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ThesisItem Open Access Physiological effect of cryoprotectants in freezing of embryonic fibroblast cells(College of Veterinary Science, Assam Agricultural University, Khanapara Campus, 2022-09) Toufiki, Faijun; Bora, ArundhatiFibroblast cells are the most common cells of connective tissue and form structural framework. In the present study duck embryonic fibroblast cells were developed up to third subcultures and were cryopreserved in three freezing media consisting of freezing medium 1 (10% DMSO), freezing medium 2 (0.9 M Trehalose) and freezing medium 3 (10% DMSO+ 0.09 M Trehalose in 1:1). The cells conforming the morphologically characteristics of fibroblast like typical fusiform shape, turgor vitalis cytoplasm, centrally located nuclei and flame like migration pattern were used for the experiment. The effect of cryoprotectant at equilibration and at different time of post thaw was assessed by their viability and post thaw characteristics. Trypan blue is an azo-based hydrophilic, tetra sulfonated blue acid dye which is used to determine the number of viable cells present in a cell suspension. A viable cell will have clear cytoplasm and non-viable cell will have a blue cytoplasm. The viability percentage before cryopreservation the viability of the duck fibroblast was 90.75±0.047. For freezing medium 1 (10% DMSO), the viability percentage at equilibration was found to be 89.75±0.047 and subsequently at 7 days 89.61±0.064, at 14 days 89.30±0.035, at 21 days 89.06 ±0.011, at 28 days 89.69±0.14. For freezing medium 2 (0.9 M trehalose), the viability percentage at equilibration was found to be 87.69±0.82 subsequently at 7 days 86.73±0.14, at 21 days 86.42±0.04, and at 28 days 86.00±0.06. The viability percentage was significantly higher (p<0.05) in freezing medium 3 (10% DMSO+0.9 M Trehalose in 1:1) followed by freezing medium 1 (10% DMSO) and freezing medium 2 (0.9 M trehalose). For freezing medium 3 (10% DMSO + 0.9 M Trehalose in 1:1), the viability percentage was found to be at equilibration 90.39±0.084 and subsequently at 7 days 89.78±0.068, at 14 days 89.78± 0.068, at 21 days 89.71 ± 0.13, at 28 days 89.68±0.021 respectively. The revival of freezing media 3 (10% DMSO + 0.9 M Trehalose in 1:1) was found to be at 24 hours 14.12±1.65, at 48 hours 26.44±1.93, at 72 hours 40.44±2.27, at 84 hours 50.64±2.89, at 96 hours 59.32 ±0.23. For freezing media 1 (10% DMSO) the confluency was found to be at 24 hours 17.68±0.97, at 48 hours 32.32±0.99, at 72 hours 43.12±1.12, at 84 hours 49.56±0.18 and at 96 hours 59.28±0.14. For freezing media 2 (0.9 M trehalose) the revivability was found to be 24 hours 9.72±0.08, at 48 hours 14.84±1.14, at 72 hours 25.20±1.20, at 84 hours 44.56±0.30, at 96 hours 53.76±0.10. The confluency of freezing medium 3 was significantly higher (p<0.05) found better than freezing medium 1 and freezing medium 2. Found that both intracellular and extracellular cryoprotectant which may favor the normal physiological process at equilibration and at thawing. NANOG, a noble pluripotent marker was found to be present in the developed fibroblast cells as well as after cryopreservation.
