Characterizing the cryobiological response of a mouse cardiac endothelial cell line to interrupted slow cooling (graded freezing)

Mouse cardiac endothelial cells play a critical role in cardiovascular research and disease; hence, high quality cryopreservation of this cell type is necessary to provide on-demand access to the cells, quality control, and consistent outcomes between different experiments and batches. However, the cryopreservation of cardiac endothelial cells has not been studied as much as other endothelial cells. The objective of this study was to characterize the cryobiological response of an immortalized line of mouse cardiac endothelial cells in suspension using an interrupted slow cooling approach (graded freezing) and to validate cryopreservation protocols that provide high post-thaw viability and function. Three different cryopreservation protocols were validated using different cryoprotectant solutions: i) 10 % dimethyl sulfoxide + 90 % fetal bovine serum, ii) 5 % dimethyl sulfoxide + 6 % hydroxyethyl starch in complete growth medium, and iii) 10 % glycerol in complete growth medium; all three protocols resulted in high post-thaw membrane integrity assessed using dual fluorescent dye Syto13/GelRed. Two functional assays: i) tube forming on Matrigel representing angiogenesis and ii) nitric oxide synthesis using DAF-FM (4-amino-5-methylamino-2',7'-difluorofluorescein) fluorescent dye revealed that cryopreserved cells retained comparable post-thaw function to non-cryopreserved cells. This study provides practical insight into successful cryopreservation of mouse cardiac endothelial cells.