Received for publication February 27, 2008.
Revised July 8, 2008.
Accepted for publication July 9, 2008.

Metabolic activity of cytochrome P450 isoforms in hepatocytes cryopreserved with wheat protein extract

Abstract

The drug discovery and development process requires adequate safety testing for drug toxicity before new drugs can be administered to patients. Hepatocytes are used in vitro to screen compounds for hepatotoxicity, induction of drug-metabolizing enzymes such as cytochrome P450 isoforms and drug-drug interactions, and to establish human relevance for metabolism. Cryopreservation makes it possible to preserve a large quantity of functional hepatocytes. Techniques for cryopreservation of hepatocytes are mainly based on dimethylsulfoxide. However, analyses of metabolic capacities of cryopreserved hepatocytes are often limited by loss of functional integrity of hepatocytes after thawing. It is therefore necessary to improve techniques of cryopreservation. We have developed a new cryopreservation technology for mammalian cells based on a wheat protein extract (WPE). We determined whether the WPE can better preserve activities of major cytochrome P450 isoforms, both in suspension and monolayer cultures of hepatocytes. This was achieved by comparing basal and inducible or metabolic activities of isoforms CYP1A1, CYP1A2, CYP2C6, CYP2D2 and CYP3A in rat hepatocytes that were cryopreserved with WPE, relative to fresh cells and those cryopreserved with DMSO. We conclusively demonstrate that rat hepatocytes cryopreserved with WPE retain their metabolic competency and their ability to respond to classical CYP inducers, when compared to freshly isolated hepatocytes. These findings clearly show that WPE are an excellent cryopreservant for rat hepatocytes. They are an efficient, non-toxic, economic natural product and universal cryoprotectant that is superior to DMSO, which has limitations due to cellular toxicity.

Key words: CYP induction, drug discovery, drug toxicity, drug-induced hepatotoxicity, hepatocytes, hepatotoxicity, toxicity testing