JPH1075775A - Bed material for cell culture - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a bed material for cell culture having solid surface with high cell adhesion property and useful e.g. for the biosynthesis of physiologically active substance with the cells of mammals by forming a hydrogel of a mixed polymer composed of polyvinyl alcohol and chitosan. SOLUTION: Polyvinyl alcohol and chitosan are homogeneously mixed with each other in the form of an aqueous solution containing acetic acid under pressure and heating, the obtained aqueous solution of a mixed polymer is formed to a prescribed form and the formed article is rieutralized in an aqueous solution of sodium hydroxide and thoroughly washed with water to obtain the objective formed article of hydrogel of the mixed polymer. The water content of the mixed polymer hydrogel is 50-95wt.%, preferably 60-80wt.% and the gel is formed in various forms such as film, cylinder, sphere, case and fiber.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cell culture flooring used for culturing and growing cells.

[0002]

2. Description of the Related Art Many physiologically active substances currently used in pharmaceuticals and the like are biosynthesized using mammalian cells. Therefore, it is necessary to grow those cells efficiently.In general, cells of mammals and other organisms do not grow in suspension, but have the property of growing only on solid surfaces. ing. In order to culture and grow cells having the property of growing in such an attached state, a solid surface on which a large amount of cells are attached, that is, a culture bed, is required. Conventionally, inorganic substances such as glass, natural polymer substances such as polysaccharides, synthetic polymer substances such as polystyrene and the like have been used as culture beds, but because cell culture cannot always be performed efficiently, The emergence of new flooring materials for cell culture is strongly desired. Cell culture is usually carried out by suspending a spherical or fibrous flooring material having a large surface area in an aqueous solution, so that the density of the flooring material is desirably about the same as that of water. It is desired that the flooring is not destroyed. It is also essential that the flooring be non-toxic. Thus, no satisfactory cell culture bed material has yet been developed.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a cell culture floor material having a solid surface with high cell adhesion in order to efficiently perform cell culture.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and have completed the present invention. That is, according to the present invention, there is provided a cell culture flooring comprising a molded article of a hydrogel of a mixed polymer comprising polyvinyl alcohol and chitosan.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The polymer gel used in the present invention is a water-containing gel of a mixed polymer comprising polyvinyl alcohol and chitosan. As the polyvinyl alcohol, those having a saponification degree of 98 mol% or more and an average polymerization degree of 1500 or more are preferably used. Chitosan is synthesized by deacetylation from chitin, and has a degree of deacetylation of 70 mol%.
The above are preferably used. The mixing ratio of chitosan is 15 to 50% by weight, preferably 20 to 40% by weight based on the mixture of polyvinyl alcohol and the water-soluble polymer.
It is. The water content of the mixed polymer hydrogel is 50%.
-95% by weight, preferably 60-80% by weight. In order to produce a molded article comprising the mixed polymer hydrogel, polyvinyl alcohol and chitosan are uniformly mixed in an aqueous solution containing acetic acid under pressure and under heating. Heating temperature is 95-140 ° C, preferably 110-125 ° C
The pressure may be any pressure at which the contents do not boil. Next, the aqueous solution of the mixed polymer is formed into a required shape, and the obtained molded product is immersed in an aqueous sodium hydroxide solution to neutralize acetic acid. After sufficiently washing the molded product with water, a molded product of the mixed polymer hydrogel can be obtained. The cell culture floor material of the present invention has a desired shape, for example, a film shape,
It has various shapes such as a tubular shape, a spherical shape, a case shape, and a fiber shape. Further, the floor material for cell culture of the present invention does not need to be entirely composed of the mixed polymer hydrogel, and only the surface portion thereof may be composed of the mixed polymer hydrogel.

[0006]

Next, the present invention will be described in more detail with reference to examples. Example 1 90 g of distilled water is added to chitosan (degree of deacetylation: 86 mol%), and 0.80 ml of acetic acid is added to 1 g of chitosan to prepare an aqueous chitosan solution. Here, polyvinyl alcohol (degree of saponification: 99.85 mol%, degree of polymerization: 17
00) was mixed at various fraction ratios so that the total weight of the polymer substance became 10 g, and the mixture was placed in a pressure-resistant glass container. The container (temperature: 20 ° C.) was gradually heated to 120 ° C. over 100 minutes (pressure = 2 × 10 ⁵ Pa), kept at this temperature for 10 minutes, and then gradually cooled to 80 ° C. over 40 minutes. The obtained homogeneous aqueous solution was cast on a glass plate and air-dried for 24 hours. Further, this was vacuum-dried at 30 ° C. for 24 hours to obtain a film sample. Thereafter, when this sample was immersed in a large amount of a 4% aqueous sodium hydroxide solution for 6 hours, the sample became a hydrogel. By replacing the aqueous solution with fresh distilled water several times, the polymer eluted from the hydrogel sample was completely removed. This sample was stored in distilled water, and its water content (the ratio of the weight of water in the gel to the weight of the hydrogel sample) was measured (Table 1). In addition, the initial weight mixing ratio of polyvinyl alcohol and chitosan is 98:
2, 95: 5, 90:10, 85:15, 80:20,
Seven types of samples of 70:30 and 60:40 were prepared. For comparison, a sample consisting of only polyvinyl alcohol was prepared. Since the water-containing gel-like film sample obtained as described above has a high water content, its specific gravity is almost 1, which is convenient for floating in water as a cell culture floor material. Table 1 shows the Young's modulus of the hydrogel film sample. The fact that the Young's modulus is high despite the high water content has the characteristic that it is not easily broken by mechanical force during use as a flooring material. The hydrogel film samples of any mixing ratio have extremely high transparency.

Example 2 The adhesion and proliferation of cells to the hydrogel film were examined. That is, mouse-derived fibroblasts (L-92) were placed on a 9 mm x 9 mm hydrogel film as a sample.
While keeping 0.3 ml of the culture solution (about 138,000 cells / ml) of 9) in contact, 37 ° C. in 5% carbon dioxide and 100% humidity
Was placed in an incubator set to 1. Eagle MEM containing 10% fetal calf serum was used as a culture solution. For comparison, a sample consisting of only polyvinyl alcohol, a collagen sample manufactured by Koken Co., Ltd., and a plastic sheet for tissue culture manufactured by Wako Pure Chemical Industries, Ltd. were tested in the same manner. After standing in an incubator for a predetermined time, the number of cells adhering to the hydrogel film as a sample was quantified by a usual nuclear staining method using crystal violet as a dye. In addition, quantification was also performed by directly counting the number of cells under a microscope. Table 1 shows the relationship between the cell growth rate (the ratio of the number of cells adhered to the gel film to the number of cells adhered to the tissue culture plastic sheet) after 30 hours of culture and the chitosan content. The cell proliferation rate increased rapidly with increasing chitosan content. The cell growth rate of the gel-like film containing 40% chitosan after culturing for 30 hours was more than 256 in the collagen sample. Furthermore, as a result of observing the morphology of the cells adhered on the sample with a microscope, the cells were spherical in the gel-like film with a low chitosan content, but were observed in vivo and on collagen as the content increased. It was found that the proportion of cells having a similar spindle shape increased.

[0008] The above results indicate that the mixture of polyvinyl alcohol and chitosan has cell adhesion growth ability.

The content (% by weight) of chitosan in the mixture, the water content, the Young's modulus, the cell growth rate after 30 hours of culture (the number of cells adhered to the gel-like film with respect to the number of cells adhered to the plastic sheet for tissue culture) The ratio is shown in the following table.

[0010]

[Table 1]

Example 3 A homogeneous aqueous solution of a mixed polymer was obtained in the same manner as in Example 1, except that the total weight of chitosan and polyvinyl alcohol was changed to 20 g instead of 10 g. This aqueous solution is extruded from a fine nozzle into methanol at -70 ° C to be dehydrated and coagulated, neutralized in a 4% aqueous sodium hydroxide solution, and washed several times with fresh distilled water to produce a fibrous cell culture. Flooring was formed.

Example 4 A spherical solution was prepared in the same manner as in Example 3 except that the aqueous solution of the mixed polymer was dropped into methanol at −70 ° C. in a fixed amount instead of being pushed out from a narrow nozzle. A cell culture floor was formed.

[0013]

The cell culture flooring material of the present invention solves the inconvenience of conventional cell culture flooring materials because of its ability to adhere and proliferate cells. is there.

Claims (1)

[Claims] 1. A floor material for cell culture comprising a molded article of a hydrogel of a mixed polymer comprising polyvinyl alcohol and chitosan.