In vitro screening system for macrosteatosis reversal in liver cells

Orthotopic liver transplantation is the only therapeutic option for end-stage liver disease, but it is limited by the scarcity of suitable grafts. Macrosteatotic livers exhibit elevated triglyceride (TG) levels in the form of large lipid droplets (LDs), elevated reactive oxygen species (ROS) and reduced adenosine triphosphate (ATP) levels in the liver's hepatocytes. These abnormalities increase sensitivity of hepatocytes to ischemia and reperfusion (I/R) stress during transplantation and to increased hepatocyte death and graft failure following transplantation. Macrosteatosis reversal several weeks prior to live-donor liver transplantation reduces graft I/R sensitivity and enables successful transplantation. However, to apply this concept to livers from deceased donors, the defatting process must be accelerated using ex-vivo perfusion with defatting agents. To explore macrosteatosis defatting phenomena and to identify potent defatting agents prior to whole liver studies, an in-vitro system simulating macrosteatosis was developed.;Primary rat hepatocyte cultures incubated for several days with elevated free fatty acid (FFA) levels exhibited characteristics of clinical macrosteatosis based on LD morphology, elevated TG levels and elevated sensitivity to hypoxia and reoxygenation (H/R) induced stress, simulating I/R. This novel system was used to explore the ability of selected defatting cocktails to reverse macrosteatosis and reduce H/R sensitivity as measured by hepatocyte viability and function.;We found that pretreatment of macrosteatotic cultures with an L-carnitine supplemented defatting cocktail under hyperoxic conditions for 48h prior to H/R induction led to a ∼82% reduction in the number of macrosteatotic LDs and to a ∼57% reduction in intrahepatic TG by promoting beta-oxidation of FFAs. Furthermore, this treatment reduced ROS stress by ∼32%, elevated the ATP levels to that of lean controls and fully abolished H/R associated hepatic death. Treated cultures maintained ∼83% viability and exhibited superior functionality compared to untreated macrosteatotic cultures as assessed by urea secretion and bile canalicular transport 48h post H/R stress.;The developed system is suitable for exploring additional defatting cocktails that may reverse macrosteatosis and elevated H/R sensitivity. The developed in-vitro defatting routine can now be scaled-up for whole organ perfusion systems to recover macrosteatotic livers in order to mitigate the persistent shortage of suitable livers for transplantation.