JPS63196283A - Base for cell culture - Google Patents

Abstract

PURPOSE:To obtain a cell-culture base having excellent adhesivity, distensibility and proliferativity of cell and enabling cell culture over a long period in high density, by supporting sugars, proteins, etc., on a polymer base covered with a multilayer film of monomolecular films. CONSTITUTION:A cell culture base composed of a polymeric material such as olefinic polymer, polyester resin, etc., and formed preferably in the form of porous material, tube, hollow fiber, etc., is covered with a multi-layer film of monomolecular films of a saturated fatty acid, higher alcohol, natural peptide or preferably synthetic polypeptide, etc. The multi-layer film is partially treated with ultraviolet radiation, electron ray or ion irradiation and at least one kind of compounds selected from sugars, proteins, lipids and their 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 allow adherent animal cells to adhere and proliferate on a culture 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 denatured 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 (see Japanese Patent Laid-Open No. 61-52280) has been proposed in which a crosslinked body of the following is laminated.

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-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 Effects for Solving the Problems In order to achieve the above object, the cell culture substrate of the present invention comprises a substrate made of a polymeric material and covered with a cumulative monomolecular film. In addition, the cumulative membrane is characterized in that at least one of sugars, proteins, lipids, and complex compounds thereof (hereinafter referred to as glycoproteins, etc.) is supported.

Note that the polymer material is preferably a porous material,
Preferably, the substrate is a hollow fiber. Further, the cumulative film is preferably partially treated with ultraviolet rays, electron beams, or ion irradiation.

Moreover, it is preferable that at least one type of glycoprotein or the like is partially supported on the cumulative monomolecular film, particularly in a lattice pattern, striped pattern, polka dot pattern, or the like.

According to the cell culture substrate having the above structure, the substrate made of a polymeric material is coated with a cumulative monomolecular film.
On the surface of the base material, there are functional groups with positive or negative charges depending on the material constituting the cumulative film and the cumulative state of the monomolecular film. It immobilizes and supports glycoproteins, etc. More specifically,
By spreading a water-insoluble organic compound on the water surface, it is possible to create a monomolecular film in which the hydrophilic and hydrophobic groups of the organic compound are oriented in a predetermined direction. By transferring it onto a substrate, the substrate can be covered with a monomolecular film in which the functional groups are oriented in a predetermined direction. By repeating the above operation, the base material can be coated with a cumulative film in which the orientation of functional groups is controlled, and the glycoprotein, etc. is supported by the cumulative film having a structure in which the orientation of functional groups is controlled. Ru. Glycoproteins and the like supported on the cumulative membrane are involved in adhesion with cells.

In addition, the structure of the cell membrane on the cell surface consists of a lipid bilayer,
Various glycoproteins and glycolipids, called intramembrane particles, are embedded in a distributed manner, and these can freely move within the lipid bilayer and adhere to functional groups on the surface of the glycoproteins and the like. Therefore, the cumulative monomolecular film strengthens the adhesion between the polymer and glycoprotein, etc., and firmly immobilizes and supports the glycoprotein. Cell adhesion can be enhanced by sites with functional groups on the surface of glycoproteins, etc.
Cell expansion and proliferation are effectively carried out.

Furthermore, when the cumulative film covering the base material is partially treated and microfabricated by irradiation with ultraviolet rays, etc., a cumulative film surface in which portions with different degrees of hydrophilicity are arranged in a fine pattern can be obtained. Therefore, glycoproteins, etc. can be immobilized and supported by controlling the orientation and distribution of glycoproteins, etc. Cell membranes with the above-mentioned structure allow cells to adhere to glycoproteins, etc., as substrates in a stable form and arrangement due to ionic bonds, hydrophobic bonds, etc., and in turn promote cell expansion and proliferation. can. In particular, items that have been treated with ultraviolet light, etc. in a certain pattern such as a checkered pattern, striped pattern, polka dot pattern, etc.
The above effects can be further enhanced.

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 present invention will be explained in detail below.

The cell culture substrate of the present invention comprises a substrate made of a polymeric material, a cumulative monomolecular film covering the surface of the substrate, and a glycoprotein etc. supported on the cumulative film.

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 2 , polyester resins such as polyphenylene oxide, polyethylene terephthalate, polybutylene terephthalate, epoxy resins, polyamides, polyimides, polysulfones, cellulose resins,
Examples include various polymers or copolymers such as 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, particles, and the like. 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 can be grown and proliferated on the hollow fiber by perfusing the culture solution into the hollow part or the outside of the hollow fiber, and sending carbon dioxide gas, air, etc. into the hollow part of the hollow fiber as necessary. can. 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.

In the cell culture substrate of the present invention, the surface of the substrate made of the polymeric material is coated with a cumulative monomolecular film in order to strengthen the adhesion between the polymeric substrate and glycoprotein, etc. ing.

The above-mentioned monomolecular film can be produced by spreading a water-insoluble organic compound on a water surface, and in the developed state, hydrophilic groups are oriented toward the water phase side and hydrophobic groups are oriented toward the gas phase side. Furthermore, by transferring the monomolecular film onto the base material by applying an appropriate pressure, the base material can be coated with the functional groups of the monomolecular film oriented in a predetermined direction. Therefore, by repeating the above operations, it is possible to produce a cumulative film in which the orientation and structure of the hydrophilic and hydrophobic groups are controlled.

In particular, since the orientation of the functional groups in the cumulative film can be controlled by adjusting the surface pressure, the cumulative film produced in this way has strong adhesion with glycoproteins, etc., and can even be used to bond with cells. useful for cell adhesion, cell spreading, and proliferation.

As the organic compound forming the monomolecular □ film, many organic compounds such as low-molecular compounds and high-molecular compounds can be used. Examples of low-molecular compounds include saturated fatty acids such as caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, dioxystearic acid, behenic acid, lignoceric acid, and montanic acid, oleic acid, elaidic acid, Fatty acids such as unsaturated fatty acids such as erucic acid, linoleic acid, lylunic acid, eleostearic acid, arachidonic acid, lysyllic acid; derivatives from the above fatty acids, e.g.
Esters with monohydric or polyhydric alcohols such as methanol, ethanol, propyl alcohol, isopropyl alcohol, ethylene glycol, diethylene glycol, polyethylene glycol, propylene glycol, polypropylene glycol, glycerin, trimethylolpropane, pentaerythritol, sorbitan; caprylic acid amide, Amides of the above fatty acids such as capric acid amide, lauric acid amide, myristic acid amide, palmitic acid amide, oleic acid amide, and stearic acid amide; higher alcohols such as lauryl alcohol, cetyl alcohol, oleyl alcohol, and stearyl alcohol; or Examples include vinyl compounds. Moreover, various polymers can be used as the above-mentioned polymer compound, but natural peptides, synthetic polypeptides, etc. are preferable, and can be appropriately selected depending on the desired functional group.

Note that the above polypeptide may be a linear or cyclic polypeptide. Among the above polypeptides, positively charged polyamino acids such as polylysine and polyornithine are particularly preferred. These promote adhesion of the above-mentioned glycoproteins etc. that have a negative charge on the surface.

In addition, the cumulative film covering the base material may be subjected to ultraviolet ray, electron beam or ion irradiation treatment, particularly partial irradiation treatment, in order to control the orientation and distribution of the glycoprotein, etc. and support the glycoprotein, etc. It is preferable to be there.

Any conventional means may be used to treat the cumulative film with ultraviolet rays, electron beams, or ion irradiation. These surface treatments can control the distribution of functional groups in the cumulative film, and introduce functional groups such as carboxyl groups, carbonyl groups, and hydroperoxide groups onto the surface of the cumulative film, thereby changing the degree of hydrophilicity. Since different parts of the glycoprotein can be formed into a fine pattern, glycoproteins, etc. can be firmly adhered to the cumulative membrane with the orientation and distribution of functional groups controlled, which in turn allows cells to spread on the surface of the glycoproteins, etc. , can promote proliferation.

The above-mentioned ultraviolet rays are those with a wavelength that causes a chemical reaction on the surface of the cumulative film that coats the base material. Among the ultraviolet rays, far ultraviolet rays of less than 200 ni have large optical energy, so they can be processed more efficiently. be able to.

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 nm, 25B, 7n
An example is a low-pressure mercury lamp that simultaneously emits light of wavelengths a+ and 184.9no+.

In addition to lasers such as Ar, He-Cd, and N2, excimer lasers that emit short-wavelength and high-output light can be used as lasers. An excimer laser is preferably used because it applies high energy to the accumulated film in a short time and can significantly modify the accumulated film chemically and physically.

The treatment with ultraviolet rays is carried out by irradiating the surface of the cumulative film 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 cumulative film, but also hydroperoxide groups and peracid groups are generated.

Further, as the electron beam source in the electron beam processing, various electron beam accelerators, Cockroft-Walton type, bump graph type, resonant transformation type, etc. can be used. Depending on the extent of treatment, various irradiation doses can be used, for example from 1 to 50 M rad.

For ion processing, a conventional ion beam irradiation device is used, and in addition to irradiating the entire sample with an ion shower, partial irradiation is performed using a mask and a focused ion beam.
It is irradiated in a fine pattern. Various ions can be used as the ions, including but not limited to He +,
Examples include ions such as A r +, C+, and N+. Further, a suitable value of ion energy is 0.
If the value is less than this value, the effect will be small, and if it exceeds this value, the carbonization of the base material will proceed significantly, which is not preferable.

The above-mentioned irradiation with ultraviolet rays, etc. may be performed on the entire surface of the cumulative film surface, but by irradiating with ultraviolet rays, etc. partially and performing microfabrication, the orientation and distribution of glycoproteins, etc. can be controlled, and glycoproteins, etc. can be immobilized and supported to increase cell spread and proliferation on surfaces such as glycoproteins. When partially irradiating ultraviolet rays, etc., the orientation and distribution of glycoproteins, etc. can be precisely controlled by irradiating fine patterns such as lattice, striped, and polka dot patterns, which is a useful method that can further enhance the above effects. surface can be formed.

In addition, excimer lasers and electron beams apply high energy to the accumulated film in a short period of time, making it possible to significantly modify the accumulated film chemically, as well as physically modifying the surface of the accumulated film into minute irregularities. This has the advantage that not only the immobilization and support of glycoproteins, etc., but also the orientation and distribution can be further controlled, and that it is also possible to promote cellular metabolism.

Note that the above-mentioned fine pattern can be formed by appropriate means, for example, by irradiating the ultraviolet rays etc. using a photomask, or by operating a laser or an electron beam.
The above-mentioned fine pattern can be drawn with desired spacing with micron-order accuracy.

Glycoproteins and the like are supported on the above-mentioned cumulative membrane. The glycoproteins used here have an affinity for cells,
Any substance that promotes cell adhesion can be used, such as oligosaccharides such as lactose and galactose, proteins such as albumin, lipids such as phospholipids, and complexes of sugars and lipids such as globosides and gangliosides. Examples include glycolipids, which are glycolipids, riboproteins, which are complexes of lipids and proteins contained in the cytoplasm and serum, and glycoproteins, which are complexes of sugars and proteins.In particular, oligosaccharides, collagen, gelatin, fibronectin, laminin, chondronectin,
Glycoproteins such as 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, after the polymer base material is coated with a cumulative film, it is immersed in a solution containing one or more of the above glycoproteins, etc.
This can be carried out by coating the base material with the monomolecular film in the same manner as described above, or by applying the solution to the surface of the accumulated film and then drying it.

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 on the entire surface of the cumulative film, but it is preferable to support them partially on the surface of the cumulative film, particularly in a patterned manner. It is possible to control the arrangement of cells adhering to glycoproteins, etc., which in turn stabilizes cell adhesion, making cell spreading, proliferation, and functional expression advantageous. 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. Glycoproteins and the like can be patterned and supported on the cumulative film by applying techniques such as screen printing, for example.

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 HeLas human fetal lung-derived cells MRC-5
, human liver-derived cells Chang Llver, human lung-derived eudiploid fibroblast cells 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

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 diagram is equipped with a support screen (2
) Both ends of the film-like cell culture substrate (1) placed on the top are held by spacers (6) provided on both sides in a housing (3) made of polycarbonate or the like. Further, the housing G) 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. The hole (4) is covered with a filter (M) 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 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 Polylysine (molecular weight 4.000-15.0
00) ammonium acetate solution (concentration 0, OLvt
%) was added dropwise, and a monomolecular film was developed while keeping the surface pressure at 0.2 dync/cm. By repeating the operation of transferring this monomolecular film onto polyethylene terephthalate (a circular film with a thickness of 100 μm and a diameter of 45 mm) by a horizontal adhesion method 20 times, a composite film in which the cumulative films were laminated was obtained. This composite film was exposed to deep UV light with a wavelength of 250 to 300 nm through a chrome mask image drawn on a quartz plate.
It was irradiated for 30 minutes using an exposure device, and a 1 μm grid pattern (
A film treated with a treated area width of 1 μM) was obtained. This treated film was set in a glass chassis with a diameter of 45 m + sφ, and after high-pressure steam sterilization, a Tris buffer solution (concentration 0.1
a+g/ml) and dried at room temperature. All these operations were performed aseptically. Chinese hamster lung-derived cells (V-79) were cultured in this petri dish. Eagle's MEM medium containing 10% by weight tallow serum was used as the culture medium.

When cultured cells were seeded at I x 104 cells per ml of culture solution and cultured for 7 days in an environment of 5% carbon dioxide and 95% air at a temperature of 37°C, 1 x I of culture solution was grown.
The average number of cells per cell was 6.4 x 106, 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 Example 1 except that the polyethylene terephthalate film was used as it was, the average number of cells was 9.0 x 105 per 1 x I of culture solution.

Example 2 Poly(γ-benzine-L-
glutamate) (molecular weight 15,000-30,000)
The cumulative film (repeated 20 times) was made into a polytetrafluoroethylene resin porous film (Fluoropore FP-010, manufactured by Judenki Kogyo ■).
was laminated to obtain a composite membrane. This film was attached to the cell culture vessel (inner diameter 47 φ) shown in the attached drawing, and after sterilizing the whole thing with high-pressure steam, a sterile collagen solution (type I, concentration 0.3%) was injected through the hole (4). After standing for 1 minute, the solution was drained and dried at room temperature. Then, from the hole (4), a suspension of human fetal foreskin-derived cells (Plov 7000) in Eagle MEM (to% tallow serum added) (cell number 2X) was added.
104 pieces/xI). The hole (4) was covered with a filter lid (7) for cutting bacteria, and fresh Eagle's MEM medium was perfused through the tube (5) and cultured at 37° C. for one week.

After the culture was completed, the number of cells attached to the film was measured, and it was found that the number of cells had grown to 5.5 x 104 cells/'11.

<Effects of the Invention> As described above, according to the cell culture substrate of the present invention, the substrate made of a polymeric material is coated with a cumulative monomolecular film, and a glycoprotein is coated on the cumulative film. Since the glycoproteins and the like are supported, the glycoproteins and the like can be firmly immobilized and supported on the base material without falling off from the base material. In addition, the above-mentioned glycoproteins and the like have excellent adhesion to cells, as well as cell spreadability and proliferation, and therefore have the unique effect of enabling high-density and long-term cell culture. 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 accompanying drawing is a sectional view showing an example of a cell culture vessel using the cell culture substrate of the present invention. (1)... Substrate for cell culture, (2)... Support screen, (3)... Housing, (4)... Hole,
(5)...Tube.

Claims (1)

[Claims] 1. The base material made of polymer material is a monomolecular film. is coated with a cumulative film of Accumulated membranes contain sugars, proteins, lipids, and Supported by at least one of the complex compounds of For cell culture, characterized by Base material. 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. The cell culture substrate according to scope 1. 4. If the cumulative film is exposed to ultraviolet rays, electron beams or ions partially treated by irradiation. Cell culture according to claim 1 Base material for use. 5. Sugars, proteins, lipids and other substances are added to the cumulative membrane. At least one of these complex compounds is The above partially claimed patent claims The cell culture substrate according to scope 1. 6. Sugars, proteins, lipids and their complexes At least one type of compound has a lattice pattern Claim No. The cell culture substrate according to item 5. 7. Sugars, proteins, lipids and their complexes At least one of the compounds has a striped pattern. Claim No. 5 as claimed above Substrate for cell culture as described in section. 8. Sugar, protein, lipid and their complexes At least one of the compounds has a polka dot pattern Claim No. The cell culture substrate according to item 5.