Method to produce epithelioid cells of light foetus of buffalo cow for cultivation of viruses

FIELD: biotechnologies.

SUBSTANCE: method is proposed to produce epithelioid cells of buffalo cow light foetus by means of long-term no-reseeding cultivation having high sensitivity to the virus of infectious rhinotracheitis of cattle, parainfluenza-3, viral diarrhea-disease of mucous membranes and adenoviral infection.

EFFECT: possibility to use in diagnostics of virus infections.

4 ex


The invention relates to biotechnology, in particular to a technology for cell cultures, and can be used in veterinary Virology in the diagnosis of viral infections of cattle.

Currently, there is a lot of material, indicating that the optimal cell systems upon receipt of viral preparations and for experimental purposes are the culture of diploid cells. Diploid cultures have several advantages over primary and permanent (stable) cultures, so long as they are able to proliferate in vitro with retention of the original properties, uniform cell structure and free from contaminants [2, 4].

Known diploid cell cultures derived from different tissues: kidney, skin, lung, heart, spleen, gimpa, segoviano gland and other organs of various animal species and humans [1-7].

A disadvantage of known methods is their complexity, which is the enzymatic and mechanical treatment of the source material that requires a lot of time, causes loss of cells, and the use of chemicals disrupts cell function. In addition, the culture obtained by prototypes enzymatic disaggregation of the tissue, were fibroblast-like Tina. It is known that a diploid cell culture fibroblast-like the IPA have a limited lifespan in culture in vitro and their sensitivity to viruses below, than to cells epitheliopathy type.

Closest to the proposed method to the technical essence and the achieved result is the method of cultivation of fibroblasts for substitution therapy, described in patent RU 2320720 [6]. In this way prompted characterized the culture of fibroblasts designed as a tool for regenerative therapy and cosmetology. The disadvantage of this method is "hard" processing of raw material (incubation with proteases of animal origin with constant stirring and pipetting), which is traumatic for the cells of the procedure, reducing the total number of viable cells in the resulting suspension dermal fibroblasts, as well as leading to violations of adhesion and receptor systems in part preserved cell viability.

The present invention represents a development in which taken Popelka get especiallyto from lung explants of fetal Buffalo, sensitive to respiratory viruses of cattle, with the use of technology for cell culture, which does not require enzymatic and mechanical treatment of the source material and culturing cells without reseeding.

The aim of the invention is to obtain epitheliopathy lung cells of the fetus Buffalo (LPB), case is valid for a wide range of respiratory viruses in cattle (cattle).

The technical result achieved by the proposed method is to simplify the process of cell epitheliopathy type with intensive reproduction and sensitivity to respiratory viruses of cattle.

To achieve the technical result in the production method of epitheliopathy cells, including the production of explants, incubation in a nutrient medium and the allocation of epitheliomata, exercise of lung tissue without enzymatic and mechanical treatment. For this lung tissue after getting in sterile conditions, cut into pieces 3...5 mm3and washed until full enlightenment supernatant. The resulting explants, washed with sterile Hanks solution with antibiotics, were placed in vials Carell, poured a nutrient medium consisting of equal volumes of 0.5%environment hydrolyzed lactalbumin (0.5% of GLA) with medium 199 with 10% serum of cattle, and incubated at 37°C. in 48-72 hours from the beginning of cultivation were changing environment. For 6-8 days of cultivation was mechanically removed explants and cells from lesions of the growth was removed with a glass of 0.02%solution of versene warmed to 35-37°C and perseval in the ratio of 1:1. Further cultured cells without reseeding for three weeks, and every 4-5 days spent changing environment by 30-50% with addition of 0.5%of the CSOs glutamine.


Epitheliopathy cells LPB was prepared as follows.

As the original fabric used is light fruit Buffalo 3...5th months of development, delivered no later than 2-3 h after slaughter of the animal. In a sterile room in the fetus removed easily and washed with sterile Hanks solution with antibiotics (200 units/ml penicillin and 200 μg/ml streptomycin). When processing authority pleura, trachea, bronchi, large blood vessels, cut the fabric into pieces 3...5 mm3and washed until full enlightenment supernatant.

Pieces of shredded tissue, rinsed with sterile Hanks solution with antibiotics, were placed in small culture flasks Carell, at a distance of 1-1 .5 cm from each other. Filled vials nutrient medium consisting of equal volumes of 0.5% of GLA with medium 199 with 10% serum of cattle, using a 10 ml pipette so that the explants were covered with medium. The vials were placed at 37°C for cultivation in thermostat.

The explants after 24 to 48 h was observed foci of cell growth, which are gradually increased. Colonies growing cells from explants were mixed (antilipogenic and fibroblast-like) type. The change of environment was performed 48-72 h from the beginning of cultivation. Later he entered the culture as follows.

After TRG is, as in vial appeared pockets of growth epitheliopathy cells (6-8 days), mechanical removal of explants and cells from lesions of the growth was removed with a glass of 0.02%solution of versene warmed to 35-37°C and perseval in the ratio of 1:1. The seeding cell concentration 300-350 thousand/ml On the 5th day of cultivation was obtained a homogeneous culture, composed mainly (with a predominance of 70-80%) of cells epitheliopathy type. This culture was used in further experiments on growing subcultures or epitheliopathy crops of the same type.

Further cultured cells without reseeding for three weeks, and every 4-5 days spent changing environment by 30-50% with the addition of 0.5%glutamine.

The result of the cultivation without reseeding observed a gradual withering away of remaining in the culture of fibroblast-like cells, and the vials were left cells epitheliopathy type, which are formed colonies of homogeneous cells. In the first passages of the multiplication of cells in the colonies was slow, and the accumulation of cells was sufficient for replanting in the same vial 1:1. Subsequent subcultures were made in the ratio 1:2, the concentration of cells was 200-250 cells/ml

The first 3-4 passage in monolayer culture were formed in 2-3 weeks. At this time every 4-5 days conducted a partial change of sredina 30-50%. Culture is stabilized at the 5th passage. Subsequent passages were performed as the formation of the monolayer, using a 0.25%solution of trypsin and 0.02%aqueous solution of versene, in the ratio of 1:9 basantapur way.

The obtained culture was subjected to subculturing many times and to date it is presented solely epitheliopathy cells. Culture was more passages 44.

Morphological characteristics. Culture has an intense growth. After 6-8 h of cultivation has been a steady growth with homogeneous cells that grew in a single layer without deposition. For 60-72 h is formed a monolayer. The coefficient reseeding is 1:2.

Ecomorphologically analysis of drugs at the level of 10, 20, 30 and 40 passages showed that culture LAB presented in monolayer cells epitheliopathy type. In stained preparations of cells had a polygonal shape, boundaries are firmly joined to each other. The nucleus of the cells is large, rounded, with 3-5 nucleoli. The structure of the nucleus had malosetti granular structure.

When karyological analysis of cells of strain modal class of chromosomes was 2n=50.

Information about contaminant. Bacteria, fungi and Mycoplasma bacteriological, radioautographic, cytological studies using fluorochrome-olivomitsina not found.

ultraline properties. Border cells are well expressed and culture good passerella. As the growth medium used: medium 199, 0.5% of GLA + 199 or 0.5% GLA + Needle in equal proportions with the addition of 10% cattle serum, inactivated at 56°C for 30 min and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin).

The monolayer of cells LAB was preserved in a thermostat at 36-37°C with medium without serum and 12-15 days, with no signs of degeneration.

Epitheliopathy cell culture LPB has found application in the work in the study of viral respiratory infections of cattle.

The described method allowed us to reliably obtain epitheliopathy culture of cells from the lung tissue of the fruit of Buffalo.

Example 1. Cell culture LAB were cultured in medium 199 with 0.5% GLA in the ratio of 1:1, with the addition of 10% cattle serum and antibiotics (100 u/ml penicillin and 100 μg/ml streptomycin). When reseeding the cells were removed from the glass 0.25%solution of trypsin and 0.02%solution of versene in the ratio (1:9), heated to 36°C. After counting the cells, the suspension was diluted to seed the concentration of nutrient medium 200-250 cells/ml

Cultural mattresses were placed in a thermostat at 37°C. the Monolayer was formed within 48-72 hours of Culture with increased monolayer used for infection with the virus of infectious bovine rhinotracheitis (RTI).

Example 2. Cell culture LAB cultivated on the environment And the La with 0.5% GLA in the ratio of 1:1, with the addition of 10% serum of cattle. After formation of the monolayer within 48-72 h of culture were used for infection with parainfluenza virus-3 (PG-3).

Example 3. Cell culture LAB were cultured in medium 199, with the addition of 10% serum of cattle. After formation of the monolayer after 48 to 72 h culture was used to infect virus viral diarrhea is a disease of the mucous membranes (BD-BS).

Example 4. Cell culture LAB were cultured in medium 199 with 0.5% GLA in the ratio of 1:1 with the addition of 10% serum of cattle. After formation of the monolayer after 48 to 72 h culture was used to infect virus adenovirus infection of cattle (ADI).

To identify the sensitivity of culture to the viruses were five serial passages of the virus. It is established that culture LAB sensitive to these viruses. Cytopathogenic action (JRC) of the virus RTIs occurred after 30 h after infection and was characterized by rounding of cells, loosening and destruction of the monolayer and subsequent rejection of the cells from the glass. In the infected culture was also mentioned in marginal chromatin in nuclei, then (after 25-30 h) is the formation of intranuclear inclusions typical of viruses of herpes group. JRS virus PG-3 occurred after 48-72 hours of culture Change, caused by a virus, characterized by the appearance of multinuclear cells (simpleton) and gradually the YM, after 72-96 h, the destruction of the monolayer. In the cytoplasm of cells was seen painted in pink cytoplasmic inclusions of different sizes. Sometimes they occupied almost the entire cytoplasm, in some cases in cell nuclei revealed a small pink inclusions, surrounded by a light area. The titer of the virus RTI reached 105,5-7,0TCD50/ml, virus NG 3 to 106,25-6,6TCD50/ml.

Cell culture LAB also susceptible to viruses VD-BS, ADVI with a characteristic expression of the JRS. For virus VD-BS is characterized by the appearance in the cells oraloader vacuoles, pikes nuclei and cells throughout infection - vacuolization of the cytoplasm. When the infection adenoviruses complete degeneration of the monolayer was accompanied by symplectomorphism, which was observed after 36-48 hours. The titer of virus VD-BS, ADVI in the culture has reached 104,75-5,4TCD50/ml and 10the 5.25-5,5TCD50/ml, respectively.

When examining one of outbreaks of respiratory infections of cattle, the maximum number of selected viral agents amounted to 35% on the primary-trypsinization culture kidneys calves, and culture LAB - 42% compared to the number of samples used for the study. Thus, culture is also suitable for the initial allocation of viruses from pathological material.

As can be seen from examples obtained epitheliopathy cell culture LAB Chu is stately to respiratory viruses of cattle.

In the dynamics of the cultivation was conducted cryogenical for long-term preservation of cells in createsite a medium consisting of 50% of media 199 with 0.5% GLA in the ratio of 1:1, 40% cattle serum and 10% DMSO. Spent freezing culture at 10, 20, 30 passages. After 15 and 30 days of snore in liquid nitrogen (-196°C) cell culture LPB held its defrost, rekultivirovanie. 48-72 h of rekultivirovanie formed a monolayer, represented by cells epitheliopathy type, which are not morphologically different from the original culture.


1. USSR author's certificate No. 1147748 from 30.03.1985.

2. Deacons L.N., Sitcov V.I. Animal cell culture (Methods and application in biotechnology). - M.: Satellite Company+. - 2000. - 400 C.

3. Look SO, Shitikova G.S., Ponomareva, NP and other fabrication and characterization of diploid lines of lung fibroblasts human embryo / Etiology and diagnosis of influenza and other acute respiratory diseases // Sat. scientific papers. - HP - H. - C.110-116.

4. Size J.F. ñ Obtaining cell cultures from animal tissues and their use in Virology. - M - 1975. - 103 C.

5. Pankov G., Sologub VK, Gololobova M.L., Rezovo TI Strain diploid lung cells of the embryo horses. Veterinary science. - 1978. No. 3. - P.42-43.

6. RF patent №2320720 from 27.03.2008. 7. Pozdnyakov A.A., Yurov C.P., MOTOZINE Z.P. and other Metadiscipline diploid strain cells testicular tissue foals and test its sensitivity to certain viruses. Bulletin of VIEW. - 1976. - VIP. - P.46-48.

The method of obtaining epitheliopathy lung cells of the fetus Buffalo for the cultivation of viruses in the implementation of which the Explant size 3-5 mm3obtained from the crushed tissue, cultured with the change of the nutrient medium within 48-72 hours from the beginning of cultivation, after 6-8 days, the explants were removed and subcultured cells grown in hotbeds of growth of explants remaining after their removal, removing them from the glass of 0.02%solution of versene warmed to 35-37°C, characterized in that the cells are cultivated without reseeding for three weeks with a change of medium every 4-5 days with addition of 0.5%glutamine.


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