Optimization of metabolic function in a bio-artificial liver

The Minnesota Bio-Artificial Liver was developed as a potential extracorporeal device for the treatment of patients suffering from fulminant hepatic failure. The liver contains porcine hepatocytes entrapped in cylindrical collagen gels. This study was performed to optimize the metabolic function of cultured porcine hepatocytes by induction of cytochrome P450 enzymes.; The induction of the cytochrome P450 family of enzymes was studied in porcine hepatocytes cultured on collagen gels. Initial studies were carried out with rifampin as an inducer of CYP3A isoforms, and with lidocaine as a substrate probe. A four-fold induction was observed in lidocaine metabolism in rifampin-induced porcine hepatocytes compared to control cultures.; Dexamethasone and rifampin were evaluated as inducers of CYP3A mediated midazolam metabolism in porcine hepatocyte cultures. There was a significant 4- to 10-fold induction of midazolam metabolism in rifampin-induced cultures compared to control cultures. Presence of dexamethasone caused no change in the metabolism of midazolam. Examination of the CYP3A mRNA levels by Northern Blotting revealed a 13-fold increase in the levels of a single band of mRNA in rifampin-induced cultures over control cultures. Western blot analysis performed on microsomes prepared from cultured porcine hepatocytes revealed in an increase in the levels of two CYP3A immunoreactive proteins in rifampin-induced cultures compared to control cultures. These results suggest that the induction of CYP3A proteins in cultured porcine hepatocytes occurs at the level of gene expression. Treatment of porcine hepatocyte spheroids with {dollar}beta{dollar}-naphthoflavone and phenobarbital caused an increase in the metabolism of ethoxyresorufin and pentoxyresorufin respectively.; The effect of culture conditions was evaluated by culturing porcine hepatocytes on collagen gels, in spinner flasks, and in a sandwich configuration between collagen gels and Matrigel. No difference was observed in the metabolism of midazolam, or levels of CYP3A proteins between the different culture systems. However, rifampin was a potent inducer of CYP3A forms in porcine hepatocytes in all culture conditions examined.; In conclusion, these results demonstrate the inducibility of P450 mediated metabolism in cultured porcine hepatocytes by putative inducers of the P450 isozymes.