly modify the base material chemically, it is also possible to physically modify the base material surface by making it microscopically uneven, which has the advantage of promoting cell metabolism.

The above-mentioned fine pattern can be formed by an appropriate means, for example, by irradiating the above-mentioned ultraviolet rays using a photomask or by operating a laser, the above-mentioned fine pattern can be formed with micron-order precision. It can be drawn in a pattern with desired spacing.

The cell culture substrate of the present invention can be used to culture various cells, and the types of cells are not particularly limited, and 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 RC-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 and Comparative Examples A circular base material (45 mm
φ) was irradiated with ultraviolet rays for 1 hour from a distance of 6 cm using a low-pressure mercury lamp. This ultraviolet treated film
After setting it in a mmφ glass petri dish and sterilizing it with high pressure steam, Chinese hamster lung-derived cells (V-79 cells) were cultured. At this time, the culture solution should be 10% by weight.
Using Eagle MEM medium containing beef tallow serum, culture solution 1
1! 1×104 cultured cells were seeded per plate. When cultured for 7 days in an atmosphere consisting of 5% carbon dioxide and 95% air at a temperature of 37°C, the average number of cells was L8 x 106 per 1'11 of the culture solution, and good growth was observed.

On the other hand, as a comparative example, cell culture was attempted in the same manner as in Example 1 above, except that a polypropylene film without UV irradiation was used.
．． The number of cells was 4×104.

Example 2 A circular base material (45 mmφ) made of polyether sulfone (manufactured by IC1, 5200p) was coated with 250 to 300 nII through an image of a chrome mask drawn on a quartz plate.
The film was irradiated with ultraviolet rays having a wavelength of 1 µm for 1 hour using a Deep UV exposure device to produce an ultraviolet-treated film in which a pattern in which treated areas and untreated areas were alternately arranged in a lattice shape at intervals of 1 μm width was formed. Using this treated film, a culture test was conducted in the same manner as in Example 1, and an average number of 4.7×10 6 cells was confirmed per 111 culture fluids.

Example 3 ArF having a wavelength of 193 nm was placed on a circular base material (45 mmφ) made of polyethylene terephthalate film.
A pulse energy of 40 mJ/c+j was irradiated using an excimer laser. The film treated in this way was set in a glass Petri dish with a diameter of 45 mm and sterilized using high-pressure steam. When a culture test was conducted in the same manner as in Example 1, an average of 4.5 x 10 cells per culture solution was obtained. number has been confirmed.

<Effects of the Invention> As described above, according to the cell culture substrate of the present invention, hydrophilic groups etc. that have excellent adhesion to cells are generated on the surface of the substrate by ultraviolet irradiation. It has excellent cell properties, cell spreadability, and proliferation, and enables high-density and long-term cell culture. In addition, with the simple treatment of ultraviolet irradiation, the surface of a substrate made of various polymers can be converted into a surface that is effective for adhesion with cells and for cell expansion and proliferation, resulting in excellent productivity and controllability. In particular, it has the unique effect of easily forming a microfabricated surface suitable for cell expansion 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. The base material made of polymer material is exposed to ultraviolet rays. The surface is treated with Substrate for cell culture. 2. The above where the polymeric material is a porous material For cell culture according to claim 1 Base material. 3. The above patent claim wherein the base material is a hollow fiber. Cell culture according to scope 1 or 2 Base material for use. 4. The base material is partially surface treated with ultraviolet light. Paragraph 1 of the above patent claim Cell culture according to any of Item 3 Base material for use. 5. The base material is UV treated in a checkered pattern. The details set forth in claim 4 above Substrate for cell culture. 6. The base material is UV treated in a striped pattern. The cell according to claim 4 above Substrate for culture. 7. The base material is treated with UV light to create a polka dot pattern. The details set forth in claim 4 above Substrate for cell culture.