A long-term culture system based on a collagen vitrigel membrane chamber that supports liver-specific functions of hepatocytes isolated from mice with humanized livers

摘要

During drug discovery, in vitro models are used to predict the in vivo pharmacokinetic and toxicological properties of drug candidates in humans. However, the conventional method of culturing human hepatocytes as monolayers does not necessarily replicate biologic reactions and does not support liver-specific functions, such as cytochrome P450 (CYP) activities, for prolonged periods. To remedy these problems and thus increase and prolong hepatic functions, we developed a culture system comprising a collagen vitrigel membrane (CVM) chamber and PXB-cells, fresh hepatocytes isolated from liver-humanized chimeric mice (PXB-mice (R)). To quantitatively assess our new system, we evaluated the activities of 5 major CYP isoforms (CYP 1 A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A), albumin secretion, and urea synthesis. First, between Days 14 and 21, the activities of all CYP isoforms tested in vitrigel culture were equal to or higher than in conventional monolayer culture system. Second, the activities of CYP3A, CYP2C9, and CYP2C19 during Days 10 through 17 were higher in vitrigel culture than in suspended PXB-cells prepared on Day 0 (suspension assay). Third, albumin secretion and urea synthesis were higher in vitrigel culture than in conventional monolayer culture. Fourth, the vitrigel-cultured PXB-cells showed the characteristic morphology of parenchymal hepatocytes and were almost all alive in monolayer. These results indicate that our vitrigel culture method is superior to the conventional monolayer method in terms of diverse liver-specific functions, including CYP activity. Our findings suggest that the vitrigel culture method could be a powerful in vitro tool for predicting the pharmacokinetic and toxicological properties of drug candidates in humans.