Development and characterization of human liver spheroid cultures for drug transporter studies and microphysiological systems

Abstract

A current problem in the drug development process today is the lack of predictable in vitro liver models for pre-clinical studies. Current in vitro methods and animal trials often fail to predict the effect of potential drugs in humans. In addition, drug-drug interactions which could lead to drug induced liver injury is often a reason for drugs not reaching the market, and it can be related to changes in efflux of metabolites from hepatocytes in liver. In this study, a two-organ-chip has been developed with aggregates formed of HepaRG cells co-cultured with stellate cells as well as pancreatic islets, and evaluated as an in vitro model. The focus of the present master thesis project was to evaluate functionality of liver aggregates used in the two-organ-chip system. As a comparison, liver aggregates from chip-system with liver aggregates only has been evaluated. The results showed a significant increase in albumin production from liver aggregates that were in the two-organ-chip system together with pancreatic islets. The liver aggregates showed no significant difference of glycogen production when cultured with or without pancreatic islets. As a conclusion, the chip-system with two organs used showed increased liver specific functions in liver aggregates. Another part of the study included evaluation of activity of the canalicular transporter multidrug resistance protein 2 (MRP2) in hepatic spheroids formed of primary human hepatocytes or hepatocytes co-cultured with stellate cells. The activity was evaluated using a substrate, 5-chloromethylfluorescein diacetate (CMFDA), which is converted to a fluorescent substrate inside cells and transported by MRP2 into bile canaliculi of spheroids. When using medium lacking of calcium it was possible to measure the amount of fluorescent substrate that was transported into bile canaliculi. The result indicated that MRP2 was active in spheroids, but the method used needs further development to reduce the large variation and the challenging experimental procedure.