Excorporeal normothermic machine perfusion resuscitates pig DCD livers with extended warm ischemia

Abstract

Background: The shortage in donor livers has led to increased use of allografts derived from donation after cardiac death (DCD). The compromised viability in these livers leads to inferior post-transplantation allograft function and survival compared with donation after brain death (DBD) donor grafts. In this study, we reconditioned DCD livers using an optimized normothermic machine perfusion system.

Methods: Livers from 12 Yorkshire pigs (20-30 kg) were subjected to either 0 min (WI-0 group, n = 6) or 60 min (WI-60 group, n = 6) of warm ischemia and 2 h of cold storage in UW solution, followed by 4 h of oxygenated sanguineous normothermic machine perfusion. Liver viability and metabolic function were analyzed hourly.

Results: Warm ischemic livers showed elevated transaminase levels and reduced ATP concentration. After the start of machine perfusion, transaminase levels stabilized and there was recovery of tissue ATP, coinciding with an increase in bile production. These parameters reached comparable levels to the control group after 1 h of machine perfusion. Histology and gross morphology confirmed recovery of the ischemic allografts.

Conclusion: Our data demonstrate that metabolic and functional parameters of livers with extended warm ischemic time (60 min) can be significantly improved using normothermic machine perfusion. We hereby compound the existing body of evidence that machine perfusion is a viable solution for reconditioning marginal organs.

FIG. 1

Ex Vivo normothermic machine perfusion system. The perfusion setup was a dual re-circulating circuit that consists of a centrifugal pump, heat exchanger, membrane oxygenator, bubble trap, venous reservoir, and organ chamber. Inflow perfusate was divided into two lines: the arterial and the portal branch. Outflow perfusate collected from the organ chamber was returned to the centrifugal pump. The entire system was kept in a laminar hood.

FIG. 2

Liver enzymes release during normothermic machine perfusion. At the start of perfusion, perfusate ALT levels detected in the WI-60 group were markedly higher compared with the base level (t = −5 min) and the level in WI-0 group [**P<0.05 versus t=−5 min (base level); *P < 0.05 versus t = 0 (WI-0 group)]. The ALT levels in the WI-60 group were elevated slightly after 1 h of perfusion and then remained stable during the rest of 3 h. There was no significant change in ALP levels observed during the perfusion.

FIG. 3

Bile production during normothermic machine perfusion. Livers from the WI-0 control group displayed a constant rate of bile production during 4-h perfusion, whereas the WI-60 group initially showed low bile production within first hour of perfusion and then picked up to the similar rate as WI-0 group during the next 3 h (*versus wit = 0 group at 1 h perfusion time).

FIG. 4

Oxygen consumption during normothermic machine perfusion. Livers from the WI-60 group showed a slightly higher oxygen consumption rate comparing to WI-0 group, but did not reach the statistical significance (P > 0.05 at 1–4 h perfusion time).

FIG. 5

Changes of tissue ATP levels during warm ischemia, cold ischemia, and normothermic machine perfusion period. In the WI-60 group, the total ATP level sharply decreased to one-third of the original level, and nearly undetectable at the end of 2 h of cold storage; 4 h NMP restored the ATP levels about 80% of initial level.

FIG. 6

Histologic changes of liver at different stages. (A) Normal liver before warm ischemia and cold storage; (B) liver after 60 min warm ischemia and 2 h cold storage; (C) liver after 4 h normothermic machine perfusion; (D) Liver gross appearance during 4 h normothermic machine perfusion. Warm ischemia resulted in prominent hepatocyte steatotic degeneration evidenced by numerous lipid vacuoles accumulation in the hepatocyte’s cytoplasma in HE staining of liver tissue. Some hepatocyte necrosis and apoptosis could also be observed (A) and (B); 4 h NMP almost completely reversed these pathologic changes (C). Some dark patchy areas were visible on the surface of the liver parenchyma at beginning of perfusion. During perfusion, these areas gradually started to disappear. At the end of the 4 h perfusion period, the aspect of the liver was homogeneous and resembled the physiologic appearance (D).