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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.

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.