Background: Whereas short‐term normothermic machine perfusion (NMP) up-to 24 hours allows for optimized preservation and monitoring of liver grafts, long-term NMP offers a previously non-existent link between basic and clinical science with the potential for modification, regeneration and repair.        
Methods: Aiming to establish a protocol for long-term NMP in a clinically approved and translatable setting, livers retrieved from domestic pigs (60-121 kg body weight) were subjected to NMP by use of a commercially available system (OrganOx Metra device) and consequences of protocol alterations on liver viability (Real-time confocal microscopy, hyperspectral imaging (HSI), high-resolution respirometry (HRR)) and function (perfusate analyte composition, quantitative and qualitive bile production) were monitored. 
Results: NMP was commenced in a total of 29 livers. Whereas priming of the NMP system with donor derived whole blood alone resulted in a stable organ function only to a maximum of 48 hours, daily substitution of the volume loss related to bile excretion could prolong NMP to 156 hours (total: 13 livers). After additional refinements of the protocol including anti-infective ameliration and stabilization of bile excretion, this approach resulted in optimal liver function and morphological integrity of eight livers after one week, even without the necessity of additional dialysis. Graft perfusion was evaluated by HSI, indicating stable oxygen saturation levels (StO2), organ hemoglobin (THI), near infrared perfusion (NIR) and tissue water (TWI). Real-time confocal microscopy (SYTO16/PI, WGA staining) revealed a stable cell viability score (day 0: 0±0 vs. day1: 0.4±0.5; day 5: 0.3±0.5; day 7: 0.3±0.5; all p>0.05 respectively). Graft functionality remained high, as observed by continuous bile production (day 1: 231.9±82.7ml vs. day 7: 320.7±179.2ml), alcaline bile pH (day 7: 7.8±0.14), decline of serum transaminases (ALT on day 1: 250.5±131.6 vs. 7: 84.4±42.9; p<0.01) and lactate levels (day 0: 40.6±21.1 vs. day 7: 13.0±18.9; p<0.05). Assessment of mitochondrial function by HRR indicated stable ATP production efficiency (P-L control efficiency: day 1: 0.80±0.08 vs. day 7: 0.79±0.09; p>0.05) and no additional damage to the outer mitochondrial membrane (cytochrome c control factor: day 0: 0.28±0.18 vs. day 7: 0.23±0.09; p>0.05).      
Conclusion: The establishment of translatable long-term preservation protocols could pave the way for NMP-based organ repair strategies.