Mavri-Damelin, Demetra; (2007) Characterizing and restoring urea cycle function in human hepatoblastoma-derived cell lines for use in bioartificial liver devices. Doctoral thesis , UCL (University College London).

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Abstract

The creation of a bio-artificial liver device could assist liver failure patients awaiting liver transplantation or regeneration by temporarily replacing liver function. The bio- artificial liver device being designed in our laboratory utilises the hepatoblastoma- derived HepG2 cell line. Despite limited liver specific functions whilst in monolayer culture, cell performance can be significantly improved by alginate encapsulation. However, urea cycle activity, which is the mechanism of ammonia detoxification, remains minimal and thus poses a problem in the removal of nitrogenous waste. The aims of this study were to i) investigate the causes for urea cycle dysfunction in the HepG2 and HepG2-C3A cell lines, and ii) investigate the possibilities of restoring urea cycle function. To accomplish these, urea cycle gene expression was determined by real time RT-PCR. Protein expression and functional enzyme activity were assessed with Western blotting, metabolic labelling and enzyme assays. To restore function, gene transfer techniques were employed using pcDNA3.1 vectors. In addition, de- methylation studies and the adaptation of HepG2 cells to different growth media were carried out. The main findings of this study showed that the lack of urea cycle function in both HepG2 and HepG2-C3A cell lines is attributable to the under-expression of two urea cycle genes, namely ornithine transcarbamylase and arginase I, although three other enzymes carbamoyl phosphate synthase I, argininosuccinate synthetase and argininosuccinate lyase are present, albeit at lower levels than in primary human hepatocytes. HepG2 cells transfected with plasmid vector constructs containing ornithine transcarbamylase and arginase I full length cDNA, led to the restoration of the urea cycle. In conclusion, this strategy could be used to restore urea cycle function in HepG2 cells by creating stable cell lines. Future work could aim at determining whether expression of the two constructs is retained over time, and whether this approach provides suitable ammonia detoxification required of a bioartificial liver device.

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