Introduction: For end-stage liver diseases, liver transplantation remains the most suitable therapy for its treatment. Nonetheless, the current gap between the patients on the waiting list and the graft supply, as well as the gold standard preservation method (Static Cold Storage or SCS), limits its usefulness. Extended Criteria Donors (ECD) has been proposed as a potential solution to increase the donor pool, although these have a greater susceptibility to ischemia-reperfusion injury (IRI), especially after SCS, causing delayed graft function and primary non-function. These side effects have fueled research into more suitable methods such as ex-situ normothermic machine perfusion (NMP).

Methods: Sixteen healthy pig livers were subjected to normothermic perfusion using blood as a perfusion solution. in 5 experiments, taurocholic acid was administrated 6 hours after perfusion initiation. Perfusate and bile were sampled before, during and after perfusion to assess the hepatocellular injury and liver function and bile duct injury and bile production. Liver samples were collected before and after perfusion to perform analysis of general damage, stellate cell activation, oxidative stress and LSEC response.

Results: AST and LDH levels increased progressively for the first 12 hours (P= 0.02 and P=0.001 respectively). Afterward, both parameters stabilized. Glucose and lactate levels decreased rapidly during the first 4 hours (p=0.003 for glucose and p=0.04 for lactate) and reached the lowest within 8 (p<0.001) and 12 hours (p<0.001) respectively. Overall, blood pH remained steady (p=0.10). Serum levels of ALKP, GGT and Bil didn’t suffer any changes during all the perfusion (p=0.99, p=0.75 and p=0.13 respectively). Bile production started within 1 hour after ex-situ perfusion. Livers supplied with taurocholic acid had a higher bile production rate, 9±3 mL/h, compared to those not supplied, 4±3 (p=0.005). No structural alterations were detected in liver samples after the Suzuki injury score assessment (p=0.33) and stellate cell activation quantification by both gene (p=0,64) and protein expression (p=0,79) and percentage of the stained area (p=0.15). Although eNOS gene and protein expression increased (p=0.01 and p=0.008 respectively), histological analysis of CD31 endothelial staining showed no differences between the baseline, 35,7±9,1 %, and the 24-hour perfusion 36,3±7,2%, of the sinusoidal area (p=0.87). This increase was associated with higher levels of MDA after the perfusion (p=0.006) as the KLF2 gene and protein expression remained invariable (p=0.80 and p=0.26 respectively).

Conclusion: Our results show that NMP, as a preservation method, is capable of maintaining the normal hepatic structure and, at the same time, supporting normal metabolic activity. However, further studies should be conducted to demonstrate post-transplant liver viability.