Primary-like Human Hepatocytes Genetically Engineered to Obtain Proliferation Competence as a Capable Application for Energy Metabolism Experiments in In Vitro Oncologic Liver Models

Abstract

Non-alcoholic fatty liver disease (NAFLD), characterized by lipid accumulation in the liver, is the most common cause of liver diseases in Western countries. NAFLD is a major risk factor for developing hepatocellular carcinoma (HCC); however, in vitro evaluation of hepatic cancerogenesis fails due to a lack of liver models displaying a proliferation of hepatocytes. Originally designed to overcome primary human hepatocyte (PHH) shortages, upcyte hepatocytes were engineered to obtain continuous proliferation and, therefore, could be a suitable tool for HCC research. We generated upcyte hepatocytes, termed HepaFH3 cells, and compared their metabolic characteristics to HepG2 hepatoma cells and PHHs isolated from resected livers. For displaying NAFLDrelated HCCs, we induced steatosis in all liver models. Lipid accumulation, lipotoxicity and energy metabolism were characterized using biochemical assays and Western blot analysis. We showed that proliferating HepaFH3 cells resemble HepG2, both showing a higher glucose uptake rate, lactate levels and metabolic rate compared to PHHs. Confluent HepaFH3 cells displayed some similarities to PHHs, including higher levels of the transaminases AST and ALT compared to proliferating HepaFH3 cells. We recommend proliferating HepaFH3 cells as a pre-malignant cellular model for HCC research, while confluent HepaFH3 cells could serve as PHH surrogates for energy metabolism studies.

A Unification of Nanotopography and Extracellular Matrix in Electrospun Scaffolds for Bioengineered Hepatic Models

“…We have therefore developed a new protocol to incorporate the perfusion of whole-organ decellularized rat liver ECM into electrospun fibers with surface depressions to explore the combined effect on human hepatic carcinoma cells (HepG2). This cell line is a proven tool for analyzing hepatocyte culture platforms due to its availability, phenotypic stability, and high degree of morphological and functional differentiation in vitro. − …”