Cryopreservation of Hepatocyte Microbeads for Clinical Transplantation

Intraperitoneal transplantation of hepatocyte microbeads is an attractive option for the management of acute liver failure. Encapsulation of hepatocytes in alginate microbeads supports their function and prevents immune attack of the cells. Establishment of banked cryopreserved hepatocyte microbeads is important for emergency use. The aim of this study was to develop an optimised protocol for cryopreservation of hepatocyte microbeads for clinical transplantation using modified freezing solutions. Four freezing solutions with potential for clinical application were investigated. Human and rat hepatocytes cryopreserved with University of Wisconsin (UW)/10% dimethyl sulfoxide (DMSO)/5% (300mM) glucose and CryoStor CS10 showed better post thawing cell viability, attachment and hepatocyte-functions than with Histidine-tryptophan-ketoglutarate (HTK)/10%DMSO/5%glucose, and Bambanker. The two freezing solutions which gave better results were studied with human and rat hepatocytes microbeads (HMBs and RMBs). Similar effects on cryopreserved microbeads morphology (external and ultrastructural), viability and hepatocyte-functions post thawing were observed over 7 days in culture. UW/DMSO/glucose, as a basal freezing medium, was used to investigate the additional effects of cytoprotectants: a pancaspase inhibitor (benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone; ZVAD), an antioxidant (desferoxamine; DFO), and buffering and mechanical protection (human serum albumin; HSA) on RMBs. ZVAD (60µM) had beneficial effect on cell viability, greater than with DFO (1mM), HSA (2%) and basal freezing medium alone. Improvements in the ultrastructure of encapsulated hepatocytes, and a lower degree of cell apoptosis were observed with all three cytoprotectants, ZVAD tending to provide the greatest effect. Cytochrome P450 activity was significantly higher in the three cytoprotectants groups than with fresh microbeads. In conclusion, developing an optimised cryopreservation protocol by adding cytoprotectants such as ZVAD could improve the outcome of cryopreserved hepatocyte microbeads for future clinical use.