JPS63196281A - Substrate for cell culture - Google Patents

Abstract

PURPOSE:To obtain a cell-culture substrate having excellent adhesivity, distensi bility and proliferativity of cell and enabling cell culture over a long period in high density, by supporting sugars or proteins, etc., on a surface of a compos ite of a polymeric material and an inorganic material. CONSTITUTION:A composite of a polymeric material and an inorganic material is produced by filling or laminating an inorganic material such as alumina, silicon dioxide, hydroxyapatite, etc., to a polymeric material composed of a polymer such as olefinic polymer, polyester resin, etc., and formed preferably in the form of porous material, tube, hollow fiber, etc. One or more components selected from sugars, proteins, lipids and their composite compounds are supported on the whole surface of said composite material or partially on the surface e.g. in the pattern of lattice, stripes, dots, etc.

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 and problems to be solved by the invention> In recent years,
BACKGROUND OF THE INVENTION Research is actively being carried out 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 substrate surface, as well as the form and arrangement of the adhered cells in a form that is effective for cell expansion and proliferation. It is necessary that the 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 a method in which collagen, which is a biopolymer, or gelatin, which is a modified product of collagen, is coated on a polymer material (see JP-A-58-71884), and a method in which soluble fibroin is coated on a polymer material. A cell culture bed in which crosslinked materials are laminated (see Japanese Patent Application Laid-Open No. 81-52280) has been proposed.

However, in the above-mentioned conventional technology, sugars and proteins are not sufficiently immobilized on the polymeric substrate and easily detach, resulting in poor cell adhesion, as well as the spreadability, proliferation, and activity maintenance of adhered cells. There is still a problem that it is not sufficient and high-density, long-term cell culture is not possible.

Purpose This invention was made in view of the above problems, and provides a cell culture substrate that has excellent cell adhesion, spreading, and proliferation, and enables high-density and long-term cell culture. The purpose is to

Means and Effects for Solving the Problems> The cell culture substrate of the present invention, which has been made to achieve the above object, has the following features:
It is characterized by supporting sugars, proteins, lipids, and complex compounds thereof (hereinafter referred to as glycoproteins, etc.).

Note that the above-mentioned composite may be one in which a polymer material is filled with an inorganic material, or one in which an inorganic material is laminated on a polymer material. Further, the composite is preferably porous, and more preferably has a hollow fiber shape. Further, it is preferable to use a complex in which glycoprotein or the like is partially supported on the surface of the complex, particularly one in which glycoprotein or the like is supported in a checkered pattern, a striped pattern, a polka dot pattern, or the like.

As mentioned above, this invention uses a composite of a polymeric material and an inorganic material, and the inorganic material is highly hydrophilic and has a high affinity for glycoproteins, etc. Can be firmly immobilized.

Furthermore, the glycoprotein etc. supported on the complex has excellent adhesion to cells, and allows cells to adhere in a stable form and arrangement. In other words, the structure of the cell membrane on the cell surface is
Various glycoproteins, glycolipids, etc. called intramembrane particles are embedded in the lipid bilayer in a distributed manner, and these can move freely within the lipid bilayer and are involved in cell adhesion. There is. The above glycoproteins etc. are covalently bonded to intramembrane particles,
Since it has a site that can be bonded by ionic bond, hydrophobic bond, etc., adhesiveness with cells is increased and cells can be held in a stable form 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.

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 flowing a culture solution or the like into the hollow part or outside the hollow fiber. Can be done.

Furthermore, when glycoproteins, etc. are partially supported on the surface of the complex, especially when glycoproteins, etc. are supported in a checkered pattern, striped pattern, polka dot pattern, etc., the position of the adhering cells is adjusted. Because the cells adhere to each other at regular intervals,
Adhesion with cells is further stabilized, and cell spreading and proliferation 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 and the like are supported on the surface of a composite made of a polymeric material and an inorganic material.

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.

Various inorganic substances can be used as the inorganic material, but in particular, alumina, silicon dioxide, silica gel, hydroxyapatite [Cac(POa)s(OH)2], tricalcium phosphate, and glass Na having the following composition. 20-Ca0-Sto, -p2os
, Me 0-Ca 0-8L Ot -ho s
, hh 20-KzO-F'a 0-Ca o-st
Ot-PtO5, etc. have good biocompatibility (they are preferably used).

The composite of the above-mentioned polymeric material and inorganic material can be produced by a conventional method. For example, by dispersing fine powder of an inorganic material in a polymeric material and molding it, a composite in which the polymeric material is filled with the inorganic material can be obtained. According to this method, since the inorganic material is filled in a polymeric material with high shapeability, the shapeability of the inorganic material is poor, and the
It compensates for the drawback that it is difficult to obtain porous molded products with high substance permeability, and has the advantage that it can be used as a base material of various shapes.

In addition, a dispersion of fine powder of inorganic material dispersed in a binder is applied to a polymer material molded into a predetermined shape and dried, or a dry process such as vacuum evaporation, ion blating, or sputtering is used. By evaporating an inorganic material or the like, a composite in the form of an inorganic material layered on a polymeric material can be obtained. In this case, the inorganic material does not necessarily need to cover the entire surface of the polymeric material, and may be scattered on the surface of the polymeric material. Note that in this lamination method as well, since the polymer material is laminated on a polymer material that has been formed into a desired shape in advance, cell culture substrates of various shapes can be obtained.

The usage ratio of the above polymeric material and inorganic material may be changed depending on the type of polymeric material or inorganic material, the form of the composite, etc., but for example, if the inorganic material is in the form of a polymeric material filled The inorganic material is 5 to 80% by weight, preferably 2
It is added in an amount of about 0 to 60% by weight. Further, in the case of a composite in which an inorganic material is laminated on a polymeric material, the inorganic material thin film is laminated at an angle of 0.01 to 10M, preferably 0.05 to 0.57711.

The cell culture substrate of the present invention can be obtained by supporting a glycoprotein or the like on the surface of the above-described composite. The glycoproteins used here can be any as long as they have an affinity for cells and promote adhesion between the substrate and cells, such as oligosaccharides such as lactose and galactose, proteins such as albumin, Lipids such as phospholipids, globosides,
Glycolipids, which are complexes of sugars such as gangliosides and lipids,
These include riboproteins, which are complexes of lipids and proteins contained in cytoplasm and serum, and glycoproteins, which are complexes of sugars and proteins, and in particular, oligosaccharides, collagen, gelatin, fibronectin, laminin, chondronectin, Glycoproteins such as vitronectin and fibrin are preferably used,
It is also useful to use two or more of these in combination. The method of supporting glycoprotein etc. on the complex is as follows.
All conventional techniques can be applied. Generally, this is carried out by immersing a complex formed into a predetermined shape in a solution containing one or more of the above-mentioned glycoproteins, or by applying the solution onto the complex and then drying it. be exposed. At this time, it is preferable to dry under conditions that do not easily denature glycoproteins and the like.

The above-mentioned glycoprotein, etc. may be supported in either a monomolecular layer or a multimolecular layer, or may be laminated on the entire surface of the complex, but
Preferably, it is supported partially on the surface of the complex, particularly in a patterned manner. By supporting it in a patterned manner, the arrangement of cells adhering to the complex can be controlled, and the adhesion of the cells can be stabilized. , can favor cell spreading, proliferation and functional expression. Furthermore, when glycoproteins and the like are partially supported as described above, a useful surface that can further enhance the above effects can be formed by supporting them in a fine pattern such as a lattice pattern, a striped pattern, a polka dot pattern, etc. Can be done. In order to support glycoproteins and the like on the complex in a patterned manner, techniques such as screen printing can be applied, for example.

The cell culture substrate of the present invention can be used in various forms, for example, in addition to molded products such as petri dishes and flasks,
Examples include forms such 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 100
A material with a diameter of about 0 μm is used.

In addition, when using the cell culture substrate of the present invention as a bead carrier in the microcarrier method,
Particles with a particle size of about 0 tones are used.

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 ■RC-90, human lymphoma-derived Namalva cells, and the like.

In addition, when culturing animal cells using the cell culture substrate of the present invention, various culture media are used depending on the type of cells to be cultured, and the optimal temperature, pH, etc. suitable for cell proliferation are used. Cultivation is carried out under these conditions.

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 the accompanying drawings.

The cell culture vessel shown in the attached drawing is equipped with support screens (2
Both ends of the film-like cell culture substrate (1) placed on ) are held by spacers (6) provided on both sides within the housing opening 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 (5) for perfusing the culture solution.
is installed. Note that the hole (4) is covered with a lid with a filter to prevent bacteria and the like 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 perfused through the tube (5) for a predetermined time under predetermined culture conditions. It will be done.

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

Example 1 and Comparative Examples Hydroxyapatite (average particle size 1.2 μm) was added to polypropylene.
) was filled at a weight ratio of 40% and formed into a film with a thickness of 300 μm. This film was set in a 45 mm diameter glass petri dish, and after sterilization using high-pressure steam, a Tris buffer solution (concentration 0.1/11) of sterile fibronectin (manufactured by Sigma, collected from bovine serum) was applied and dried at room temperature. I let it happen. All these operations were performed aseptically. In this petri dish, Chinese hamster lung-derived cells (V-7
9) was cultured. Eagle's MEM medium containing 10% by weight tallow serum was used as the culture solution. I x 104 cultured cells were seeded per 111 culture fluids, and 5% carbon dioxide gas was added.
When cultured for 7 days in a 95% air atmosphere and a temperature of 37°C, an average of 6.1 x 1
The number of cells was 0.06, confirming good proliferation.

On the other hand, in a comparative example in which the test was conducted in the same manner as in Example 1 except that a polypropylene film not filled with hydroxyapatite was used, an average of 8.4 × 1
The number of cells was 0.05.

Example 2 A porous film made of tetrafluoroethylene resin (Fluorobor FP-022, manufactured by Sumitomo Denki Kogyo Co., Ltd.) was placed in a sputtering apparatus, and alumina was sputter-deposited using a target. The processing conditions were 100 W for 5 minutes using a 13.58 MHz high frequency power source in nitrogen gas. Next, using a metal screen on this treated film, collagen (type I, concentration 0.
After forming a striped pattern with a treated width of 40 μm and an untreated width of 40 μm (3%), the striped pattern was dried to support collagen on the striped pattern. The above composite film as a cell culture substrate was used in a polycarbonate cell culture vessel (inner diameter 47 m) shown in the attached drawings.
mφ), the entire body was sterilized with ethylene oxide gas, flushed with excess sterile water and culture solution, and then human fetal foreskin-derived cells (Flow 7
000) Eagle MEM (1096 tallow serum supplemented) suspension (2 x 104 cells/cell) was filled. The hole (4) has a lid (7) with a filter to cut out bacteria.
and perfuse with fresh Eagle medium through tube (5).
Culture was performed at 37°C for one week. After culturing, the number of cells attached to the film was measured and found to be 5.2 x 10
It was found that the number of cells had grown to 4/yl.

<Effects of the Invention> As described above, according to the cell culture substrate of the present invention, glycoproteins and the like are supported on the surface of the composite of a polymeric material and a highly hydrophilic inorganic material. A base material with excellent adhesion to glycoproteins etc. can be obtained.

Furthermore, glycoproteins have excellent affinity for cells and can increase adhesion, as well as allow cells to adhere in a form and arrangement suitable for expansion and proliferation, allowing for high-density and long-term cell culture. It has the unique effect of making it possible. 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 be used, for example, in adhering insulin-producing cells to the composite surface.
It can be used to construct artificial organs, such as the formation of an artificial pancreas by culturing.

[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, ■... Support screen, (3)... Housing, (4)... Hole, (5
)···tube.

Claims (1)

[Claims] 1. Composite consisting of polymer material and inorganic material sugars, proteins, lipids and One or more of those complex compounds A thin piece characterized by the fact that the top is carried Substrate for cell culture. 2. Composite material made of polymeric material filled with inorganic material Claim 1 above which is a combination Substrate for cell culture. 3. Inorganic material layered on polymer material Claim 1 above which is a composite body The described cell culture substrate. 4. The above patent claim in which the composite is porous Recorded in any of the range items 1 to 3. Substrate for cell culture. 5. The above patent claim in which the composite is a hollow fiber Recorded in any of the range items 1 to 4. Substrate for cell culture. 6. Sugar, protein, lipid on the surface of the complex and any one of their composite compounds Above types are partially carried Claims 1 to 5 The cell culture substrate according to any one of the above. 7. Sugars, proteins, lipids and their complexes One or more of the compounds has a lattice pattern. The above claims are carried as follows: The cell culture substrate according to item 6. 8. Sugar, protein, lipid and their complexes One or more of the compounds has a striped pattern Claim No. The cell culture substrate according to item 6. 9. Sugars, proteins, lipids and their complexes One or more of the compounds has a polka dot pattern. The above claims are carried as follows: The cell culture substrate according to item 6.