JPS6030508B2 - Cell culture container - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a cell culture container made of synthetic polymer material, glass, or the like.

Conventionally, glass surfaces or treated surfaces of synthetic polymers have been used to grow cells, and various containers made of polystyrene and subjected to surface treatment are already in widespread use as containers for cell culture. In recent years, problems such as the soaring price of serum, labor shortages, variations in serum lots, and removal and purification of serum components after manufacturing vaccines and interferon have resulted in products containing no serum (serum-free) or serum-containing products containing only 1.
% or less (low serum) is gaining attention, and a number of culture medium formulations have been devised.

However, in these serum-free (low) serum-free beaches, depending on the type of cells, the cell culture containers currently available on the market have poor cell growth compared to normal serum-containing media containing 5 to 20%. When a small number of cells are cultured, there is almost no proliferation and no colony formation is observed. It is said that this is not due to a lack of growth factors contained in serum, but is often due to a lack of wall-adhering factors on the culture surface. For example, fibronectin is a major wall-adhesion factor for serum, and it is coated on the culture surface, and when added to the culture medium, serum can be increased by 5 to 20% even in cultures without (or little) serum.
It has been reported that growth almost equivalent to that of the containing medium can be obtained.

Although this fibronectin is purified from serum, it has drawbacks such as high cost and difficulty in handling such as sterilization. Therefore, instead of fibronectin, various natural or synthetic polymers such as polyarginine, polylysine, collagen,
Although there are reports of culturing by coating the culture surface with polyethyleneimine, etc., very good results have not been obtained. As a result of intensive research on various coating treatment methods, the present inventors discovered that gelatin, which is a hydrolyzate of collagen, is a wall-adhesive factor equal to or greater than fibronectin. We obtained very good results by coating the culture surface of a cell culture container and culturing cells in a serum-free (low) serum medium.

Alternatively, it is very difficult to grow a small number of cells in normal serum-free (low) serum culture, but when a gelatin-coated culture surface is used, they easily grow and form colonies. Furthermore, when using gelatin, it was confirmed that it is an easy-to-handle material that hardly loses its effect even if it is sterilized by autoclaving (for example, 12 RO, 18 minutes).

Cell culture containers made of synthetic polymer materials or glass coated with gelatin-containing solutions are not only very suitable for culture without (or little) serum, but can also be used in normal media with the addition of serum. It was concluded that the amount could be reduced. The synthetic polymer materials mentioned here include polystyrene, polyethylene, polypropylene, cellulose acetate, polycarbonate, polyester, nylon, polyacrylate, polyvinyl chloride, and the like.

Further, the cell culture containers mentioned here include petri dishes, various plates, flasks, culture tubes, culture bottles, large-scale culture beds, and the like. Gelatin used in the present invention includes, for example, skin, knee,
It is a denatured protein obtained by hydrolyzing collagen, which is a hard protein of bones and the like, from hot hollywood, and preferably has a molecular weight of 100,000 to 200,000 and is a mixture containing simple amino acids such as glycine, alanine, valine, and leucine.

These gelatins may be used alone or in combination with various known coating agents. Although gelatin coating is effective even when treated at a low temperature of around 4° C. for a short period of time, treating just one powder, it is more effective if the gelatin coating is desirably treated at a high temperature of 30° C. or higher or for a long period of time, overnight or longer.

Also, both gelatin-coated products that remain wet and those that have been dried to form a gelatin film have similar effects. 0 again
．． Even if cornering is performed using a gelatin solution with a low concentration of up to 0.0002%, the effect will not be lost. Examples are shown below.

Example 1 A phosphate buffer solution containing 1% gelatin was heated at 121°C for 15 minutes.
Sterilize by autoclaving for 0.5 minutes and place in a 600 plastic petri dish (made of polystyrene) for cell culture for 0.5 minutes.
The cells left at 370 for 15 minutes were used for culture.

Example 2' A phosphate buffer solution containing 1% gelatin was heated at 121°C for 18
Sterilize by autoclaving for 1 minute, then place 1 skin in a 600 plastic petri dish (made of polystyrene) for cell culture.
The cells were left overnight at ℃ and used for culture.

Example 3 Purified distilled water containing 1% gelatin was sterilized by autoclaving at 12 RO for 18 minutes, and this was sterilized by autoclaving at 60°C for cell culture. The cells were placed in a plastic petri dish (made of polystyrene) at a concentration of 0.5 mm and dried at 37° C. until a gelatin film was formed, and used for culture.

Example 4 The petri dish of Example 1 was then washed with physiological saline and used for culture.

Example 5 Phosphate buffer containing 0.0002% gelatin was
ro, sterilize the gelatin for 18 minutes and use it for cell culture.
The cells were placed in a plastic Petri dish (made of polystyrene) with 0.5 secretions and left for 370 minutes for 15 minutes, which was then used for culture.

Example 6 Y-ray sterilization of the petri dish of Example 3 (2.
d) was used for culture.

Example 7 The petri dish of Example 3 was EOG sterilized (300 p/night, 5
0qo, 2 hours) was used for culture.

Comparative example 1 For cell culture without coating on the surface 6
A plastic Petri dish (made of polystyrene) was used as it was for culture. Comparative Example 2 Cell culture was carried out in a 604 plastic Petri dish for cell culture (made of polystyrene) using Eagle M old M medium containing 10% of normal calf serum.

Table 1 shows the results of culturing under the following conditions using the cell culture containers of each Example and Comparative Example.

The culture conditions are as follows: {1} Cells V7 stage convoluted ■ Culture medium SF Toji* {3} Culture conditions C02 incubator (5% C0
2) for 370, 7 days.

{4} Culture method V79 cells passaged in Eagle M old M toji containing 10% calf serum were cultured at 10 cells per 600 petri dishes.
Plant 0.

*SF medium is Eagle MEM medium A 5/
So, insulin 20 units/night, pyruvate 110/9/y, ipoxanthine 4/9, thymidine 2.4/9/2, and dispensable amino acids added.

Table 1※※ Day: Colony formation is good (colony formation rate 50% or more) Ten: Colony formation is observed (colony formation rate 0-50%) -...No colony formation (colony formation rate 0%) ) As is clear from Table 1, the cell culture container of the present invention exhibits good cell culturability even in a serum-free medium.

Claims (1)

[Claims] 1. A cell culture container made of a synthetic polymer material, glass, etc., coated with a gelatin-containing solution.