Development of a Liver-on-a-chip System to Evaluate Drug Metabolism and Hepatotoxicity based on HepaRG 3D Spheroids

Abstract

Cytochrome P450 3A4 (CYP3A4) is a liver enzyme that metabolizes more than 50 % of therapeutic drugs. Therefore, measuring CYP3A4 activity is important in hepatotoxicity evaluation and drug development. HepG2 cells are widely used in hepatotoxicity evaluation, but their CYP3A4 activity is low. Due to the limitations of HepG2 cells, HepaRG cells with abundant CYP3A4 enzymes are widely used in drug metabolism research. However, in vitro experiments are difficult to predict the flow and metabolism of drugs because they do not mimic the human liver environment. To overcome this, 2D cell culture systems have evolved into 3D cell culture systems and microchips are being studied to mimic the in vivo environment. The goal of this study is to develop a liver-on-a-chip based on HepaRG 3D spheroids for hepatotoxicity evaluation and drug metabolism confirmation. We optimized the differentiation and metabolism conditions of HepaRG 3D spheroids and conducted hepatotoxicity evaluation on chips. We used testosterone, one of the drugs metabolized by CYP3A4, to confirm the metabolism ability of HepaRG 3D spheroids. Testosterone metabolism was analyzed using High Performance Liquid Chromatography (HPLC). CYP3A4 activity was higher in HepaRG 3D spheroids than in HepG2 cells. In addition, viability, CYP3A4 activity and albumin were maintained for a week in HepaRG 3D spheroids. Drugs with hepatotoxicity were treated with HepaRG 3D spheroids to confirm the effects of toxic drugs. The toxicity was more affected by the chip than by the cell culture plate. These results show that liver-on-a-chip based on HepaRG 3D spheroids is a good model for drug development and hepatotoxicity prediction. In addition, during the process of confirming testosterone metabolism, it was confirmed that its metabolite, 6β-hydroxytestosterone, flowed up and down on inserts on the chip and on both sides of the chip. This indicates that it can be used as a model to confirm interactions between organs by culturing cells from different organs such as intestines and kidneys as well as liver cells in three chip inserts.