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Since there is a shortage of transplantable livers, the donor pool needs to be increased. One method to accomplish this is to recondition extended criteria donor grafts, a large portion of which are moderate to severe macrosteatotic livers. Transplantation of these livers often leads to primary nonfunction caused by an increased susceptibility to the effects of ischemia reperfusion injury (I/R) that result from the harvesting, transportation, and transplantation of the liver. Our lab has developed a novel procedure to recondition these livers through an ex vivo perfusion that will reduce the hepatic triglyceride content before the onset of the effects of I/R injury. To be feasibly performed in a clinical setting, the defatting must be completed in a matter of hours. While attempts to identify the ideal defatting cocktail in static culture only resulted in defatting livers after days of treatment, the rate of defatting increases to more a clinically relevant timeframe in a flow environment. My project focuses on understanding the differences between a static and flow environment and using this information to develop the ideal parameters for defatting. I hypothesize that using flow and appropriate defatting agents, steatotic hepatocytes can be defatted in a clinically relevant time of hours, without harming viability or function, to a level that reduces or eliminates increased sensitivity to I/R injury. In order to prove the hypothesis I will use the following 3 specific aims: 1. Develop a metabolic flux analysis model to explore defatting metabolic pathways. 2. Develop a computational fluid dynamics and kinetic model of a vitro flow reactor to optimize key transport parameters. 3. Validate the optimal conditions of flow has on the viability and function in hepatocyte cell lines.

Macrosteatotic livers exhibit elevated intrahepatic triglyceride (TG) levels in the form of large lipid droplets (LDs), reduced adenosine triphosphate (ATP) levels, and elevated reactive oxygen species (ROS) levels, and this contributes to their elevated sensitivity to ischemia/reperfusion injury during transplantation. Reducing macrosteatosis in living donors through dieting has been shown to improve transplant outcomes. Accomplishing the same feat for deceased donor grafts would require ex vivo exposure to potent defatting agents. Here we used a rat hepatocyte culture system exhibiting a macrosteatotic LD morphology, elevated TG levels, and an elevated sensitivity to hypoxia/reoxygenation (H/R) to test for such agents and ameliorate H/R sensitivity. Macrosteatotic hepatocyte preconditioning for 48 hours with a defatting cocktail that was previously developed to promote TG catabolism reduced the number of macrosteatotic LDs and intracellular TG levels by 82% and 27%, respectively, but it did not ameliorate sensitivity to H/R. Supplementation of this cocktail with l-carnitine, together with hyperoxic exposure, yielded a similar reduction in the number of macrosteatotic LDs and a 57% reduction in intrahepatic TG storage, likely by increasing the supply of acetyl coenzyme A to mitochondria, as indicated by a 70% increase in ketone body secretion. Furthermore, this treatment reduced ROS levels by 32%, increased ATP levels by 27% (to levels near those of lean controls), and completely abolished H/R sensitivity as indicated by approximately 85% viability after H/R and the reduction of cytosolic lactate dehydrogenase release to levels seen in lean controls. Cultures maintained for 48 hours after H/R were approximately 83% viable and exhibited superior urea secretion and bile canalicular transport in comparison with untreated macrosteatotic cultures. In conclusion, these findings show that the elevated sensitivity of macrosteatotic hepatocytes to H/R can be overcome by defatting agents, and they suggest a possible route for the recovery of discarded macrosteatotic grafts.