EFFECTIVE CRYOPRESERVATION AND LONG-TERM STORAGE OF PRIMARY HUMAN HEPATOCYTES WITH RECOVERY OF VIABILITY, DIFFERENTIATION, AND REPLICATIVE POTENTIAL

Despite reports of successful cryopreservation of primary human hepato cytes, existing methods do not produce sufficient recovery of viable c ells to meet the needs of basic research or clinical trials of hepatoc ellular transplantation. We now describe a protocol for efficient cryo preservation of primary human hepatocytes using University of Wisconsi n (UW) solution, fetal bovine serum, and dimethyl sulfoxide (DMSO), Th is method provides >90% viability of differentiated, primary human hep atocytes 8 mo after cryopreservation as measured by trypan blue exclus ion, preserves hepatocyte morphology, liver-specific gene expression ( cu, antitrypsin), and replication. The effectiveness of UW solution as a cryopreservative agent suggests that metabolic as well as ultrastru ctural factors may be important in the effective cryopreservation of p rimary human hepatocytes. The present method represents an effective p rotocol for cryopreserving differentiated primary human hepatocytes fo r research. This method may allow characterization and banking of huma n hepatocytes for clinical applications, including hepatocellular tran splantation and hepatic assist devices.