Effect of cryopreservation on metabolic activities of hepatocytes

Abstract

AIM: To study the effect of cryopreservation on metabolic activities and cytochrome P450 (CYP) mRNA expression in hepatocytes and provide support for application of cryopreserved hepatocytes in experimental research. METHODS: Freshly isolated rat hepatocytes were cryopreserved with rate-controlled freezer, and thawed after 1 month. Real-time quantitative PCR was used to detect expressions of CYP1A2, CYP2B1 and CYP3A1 mRNA, and LC-MS/MS was used to measure contents of metabolites of midazolam-1′-hydrxylation (OH-Mid), diclofenac-4′-hydroxylation (OH-Dic) and dextromethorphan-O-demethylation (Dex) in hepatocytes, respectively. RESULTS: There was no significant difference in cell viability between fresh and cryopreserved hepatocytes. The cryopreserved hepatocytes attached and established extensive cell-cell contact, with round and bright nucleus. CYP1 A2 and CYP2B1 mRNA expressions induced by β-naphthoflavone and phénobarbital in cryopreserved hepatocytes were similar to that in the fresh primary cells. However, CYP3A1 mRNA expression did not induced by pregnenolone-16α-carbonitrile in cryopreserved hepatocytes. In cryopreserved hepatocytes, the content of OH-Mid was remained as almost the same as the fresh primary hepatocytes, while contents of OH-Dic decreased approximately as a half, and Dex was double as fresh hepatocytes. CONCLUSION: Cryopreservation exerts different effects on metabolic activities of hepatocytes. To acquire objective and appropriate results, it is necessary to consider the different influence of cryopreservation on cell metabolic activity in drug metabolic research.