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Background & Aims End-ischemic hypothermic oxygenated machine perfusion (HOPE) of the donor liver for 1–2 h mitigates ischemia-reperfusion injury during subsequent liver transplantation. Extended preservation time may be preferred to facilitate difficult recipient hepatectomy or to optimize logistics. We therefore investigated whether end-ischemic dual HOPE (DHOPE) could extend preservation time for up to 24 h using a porcine liver reperfusion model. Methods Following 30 min warm ischemia, porcine livers were subjected to 2 h static cold storage (SCS), followed by 2 h, 6 h, or 24 h DHOPE (n = 6 per group). Subsequent normothermic reperfusion was performed for 4 h using autologous blood. Two livers preserved by 24 h SCS served as additional controls. A proof of principle confirmation was carried out in 2 discarded human livers subjected to extended DHOPE. Hepatocellular and cholangiocyte injury and function were assessed. Oxidative stress levels and histology were compared between groups. Results Perfusion flows remained stable during DHOPE, regardless of duration. After normothermic reperfusion, livers perfused for 24 h by DHOPE had similar lactate clearance, blood pH, glucose, and alanine aminotransferase levels, and biliary pH, bicarbonate, and LDH levels, as livers perfused for 2 h and 6 h. Levels of malondialdehyde and high-mobility group box 1 in serum and liver parenchyma were similar for all groups. Histological analysis of bile ducts and liver parenchyma revealed no differences between the groups. Extended DHOPE in discarded human livers preserved hepatocellular and cholangiocyte function and histology after reperfusion. In contrast, livers preserved by 24 h SCS were non-functioning. Conclusion Extended end-ischemic DHOPE enabled successful preservation of porcine and discarded human donor livers for up to 24 h. Extended DHOPE enables safe extension of preservation time, which may facilitate allocation and transplantation from a logistical perspective, and further expand the donor pool. Lay summary It has been suggested that preserving liver grafts with a technique called (dual) hypothermic oxygenated machine perfusion ([D]HOPE) leads to better outcomes after transplantation than if livers are stored on ice, especially if an organ is of lesser quality. In this study, we showed that DHOPE could be used to preserve liver grafts for up to 24 h. This extended procedure could be used globally to facilitate transplantation and expand the donor pool., Graphical abstract, Highlights • DHOPE can be extended for up to 24 h to prolong donor liver preservation time. • Hepatocellular and biliary function is maintained after ex situ preservation by 24 h DHOPE. • Extension of DHOPE for up to 24 h does not induce more injury compared to shorter preservation times. • The initial results of extended preservation by DHOPE for discarded human livers are promising.

To improve preservation of donor livers, we have developed a portable hypothermic machine perfusion (HMP) system as an alternative for static cold storage. A prototype of the system was built and evaluated on functionality. Evaluation criteria included 24 h of adequate pressure controlled perfusion, sufficient oxygenation, a maintained 0-4 degrees C temperature and sterile conditions. Porcine livers were perfused with pump pressures that were set at 4 mmHg (continuous, portal vein) and 30/20 mmHg, at 60 BPM (pulsatile, hepatic artery). Control livers were preserved using the clinical golden standard: static cold storage. In the HMP group, pressure, flow and temperature were continuously monitored for 24 h. At time-points t = 0, 2, 4, 8, 12, and 24 h samples of University of Wisconsin machine preservation solution were taken for measurement of partial oxygen pressure (pO(2)) and lacto-dehydrogenase. Biopsies in every lobe were taken for histology and electron microscopy; samples of ice, preservation solution, liver surface, and bile were taken and cultured to determine sterility. Results showed that temperature was maintained at 0-4 degrees C; perfusion pressure was maintained at 4 mmHg and 30/20 mmHg for portal vein and hepatic artery, respectively. Flow was approximately 350 and 80 ml/min, respectively, but decreased in the portal vein, probably due to edema formation. Arterial pO(2) was kept at 100 kPa. Histology showed complete perfusion of the liver with no major damage to hepatocytes, bile ducts, and non-parenchymal cells compared to control livers.The machine perfusion system complied to the design criteria and will have to demonstrate the superiority of machine perfusion over cold storage in transplant experiments.

To overcome the present shortage of liver donors by expansion of the existing donor pool and possibly lengthening of the storage time, hypothermic machine perfusion of the liver as a dynamic preservation method is revisited. The three most important aspects are defined to be the type of preservation solution, the characteristics of perfusion dynamics, and the oxygen supply. Reviewing hypothermic liver machine perfusion experiments, the University of Wisconsin machine preservation solution is the solution most used. It is also found that nothing conclusive can be said about the optimal perfusion characteristics, since either perfusion pressure or perfusion flow is reported. The best estimation is perfusion of the liver in a physiological manner, i.e. pulsatile arterial perfusion and continuous portal venous perfusion. The applied pressures could be chosen to be somewhat lower than physiological pressures to prevent possible endothelial cell damage. Oxygen supply is necessary to achieve optimal preservation of the liver. The minimal amount of partial oxygen pressure required is inversely related to the normalized flow. Incorporating these features in a system based on existing standard surgical and organ sharing procedures and which is able to work stand-alone for 24 It, weighing less than 23 kg, could successfully implement this technique into every day clinical practise.