Cryopreservation of chicken blastodermal cells and their quality assessment by flow cytometry and transmission electron microscopy

The goal of this study was to evaluate effect of slow freezing and vitrification methods on the viability of chicken blastodermal cells (BCs). Proper aliquot of isolated BCs were diluted in the freezing medium composed of 10% DMSO and frozen in the freezing vessel BICELL to reach desired temperature up to -80 degrees C. Then samples were immersed in liquid nitrogen. Other cell aliquot was vitrified in solution containing 10% DMSO and samples were immediately immersed in the liquid nitrogen. The viability of fresh and frozen/thawed BCs was evaluated using Trypan blue method and flow cytometry. Flow cytometry analysis was provided by DRAQ5 dye in combination with Live-Dead kit. Overall, this technique provides both quantitative and qualitative information about BCs. Results obtained from Trypan blue method showed significant differences (P<0.05) between control (8.37 +/- 1.04%) slow freezing (83.73 +/- 2.72%) and vitrification group (84.39 +/- 1.77%) in the percentage of Trypan blue positive (necrotic) BCs. Moreover, differences (P<0.05) between control and slow freezing (5.08 +/- 1.94%, 73.31 +/- 3.90%) and control and vitrification group (2.97 +/- 0.30%, 79.02 +/- 1.56%) in results on portion of necrotic cells (DRAQ5(+)/LD+) analyzed by flow cytometry were also observed. The large percentage of necrotic BCs was found in all freezing methods. However, based on ultrastructural analysis, our study showed, that BCs contain lipid granules which prevent successful freezing even though different methods of cryopreservation were used. Thus, freezing of BCs probably required subsequent culture to eliminate lipid droples and yolk granules in the cells, which could possibly improve the success. (c) 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:778-783, 2018