Activation of Mitochondrial-Associated Apoptosis Contributes to Cryopreservation Failure

Abstract

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-X_L) 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, and 24 h post-thaw, and Bcl-X_L and Bax levels were analyzed via Western blotting. Results: (1) utilization of CryoStor™ CS5 yielded an increase in cell survival over that of media +5% DMSO (61% vs. 37%, 24 h post-thaw). (2) Cryopreservation resulted in the disruption of the pro-/anti-apoptotic ratio, shifting towards pro-death signaling during the recovery period. (3) Utilization of CryoStor™ CS5 decreased the shift in the pro-/antiapoptotic ratio compared with storage in Media +5% DMSO (severalfold). In conclusion, examination of mitochondrial protein levels following CP revealed distinct temporal profiles between differing CP protocols. This study demonstrates that activation of the mitochondrial-associated apoptotic pathway plays an integral role in the execution of CP-induced DOCD. This study further illustrates that the control of apoptotic cell death, particularly mitochondrial associated, may facilitate further enhancement in cell survival following CP.