Ali S. Cultivation and cryopreservation of cells and multicellular spheroids from the dorsal root ganglia of neonatal piglets

The object of the research is the formation of monolayer culture and multicellular spheroids (MS) from the cells of the dorsal root ganglia of neonatal piglets under different conditions of in vitro cultivation before and after cryopreservation with different concentrations of the cryoprotectant dimethyl sulfoxide (DMSO). The goal of the research is the obtaining of cell culture of dorsal root ganglia of neonatal piglets in the form of monolayer and MS, study of their morphological and functional as well as immunophenotypic characteristics, and assessment of the effect of cryopreservation in the cryoprotectant media with different concentrations of DMSO on cell culture features. Research methods: methods of enzymatic cell isolation from dorsal root ganglia; monolayer (2D) and three-dimensional (3D) cultivation; cryopreservation; immunocytochemical staining; light and fluorescence microscopy; morphometric analysis; methods of statistical analysis. For the first time it was established that the addition of serum to the culture medium was a decisive condition for the production of dorsal root ganglia cell culture in the form of monolayer or MS. Regardless of the method of obtaining, the culture consisted of 3 cell types that expressed a marker of satellite glial cells glutamine synthetase, a neuroblast marker β-III-tubulin, but did not express a marker of Schwann cells S100. For the first time it was established that cryopreservation of culture both as a single cell suspension and as a part of MS in the media containing DMSO at concentrations of 5, 7.5, and 10%, using a three-stages cooling regimen, made it possible to preserve the main morphological, immunophenotypic and functional properties of the culture. Field of Application: cryobiology, biology, medicine, biotechnology, veterinary, pharmacy.