JP2596932B2 - Cell culture substrate - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a cell culture substrate. More specifically, the present invention relates to a cell culture substrate used for culturing animal cells.

<Problems to be Solved by the Prior Art and the Invention> In recent years, biologically active substances, for example, vaccines, hormones,
Research for producing interferon and the like has been actively conducted.

In such a method, conventionally, culture of the adherent animal cells has been performed using a glass or plastic petri dish, a test tube, a culture bottle, or the like. Recently, attempts have been made to use a microcarrier or a hollow fiber as a substrate for culture to perform higher-density culture or long-term culture. In order to adhere and grow the adherent animal cells on the culture substrate, the cells have good adhesion to the substrate surface, and the form and arrangement of the adhered cells are effective for cell extension and growth. It is necessary that it is in a proper form. However, although the polymer material conventionally used as a cell culture substrate is excellent in shapeability and durability, it is unsuitable in terms of the adhesiveness and the like, and performs high-density and long-term cell culture. And none of them have achieved satisfactory results.

In order to improve this point, collagen which is a biopolymer or gelatin which is a denatured product thereof is coated on a polymer material (see JP-A-58-71884). A cell culture bed on which a crosslinked body has been formed (see JP-A-61-52280) has been proposed.

However, when collagen is used as a natural polymer material in a cell culture substrate obtained by complexing a natural polymer with the above-mentioned polymer material, the adhesion and proliferation of cells can be improved, but collagen is There is a problem that handling is difficult and reproducibility is poor because the solubility under acidic conditions with good stability is very small, and denaturation at room temperature is likely to occur. Further, when another natural polymer material is used, there is a problem that the adhesiveness of the cells and the extensibility, proliferation and activity of the adhered cells are not yet sufficient.

<Objective> The present invention has been made in view of the above problems,
An object of the present invention is to provide a cell culture substrate that is easy to handle, has excellent adhesion to cells, and has excellent cell spreading, growth and activity maintenance, and enables high-density, long-term culture.

<Means and Actions for Solving the Problems> The cell culture substrate of the present invention, which has been made in order to achieve the above object, has a structure in which a side chain amino group is a guanidyl group on the surface of a polymer substrate. Exchanged gelatin (hereinafter, referred to as guanidylated gelatin) is supported.

The present invention has the above constitution, and gelatin is a kind of derived protein which is irreversibly changed to water-soluble by boiling with collagen and water, and guanidylated gelatin in which the amino group of the side chain of this gelatin is converted into guanidyl group is It has characteristics of high solubility in water, good stability and excellent affinity with cells. That is, the structure of the cell membrane on the cell surface is such that various glycoproteins, glycolipids, and the like called intramembrane particles are embedded in the lipid bilayer with distribution, and these freely flow in the lipid bilayer. Can be involved in cell adhesion. Since the above-mentioned guanidylated gelatin has a site capable of binding to particles in the membrane by ionic bond, hydrophobic bond, or the like, it has high affinity with cells and can secure cells in a stable form and arrangement. Therefore, in the cell culture substrate of the present invention in which guanidylated gelatin is supported, the guanidylated gelatin serves to connect the cells and the substrate, and the cells have excellent adhesiveness and the cells impair the higher-order structure. The cells can be adhered in a stable form and arrangement without causing any damage to cell spreading and proliferation.

In addition, it is preferable that the above-mentioned polymer base material is chemically and physically surface-treated, or a porous material. Further, the base material is preferably a hollow fiber. When the polymer substrate is chemically and physically treated, the wettability is improved, the adhesiveness between the guanidylated gelatin and the substrate is excellent, and the polymer substrate is porous. When the material is a porous material, the metabolism of the substance is facilitated through the pores of the porous material, and the cells can be cultured for a long period of time. In particular, when the polymer substrate is a hollow fiber, cells can be grown and grown on the hollow fiber at a high density by perfusing a culture solution or the like inside or outside the hollow fiber.

Further, it is preferable that guanidylated gelatin is partially supported on the base material, and particularly preferable is that the guanidylated gelatin is supported on a lattice pattern, a striped pattern, a polka dot pattern, or the like. Those in which guanidylated gelatin is partially supported on the base material, in particular, those in which guanidylated gelatin is supported in a lattice pattern, a striped pattern, a polka dot pattern, etc. Adhere at predetermined intervals, so that the adhesion to cells is further stabilized, and the extension and proliferation of cells can be further increased.

 Hereinafter, the present invention will be described in detail.

The cell culture substrate of the present invention comprises a polymer substrate and guanidylated gelatin supported on the substrate.

The above guanidylated gelatin can be easily obtained by reacting commercially available gelatin with a guanidylating agent such as O-methylisourea or 1-guanyl-3,5-dimethylpyrazole in the presence of a basic compound. This reaction is generally performed in a solvent, and water, hydroalcohol, or the like is used as the solvent. Examples of the basic compound include alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, and alkali metal carbonates and hydrogen carbonates such as sodium carbonate, potassium carbonate and sodium hydrogen carbonate. The reaction is carried out at room temperature to heating, and after the reaction, reprecipitation,
The guanidylated gelatin can be separated and purified by a conventional method such as dialysis. The amount of guanidyl groups introduced into the guanidylated gelatin used in the present invention is not particularly limited, and those having various degrees of substitution can be used. The amount of the guanidyl group introduced can be adjusted by appropriately selecting the amount of the guanylating agent to be used, the reaction time, and the like.
The guanidylated gelatin thus obtained has high solubility in water and excellent stability, and is easy to handle.

Further, as the material of the polymer base material, any material having shapeability and mechanical strength can be used, for example, polyethylene, polypropylene, chlorinated polyethylene, olefin polymers such as ionomers, and polyolefins. Tetrafluoroethylene, 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, polyphenylene oxide, polyethylene terephthalate, polyester resin such as polybutylene terephthalate, epoxy resin, polyamide, polyimide, polysulfone, cellulose resin, silicone resin, polyurethane, etc. Or a blend thereof.

The polymer substrate can be formed in various forms, for example,
In addition to molded articles such as petri dishes and flasks, examples include forms of films, tubes, hollow systems, fibers, fine particles, and the like. Of these forms, for long-term cell culture, a porous polymer substrate having pores that facilitate substance metabolism is preferable, and for high-density culture, a tube or hollow fiber shape is preferable. Is preferred. In particular, a hollow fiber made of a porous polymer base material that facilitates substance metabolism and can perform high-density culture for a long period of time is preferable. When using this hollow fiber, the culture solution
The cells can be grown and grown on the hollow fiber by perfusing the hollow fiber or the outside of the hollow fiber and sending carbon dioxide gas, air or the like to the hollow fiber or the like as necessary. The hollow fiber may be of various sizes, for example, having an inner diameter of about 50 to 1000 μm.

When the cell culture substrate of the present invention is used as a bead carrier in a microcarrier method, the polymer substrate has a particle size of about 100 to 300 μm.

The polymer substrate may be untreated,
In order to improve the adhesiveness to guanidylated gelatin, it may be physically or chemically treated. Examples of the treatment include plasma treatment, radiation treatment by irradiation with ultraviolet rays (including laser), electron beam, α-ray, β-ray, γ-ray, ion treatment by ion sputtering, ozone treatment in an ozone atmosphere, or peroxidation. Hydrogen, potassium persulfate,
Chemical treatments such as introduction of an amino group which is oxidized, nitrated and reduced by chromate or the like, sulfonation with chlorosulfonic acid, etc., improve the wettability between the guanidylated gelatin and the base material and improve the adhesiveness. In addition, among the above surface treatments, the laser treatment can chemically modify the base material, and can also perform physical modification to process the base material surface into fine irregularities, so that the metabolism of cells can be reduced. There is an advantage that it can be promoted.

The cell culture substrate of the present invention is obtained by supporting a guanidylated gelatin on a polymer substrate.For example, if necessary, a polymer substrate that has been physically or chemically pretreated is converted into guanidyl. It is obtained by immersing in a solution containing gelatinized gelatin, or applying the solution on a substrate, followed by drying.

The supporting of the guanidylated gelatin may be either a monomolecular layer or a multimolecular layer, and may be supported on the entire surface of the base material. Preferably, by carrying such a pattern, it is possible to control the arrangement of the cells adhering to the substrate, thereby stabilizing the adhesiveness of the cells, and making it possible to favor cell spreading, proliferation and functional expression. . further,
When the guanylated gelatin is partially supported as described above, a useful surface capable of further enhancing the above effects can be formed, particularly by supporting the guanylated gelatin in a fine pattern such as a lattice, a striped pattern, and a polka dot pattern. . In order to carry the guanidylated gelatin in a pattern on the substrate, for example, a technique such as screen printing can be applied.

The substrate for cell culture of the present invention can be used for culturing various cells, and the type of cells is not particularly limited,
Epithelial cells are particularly exemplified.

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 growth are used. The culture is carried out.

<Example> Hereinafter, the present invention will be described in more detail based on examples. The guanidylated gelatin was prepared by the method of the following Reference Example.

Reference Example 15 ml of distilled water adjusted to pH 11 using sodium hydroxide
In addition, 0.2 g of gelatin and O-methylisourea at 40 ° C.
Dissolve 2 g. Thereafter, the reaction was allowed to proceed at room temperature for 3 days to perform guanidylation of gelatin. Next, the reaction solution was charged into a dialysis membrane and dialyzed in distilled water to obtain a guanidylated gelatin solution.

Example 1 and Comparative Examples 1 to 3 The guanidylated gelatin solution obtained in the above reference example was poured into a polystyrene petri dish so as to cover the entire petri dish, and dried at a distance of about 30 cm under an ultraviolet lamp on a clean bench for a day and a night. To obtain a petri dish coated with guanidylated gelatin.

On the other hand, as comparative examples, a Petri dish obtained by treating gelatin in the same manner as described above using a solution prepared by adjusting gelatin to the same concentration as the guanidylated gelatin solution (Comparative Example 1), and a commercially available surface-treated polystyrene Petri dish for cell culture ( Comparative Example 2)
And an untreated polystyrene dish (Comparative Example 3).

In each of these dishes, a culture medium containing 10% fetal bovine serum added to an Eagle's MEM solution was used to prepare Chang from human normal hepatocytes.
After injecting a suspension prepared by adjusting Liver cells to a concentration of 5 × 10 ⁴ cells / ml, incubate at 37 ° C. for 1 hour in a carbon dioxide incubator in an atmosphere of 5% carbon dioxide and 95% air. did. Next, the culture solution in the Petri dish is discarded, and further washed sufficiently with a phosphate buffer containing no Ca ⁺⁺ and Mg ⁺⁺ to remove non-adhered cells, followed by a solution containing trypsin-EDTA. The adherent cells were peeled off, the number of adherent cells was examined with a hemocytometer, and the cell adhesion rate was determined. The results shown in the following table were obtained.

Example 2 A plurality of petri dishes used as Example 1 and Comparative Examples 1 to 3 were prepared, and Chang Live was prepared in the same manner as in Example 1.
r The cells were cultured, the number of cells adhered to the incubator and proliferated was measured over time using a hemocytometer, and cell proliferation was examined. The results are shown in the accompanying drawings.

As is clear from the above table and the attached drawings, the petri dishes coated with guanidylated gelatin showed excellent cell adhesion and proliferation.

Example 3 A porous film of a tetrafluoroethylene resin porous film (Fluoropore FP-022, manufactured by Sumitomo Electric Industries, Ltd.) was subjected to plasma treatment in an ammonia gas atmosphere. At this time, the discharge output was 100 W, the pressure was 0.3 Torr, the ammonia gas flow rate was 7 cc / min, and the processing time was 10 minutes. The treated film was set on a 45 mm φ glass Petri dish, sterilized by high pressure steam, added with a guanidylated gelatin solution, and dried to coat the guanidylated gelatin. Then, human normal hepatocyte-derived
A Chang Liver cell suspension (5 × 10 ⁴ cells / ml in an Eagle's MEM culture medium supplemented with 10% fetal bovine serum) is added to a Petri dish,
The cells were cultured in an air atmosphere containing carbon dioxide at a temperature of 37 ° C. for 6 days. After culturing, the average number of cells is 6.5 × 10 ⁵ cells / ml
And good growth was observed.

<Effect of the Invention> As described above, according to the substrate for cell culture of the present invention,
Guanidylated gelatin is supported on the surface of the polymer substrate, and guanidylated gelatin has high solubility and good stability, so it is easy to handle, and has excellent affinity with cells and adhesion. Since it is possible to increase the density, a unique effect that high-density and long-term cell culture can be performed is exhibited. Therefore, the cell culture substrate of the present invention,
The present invention can be used for a production system for producing useful substances such as hormones by culturing animal cells, and also for constructing an artificial organ so that an artificial pancreas can be formed by, for example, adhering and culturing insulin-producing cells on a substrate surface.

[Brief description of the drawings]

The accompanying drawings are diagrams showing cell proliferative properties of the cell culture substrates of the present invention and comparative examples.

Claims (8)

(57) [Claims] 1. A substrate for cell culture, characterized in that gelatin having a side chain amino group exchanged for a guanidyl group is carried on the surface of a polymer substrate. 2. The cell culture substrate according to claim 1, wherein the polymer substrate is chemically or physically surface-treated. 3. The cell culture substrate according to claim 1, wherein the polymer substrate is a porous material. 4. The cell culture substrate according to any one of claims 1 to 3, wherein the polymer substrate is a hollow fiber. 5. The method according to claim 1, wherein gelatin having a side chain amino group converted to a guanidyl group is partially supported on the surface of the polymer base material. The substrate for cell culture according to item 1. 6. The cell culture substrate according to claim 5, wherein the gelatin in which the amino group in the side chain is converted to a guanidyl group is carried in a lattice pattern. 7. The cell culture substrate according to claim 5, wherein the gelatin in which the amino group in the side chain is converted to a guanidyl group is carried in a striped pattern. 8. The cell culture substrate according to claim 5, wherein the gelatin in which the amino group in the side chain is converted to a guanidyl group is carried in a polka dot pattern.