Genetically engineered hepatocytes as a capable application for in vitro liver cell systems

Background:

The use of primary human hepatocytes (PHH) in biomedical research is still considered as the gold standard. Their application possibilities encompass identifications of pathophysiological alterations and new biochemical pathways in liver diseases, e.g. steatosis. The main problems are the limited availability of liver tissue and donor variability. Therefore the development of alternative liver models has gained a lot of interest in recent years. Beside hepatic cell lines and stem cell derived hepatocyte like cells, genetically engineered cells have become a new promising approach. Aim of the present study was the characterization of a genetically engineered hepatocyte culture in comparison to conventional liver model systems.

Methods:

PHH were isolated from human liver resectates using a two-step collagenase perfusion technique. A lentiviral vector system was used to transfer proliferation genes (Upcyte® genes) to generate a stable hepatocyte culture (HepaFH3). PHH, HepG2 and HepaFH3 were analyzed for their capability of lipid and glucose storage using Oil-Red-O staining and a biochemical assay for glycogen quantification, respectively. Energy consumption was investigated by measurement of mitochondrial activity using the MTT assay. For evaluation of hepatic functions: albumin synthesis, urea formation and transaminase activities were determined.

Results:

The quantification of triglyceride storage revealed that lipid accumulation was two times higher in PHH and comparable in proliferating cell lines. Regarding comparison of energy consumption PHH showed lowest rates whereas two fold higher rates in HepaFH3 and up to ten times higher rates in HepG2 were detected. In opposite to hepatic cell lines PHH showed performance of gluconeogenesis, while HepG2 cell medium was completely depleted of glucose after 24h. Determination of glycogen storage revealed a fourfold lower rate in differentiated HepaFH3 than in PHH, a 100 fold lower value in proliferating HepaFH3 and was not detectable in HepG2 cells. Investigation of hepatic functions displayed a diminished capability of cell lines to synthesize urea and albumin, as well as diminished transaminases activities (especially ALT). However, in comparison with cell line HepG2, HepaFH3 loses capability of albumin and urea synthesis with increasing number of cell passages.

Conclusions:

Even if HepaFH3 showed overall lower values in the investigated parameters these cells are more consistent with PHH than with the tumor cell line HepG2. The latter miss gluconeogenesis and glycogen storage, propably to their high cell division rate and its enhanced energy metabolism. Therefore HepaFH3 seem to be a more appropriate substitution for PHH in in vitro experiments concerning energy metabolism.