Introduction: Prolonged ex situ normothermic machine perfusion (NMP) can serve as a platform to recover marginal livers. Previous long-term protocols do not incorporate perfusate substitution and require open systems, custom made or modified machines. We herein test a perfusion protocol introducing a bile volume based perfusate replacement recipe using a commercially available, fully automated NMP device without further modifications in declined human livers. 
Method: Human livers (N=22) declined for transplantation were exposed to NMP for up to 7 days or until perfusate lactate reached 22 mmol/L. For all perfusions, the fully automated NMP device OrganOx Metra® was utilized without modifications. In the control group (N=6) livers were perfused without modification of the perfusate. In the experimental group (N=16) a novel perfusion protocol for long-term porcine NMP was applied, which includes packed red blood cell (RBC)-based perfusate addition (300 mL/24h). This equals bile volume loss. Perfusate samples were taken every 12 hours for blood gas analysis and biochemistry. Wedge biopsies were collected at the start and end of NMP and analyzed for bioenergetic function by high-resolution respirometry (HRR). For assessment of cell viability and tissue integrity, real-time confocal microscopy (RTCM) and H&E histology were applied.
Results: Control livers (without RBC addition) were perfused with a median NMP duration of 66 [48 – 84] hours until organ failure, while hemoglobin and hematocrit decreased significantly after 36 hours (p<0.0001 and p<0.0001, respectively). In contrast, median perfusion time in the experimental group (with RBC substitution) was 132 [96 – 165] hours, with ten livers being successfully perfused for 5 days and another four livers for 7 days. Bile production was present throughout NMP (16.67 [13.22 – 18.99] mL/h; median over time), and hemoglobin and hematocrit levels remained stable during perfusion. RTCM analysis and H&E histopathology confirmed excellent tissue viability and integrity after 7 days. Common bile ducts showed no signs of injury by then. The bioenergetic function in liver biopsies was stable indicated by adequate ATP production efficiency and outer mitochondrial membrane integrity. 
Conclusion: 7 day preservation of previously discarded human livers using a novel perfusion protocol in a fully automated commercial perfusion system is feasible. Livers showed preserved viability and organ function as indicated by lactate clearance, bile production, bioenergetic competence and tissue integrity.