Ischemia-reperfusion injury (IRI) constitutes a significant source of morbidity and mortality after orthotopic liver transplantation (OLT). The allograft is metabolically impaired during warm and cold ischemia and is further damaged by a paradox reperfusion injury after revascularization and reoxygenation. Short-term and long-term complications including post-reperfusion syndrome, delayed graft function, and immune activation have been associated with IRI. Due to the current critical organ shortage, extended criteria grafts are increasingly considered for transplantation, however, with an elevated risk to develop significant features of IRI. In recent years, ex vivo machine perfusion (MP) of the donor liver has witnessed significant advancements. Here, we describe the concept of hypothermic (oxygenated) machine perfusion (HMP/HOPE) approaches and highlight which allografts may benefit from this technology. This review also summarizes clinical applications and the main aspects of ongoing randomized controlled trials on hypothermic perfusion. The mechanistic aspects of IRI and hypothermic MP—which include tissue energy replenishment, optimization of mitochondrial function, and the reduction of oxidative and inflammatory damage following reperfusion—will be comprehensively discussed within the context of current preclinical and clinical evidence. Finally, we highlight novel trends and future perspectives in the field of hypothermic MP in the context of recent findings of basic and translational research.

Increasing amounts of human hepatocytes are needed for clinical applications and different fields of research, such as cell transplantation, bioartificial liver support, and pharmacological testing. This demand calls for adequate storage options for isolated human liver cells. As cryopreservation results in severe cryoinjury, short-term storage is currently performed at 2-8°C in preservation solutions developed for the storage of solid organs. However, besides slowing down cell metabolism, cold also induces cell injury, which is, in many cell types, iron dependent and not counteracted by current storage solutions. In this study, we aimed to characterize storage injury to human hepatocytes and develop a customized solution for cold storage of these cells. Human hepatocytes were isolated from material obtained from partial liver resections, seeded in monolayer cultures, and, after a preculture period, stored in the cold in classical and new solutions followed by rewarming in cell culture medium. Human hepatocytes displayed cold-induced injury, resulting in >80% cell death (LDH release) after 1 week of cold storage in University of Wisconsin solution or cell culture medium and 3 h of rewarming. Cold-induced injury could be significantly reduced by the addition of the iron chelators deferoxamine and LK 614. Experiments with modified solutions based on the new organ preservation solution Custodiol-N showed that ion-rich variants were better than ion-poor variants, chloride-rich solutions better than chloride-poor solutions, potassium as main cation superior to sodium, and pH 7.0 superior to pH 7.4. LDH release after 2 weeks of cold storage in the thus optimized solution was below 20%, greatly improving cold storage of human hepatocytes. The results were confirmed by the assessment of hepatocellular mitochondrial membrane potential and functional parameters (resazurin reduction, glucagon-stimulated glucose liberation) and thus suggest the use of a customized hepatocyte storage solution for the cold storage of these cells.

Cell-based extracorporeal liver support is an option to assist or replace the failing organ until regeneration or until transplantation can be performed. The use of porcine cells or tumor cell lines is controversial. Primary human liver cells, obtained from explanted organs found to be unsuitable for transplantation, are a desirable cell source as they perform human metabolism and regulation. The Modular Extracorporeal Liver Support (MELS) concept combines different extracorporeal therapy units, tailored to suit the individual and intra-individual clinical needs of the patient. A multi-compartment bioreactor (CellModule) is loaded with human liver cells obtained by 5-step collagenase liver perfusion. A cell mass of 400 g – 600 g enables the clinical application of a liver lobe equivalent hybrid organ. A detoxification module enables single pass albumin-dialysis via a standard high-flux dialysis filter, and continuous venovenuous hemodiafiltration may be included if required. Cells from 54 human livers have been isolated (donor age: 56 ± 13 years, liver weight: 1862 ± 556 g resulting in a viability of 55.0 ± 15.9%). These grafts were not suitable for LTx, due to steatosis (54%), cirrhosis (15%), fibrosis (9%), and other reasons (22%). Out of 36 prepared bioreactors, 10 were clinically used to treat 8 patients with liver failure. The overall treatment time was 7–144 hours. No adverse events were observed. Initial clinical applications of the bioreactor evidenced the technical feasibility and safety of the system.

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