The role of mitochondria in human liver cell line differentiation for bioartificial livers

Bioartificial livers (BALs) have been developed to provide liver support to patients with end-stage liver failure and bridge them either to liver transplantation or native liver recovery. BALs are extracorporeal devices through which plasma is circulated over liver cells embedded in a bioreactor. Hepatic functions required for BAL-support include ammonia elimination, preferably through urea cycle activity, biotransformation, lactate elimination and blood protein synthesis. In this thesis, we analyzed the performance of human liver cell lines utilized as biocomponents of BALs and increased their applicability by optimizing culturing conditions and/or overexpressing critically low expressed genes.
The functionality of human liver cell lines HepaRG and C3A was compared in conventional monolayer culture and in BAL-culture. The functionality of both cell lines was highly improved under BAL-culturing with HepaRG cells being most promising. The positive effects of BAL-culturing were highly associated with increased mitochondrial biogenesis, which was stimulated by 3D-configuration, medium flow and high oxygenation. With this knowledge, we improved the monolayer culture procedures of both cell lines by culturing in 40%O₂ (hyperoxia) instead of conventional normoxia (20%O₂) or by shaking instead of static culturing. Both strategies stimulated hepatic functions and mitochondrial energy metabolism. Hypoxia (5%O₂), on the other hand, increased the stability of the HepaRG cell line.
By overexpressing the constitutive androstane receptor (CAR), a liver-enriched transcription factor, the biotransformation, stability, and mitochondrial energy metabolism were improved in the HepaRG cell line. In parallel, urea cycle activity was improved by shaking and overexpressing the critically low expressed Carbamoyl Phosphate Synthetase gene