Effect of Cryopreservation on Different Passages of Porcine Dorsal Root Ganglion Cell Culture

Abstract

The cell culture obtained from dorsal root ganglia (DRG) is a valuable model used in biology or medical research. However, the effect of cryopreservation on the properties of DRG-derived cell culture of different passages remains unclear up to date. The objective of the study is to assess the effect of cryopreservation with various concentrations of cryoprotectant dimethyl sulfoxide (DMSO) on the viability and morphological features of porcine neonatal DRG cell culture of different passages. Cell suspension was obtained from DRG of neonatal piglets and cultured in an α-MEM nutritional medium supplemented with 10% fetal calf serum (0, 1st, and 2nd passages). Cells of different passages were cryopreserved at a cooling rate of 0.5°C/min to – 20°C (step 1) and 1°C/min to – 80°C (step 2) followed by immersion into liquid nitrogen. Cryoprotective solutions based on α-MEM nutrient medium and 25% fetal calf serum (FCS) containing 5%, 7.5%, and 10% DMSO were used. It was established that the primary culture (passage 0) consisted of four cell morphological types: large rounded cell bodies of sensory neurons (SN) and three types of non-neuronal cells, namely, polygonal cells with pronounced elongated processes (type 1), spindle-shaped cells (type 2), and multipolar flattened fibroblastoid cells (type 3). As the number of passages increases, an elimination of SN from the culture, a decrease in the relative number of 1st and 2nd cell types, and expansion of 3rd cell type were observed. DRG cell culture had sufficiently high resistance to cryopreservation as cell viability was in the range from 83% to 90% using different concentrations of DMSO. The cells of passage 1 were more resistant to cryopreservation in comparison with primary culture cells (passage 0). The best result was achieved by freezing the culture of passage 1 in the cryoprotective medium with 7.5% DMSO, where 90.6% of viable cells were observed after thawing.

Biologically Active Compositions Containing Neurotrophic Factors Change the Contractile Activity of Detrusor of Rats with Infravesical Obstruction

“…These eff ects are likely due to the action of NF coming from the SG and from the MG culture after freezing and warming. It has been proven that after cryopreservation the ratio of morphological types of cells changes, namely, their production of NFs as well [1]. The fi ndings indicate a positive eff ect of BACs on the course of IVO in rats, which may be implemented by the UB neuromuscular apparatus.…”

“…Клітини СГ, отримані ферментативним способом, культивували 20 діб у базовому середовищі культивування (БСК), яке містить α-МЕМ, із додаванням 10% фетальної телячої сироватки (ФТС, «BioSera», Франція); 100 мкг/мл гентаміцину («ПанЕко», Росія); 100 мкг/мл цефотаксиму («Біосинтез», Росія); 2,5 мкг/мл амфотерицину В («Biowest», Франція). Клітини культивували в пластикових чашках Петрі з площею 9 см 2 , які було оброблено полі-D-лізином («Orange Scientifi c», Бельгія) при 37°С в атмосфері з 5% СО 2 [1]. Заміну середовища проводили кожні 3 доби.…”

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“…Based on the foregoing, it can be assumed that, in the future, it is advisable to use NFs obtained from SG and from the MG culture after freezing-warming, due to their wide spectrum of action on the morphological and functional properties of UB. This is possible due to the fact that after cryopreservation the ratio of morphological cell types changes, that is, their production of NFs [25]. At the same time, the use of neurotrophic drugs such as cortexin is less justified due to their predominant effect mainly on the nervous system.…”