JPH0542258B2 - - Google Patents

Description

[Detailed description of the invention] [Industrial application field]

The present invention relates to a novel cell culture substrate that has excellent safety, biocompatibility, and mechanical strength.

[Conventional technology]

Glassware has been used as a substrate for cell culture for a long time. Other examples include plastic shear whose wettability has been changed by chemical etching or electrical discharge machining, microcarriers with electrical charges introduced into cross-linked dextran, microcarriers with electrical charges introduced into various synthetic polymers, and Examples include chemically modified collagen gel. All of these substrates were discovered by changing physicochemical parameters such as surface wetting properties, water content, ionic interactions, and microdomain structure, or by using biochemically specific binding reactions. be. However, materials such as plastic shear cannot withstand high-pressure steam sterilization, and dextran crosslinked products, collagen gel, etc. have the disadvantage of low mechanical strength. Furthermore, the production of microcarriers using synthetic polymers requires sophisticated technology, and this artificial substrate is inferior in terms of compatibility with living organisms. Therefore, there is currently a high demand for a substrate that can withstand high-pressure steam sterilization and has excellent strength and is free from cytotoxic substances eluated from the material itself. Until now, there had been no idea of applying cross-linked chitosan molded products as substrates for cell culture.

[Problems to be solved by the invention]

The present invention solves the above-mentioned drawbacks of previously known cell culture substrates. Chitosan is a substance obtained by deacetylating chitin found in natural crustaceans such as shrimp and crabs, and since it is derived from living organisms, it has low cytotoxicity and is well compatible with living organisms. It is extremely safe as it does not contain any toxic residual monomers found in synthetic polymeric substances. In addition, chitosan has a suitable crystal structure, so it has high mechanical strength and high resistance to high-pressure steam sterilization.Chitosan has an amino group in its molecular structure, so it has antithrombotic properties by introducing a charge and chemical modification. It also has the property of being easy to apply biochemical treatments such as immobilization of enzymes and proteins. Therefore, the present inventors have created the present invention by paying attention to the above-mentioned characteristics of chitosan, and by using chitosan, which has excellent safety and biocompatibility that are not found in synthetic polymers, A novel cell culture substrate with high mechanical strength not found in other biologically derived artificial substrates has been obtained.

[Means to solve the problem]

The cell culture substrate of the present invention can be obtained by using chitosan as a raw material, dissolving it in an acidic aqueous solution, shaping it into a membrane, fiber, sphere, etc. in a basic solution, and crosslinking it with a crosslinking agent. It will be done. Although the chitosan used is not particularly limited, it is preferable to use a low molecular weight chitosan having an average molecular weight of 10,000 to 230,000. Chitosan is dissolved in an aqueous solution of acetic acid, dichloroacetic acid, formic acid, etc. alone or in a mixture. The concentration of the chitosan acidic aqueous solution can be arbitrarily selected within a range suitable for handling, and the chitosan acidic aqueous solution contains sodium hydroxide, potassium hydroxide,
Coagulate in a basic aqueous solution containing alkaline substances such as sodium carbonate, potassium carbonate, ammonia, and ethylenediamine. A polar alcohol such as methanol or ethanol may be added to the basic aqueous solution. The cell culture substrate according to the present invention can have any shape such as membrane, fiber, or spherical shape. In order to form a film, methods such as applying an acidic chitosan aqueous solution onto a solid and immersing it in a basic aqueous solution, or extruding it into a film into a basic solution, etc. are used.
In the case of fibrous or spherical shapes, the chitosan acidic aqueous solution is supplied under pressure from a discharge port into a coagulation bath consisting of a basic aqueous solution, either continuously or in a fixed amount, and is obtained by coagulating the chitosan acidic aqueous solution. The chitosan molded product obtained as above is
Crosslinking treatment is performed using an organic diisocyanate compound, epihalohydrin, glutaraldehyde, or organic halide. By crosslinking, the chitosan molded product has increased mechanical strength and becomes convenient to handle. When performing a crosslinking reaction using an organic diisocyanate compound, the reaction is performed in a polar solvent. As the polar solvent, alcohols such as methanol, ethanol and isopropyl alcohol, ketones such as acetone and methyl ethyl ketone, and amides such as dimethylformamide and dimethylacetamide can be used. Examples of organic diisocyanate compounds include 4,4'-diphenylmethane diisocyanate, 1,4-phenylene diisocyanate, 2,4-tolylene diisocyanate, naphthalene diisocyanate, 1,4-cyclohexane diisocyanate, 4,4'- Examples include dicyclohexylmethane diisocyanate, xylylene diisocyanate, isophorone diisocyanate, hexamethylene diisocyanate, and the like. The cell culture substrate according to the present invention includes interferon, tumor antigen, urokinase, insulin,
It can be used not only as a substrate for mass culture of cells when separating and purifying monoclonal antibodies, etc., but also as a hybrid artificial organ, which is attracting attention as an artificial organ that allows cells to engraft and makes use of the natural abilities of living organisms.

【Example】

Next, the present invention will be explained with reference to Examples. Cell suspension, culture and counting methods in Examples are as follows. (1) Eagle's minimum essential medium supplemented with 10% fetal bovine serum was used as the culture medium. (2) The cells used were L929, a mouse connective tissue-derived fibroblast, and Vero, an African green monkey kidney-derived fibroblast. (3) Add 1.0×10 ⁵ cells/ml to the culture solution in (1).
Each cell in (2) was suspended. (4) Dispense 5 ml of cell suspension into each dish and heat at 37°C.
The number of cells was counted after culturing for 1, 2, and 4 days in an atmosphere of 5% CO ₂ . (5) The counting method is to remove floating cells (cells that are floating without engrafting) and the culture medium with a phosphate buffer, detach the engrafted cells with a trypsin-EDTA solution treatment, and
After that, the liquid was centrifuged at 700 rpm for 6 minutes,
After removing the trypsin-EDTA solution, etc., the precipitated cells were suspended in 2 ml of phosphate buffer and counted using a hemocytometer. ◇Example 1 Chitosan with a molecular weight of 200,000 and a degree of deacetylation of 80% and 95% was made into a 0.5% acetic acid aqueous solution, and 2 ml of this was uniformly injected into the bottom of a glass shear dish with a diameter of 60 mm, and neutralized with 5% ammonia water to form a solution. The membrane was washed with pure water, hot water, and dried at 40°C. 10 ml of 5% epichlorohydrin in ethanol was poured onto the film on the shear plate, left to stand at 40°C for 4 hours, washed 3 times with ethanol, then rinsed with pure water, and then washed once with hot water at 80°C.
The samples were sterilized using high-pressure steam at 121°C for 20 minutes, and washed with phosphate buffer under aseptic conditions to obtain culture substrates of sample numbers 1 and 2. In addition, a film was made on Shearle as described above using chitosan with a molecular weight of 200,000 and 43,000 and a degree of deacetylation of 95%, and 10 ml of a pure aqueous solution of 2.5% glutaraldehyde was poured into it, and the film was allowed to stand at room temperature for 2 hours.
Rinse with pure water, then rinse with hot water at 80℃ for 1 hour.
The samples were sterilized with high-pressure steam at ℃ for 20 minutes and washed with phosphate buffer under aseptic conditions to obtain culture substrates of sample numbers 3 and 4.
In addition, a film was made on Shearle as described above using chitosan with a molecular weight of 200,000 and a degree of deacetylation of 80% and 95%, and 10 ml of a 5% hexamethylene diisocyanate (HDI) solution in dimethylformamide was added to the film and the mixture was heated at room temperature for 1.5 min. Let it stand for an hour, wash it three times with dimethylformamide, wash it with pure water, wash it with hot water at 80℃ for 30 minutes, autoclave it at 121℃ for 20 minutes, wash it with phosphate buffer under aseptic conditions, and sample number 5. , 6 culture substrates were obtained. In addition, a film was made on Shearle as described above using chitosan with a molecular weight of 43,000 and 200,000 and a degree of deacetylation of 95%, and 10 ml of a 1% solution of 4,4' diphenylmethane diisocyanate (MDI) in dimethylformamide was poured. Leave it at room temperature for 1 hour, wash it 3 times with dimethylformamide, and then with pure water.
Washed in hot water at ℃ for 30 minutes. This was autoclaved at 121°C for 20 minutes, washed with phosphate buffer under aseptic conditions,
Culture substrates of sample numbers 7 and 8 were obtained. In addition, the degree of deacetylation is 95 with a molecular weight of 43,000 and 200,000.
% of chitosan was used as described above, 10 ml of 5% cyanuric chloride in dimethylformamide was poured into the film, and the film was left to stand at 80°C for 2 hours, washed three times with dimethylformamide, and then washed with pure water. and 80
The samples were washed with hot water at 121°C for 1 hour, sterilized with high-pressure steam at 121°C for 20 minutes, and washed with phosphate buffer under aseptic conditions to obtain culture substrates of sample numbers 9 and 10. For these sample numbers 1 to 10, cells L929 were cultured on these substrates. 1, 2,
The number of cells after 4 days is shown in Table 1 together with Comparative Example 1 in which conventionally used glass shears were used as the culture substrate. The culture substrate according to the present invention had increased strength due to the crosslinking reaction, was easy to handle, and had excellent performance as a culture substrate.

[Table] Also, using the culture substrate used for sample numbers 4 and 8 above with a molecular weight of 200,000 and a degree of deacetylation of 95%,
The results of culturing Vero cells as sample numbers 11 and 12 are shown in Table 2 together with the cell counts of Comparative Example 2 using glass shear as the culture substrate. The chitosan culture substrate subjected to the crosslinking reaction showed excellent performance as a culture substrate, as in the case of cell L929.

[Table] ◇Example 2 A 5% acetic acid aqueous solution of chitosan with an average molecular weight of 42,000 and a degree of deacetylation of 80% was mixed with 10% NaOH and 30%
After forming into a sphere by dropping N ₂ into a basic aqueous solution consisting of CH ₃ OH and water through a nozzle with a nozzle diameter of 0.24 mm at a pressure of 2 kg/cm ² , the spherical object was washed with water until the washing liquid became neutral. Spherical substrates with a particle size of 42-80 mesh were obtained. Take out 100 ml of this spherical material, perform dimethylformamide substitution four times, and then add 100 ml of 10% 4,4' diphenylmethane diisocyanate (MDI).
was added and stirred at room temperature for 1 hour. After that, after replacing with dimethylformamide three times, washing with water, washing with hot water at 80℃ for 1 hour, replacing with phosphate buffer, autoclaving at 121℃ for 20 minutes, washing with phosphate buffer under aseptic conditions, and using sample number 17. A culture substrate was obtained. Separately, 100 ml of 2.5% glutaraldehyde was added to 100 ml of the spherical material, stirred at room temperature for 2 hours, washed with pure water, and then washed with hot water at 80° C. for 1 hour. After replacing this with phosphate buffer, it was sterilized with high pressure steam at 121℃ for 20 minutes.
It was washed with phosphate buffer under sterile conditions and used as a culture substrate for sample number 18. A predetermined amount of the culture substrates of Sample Nos. 13 and 14 were placed in a shear dish, and cells L929 were cultured.
The results of this culture are shown in Table 3 together with the results obtained using a commercially available culture substrate (Cytodex type, manufactured by Pharmacia, Sweden) as Comparative Example 3. These results revealed that the spherical chitosan substrate also has excellent performance as a culture substrate.

【table】

【Effect of the invention】

As is clear from the description of the above examples, the cell culture substrate of the present invention can be sterilized with high pressure steam,
Since the molded chitosan culture substrate is cross-linked, its mechanical strength is increased and it can be easily handled. The cell culture substrate according to the present invention has excellent performance for cell culture, which is exactly the same as that of conventionally used substrates. This provides a very useful and safe substrate in terms of toxicity and the like.

Claims (1)

[Scope of Claims] 1. A substrate for cell culture, characterized in that chitosan is dissolved in an acidic aqueous solution, re-molded in a basic solution, and then cross-linked with a cross-linking agent. 2. The cell culture substrate according to claim 1, wherein the crosslinking agent that performs the crosslinking treatment is any one of organic diisocyanate, epihalohydrin, glutaraldehyde, and organic halide.