RU2488631C1 - CELL LINE OF CALF KIDNEY Bos taurus RBT (Rene Bos Taurus) FOR REPRODUCTION OF ANIMAL VIRUSES - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: permanent cell line of calf kidney Bos taurus RBT, deposited in 2010 into the Russian Collection of Cell Cultures (V) (Russian Academy of Agricultural Sciences, agricultural animals) under the No.74. The RBT line is produced from primarily trypsinized kidney cells of a 2-month calf in process of 65 serial passages in a single layer in the medium of 0.4 GLA on Earl with 10% foetal serum of cattle. Forms a population of epithelial cells, which is homogeneous by morphology, with considerable polymorphism in karyotype. At the same time population of cells with 51 chromosomes makes 32%, in contrast to normal karyotype Bos taurus (60 chromosomes), in RBT there are 9 metacentric and 1 submetacentric chromosomes, the number of polyploids is not more than 2%.

EFFECT: RBT line has high sensitivity to a corona virus, a virus of infectious rhinotracheitis, a plague virus and virus diarrhoea, and also high proliferative activity and ability for growth in rotary vessels.

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Description

The invention relates to the field of biotechnology and virology, and for the production of a new cell line of the calf kidney (Bos taurus), intended for the reproduction of animal viruses for the purpose of their detection, isolation, virological studies, as well as for the manufacture of diagnostic and preventive veterinary biological products.

Permanent cattle kidney cell lines (cattle) are widely used in scientific research and biotechnology [1, 2]. They are obtained from embryos, newborns and adult animals [3]. The following cell lines of kidneys and other organs of cattle are known:

- calf kidneys - MDBK - Madin-Darby Bovine Kidney cells;

- calf kidney - Taurus - 1;

- cattle kidney - PT-80;

- kidneys of a bull embryo - AUBEK;

- gobies embryo trachea - FBT.

These and other cell lines of calf kidneys and other cattle organs are used for the cultivation of diarrhea, rhinotracheitis [4] viruses, influenza, parainfluenza-III, coronaviruses, adenoviruses, etc. (A.S. USSR No. 1347448, 1985; A.S. USSR No. 1212043, 10/15/1985 [5]). Cell lines differ in terms of proliferation, adhesion, virus sensitivity, ability to reproduce in a particular nutrient medium, antigenicity, cytogenetic characteristics, and morphology. Permanent cell lines obtained from kidneys and other organs of cattle are divided according to the degree of chromosomal transformation into 2 groups: the first group with a near diploid set of chromosomes (58-62 chromosomes) and without significant chromosomal rearrangements, which include Taurus-1, PT- cell lines 80, AUBEK [5, 6, 7]; the second group with a hypoploid modal class (50-52 chromosomes) and the presence of 8 to 11 marker, transformed chromosomes, which include cell lines MDBK, FBT [8, 9].

During prolonged passage, the above presented constant cell lines, usually heterogeneous in cytogenetic, morphological and physiological characteristics, have limited application in virological work, which necessitates the expansion of the arsenal of animal cells by obtaining new cell lines with stable cultural properties and a wide range of use in vaccine production.

The closest cell line according to cytomorphological, karyological and cultural characteristics is MDBK, which was obtained in 1958 [8] and has several trophovariants [9]. In terms of biotechnological parameters, it is inferior to the newly obtained line: the average productivity of MDBK from a wedge mattress is 100 ÷ 130 million cells, and from a 3-liter roller, 120 ÷ 150 million cells. Also, this cell line is rapidly eliminated after a decrease in serum less than 5%.

The problem to which this invention is directed, was to obtain a new cell line from a calf kidney with improved biotechnological characteristics, suitable for the detection and isolation of a wide range of viruses, as well as for the manufacture of diagnosticums and specific prevention of animal viral diseases.

The problem is solved by obtaining a cell line of the kidney calf (Bos Taurus) RBT - Rene Bos Taurus.

The essence of the invention is illustrated by the following graphic materials:

Figure 1 - Metaphase plate of the cell line RBT - 51 chromosome;

Figure 2 - The idiogram of the RBT karyotype;

Figure 3 - Monolayer cell line RBT in the logarithmic phase of growth;

Figure 4 - Diagram of the spectrum of LDH isoenzymes of primary culture (PT) and constant culture (RBT).

Obtaining a calf kidney cell line (Bos Taurus)

RBT - Rene Bos Taurus

We obtained a new line of RBT cells from primary trypsinized kidney cells of a 2-month-old calf. A monolayer of primary culture cells from a calf kidney was formed on the fifth day after plating of primary trypsinized cells at a concentration of 300,000 cells in 1 ml (with a change of medium after 2 days of cultivation). The primary culture was grown using a nutrient medium based on Earl salt solution with 0.4% lactalbumin hydrolyzate (Peptone from lactalbumin ensimatic, readilly soluble Serva), 0.3 g / l glutamine and vitamins, supplemented with 10% fetal bovine serum. After the first subculture, a significant potential for proliferation remained: in the first passage, a monolayer formed on 3-4 days at an inoculum concentration of 150,000 cells per milliliter. The initial passages of the subculture were conducted only on the medium of 0.4% GLA on Earl with the addition of 10% fetal serum. On synthetic and semi-synthetic media (Migla MEM medium and PSP), a sharp decrease in the mitotic activity of cells occurred. Until the 30th passage, cultivation was performed on 0.4% GLA in Earl. Until the 11th passage, the calf kidney subculture was distinguished by polymorphism: there were fibroblast-like, epithelioids, spindle-shaped and polygonal cells. Productivity was negligible - 15 ÷ 20 million cells from a wedge mattress. Sieving was carried out once a week in a ratio of 1: 2, 1: 1.

Towards the 30th passage, tendencies toward isomorphism and an increase in the growth activity of the subculture appeared: fibroblast-like cells began to enlarge and eliminate. Groups of small epithelial-like cells appeared (up to 15 μm in the adhered state), which actively grew into large colonies and replaced fibroblast-like large cells. In 10-15% of the cell population, 1-2 metacentric chromosomes began to appear (except for the X chromosome). By the same period, the productivity of the subculture began to increase (up to 20–25 million cells from the wedge mattress). After each reseeding, the epithelial-like cells became larger, and fibroblast-like cells enlarged and destroyed under the influence of trypsin. From this period, cultured cells began to adapt to the medium Needle MEM and PSP. One condition remained unchanged - the use of fetal bovine serum free of mycoplasmas and latent viruses.

The following detailed testing of the subculture was carried out at 50-52 passages. By this period, the culture was fully adapted to the media Needle, PSP, MEM; cell productivity from a wedge mattress increased to 50 million. The cell population mainly consisted of epithelial (polygonal) cells, fibroblast-like cells were almost completely eliminated, but giant cells appeared (up to 10% of the total monolayer area), which were stored in passages. Significant changes in the karyotype occurred: 6–7 meta- and submetacentric autosomes appeared; the modal class was formed within 53–54 chromosomes; polyploids and giant cells comprised 6–8%.

Further study of the culture was carried out at passage 65, when the productivity of the cells from the wedge mattress increased to 100 million or more, and the sieving of the cells could be carried out in proportions of 1: 4 ÷ 1: 8. The ability of the cell line to grow in rotating 3-liter vessels was also revealed.

The formed cell population, at this stage of its existence, according to the above signs, was designated as a constant (transplantable) cell line. The karyological study of cells at 65 and 81 passages showed that the modal class stabilized at the level of 51 chromosomes, 10 meta- and submetacentric chromosomes appeared, which are markers of a new cell culture (Figs. 1, 2).

The cells of the formed monolayer differ in isomorphism of epithelial-like and spindle-shaped cells, the number of polyploids (giant cells) significantly decreased and amounted to less than 1%. The nuclei of the cells are mainly rounded, contain 1-2 large nucleoli (Figure 3). In the stationary phase of growth, cell membranes are weakly contrasted (pseudosyntsium forms). At the same time, the cell culture is well trypsinized to single cells.

The analysis of LDH isoenzymes confirmed the species origin of this cell culture from cattle Bos taurus (Figure 4). The spectrum of LDH isoenzyme lines shown in the diagram for the primary PT cell culture (calf kidney) is completely identical to that for the new transplanted calf kidney cell culture, RBT.

Sowing on bacterial media at all stages of the formation of cell culture showed the absence of contamination with bacteria, yeast, fungi and mycoplasmas.

Cells underwent a total of 65 passages before being converted from a subculture into a highly productive, constant cell line with the above properties. Their samples, taken in 67-94 passages, were stored in the cryobank of the All-Russian Scientific Research Institute for Scientific Research under the author's name “RBT Rene Bos Taurus Calf Kidney Cell Line (Bos Taurus)”.

A sample of a constant line of calf kidney cells (Bos Taurus) RBT - Rene Bos Taurus, taken at passage 78, was deposited in 2010 in the collection of somatic cells of agricultural and commercial animals of the All-Russian Scientific Research Institute of Experimental Veterinary Medicine of the Red Cross (RKKK) (CJS RAAS) No. 74.

Biotechnological characteristics

RBT cells are grown at pH 6.9 ÷ 7.2 and a temperature of 36 ÷ 38 ° C in a monolayer, in plastic or glass vessels with a capacity of 50 to 1500 ml and in rotating vessels of 2000-3000 ml with a ratio of medium and volume of the vessel within 1 : 6 ÷ 1: 10. In this case, MEM medium with 0.25% lactalbumin hydrolyzate or PSP in Earle's salt solution with the addition of 10% cattle serum is used. Table 1 presents data on the productivity of the RBT cell culture when cultured in different culture containers.

With a screening ratio of 1: 4 ÷ 1: 8, a monolayer is formed after 2-4 days of cultivation without changing the medium. Subculturing is carried out by the generally accepted method, causing the monolayer to disperse with a mixture containing 0.02% versene (ethylenediaminetetraacetate) and 0.25% trypsin in a 1: 1 ratio. The proportion of viable cells is determined by the method of supravital staining of the removed suspension with trypan blue, as well as by the results of observation of the growth dynamics of the seeded culture. A study of the kinetic characteristics of the RBT line at passage 94 showed that the maximum yield of 150–200 million cells in a 1500 ml mattress is provided after 4 days at an inoculum density of 100 thousand cells per ml (table 1). Productivity in roller vessels reaches 400 million / cl. In terms of area, productivity from 1 cm ² reaches 660 thousand cells.

Under standard culture conditions, the cell line has a high level of glycolysis. With acidification of the environment, degeneration of cell culture begins on 5-7 days. Complete destruction of the monolayer at 37 ° C occurs on 10-12 days of growth.

In addition to standard cultivation parameters, the RBT cell line stably grows for 10 or more passages using 2% cattle serum instead of 10%. A decrease in cell proliferation occurs after 5 consecutive passages using 0.6% serum.

Ampoules with RBT cells concentrated to a density of 5.0 ÷ 10.0 × 10 ⁶ cells / ml in fresh growth medium with 10% dimethyl sulfoxide are stored in liquid nitrogen. Cells are thawed by thawing ampoules in a water bath at a temperature of 37 ÷ 38 ° C. The optimal level of proliferative activity of the cell culture, noted before freezing, is reached already at the 2nd passage after thawing.

The monolayer formed by RBT cells is characterized by the ordered orientation of elongated polygonal epithelial cells. Cell nuclei, as a rule, are round or oval in shape, of various sizes (depending on the stage of the cell cycle), with 1-2 large and several small nucleoli.

In the process of obtaining a constant RBT cell line, the species affiliation was checked by methods and karyological analysis of 11, 30, 50, 65, 81 passages. In all cases, the signs of the species Bos Taurus were detected by the spectrum of LDH isoenzymes. Stabilization occurred in the cell line karyotype: a modal class of cell population with 51 chromosomes (32%) was formed; cells with 50-52 chromosomes make up 77%; polymorphism from 40 to 65 chromosomes. There are 9 metacentric and one submetacentric chromosomes in the karyotype, the Y chromosome is eliminated, polyploids less than 1%.

When examining cells with cytochemical methods (staining with acridine orange or olivomycin), as well as when plating on bacterial media (MPB, MPA, TGS and 0.3% PPLO agar), when examined by electron microscopy and PCR, the presence of bacteria, fungi and mycoplasmas does not revealed.

RBT cells are designed to isolate and accumulate animal viruses, to determine their infectious activity, to study genetic and phenotypic properties, as well as for the manufacture of diagnostic and vaccine preparations, including viruses or their components. These cells are sensitive to infection by viruses of various families of infectious rhinotracheitis (fam. Herpes viruses), plague (fam. Paramyxoviruses) and coronavirus (fam. Coronaviruses) of cattle, viral diarrhea - diseases of the mucous cattle (fam. Flaviviruses).

The essence of the invention is illustrated by examples of its use, which do not limit the scope of the invention.

Example 1

Reproduction of cattle coronavirus in cell cultures

A suspension of cells with a concentration of 50-150 thousand cells / ml was scattered into stationary or rotating vessels. Cells were cultured in Eagle’s medium with 0.25% GLA or PSP with 10% cattle serum at a temperature of 36–37 ° C at pH 6.9–7.1 without changing the medium until a complete dense monolayer was formed. To propagate the virus, a cell monolayer was used that did not show signs of contamination and degeneration.

Infection of the culture was carried out by contacting a concentrated suspension of the virus with a monolayer in wedge mattresses, previously removing the growth medium used and rinsing the cells twice with a heated Hanks solution. The virus contacted the cells for 1 hour with 5–8 ml of the virus suspension at 37 ° C, added in an amount that ensured a multiplicity of infection of 1.0–5.0 TCD ₅₀ / ml per cell. This portion of the virus was uniformly irrigated with a monolayer, slightly shaking the culture vessel. To ensure the specified multiplicity of infection, the seed virus concentrate and the support medium were mixed, as a rule, in a ratio of 1: 30 ÷ 1: 50. Then, serum-free PSP medium was added to the culture vessels and incubated at pH 7.2–7.3 for 45–96 hours at 37 ° C until the complete lysis of the infected culture.

Thus obtained viral material was frozen at a temperature of minus 40 ° C to extract the maximum viral yield. Then it was titrated in cell culture RBT and Taurus-2 in penicillin vials. The virus titer obtained on the RBT culture was in the range of 8.0 ÷ 9.0 lg TCD ₅₀ / ml (table 2).

In subsequent experiments, it was found that upon infection of RBT cells at any level after passage 65 with strains of cattle coronavirus, its yield was stably reproduced with titers of infectious activity that were within the same range.

Example 2

Reproduction of cattle infectious rhinotracheitis virus

Cells and the virus were treated in the same manner as described in Example 1. Titration was performed on RBT, MDBK, YaDK-04 cell cultures. Virus reproduction in an RBT cell culture occurred within 24–48 hours, faster than in other cultures. The titer reached 7.0 ÷ 8.0 lg TCD ₅₀ / ml (table 3).

Example 3

Reproduction of viral diarrhea - cattle mucosal disease

The cells and the virus were treated in the same manner as described in Example 1. The difference was that upon contact of the virus with a monolayer of cells, trypsin at a concentration of 0.01% was added to the medium with the viral suspension. Titration was performed on RBT and Taurus-2 cell cultures. The titer of infectious activity in the RBT cell culture reached 6.0 ÷ 7.0 lg TCD ₅₀ / ml and 1: 100 in ELISA (table 4). The proliferative activity of the RBT cell culture is 2-3 times higher than that of Taurus-2, therefore, the production of vaccines based on it is more cost-effective.

Example 4

Reproduction of cattle plague virus

Reproduction of cattle plague virus occurs within 96 ÷ 120 hours. For the cultivation of the virus used medium PSP with 2.5% serum of cattle. Titration was performed on constant RBT, Taurus-2 cell lines and a PT subculture. The titer of infectious activity during virus cultivation in RBT cell culture reached 7.0 lg TCD ₅₀ / ml (table 5).

Studies on the cultivation of coronavirus and rhinotracheitis cattle virus in the RBT cell line indicate a high sensitivity of this culture to the above viruses - 8.0 ÷ 9.0 lg TCD ₅₀ / ml. In the case of cattle rhinotracheitis virus, RBT cells have proven to be a convenient system for titration of this virus.

A comparative study of the reproduction of coronavirus, rhinotracheitis, diarrhea and cattle plague viruses in various cultures suggests that the proposed new constant line of RBT cells (obtained from a calf kidney) is highly sensitive to the above viruses and, in combination with high proliferative activity and growth potential in rotating vessels, makes it suitable for the production of prophylactic antiviral drugs.

Information sources

1. Syurin V.N., Samuilenko A.Ya., Soloviev B.V., Fomina N.V. - Viral diseases of animals. M .: VNIIT and BP, 1998.

2. Spier R.E., Griffiths J.B. - Biotechnology of animal cells. M .: Agropromizdat, 1989 .-- 185 p.

3. Dyakonov L.P., Sitkov V.I. - An animal cell in culture. Methods and their application in biotechnology. M .: Sputnik +, 2009, 656 p.

4. Kozyrenko T.I., Dyakonov L.P., Pozdnyakov A.A. Studying the sensitivity of transplanted cells to the cattle infectious rhinotracheitis virus // Scientific principles of the technology for the industrial production of veterinary drugs: thesis. doc. Second All-Union. conf. M. 1981. - S.20-21.

5. CATALOG. Specialized collection of transplantable somatic cell cultures of agricultural and commercial animals RKKK (P), (SKHZh RAAS), Moscow, 2006, 115 pp.

6. Mironova L.L., Preobrazhenskaya N.K., Shitikova G.S. and other Domestic lines of transplantable cells of calf kidneys - a substrate for biotechnology - Biotechnology, 1998, No. 5, p. 25-31.

7. Mironova L. L., Popova V. D., Konyushko O. I., Khapchaev V. D., Zybin D. V., Akopyan A. S. The experience of creating a bank of copyright lines of transplantable cell lines and their use in virological practice - Biotechnology, 2000, No. 6, P.41-47.

8. Madin S.H., Darby N.B. Established Kidney Cell lines of Adult Bovine and Ovine Origin // Proc. Soc. Exp. Biol. Med. - 1958. - Vol. 98. - P.574-576.

9. CATALOG. Russian collection of cell cultures, ed. G.P. Pinaeva, St. Petersburg, 2004, 315 p.

Table 1 The dynamics of proliferation of the RBT cell line in different culture vessels The conditions for growing cells in a monolayer Sowing concentration (mln / ml) The cultivation time (day) Cell productivity from a culture vessel (million) Growth rate Glass culture vessels (wedge mattresses) with a volume of 1500 ml 0.1 2 97.0 ± 0.4 3.8 3 120.0 ± 0.1 4.8 four 200.0 ± 0.1 8.0 0.05 2 55.0 ± 0.2 4.2 3 90.0 ± 0.1 7.0 four 120.0 ± 0.2 9.6 0,025 2 - - 3 - - four 60.0 ± 0.1 9.8 3000 ml rotating vessels 0.1 2 150.0 ± 0.2 5,0 3 200.0 ± 0.5 6.6 four 350.0 ± 0.4 11.6 0.15 2 180.0 ± 0.2 4.0 3 250.0 ± 0.1 5.5 four 400.0 ± 0.3 8.8

table 2 Reproduction of cattle coronavirus in cell cultures Cell culture Cultivation time (hour) The titer of infectious activity (lg TCD ₅₀ / ml) Electron microscopy (particles per ml) MDBK 96-120 2.0-3.0 10 ^6.5-7 Taurus-2 96-120 3.0-4.0 10 ^7-7.5 RBT (in wedge mattresses) 40-48 6.0-7.0 10 ^6.5-7 RBT (in scooters) 40-48 8.0-9.0 10 ^8-8.5

Table 3 Reproduction of cattle infectious rhinotracheitis virus in cell cultures Cell culture Cultivation time (hour) The titer of infectious activity (lg TCD ₅₀ / ml) YADK-04 48-72 7.0-8.0 MDBK 72-80 7.0 RBT 24-48 7.0-8.0

Table 4 Reproduction of viral diarrhea - cattle mucosal disease in cell cultures Cell culture Cultivation time (hour) The titer of infectious activity in ELISA The titer of infectious activity (lg TCD ₅₀ / ml) Taurus-2 48-96 1: 100-1: 500 7.0 RBT (in wedge mattresses) 96-120 1: 100 6.0 RBT (in scooters) 48-96 1: 100 6.0-7.0

Table 5 Reproduction of cattle plague virus in cell cultures Cell culture Cultivation time (hour) The titer of infectious activity (lg TCD ₅₀ / ml) PT (primary calf kidney culture) 72-96 5.75-6.0 Taurus-4 72-96 6.0-6.5 SPEV 96-120 7.0-7.5 RBT 96-120 6.0-7.0

Claims (1)

The constant line of cells of the kidney calf Bos taurus RBT, deposited in 2010 in RKKK (P) (SCW RASHN) under No. 74, for the reproduction of animal viruses, characterized in that it:
- obtained from primary trypsinized kidney cells of a 2-month-old calf during 65 consecutive passages in a monolayer on 0.4 GLA medium on Earle with 10% fetal bovine serum;
- forms a morphologically homogeneous population of epithelial cells with significant polymorphism in the karyotype, while the population of cells with 51 chromosomes is 32%, in contrast to the normal Bos taurus karyotype (60 chromosomes), RBT has 9 metacentric and 1 submetacentric chromosomes, the number of polyploids is not more than 2%;
- when cultivated in a monolayer in a cyclic manner, it reproduces cattle coronavirus with a titer of 8.0-8.5 lg TCD ₅₀ / ml, cattle rhinotracheitis virus with a titer of 7.0-8.0 lg TCD ₅₀ / ml, cattle plague virus with a titer of 6 , 0-7.0 lg TCD ₅₀ / ml, the titer of the infectious activity of viral diarrhea of calves in ELISA reaches 1: 100.

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