Development of a defatting protocol for steatotic livers to increase the number of organs available for transplantation.

Introduction: For individuals with chronic liver disease, liver transplantation is the only viable treatment option. Currently, there is an insufficient level of organ donation, with a high rate of discarded donations1. Liver steatosis (fat accumulation) is the primary reason for discard, accounting for 40-60% of unused livers2,3. This is evident in the United States due to the ongoing obesity epidemic, which contributes to the prevalence of steatotic liver disease. Steatotic livers are discarded due to their high susceptibility to ischemia-reperfusion injury following transplantation. With a 2020 study reporting that 23.8% of individuals who declined a steatotic donor liver died while waiting for a transplant4,5, there is a critical need to defat steatotic livers to increase the number of viable livers available for transplantation. Current defatting strategies have relied on the usage of murine models, with attempts to translate these findings to humans yielding limited success. The inclusion of potentially carcinogenic compounds in these defatting strategies renders them unviable for application in transplanted human livers. Further, there is large variability in individuals’ responses to lipolytic agents, highlighting the need for a more comprehensive approach to defatting6. To address this, we employ a multi-faceted approach that targets multiple pathways involved in lipid metabolism, aimed at achieving consistent and efficacious outcomes across different individuals’ organs. We hypothesize that a cocktail of reagents targeting various lipolytic pathways (Figure 1A) will have synergistic defatting effects, measured as a greater reduction in lipid quantity, than that of the individual factors when applied on the steatotic human organoid.

Materials and

Methods: Here, we generated human hepatic organoids containing endothelial cells, stellate cells, and primary hepatocytes. We exposed these hepatic organoids to environmental factors implicated in the pathogenesis of steatosis, including, free fatty acids. Upon acquisition of an appropriate steatosis model, reagents targeting different pathways (Figure 1A), including lipophagy, lipolysis, and beta-oxidation were tested in varying concentrations for 48 hours on hepatic organoids developed from three separate patient’s hepatocytes. The viability of the treated organoids was assessed through a live/dead assay, subsequent confocal microscopy imaging, and manual determination of the number of live and dead cells. The defatting potential of the reagents was assessed by visualizing the lipid quantity in the organoids through staining with a lipophilic stain, BODIPY, and a nuclear stain, Hoescht 33342. Following visualization, the most promising defatting candidates were determined as those that decreased lipid accumulation to within physiological levels while maintaining high viability (>80%). Cocktails were then formulated through the inclusion of the most potent reagents that maintained viability within each pathway. Single reagent exclusion was performed to identify the most effective cocktail with the fewest number of reagents. Viability and lipid visualization were conducted as previously discussed. Steatotic organoids were then treated with the most effective cocktail and characterized using cellular identity markers, ROS staining, and mechanistic assays.

Results, Conclusions, and Discussions: Results and Discussion
Hepatic organoids loaded with free fatty acids acquired a steatotic phenotype similar to that seen in discarded livers (favoring macrosteatosis over microsteatosis). These organoids maintained their viability, but produced reactive oxygen species (ROS), which are known stimulators of steatosis while also being major contributors toward post-transplant graft failure of steatotic livers. Individual defatting reagents were applied to these steatotic organoids at varying dosages. Each reagent had a dose-dependent and patient-specific effect (Figure 1B), prompting different combinations of these reagents (at their lowest effective dosage) into six different cocktails to delineate any additive or synergistic defatting effects (Figure 1C, D). It was apparent that after 48 hours of treatment with the various cocktails, organoid viability was maintained (Figure 1C). All defatting cocktails resulted in a reduction in intrahepatic lipid quantity when compared to the control organoids (Figure 1D). Additionally, the reagents’ defatting effects were greater when applied as cocktails compared to the individual applications. Cocktail number 1, consisting of Carbamazepine, Isoproterenol, Elafibranor, Bezafibrate, and L-Cartinine, proved to be most effective at defatting across all three patients, partially attributed to enhanced beta-oxidation. However, the excessive production of ROS was not alleviated, prompting further investigation into incorporating antioxidant and mitochondrial biogenic reagents into cocktail number 1 to support the defatting process.

Conclusions
Here, we utilize a high throughput model to screen reagents for their potential defatting capabilities. Reagents were tested at various concentrations to assess their defatting efficacy and toxicity toward human steatotic liver organoids over the course of 48 hours. The most effective reagents were combined in order to optimize the defatting effectiveness in all patients, demonstrating that including stimulants for multiple pathways can improve outcomes across highly variable patients. Future work to improve upon this defatting cocktail will focus on not only reducing steatosis to healthy levels but also alleviating pro-inflammatory side effects, such as ROS, that are known to exacerbate steatotic liver related transplant injury.

Acknowledgements (Optional): The authors would like to thank the Mayo Clinic and Carnegie Mellon Transforming Transplant initiative for funding this work. Additionally, we would like to thank the University of Pittsburgh Liver Research Center for the primary cells utilized in this study.