RU2348688C1 - Constant sso-3 pool cell line of parenchymic organs of siberian sturgeon acipenser baeri - Google Patents

Abstract

FIELD: chemistry; biochemistry.

SUBSTANCE: invention concerns biotechnology, particularly obtaining interweaving lines of fish cells, applicable for virus extraction and research for various taxonomic fish groups. The line can be applied as test culture for extraction, accumulation, titration and research of Siberian sturgeon herpes virus (SSHV), spring viperemia of carp virus (SVCV), viral hemorrhagic septicemia virus (VHSV), infection hemopoietic tissue necrosis virus (IHNV), European ell rhabdovirus (Rhabdovirus anguilla), pike fishling rhabdovirus (PFRV) and European lake trout rhabdovirus (ELTV).

EFFECT: obtaining interweaving lines of fish cells, applicable for virus extraction and research for various taxonomic fish groups.

Description

The invention relates to biotechnology, in particular to the production of transplantable fish cell lines. suitable for isolation and study of fish viruses of different taxonomic groups bred in aquaculture.

Viral diseases are one of the main factors inhibiting the development of world aquaculture. Today, about 400 fish viruses are known, and their number continues to grow. Ten viruses have been detected in sturgeons abroad. Therefore, the task of obtaining constant (transplantable) cell lines sensitive to a wide range of viruses of cultivated fish is currently very urgent. Siberian sturgeon (Acipenser baeri) is one of the most popular breeding sites in aquaculture, however, virological research for its disease is hampered by an acute shortage of cell lines obtained from sturgeons of this species.

Only one cell line of the Siberian sturgeon obtained from dorsal fin cells is known [1]. However, its sensitivity to fish viruses has not been determined, which does not allow its use in work on the isolation and study of viruses that cause dangerous fish diseases.

The aim of the invention is to obtain a constant cell line of Siberian sturgeon, suitable for virological studies of sturgeon of this species and sensitive to viruses of other fish species.

The goal is achieved by obtaining a constant cell line SSO-3 pool of parenchymal organs of Siberian sturgeon yearlings, Acipenser baeri, used in virological studies and sensitive to 7 viruses of different fish species.

The constant SSO-3 cell line was obtained by repeatedly passaging a primary trypsinized cell culture from a pool of parenchymal organs (kidney, liver, spleen) of two yearlings of the Siberian sturgeon Acipenser baeri.

The constant SSO-3 cell line is characterized by the following features.

Cultural properties. The life cycle of the SSO-3 cell line, in which its basic biological parameters are preserved, is 251 passages. At an inoculum concentration of 60 × 10 ³ cells / cm ^{2, the} monolayer is formed after one day of cultivation at 19-21 ° C and remains without changing the medium for 14-20 days, if 2-3 days after its formation, the incubation is continued at 15 ° C. Reseeding ratio 1: 4.

The SSO-3 cell line was cultured in monolayer cultures in Eagle MEM with a double set of amino acids and vitamins (2MEM) with 5% serum of cow embryos. A mixture of a 0.25% trypsin solution and a 0.02% versene solution in a 1: 1.5 ratio at room temperature is used to disperse the cell monolayer during transfers. The harvest of cells from a vial with an area of 20 cm ² is about 6 million cells.

For cryopreservation at a temperature of -196 ° C, a mixture consisting of 70% of 2MEM nutrient medium, 20% of serum of cow embryos and 10% of dimethyl sulfoxide, is used at a cell concentration of 2-3 × 10 ⁶ in ml. Cell viability according to the test of vital staining with trypan blue after storage in liquid nitrogen is 70-80%. Cells restore their original growth and morphological properties within 2 passages.

Morphological signs. When inoculated, SSO-3 cells are evenly distributed over the surface of the substrate. The cells are homogeneous epithelial-like, polygonal in shape with long cytoplasmic processes. As the monolayer condenses, the processes are shortened, and the shape of the cells approaches cubic. The nuclei are large, oval. The number of nucleoli from 1 to 3, often 1-2.

Karyological characteristic. (2n = 248 ± 5). The range of variation of chromosomes is from 168 to 414. The modal number is 196 chromosomes. The value of the modal class is 60%.

Species identity is confirmed by karyological methods.

Control of the SSO-3 cell line for extraneous contaminants. When studying the SSO-2 cell line for sterility (the presence of bacteria, fungi, mycoplasmas), negative results were obtained.

The SSO-3 cell line can be used to isolate, accumulate, titrate and study the following fish viruses: Siberian sturgeon herpesvirus (SSHV), carp spring viremia viruses (SVCV), viral hemorrhagic septicemia (VHSV), hematopoietic tissue infectious necrosis (IHNV), European eel rhabdovirus (Rhabdovirus anguilla), pike fry rhabdovirus (PFRV) and European lake trout rhabdovirus (ELTV).

The permanent cell line SSO-3 was deposited in the Specialized Collection of Transplantable Somatic Cell Cultures of Agricultural and Commercial Animals of the Russian Cell Culture Collection at the All-Russian Scientific Research Institute of Experimental Veterinary Medicine under the number 68.

Bibliography

1. Fontana F., Rossi R., Lanfredi M., Arlati G., Bronzi P. Cytogenetic characterization of cell lines from three sturgeon species // Caryologia. - 1997. - V. 50. - N 1. - P.91-95.

Claims (1)

The permanent cell line SSO-3 of the pool of parenchymal organs of Siberian sturgeon yearlings, Acipenser baeri, used in virological studies and sensitive to 7 viruses of different fish species, was deposited in the Specialized Collection of Transplantable Somatic Cell Cultures of Agricultural and Commercial Animals of the Russian Collection of Cell Culture at the All-Russian Scientific Research Institute of Experimental Veterinary Medicine (SCW RASHN) under the number 68.