Activation of Mitochondrial-Associated Apoptosis Contributes to Cryopreservation Failure

Post-thaw viability following cryopreservation (CP) remains suboptimal for many cell systems. Recent focus on delayed-onset cell death (DOCD) is providing an explanation for this observed failure. One potential avenue for the activation of DOCD involves the intrinsic mitochondrial-apoptotic pathway. Specifically, stressors can disrupt the pro-/anti-apoptotic protein balance associated with the mitochondria and the related Bcl-2 protein family. We hypothesized that CP-dependent disruption of the pro-/anti-apoptotic ratio (specifically, Bax: Bcl-XL) contributes to the activation and progression of DOCD. In this study, human dermal fibroblasts were cryopreserved in various freeze media (media +5% DMSO or CryoStor™ CS5), frozen at 1°C · min−1to −80°C and stored in LN2. Following storage, cells were rapidly thawed and plated in culture media. Viability was assessed daily using a metabolic indicator (alamarBlue) and a nucleic acid probe (SytoDye). Total cellular protein was isolated from samples at 0, 6, 12, an...