SU1655982A1 - Method for decontamination of transplanted cell culture from mycoplasma - Google Patents

Abstract

The invention relates to virology and can be used to obtain mycoplasma-free cell cultures used in virological work. The purpose of the invention is to increase the degree of decontamination of cell cultures from mycoplasmas. The essence of the method consists in the combined treatment of transplantable cell cultures with tiamulin (10-15 µg / ml) and etonium (5-7 µg / ml) for three lases. Etonium is applied directly to the monolayer. Recording of the results on decontaminocyto is carried out by seeding on elective media, staining with DNA-fluorochrome-olivomycin, and setting ELISA. Cell cultures are free of mycoplasmas for 15 passages (observation period), C

Description

The invention relates to virology and can be used to obtain mycoplasma-free cell cultures used in virological work.

The aim of the invention is to increase the degree of decontamination of cells from mycoplasma achieved by treating the cell monolayer with etonium at a concentration of 5-7 µg / ml and with the antibiotic tiamulin at a concentration of 10-15 µg / ml for three lases.

Example 1 „Transferable cell line LEK at a concentration of 100 thousand / ml

Sown in 250 ml of matrasiki. The cells are cultured on FGM-s enriched medium with 10% serum bodies. Thiamulin 10 µg / ml is added to the growth medium, and after forming a monolayer, 5 µg / ml is injected into the growth medium. The next day, the cells are subcultured, and the treatment cycle is repeated three times. After the third cycle of treatment, the cell culture is checked for the presence of mycoplasmas on elective nutrient media: 2-3% PGM-C and medium 1-8, DNA-Llorochromation, ELISA.

As a result, cell lines free of mycoplasmas are obtained. 0 For &

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We do not cultivate for 2 months (20 passages) without preparations; cells are free of mycoplasmas (observation period). c

Example 2 “The transplanted PT-80 cell line at a concentration of 80 thousand / ml is seeded in 250 ml of mattress. The cells are cultured on medium 199 and GLA (0.5% lactal hydrolyzate | 0 bumin hydrolyzate solution) in equal ratios with 10% bovine serum, 13 μg / ml tiamulin is added to the growth medium, and after the formation of a monolayer Etonium 7 mcg / ml. Pa-15 the next day, the cells are seeded, the treatment cycle is repeated three times. After the third cycle, cell culture was checked for mycoplasma.

As a result, lines of 20 t0k free from mycoplasmas are obtained.

With further cultivation for 2 months (20 passages), the bps of drugs of the cell are free of mycoplasmas (observation period) “25

PRI me R S Zootlivaemy cell line of AA in a concentration of 90 thousand / ml is seeded in 250 ml of matrasiki. The cells were cultivated on medium 199 with 10% syrvolta of cattle. Tiamulin 10 µg / ml was added to the growth medium; and after forming the monolayer, etonium was introduced at 5 µg / ml. The next day, cells reseed t, the processing cycle

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negative) With further cultivation for 2 months (15 passages) without preparations, the cells are free from mycoplasmas (observation period) „

The method of decontamination of transplanted cultures of animal cells from mycoplasmas will be used in virological and biotechnological studies, will allow for the timely diagnosis of cell cultures and to obtain cell cultures free of mycoplasma

Claims (1)

Invention Formula The method of decontamination of transplanted cultures of animal cells from mycoplasmas, including the treatment of cell cultures with 10% bovine serum, etonium surfactant and antibiotics, carrying out successive passages and recording results for decontamination by seeding on the elective nutrient medium and DNA staining fluorochrome, characterized in that, in order to increase the degree of decontamination, etonium is taken in an amount of 5-7 µg / ml and added directly to the monolayer, and as an antibiotic it is used tiamulin comfort in an amount of 10-15 mcg / mL, processing monolayer preparations carried out over a period of three passages, and coloring is carried out a DNA fluorochrome - olive om about omitsin three times After the 3rd treatment cycle, 2–3% FGM-s, medium No. 8, DNA fluorochromination (olive oil Invention Formula The method of decontamination of transplanted cultures of animal cells from mycoplasmas, including the treatment of cell cultures with 10% bovine serum, etonium surfactant and antibiotics, carrying out successive passages and recording results for decontamination by seeding on the elective nutrient medium and DNA staining fluorochrome, characterized in that, in order to increase the degree of decontamination, etonium is taken in an amount of 5-7 µg / ml and added directly to the monolayer, and as an antibiotic it is used tiamulin comfort in an amount of 10-15 mcg / mL, processing monolayer preparations carried out over a period of three passages, and coloring is carried out a DNA fluorochrome - olive om about omitsin