Physiological effect of cryoprotectants in freezing of embryonic fibroblast cells

Abstract

Fibroblast cells are the most common cells of connective tissue and form structural framework. In the present study embryonic fibroblast cells up to three subcultures were cryopreserved and their viability was studied using three freezing media. The viability at equilibrium of freezing media-3 (10% DMSO + 0.9 M Trehalose), 1(10% DMSO), 2 (0.9M Trehalose) was found to be 90.39±0.084, 89.75±0.047 and 87.69±.082 respectively by using trypan blue exclusion dye test. 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. Media 3 reach confluency 60% after post thaw re-cultured in lower time of 4 days as compared to freezing media 1 and 2. The confluency timing was consequently found better from 2 days onwards in both freezing media -1 and freezing media - 2. Morphologically the isolated cells had fibroblast character like typical fusiform shape, turgor Vitalis cytoplasm, centrally located nuclei and flame like migration pattern.