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Establishment, cultural and morphological characterization of primary cell cultures from clawed frog (Xenopus laevis)

The method of cell culture is a fundamental method, whose appearance at the time led to the rapid development of virology today remains one of its main tools also. In the world's largest cell banks such as ECACC, ATCC, DSMZ and several collections of cell cultures institutes of international importance, which account for tens of thousands of a variety of cell cultures, at present remains very limited number of cell lines amphibians. Most of them received in the middle of the last century and preserved to the now days. However, despite on existing cell lines frogs they cannot always meet the growing needs of researchers. Ukrainian banks collections and cell cultures contain not only a single domestic cell line of amphibians, and even strains from international collections. Also worth noting that diseases of amphibians in the last few years caused an increased interest not only because of a sharp decline in their numbers due to destruction of their natural habitat, as well as due to sudden outbreaks of diseases such as infection with Batrachochytrium dendrobatidis and infection with ranavirus lead to high morbidity and mortality among of populations of these animals in industrial farms and zoological collections, but also in the minority populations of rare and endangered species in nature. And therefore from 2009 these diseases are included in OIE Listed diseases. According to the guidelines of the OIE Manual of Diagnostic Tests for Aquatic Animals (2012) cell culture is the gold-standard test for agent isolation and identification. There are many fish cell lines are used to amphibian ranavirus diagnostic isolation and identification. But for better understanding ecological and evolutionary features of the pathogen, as well as interaction pathogen-host species the amphibian cell lines are more preferable. 

As the donor tissue for primary cell culture young clawed frog (Xenopus laevis) were used. The hind limbs from two animals were washed properly with Decasanum 0,02 % after collection, dissected with forceps and then washed three times with 80 % DMEM (SH30243.01; HyClone), 20 % sterile demineralized water, supplemented with 200 IU/mL penicillin (Sigma), 200 µg/mL streptomycin (Sigma), 400 µg/mL gentamicin (Arterium) and fluconazole 40 µg/mL (Arterium). The dissected tissue were incubated with trypsin 0,25 % (Biotestlab) at room temperature approximately one hour with periodic shaking.
The isolated cells and the remaining tissue pieces were then washed with culture medium, centrifuged at 100g for 5 min. After discarding supernatant, the cell pellet was re-suspended at 3–4 ml of growth media (the same media, as for cell obtaining, supplemented with 15 % fetal bovine serum (Hyclone) and seeded in flasks (Sarstedt, TPP and glass). Previously treated and untreated flasks were used. Explants and cell suspension were arranged evenly on the growth surface, and flasks were maintained in working position without additional media for 30-40 min at 37 °C. Growth media was added very carefully, did not disturbed explants, which have been attached. The flasks were tightly closed and cultured at 28 °C.

Suitability of the treated and untreated substrates was evaluated by the seeding (attachment) efficiency. Counting was performed in 10 fields of view at x100 magnification. The TPP flasks with treated and untreated surfaces have demonstrated almost the same results, such as attachment and proliferating of cells were noticed at 6–8 from 10 fields of view. The Sarstedt flasks have promoted cells and explants attachment and proliferating also. Seeding (attachment) efficiency was 7 from 10 fields of view in the treated, and 6 from 10 in the untreated flasks. The glass flasks almost did not support cells proliferating and growing: 3 and 1 from 10 fields of view for the treated and untreated surfaces accordingly.

Established primary cell culture from Xenopus laevis demonstrated fibroblast morphology. The strongly flattened, elongated, fusiform cells had the oval, centrifugal located nucleus and surrounded by unclearly expressed cytoplasm. The cells, which migrate from tissue explants, attached form bundles that randomly intertwined, compact adjoining to each other.

Key words: cеll culture, amphibian, frog (Xenopus laevis), fibroblast cultural substrate, nutrient medium.

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