Cryopreserved primary hepatocytes as a constantly available in vitro modelfor the evaluation of human and animal drug metabolism and enzyme induction

The use of primary hepatocytes is now well established for both studies of drug metabolism and enzyme induction. Cryopreservation of primary hepatocyt es decreases the need for fresh liver tissue. This is especially important for research with human hepatocytes because availability of human liver tis sue is limited. In this review, we summarize our research on optimization a nd validation of cryopreservation techniques. The critical elements for successful cryopreservation of hepatocytes are (1 ) the freezing protocol, (2) the concentration of the cryoprotectant [10% d imethylsulfoxide (DMSO)], (3) slow addition and removal of DMSO, (4) carbog en equilibration during isolation of hepatocytes and before cryopreservatio n, and (5) removal of unvital hepatocytes by Percoll centrifugation after t hawing. Hepatocytes of human, monkey, dog, rat, and mouse isolated and cryo preserved by our standard procedure have a viability greater than or equal to 80%. Metabolic capacity of cryopreserved hepatocytes determined by testo sterone hydroxylation, 7-ethoxyresorufin-O-deethylase (EROD), 7-ethoxycouma rin-O-deethylase (ECOD), glutathione S-transferase, UDP-glucuronosyl transf erase, sulfotransferase, and epoxide hydrolase activities is greater than o r equal to 60% of freshly isolated cells. Cryopreserved hepatocytes in susp ension were successfully applied in short-term metabolism studies and as a metabolizing system in mutagenicity investigations. For instance, the compl ex pattern of benzo[a]pyrene metabolites including phase II metabolites for med by freshly isolated and cryopreserved hepatocytes was almost identical. For the study of enzyme induction, a longer time period and therefore cryo preserved hepatocyte cultures are required. We present a technique with cry opreserved hepatocytes that allows the induction of testosterone metabolism with similar induction factors as for fresh cultures. However, enzyme acti vities of induced hepatocytes and solvent controls were smaller in the cryo preserved cells. In conclusion, cryopreserved hepatocytes held in suspension can be recommen ded for short-term metabolism or toxicity studies. Systems with cryopreserv ed hepatocyte cultures that could be applied for studies of enzyme inductio n are already in a state allowing practical application, but may be further optimized.