JPS6337625B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel Dulbecco's modified Eagle medium (hereinafter referred to as DME medium) that can be sterilized under high pressure. DME medium was prepared by Earl Dulbetsko (R.
This is a medium developed by Dulbecco et al.
By doubling the amino acid concentration and quadrupling the vitamin concentration of the known Eagle MEM, we enriched the nutrients necessary for the growth of cultured cells, and further added glycine, serine, pyruvate, and iron ions. This is an improved type of Eagle MEM, which is widely used for primary culture and single cell culture of various cells. In particular, DME medium is widely used as a medium that can maintain cells for a long time even if cells are infected with a virus and then changed to a serum-free medium. This DME medium was initially a liquid medium, but
The manufacturing method has since been improved, and it is now available as a powdered medium that can be stored for a long period of time. However, the powdered medium must be dissolved and sterilized prior to its use.
Currently, the most effective and easy-to-operate method for sterilizing culture media is high-pressure sterilization. However, traditional
When a DME medium is subjected to high-pressure sterilization, its supporting capacity for cell growth is significantly reduced, so sterilization using a bacterial strainer has been unavoidable. However, the sterilization method using a bacterial strainer has the disadvantage that it has insufficient bactericidal effects and requires a lot of labor and time, and there has been a desire for the development of a DME medium that can be sterilized under high pressure. Therefore, the inventor of the present invention conducted intensive research to solve these problems, and as a result, removed or replaced the heat-labile substances in the formulation of the medium, and added an organic acid buffer. The present invention was completed based on the discovery that a DME medium that does not lose its supporting capacity for cell growth even after high-pressure sterilization can be obtained by containing the agent. That is, the present invention provides L from the formulation of DME medium.
- High-pressure sterilization type that removes glutamine and L-cysteine and instead contains L-cysteine and an organic acid buffer consisting of a combination of an organic acid and its salt.
It provides a DME medium. The organic acid buffer used in the present invention includes PH
is preferably 4.25, such as acetic acid and sodium acetate, succinic acid and sodium succinate, etc. A typical formulation of the autoclaved DME medium of the present invention (hereinafter referred to as the medium powder of the present invention) is as follows. Sodium chloride 6400.0 (mg) Potassium chloride 400.0 Calcium chloride 200.0 Magnesium sulfate 97.7 Sodium dihydrogen phosphate (anhydrous) 108.0 Ferric nitrate (nonahydrate) 0.1 Glucose 1000.0 Sodium pyruvate 110.0 Succinic acid 106.0 Sodium succinate (hexahydrate) ) 27.0 L-arginine hydrochloride 84.0 L-cysteine hydrochloride (monohydrate) 70.3 Glycine 30.0 L-histidine hydrochloride (monohydrate) 42.0 L-isoleucine 104.8 L-leucine 104.8 L-lysine hydrochloride 146.2 L-methionine 30.0 L-phenylalanine 66.0 L-serine 42.0 L-threonine 95.2 L-tryptophan 16.0 L-tyrosine disodium 89.5 L-valine 93.6 Choline bitartrate 7.2 Folic acid 4.0 Nicotinamide 4.0 Calcium pantothenate 4.0 Pyridoxal hydrochloride 4.0 Riboflavin 0 .4 Thiamin hydrochloride Salt 4.0 i-inositol 7.2 Phenol red 5.0 Total amount 9503.0 The autoclaved DME medium of the present invention is produced by mixing the above components in a conventional manner. still
DME medium generally contains 10
When the amount of serum added is small, the advantage of the medium of the present invention containing L-cysteine instead of L-cysteine in autoclaving is particularly remarkable. Next, an example will be given and explained. Example 1 10.0 g of DME medium powder (commercially available) was dissolved in 1 distilled water, and 1.2 g of sodium bicarbonate was added to completely dissolve it. This has a pore size of 0.2μ.
The DME medium (hereinafter referred to as conventional medium) was obtained by over-sterilizing with a filter. Dissolve 9.5 g of the present invention medium powder in 1 distilled water, autoclave at 121°C for 15 minutes, and after cooling,
A medium (referred to as the medium of the present invention) was obtained by adding 0.584 g of dissolved and oversterilized L-glutamine and 1.2 g of sodium bicarbonate. A medium (comparison medium ) was obtained. Newborn calf serum was added to the three media obtained above to a concentration of 2%, and the cell line Hela-S3 derived from human uterine cancer cells was cultured under the following conditions, and its proliferation was examined. . φ6cm plastic jar (medium volume: 5ml), number of implanted cells: 5 x ¹⁰⁴ cells/dish, 37°C, 5% carbon dioxide gas. The results are shown in FIG. Example 2 Cell line Hela-S3 derived from human uterine cancer cells was cultured in each culture medium prepared in the same manner as in Example 1 to examine the colony formation rate. However, the serum concentration was investigated at 5% and 2%.
The following table shows the results of each measurement using five sheets of shear glass. 【table】

[Brief explanation of the drawing]

FIG. 1 shows the growth state of HeLa-S3 strain in an autoclaved medium of the present invention to which 2% serum was added.

Claims (1)

[Claims] 1 L from the formulation of Dulbecco's modified Eagle's medium
- An autoclaved Dulbecco's modified Eagle medium characterized in that glutamine and L-cystine have been removed and, instead, an organic acid buffer consisting of L-cysteine and a combination of an organic acid and its salt is contained. 2. The autoclaved Dulbecco's modified Eagle medium according to claim 1, wherein the organic acid buffer is a combination of succinic acid and sodium succinate.