Induction of cytochrome-P450 in cryopreserved rat and human hepatocytes

Our laboratory has been routinely using suspended and cultured human hepato cytes for predicting drug metabolism and enzyme induction by drug candidate s to aid drug discovery. Increasing limitation and irregular availability o f human tissue has indicated the need for maximizing the use of this valuab le resource. Cryopreservation of surplus hepatocytes after isolation would greatly increase the potential of this model. However, cryopreservation of hepatocytes by Various methods has resulted in cells with poor metabolic ac tivity and unacceptably low survival rates in culture. Recently, Zaleski et al. (Biochem. Pharmacol. 46 (1993) 111-116) reported that cryopreserved ra t hepatocytes retained metabolic capacity similar to fresh hepatocytes when the cells were preincubated for 30 min at 37 degrees C in Krebs-Ringer bic arbonate buffer prior to freezing. To further explore this methodology, bot h the functional capacity of the cells in culture as well as their ability to retain CYP inducibility were investigated with thawed cryopreserved hepa tocytes. Although human hepatocytes were used in this study the initial wor k focused on rat hepatocytes as a cell model. Our results showed that while the preincubation step did not appear to effect the initial viability of c ryopreserved hepatocytes, survival of the cells in culture was greatly enha nced. Plating efficiencies for nonpreincubated cryopreserved hepatocytes we re decreased to approximate to 15% of fresh cells after 48 h in culture. In contrast, cells that had been preincubated prior to freezing had an excell ent plating efficiency (similar to 60%) and responded to classical CYP indu cers dexamethasone, beta-naphthoflavone and phenobarbital in a manner indis tinguishable from that of fresh hepatocytes. Experiments with human hepatoc ytes have also demonstrated similar results. This is the first time to our knowledge that cryopreserved hepatocytes from both rat and human have been shown to reproducibly respond to CYP inducers in culture. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.