Jantje Wiersema-Buist

Ex vivo Normothermic Machine Perfusion and Viability Testing of Discarded Human Donor Livers

In contrast to traditional static cold preservation of donor livers, normothermic machine perfusion may reduce preservation injury, improve graft viability and potentially allows ex vivo assessment of graft viability before transplantation. We have studied the feasibility of normothermic machine perfusion in four discarded human donor livers. Normothermic machine perfusion consisted of pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous portal perfusion for 6 h. Two hollow fiber membrane oxygenators provided oxygenation of the perfusion fluid. Biochemical markers in the perfusion fluid reflected minimal hepatic injury and improving function. Lactate levels decreased to normal values, reflecting active metabolism by the liver (mean lactate 10.0 ± 2.3 mmol/L at 30 min to 2.3 ± 1.2 mmol/L at 6 h). Bile production was observed throughout the 6 h perfusion period (mean rate 8.16 ± 0.65 g/h after the first hour). Histological examination before and after 6 h of perfusion showed well‐preserved liver morphology without signs of additional hepatocellular ischemia, biliary injury or sinusoidal damage. In conclusion, this study shows that normothermic machine perfusion of human donor livers is technically feasible. It allows assessment of graft viability before transplantation, which opens new avenues for organ selection, therapeutic interventions and preconditioning.

Oxygenated Hypothermic Machine Perfusion After Static Cold Storage Improves Hepatobiliary Function of Extended Criteria Donor Livers

Background The mechanism through which oxygenated hypothermic machine perfusion (HMP) improves viability of human extended criteria donor (ECD) livers is not well known. Aim of this study was to examine the benefits of oxygenated HMP after static cold storage (SCS). Methods Eighteen ECD livers that were declined for transplantation underwent ex situ viability testing using normothermic (37°C) machine perfusion (NMP) after traditional SCS (0°C-4°C) for 7 to 9 hours. In the intervention group (n = 6), livers underwent 2 hours of oxygenated HMP (at 12°C) after SCS and before NMP. Twelve control livers underwent NMP without oxygenated HMP after SCS. Results During HMP, hepatic ATP content increased greater than 15-fold, and levels remained significantly higher during the first 4 hours of NMP in the HMP group, compared with controls. Cumulative bile production and biliary secretion of bilirubin and bicarbonate were significantly higher after HMP, compared with controls. In addition, the levels of lactate and glucose were less elevated after HMP compared with SCS preservation alone. In contrast, there were no differences in levels of hepatobiliary injury markers AST, ALT, LDH, and gamma-GT after 6 hours of NMP. Hepatic histology at baseline and after 6 hours of NMP revealed no differences in the amount of ischemic necrosis between both groups. Conclusions Two hours of oxygenated HMP after traditional SCS restores hepatic ATP levels and improves hepatobiliary function but does not reduce (preexisting) hepatobiliary injury in ECD livers.

Hypothermic Machine Perfusion Of The Liver And The Crucial Balance Between Perfusion Pressures And Endothelial Injury

Hypothermic machine perfusion (HMP) provides better protection against cold ischemic injury than cold storage in marginal donor kidneys. Also, in liver transplantation a switch from static cold storage to HMP could be beneficial as it would allow longer preservation times and the use of marginal donors. A critical question concerning application of HMP in liver preservation is the crucial balance between perfusion pressure and occurrence of endothelial injury. Rat livers were cold-perfused for 24 hours to study perfusion pressures for both hepatic artery and portal vein. Cold storage served as control and was compared to HMP-preserved livers using a mean arterial perfusion pressure of 25 mm Hg and a portal perfusion pressure of 4 mm Hg (25% of normothermic liver circulation) and to HMP at 50 mm Hg and 8 mm Hg perfusion, respectively (50% of normothermic liver circulation). UW solution was enriched with 14.9 micromol/L propidium iodide (PI) to stain for dead cells and with an additional 13.5 micromol/L acridine orange to stain for viable hepatocytes. A low PI-positive cell count was found using HMP at 25% of normal circulation compared to cold storage. The PI count was high for the HMP group perfused at just 50% of normal circulation compared to HMP at 25% and compared to cold storage. In summary, for liver HMP, perfusion at 25% showed complete perfusion with minimal cellular injury. HMP using perfusion pressures of 25 mm Hg for the hepatic artery and 4 mm Hg for the portal vein is feasible without induction of endothelial injury.

Oxygenation during hypothermic rat liver preservation

Hypothermic machine perfusion (HMP) of abdominal organs is shown to be superior compared to cold storage. However, the question remains if oxygenation is required during preservation as oxygen is essential for energy resynthesis but also generates toxic reactive oxygen species (ROS). To determine if oxygenation should be used during HMP, urea‐synthesis rate, adenosine triphosphate (ATP), and generation of ROS were studied in an in vitro model, modeling ischemia‐reperfusion injury. Furthermore, expression of uncoupling protein‐2 (UCP‐2) mRNA was assessed since UCP‐2 is a potentially protective protein against ROS. Rat liver slices were preserved for 0, 24, and 48 hr in University of Wisconsin machine perfusion solution (UW‐MP) with 0%, 21%, or 95% oxygen at 0‐4°C and reperfused for 24 hours. In the 0% and 95% groups, an increase of ROS was found after cold storage in UW‐MP. After slice reperfusion, only the 0% oxygen group showed higher levels. The 0% group showed a lower urea‐synthesis rate as well as lower ATP levels. mRNA upregulation of UCP‐2 was, in contrast to kidney mRNA studies, not observed. In conclusion, oxygenation of UW‐MP gave better results. This study also shows that ROS formation occurs during hypothermic preservation and the liver is not protected by UCP‐2. We conclude that saturation of UW‐MP with 21% oxygen allows optimal preservation results. (Liver Transpl 2005;11:1403–1411.)