(93x) Analysis of Apoptotic Cell Death in Insulin Secreting Cells Due to Cryopreservation

Cryopreservation, the freezing of cells or tissues, is considered to be an increasingly important form of long-term storage of biological materials. Depending on cell and tissue type, current freezing methods can induce significant amounts of cell death and tissue damage. It has been shown that the observed cell death is not only necrosis due to physical injury from ice, but may also result from apoptosis, a highly regulated biochemical process of programmed cell deletion. Although there have been studies on freeze-induced apoptosis, there is still a lack of information on the extent of apoptotic cell death during the cryopreservation process. The objective of this study was to investigate the extent of apoptosis induced by the various steps of cryopreservation on insulin-secreting βTC tet cells. Assays were used to examine the main hallmarks of early, middle, and late stage of apoptosis. These hallmarks are activation of a cell-protein digesting enzyme (Caspase-8), externalization of phosphatidylserine to the outer leaflet of the cell membrane, and fragmentation of DNA, respectively. It was found that the addition of dimethyl sulfoxide, a common cryoprotectant, did not induce apoptosis to the cells; however, there was an increase in apoptotic activity after the freeze-thaw process. The significance of this work is to not only gain a fundamental understanding of the process of apoptosis as it is induced by cryopreservation in insulin-producing cells, but also apply the knowledge obtained to enhance current cryopreservation protocols to achieve higher post-thaw cell viability and retention of cellular function.