Metabolomics of an in vitro liver model containing primary hepatocytes assembling around an endothelial cell network: Comparative study on the metabolic stability and the effect of acetaminophen treatment

Recently, a novel culture system consisting of primary hepatocytes structured over a network of endothelial cells on the Engelbreth-Holm-Swarm (EHS) gel has been reported. This in vitro liver model on the EHS gel (IVL_EHS) has been shown to maintain the expression of hepatic genes and their functional activity. Moreover, the IVL_EHS was more sensitive to xenobiotics than hepatocyte monocul-tures, suggesting the potential utility of this culture system for compound hepatotoxicity screening. However, the effect of this three-dimensional structure formation on the cellular metabolic profile of hepatocytes in the IVL_EHS is not well understood. To address this concern, we performed metabolome analysis using capillary electrophoresis-time of flight mass spectrometry. Between the IVL_EHS and mono-cultured hepatocytes on the EHS gel, there was no significant difference in the levels of metabolites of the urea cycle and the tricarboxylic acid cycle, essential amino acids, and adenylate energy charge (AEC) which is an important indicator of cellular energy status. On the other hand, acetaminophen-dependent decrease of the AEC in the IVL_EHS was greater than that in the monoculture, suggesting the higher sensitivity of IVL_EHS to acetaminophen-induced hepatotoxicity which is caused by metabolic activation of this drug. Further analysis showed that the levels of taurocholate, one of the major conjugated bile acids, were higher in the IVL_EHS than in the monoculture. Considering that the construction of the IVL_EHS did not seem to disturb the major cellular metabolism, our findings would strengthen the concept that IVL_EHS would have beneficial effects on the maintenance of hepatic functions.