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Normothermic ex vivo liver machine perfusion (NEVLP) has been developed to address the increasing organ shortage in liver transplantation, through optimal preservation, assessment, and conditioning of grafts from extended criteria donors. There remains a need to establish simple and standardized animal models that simulate clinical NEVLP to test novel therapies. Liver grafts from 36 Sprague-Dawley rats were perfused for 6 h in a dual-vessel NEVLP system with a Dulbecco's modified Eagles medium-based perfusate supplemented with rat plasma and erythrocytes. Varying doses of the clinically used vasodilator epoprostenol, Kupffer cell inhibitor glycine, and a Steen™-based perfusate were assessed. Perfusion pressures and bile production were recorded, and perfusate was analyzed for transaminase secretion. Tissue samples were evaluated histologically, and levels of cytokines and 8-Isoprostane were measured. Increasing levels of epoprostenol and the addition of glycine resulted in a stepwise decrease of transaminase secretion and improved bile production. Steen further decreased transaminase release and interleukin 1 beta levels. Liver grafts perfused with the optimized Steen-based protocol exhibited lowest levels of oxidative stress and best-preserved liver integrity. In conclusion, epoprostenol seemed to ameliorate liver function and prevent cellular damage beyond its vasodilatory effect, with glycine acting synergistically. The anti-inflammatory and antioxidative properties of Steen further improved the outcome of perfusion. Our rodent NEVLP system may be used to rapidly test new agents for the pharmacologic conditioning of livers and help translate findings from bench-to-bedside.

Primary human hepatocytes isolated from surgically resected liver tissue are an essential resource for pharmaceutical and toxicological studies. Patients undergoing partial liver resections have often received preoperative chemotherapy. The aim of our study was to investigate whether preoperative chemotherapy has effects on the outcome of cell isolation or the metabolic function of cultured hepatocytes. Liver specimens from 48 patients were used for hepatocyte isolation. Out of these, 21 patients had prior chemotherapy, with fluoropyrimidine-based regimen in 14 patients. Viability and cell yield as parameter for the outcome of isolation, as well as transaminase levels, urea or albumin secretion to the culture medium were not different between hepatocytes from pretreated and untreated donor. Furthermore, the transcription levels of cytochrome P450 (CYP) 1A2, CYP 2B6, and CYP 3A4 of cultured hepatocytes were not affected by prior chemotherapy of the tissue donors. In conclusion, hepatocytes from tissue donors that underwent fluoropyrimidine-based chemotherapy regimens before isolation seem to perform as well as hepatocytes without preoperative chemotherapy exposure. Our results suggest that hepatocytes from patients who received combination chemotherapy before liver resection are an uncompromised resource for pharmacological and toxicological studies. Impact statement Isolated primary human hepatocytes are an essential resource for pharmacological and toxicological studies. Our results present further evidence that isolated hepatocytes from patients who received combination chemotherapy before liver resection are an uncompromised resource for pharmacological and toxicological studies-especially when fluoropyrimidine-based regimens are used.

The most common preservation technique for liver grafts is static cold storage. Due to the organ shortage for liver transplantation (LT), extended criteria donor (ECD) allografts are increasingly used-despite the higher risk of inferior outcome after transplantation. Ex vivo liver machine perfusion (MP) has been developed to improve the outcome of transplantation, especially with ECD grafts, and is currently under evaluation in clinical trials. We performed a literature search on PubMed and ISI Web of Science to assemble an overview of rodent and porcine animal models of ex vivo liver MP for transplantation, which is essential for the present and future development of clinical liver MP. Hypothermic, subnormothermic, and normothermic MP systems have been successfully used for rat and pig LT. In comparison with hypothermic systems, normothermic perfusion often incorporates a dialysis unit. Moreover, it enables metabolic assessment of liver grafts. Allografts experiencing warm ischemic time have a superior survival rate after MP compared with cold storage alone, irrespective of the temperature used for perfusion. Furthermore, ex vivo MP improves the outcome of regular and ECD liver grafts in animal models. Small and large animal models of ex vivo liver MP are available to foster the further development of this new technology. Impact Statement Ex vivo machine perfusion is an important part of current research in the field of liver transplantation. While evidence for improve storage is constantly rising, the development of future applications such as quality assessment and therapeutic interventions necessitates robust animal models. This review is intended to provide an overview of this technology in common large and small animal models and to give an outlook on future applications. Moreover, we describe developmental steps that can be followed by others, and which can help to decrease the number of animals used for experiments based on the replace, reduce, refine concept.

Ex vivo liver machine perfusion (MP) is a promising alternative for preservation of liver grafts from extended criteria donors. Small animal models can be used to evaluate different perfusion conditions. We here describe the development of a miniaturized ex vivo MP system for rat liver grafts, evaluating cell-free and erythrocyte-based perfusion solutions, subnormothermic and normothermic temperatures, and dialysis. A perfusion chamber was designed after a suitable liver position was identified. Normothermic ex vivo liver perfusion (NEVLP) required supplementation of erythrocytes to reduce cell damage. Perfusion with erythrocytes led to rising potassium levels after 12 h (NEVLP, 16.2 mM, interquartile range [IQR]: 5.7 and subnormothermic ex vivo liver perfusion [SNEVLP], 12.8 mM, IQR: 3.5), which were normalized by dialysis using a laboratory dialysis membrane (NEVLP, 6.2 mM, IQR: 0.5 and SNEVLP, 5.3 mM, IQR: 0.1; p  = 0.004). Livers treated with NEVLP conditions showed higher bile production (18.52 mg/h/g, IQR: 8.2) compared to livers perfused under SNEVLP conditions (0.4 mg/h/g, IQR: 1.2, p  = 0.01). Reducing the perfusion volume from 100 to 50 mL allowed for higher erythrocyte concentrations, leading to significantly lower transaminases (15.75 U/L/mL, IQR: 2.29 vs. 5.97 U/L/mL, IQR: 18.07, p  = 0.002). In conclusion, a well-designed perfusion system, appropriate oxygen carriers, dialysis, and miniaturization of the perfusion volume are critical features for successful miniaturized ex vivo liver MP. Impact Statement Ex vivo liver machine perfusion (MP) is an emerging preservation alternative to static cold storage. Even though clinical studies have shown benefits for extended criteria donor grafts, standardized systems for perfusion of rat liver grafts are not available, which are inevitable for large-scaled studies on liver reconditioning by ex vivo MP. We here comprehensively describe the development of an ex vivo rat liver perfusion system that can be used as modular setting in various approaches of liver MP. We describe pitfalls and techniques that might be of interest when establishing such perfusion systems for basic and translational research.