Generation of Scalable Hepatic Micro-Tissues as a Platform for Toxicological Studies

Abstract

Background: Currently, there is an urgent need for scalable and reliable in vitro models to assess the effects of therapeutic entities on the human liver. Hepatoma cell lines, including Huh-7, show weakly resemblance to human hepatocytes, limiting their significance in toxicity studies. Co-culture of hepatic cells with non-parenchymal cells, and the presence of extracellular matrix have been shown to influence the biological behavior of hepatocytes. The aim of this study was to generate the scalable and functional hepatic micro-tissues (HMTs). Methods: The size-controllable HMTs were generated through co-culturing of Huh-7 cells by mesenchymal stem cells and human umbilical vein endothelial cells in a composite hydrogel of liver-derived extracellular matrix and alginate, using an air-driven droplet generator. Results: The generated HMTs were functional throughout a culture period of 28 days, as assessed by monitoring glycogen storage, uptake of low-density lipoprotein and indocyanine green. The HMTs also showed increased secretion levels of albumin, alpha-1-antitrypsin, and fibrinogen, and production of urea. Evaluating the expression of genes involved in hepatic-specific and drug metabolism functions indicated a significant improvement in HMTs compared to two-dimensional (2D) culture of Huh-7 cells. Moreover, in drug testing assessments, HMTs showed higher sensitivity to hepatotoxins compared to 2D cultured Huh-7 cells. Furthermore, induction and inhibition potency of cytochrome P450 enzymes confirmed that the HMTs can be used for in vitro drug screening. Conclusion: Overall, we developed a simple and scalable method for generation of liver micro-tissues, using Huh-7, with improved hepatic-specific functionality, which may represent a biologically relevant platform for drug studies.