Caspase inhibition reduces apoptotic death of cryopreserved porcine hepatocytes

Cryopreserved porcine hepatocytes are a ready source of metabolic function for use in a bioartificial liver (BAL). However, cryopreservation is associated with a loss of hepatocyte viability. The mechanism of cell death during cryopreservation is incompletely understood, but may involve apoptosis through caspase activation. This study evaluates the cytoprotective effect of a global caspase inhibitor, benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (ZVADfmk) during cryopreservation of porcine hepatocytes. Freshly isolated porcine hepatocytes (viability, 97.4% ± 0.9%) were cryopreserved in 60 μmol/L ZVAD-fmk (+ZVAD group) or without ZVAD-fmk (-ZVAD group) for 24 to 72 hours. Apoptotic and necrotic death were both observed after thawing and after 24 hours of culture. Caspase 3-like activity was significantly reduced by ZVADfmk, and was associated with improved viability and reduced apoptotic death of porcine hepatocytes after cryopreservation. Mitochondrial membrane potential (MMP) was increased in cultures of porcine hepatocytes that were cryopreserved in ZVAD-fmk. These results demonstrate the following: 1) Caspase 3-like protease activation and apoptosis occurs in porcine hepatocytes during cryopreservation; and 2) mitochondrial injury in this process is reduced by caspase inhibition.