RU1347448C - Strain taurus-1 of heteroploid cells of calf kidney used for virus cultivation - Google Patents

Abstract

FIELD: virology. SUBSTANCE: strain is prepared from calf kidney cells by trypsin treatment of minced organ pieces and adaptation of prepared cellular suspension for the growth in vitro. Strain is deposited in Collection of cellular cultures VNII-influenza of Russia Ministry Health at number VSKK (DP) 002. EFFECT: preparing of new cellular strain of calf kidney showing sensitivity to the broad virus spectrum.

Description

 The invention relates to the field of virology and can be used to obtain virus-containing material on cellular substrates.

 The purpose of the invention to obtain a new cell strain of the kidney of a calf sensitive to a wide range of viruses.

 This strain was obtained from cells of a calf kidney by trypsinization of crushed pieces of organs and adaptation of the resulting cell suspension to in vitro growth. It is stored in the cell culture collection of the All-Russian Research Institute of Influenza of the USSR Ministry of Health under the number VSCK (DP) N 002.

 The strain of heteroploid cells of a calf kidney is characterized by the following features.

 Morphological signs.

 Organotypic growth is noted in the primary culture and in the passage transplanted to 6–7: islands of epithelial cells (EC) alternate with strands consisting of fibroblast-like cells (FC). Both types of cells occupy approximately the same area.

 In the course of further inoculations, the amount of EC gradually increases, and FC decreases. Mixed cell composition in different cultures lasts up to 25-27 passage. Only after fractional trypsin treatment is a homogeneous EC culture obtained. On fixed and stained culture preparations of 34-99 passages, it was found that the cell layer on the 3-4th day of incubation consists of polygonal cells with closely defined boundaries with clearly defined boundaries.

 When electron microscopic radiation of strain Taurus-1 at passage 69, it was found that the culture is represented by epithelioid cells, which have an oval or slightly elongated shape with a large centrally located nucleus. At the periphery of the nucleus, the rim of a wall-mounted chromatin is clearly pronounced. The contours of the nucleus are even, but sometimes invagination of the nuclear membrane is observed. In the cytoplasm, relatively large mitochondria with a low-contrast matrix and a small number of cristae are found, enlarged tubules of the granular endoplasmic reticulum, and a well-developed Golgi complex.

 Karyological signs.

 At passage 65, the heteroploidy of Taurus-1 strain was established. The modal number of chromosomes is 57 (74%). Aneuploid cells accounted for 24%. All 98% of the cells contain one to three two-arm markers; 2% of the cells were tetraploid with four two-arm marker chromosomes.

 Cultural signs.

The strain is maintained on the environment of the Needle MEM with 10% serum of cattle. Cells were incubated at ³⁷ ° C, multiplicity sieving 1: 2; 1: 3. A characteristic feature of cells is their ability at the level of 1-40 passages to remain on glass in a viable state for up to 3 months. without changing the environment, but with a change of environment up to 1 year. The number of cells in a monolayer of 1.5 mattresses reaches 80-90 million.

 Isozyme characteristic.

 The cells of the strain possess glucose-6-phosphate-dehydrogenase and lactate dehydrogenase activity, characteristic of cow cells.

 When examining the cells of the Taurus-1 strain for sterility (the presence of fungi, bacteria, mycoplasmas), negative results were obtained at levels 5, 8, 9, 15, 20, 27, 34, 40, 45, 55, 65, 7, 77, 81, 86, 89, 94, 100 passages.

 In the study of the culture fluid and the cells themselves at levels 8 and 75 of the passages in sensitive cell systems, as well as in hemedesorption reactions, no contaminants were detected.

 When examining cells of the Taurus-1 strain at the same levels of passages in experiments on adult and suckers of white mice, rabbits, guinea pigs, as well as cells at levels 9 and 81 in experiments on chicken embryos and immunosuppressed mice of the CBA line of extraneous agents, including and oncogenic, not detected.

 The cells of the Taurus-1 strain retain viability (at least 85%) when preserved in liquid nitrogen.

 A seed bank was created at passage 65 at a rate of 340 ampoules of 10 million each. This pool of cells is monitored for safety.

 The Taurus-1 calf kidney cell strain is susceptible to infection by human and animal viruses.

 PRI me R 1. The cultivation of influenza A.

A mixture of solutions of 0.25% trypsin and 0.02% versene in an amount of 20 ml is introduced into 1-liter mattresses with a monolayer of Taurus-1 strain cells at passage level 90. Rinse the monolayer with gentle swaying and the solution is drained. After 5-10 minutes, 5-10 ml of growth medium is introduced into the mattress, and the cells are thoroughly pipetted. The number of cells is determined and diluted with medium to a seed concentration: 100 thousand cells in 1.0 ml for stationary cultivation; 150 thousand cells in 1 ml when cultured in test tubes and in roller bottles. As a growth medium, Eagle’s medium with 10% calf serum is used. The cell suspension is poured into test tubes, mattresses with a volume of 1 l, a roller of 3.0 or 5.0 l and cultured at 37 ^about C for 48-72 hours until a monolayer is formed. The resulting culture is washed with a Hanks solution or phosphate-buffered saline (pH 7.2-7.4) from the growth medium, after which it is used to accumulate the virus or titrate its infectious activity in test tubes.

For the accumulation of influenza A virus (Leningrad) 385/80, 5.0-7.0 ml of the infectious virus at a concentration of 6.0-7.0 lg TCD is added to a monolayer culture grown on 3.0 and 5.0 l roller bottles ₅₀ / ml. Virus adsorption is carried out for an hour, then a support medium is introduced. The cultures are incubated at 37 ° ^C. After 5-7 days after infection develop cytopathic changes. The level of virus accumulation is determined by its infectious titer and hemagglutinating activity. An infectious titer is determined by titration of a virus-containing fluid in 10-11 day old chicken embryos. The level of hemagglutinating activity of the virus is determined in the hemagglutination reaction with a 1% suspension of freshly prepared chicken red blood cells and is expressed as the reciprocal of the highest dilution that causes their agglutination.

The infectious titer of influenza A virus reproduced on the cells of Taurus-1 strain is 8.5 lg TCD ₅₀ / ml, while its hemagglutinating activity is 1:64, respectively.

 PRI me R 2. The cultivation of human adenovirus.

To propagate type 1–7 adenoviruses, 3.0–5.0 ml of the infectious virus at a concentration of 5.0 lg TCD ₅₀ / ml . Adsorption is carried out for half an hour, then a support medium is introduced (Eagle’s medium without serum). The cultures are incubated for 6-7 days with daily viewing under a small magnification of the microscope. The cytopathic effect develops under the influence of all types of adenovirus, except 5. The level of viral reproduction is determined in parallel titration of infected materials in a test tube culture of cells of strain Taurus-1 and in comparison with the line of Hep-2.

 Analysis is carried out according to the cytopathic effect, which occurs on the 8-10th day.

The infectious titer on the cells of the Taurus-1 strain of adenoviruses 1, 2, 3, 4, 6, 7 types is 4.0-6.5 lg TCD ₅₀ / ml, on the Ner-2 line, respectively 5.0-67.0 lg TCD ₅₀ / ml (and the types are as follows: 1 6.5 and 7.0; 2 6.0 and 7.0; 3 5.2 and 5.0; 4 5.0 and 7.0; 6 6.0 and 7.0; 7 4.0 and 7.0).

 PRI me R 3. The method of cultivation of the virus of infectious rhinotracheitis in cattle.

The culture vessel with cells Taurus-1 strain at passage level 95 administered 0.25% strength trypsin solution warmed to 37 C. After ^about 1-1.5 minutes, the solution was removed, and the vessel containing the cells were placed in a thermostat at a temperature of ^about 37 C for 2-3 minutes. After that, a small amount of the nutrient medium is introduced into it and the process of separation of cells from the glass is completed with vigorous shaking. Then prepare a cell suspension in the medium Needle MEM with 10% SCRS and explanted in 1 ml tubes containing 200 thousand cells. Cultures were incubated at ³⁷ ° C for 3 days until the formation of a monolayer.

If the virus is infected (isolate 4016), the medium is removed from the tubes, the cells are washed once with Hanks solution and 0.2 ml of virus-containing liquid is introduced at the rate of 1 TCD ₅₀ / ml per cell. The adsorption of the virus is carried out for 1 h at 37 ^about C. Then add 1 ml of the same medium without serum. The cultures are incubated at ³⁷ ° C. Collect the virus is carried out for 5-6 days. The virus titer is determined by the development of a cytopathic effect. The titer of the virus is 6.5 lg TCD ₅₀ / ml.

 PRI me R 4. The method of cultivation of adenovirus cattle.

The cells of the Taurus-1 strain at the passage level are prepared for infection with the virus according to the method described in Example 3. Viruses (isolates Rus 18/81 and Moscow 3056) are introduced at the rate of 0.03 TCD ₅₀ / ml per cell. The virus is collected after 12 days.

The virus titer for both isolates is 5.5 lg TCD ₅₀ / ml.

Claims (1)

 The strain of heteroploid cells of the kidney of a calf Taurus-1 VSCK (DP) N 002 (collection of cell cultures of the All-Russian Research Institute of Influenza of the USSR Ministry of Health) used for the cultivation of viruses.