Histological assessment of the perfused organs.

Panel A shows two haematoxylin—eosin (H&E)-stained paraffin-embedded sections at the end of the perfusion run. The left-hand picture is a large portal tract showing well-preserved bile duct (arrow), artery (circle) and portal vein (arrowhead). A similar picture was seen for smaller intrahepatic portal tracts in the right-hand picture, where the same well-preserved structures can be identified; and, interestingly, the presence of the Hemopure®-based perfusate can be visualized (asterisk) in the vein, artery and even in the sinusoids. Panel B shows the change in glycogen content over time. The first figure on the left shows one liver from the cold-to-warm group at time 0 with severely depleted glycogen stores; at the end of 6 hours of perfusion, this was slightly replenished. The graph compares the dynamic changes in glycogen content between groups.</p

Vascular parameters throughout the perfusion protocols.

Panel A shows hepatic artery vascular parameters. The flow was higher for the HOPE+NMP group, despite slightly higher pressures used for the cold-to-warm group in an attempt to overcome the higher vascular resistance. A similar trend was seen for the portal vein vascular parameters (Panel B). The cold-to-warm group had higher vascular resistance mainly during the hypothermic phase, which improved during the rewarming. The higher vascular resistance required increases in the perfusion pressures, although the vascular flows were still lower during the initial period of the NMP phase. In the graphs, dots represent individual organs at the time points and the line is the median of the values for each group.</p

Donor demographics, liver characteristics and machine perfusion data.

Donor demographics, liver characteristics and machine perfusion data.</p

Mitochondrial and hepatic functional markers during the normothermic phase.

Panel A shows a similar incremental rate of oxygen consumption during the rewarming period, which finally culminated in a higher peak at the beginning of the normothermic phase for the cold-to-warm group. The rate of carbon dioxide (CO2) release in the perfusate followed a similar trend to the oxygen consumption, and the adenosine triphosphate (ATP) levels reached higher figures at the end of the 6 hours of perfusion in the cold-to-warm group than with the HOPE+NMP. Panel B represents parameters of metabolic function of the organs during the perfusion. During the hypothermic phase, lactate levels increased slightly for the livers that had D-HOPE and were constant throughout HOPE. After rewarming, the lactate peak was comparable between groups, and then the lactate clearance was more effective in the cold-to-warm group resulting in similar levels at the end of the perfusion. There was evidence of glycogenolysis at the beginning of the D-HOPE perfusion, and thereafter the organs start to consume glucose during the NMP phase. For the HOPE+NMP group, there was a sudden drop related to perfusate change at 2-hour perfusion and then the livers start to metabolise glucose during the NMP phase. Perfusate pH was similar between groups during the perfusion. Three livers in each group produced bile during the perfusion (Panel C). The bile pH was comparable between them, as was the glucose content. In the graphs, dots represent individual organs at the time points, and the line is the median of the values for each group. For the bar graph, the median and interquartile range are represented.</p

Study design.

Donor human livers had standard procurement, they were cold flushed and stored. Once rejected for transplantation, they were offered for research and consecutively allocated to the two experimental groups. Image A shows the protocol for the HOPE+NMP group, livers had hypothermic oxygenated perfusion (HOPE) using Belzer MPS® UW Machine Perfusion Solution for 2 hours. HOPE was performed at 10 °C via the portal vein only, as represented on image C. After 2 hours, the perfusate was changed to an acellular haemoglobin-based oxygen carrier (HBOC) Hemopure® (HBOC-201, Hemoglobin® Oxygen Therapeutics LLC, Cambridge, USA)-based perfusate for the rewarming and normothermic machine perfusion (NMP). The livers from the cold-to-warm group (Image B) were fully cannulated at the start of the perfusion, including portal vein, hepatic artery and common bile duct (Image D). They received the HBOC-based perfusate from time 0. For this group, the livers had 2 hours of dual hypothermic oxygenated perfusion (D-HOPE) at 10 °C followed by 1 hour of slow rewarming to 20 °C (controlled oxygenated rewarming [COR]) and then NMP. Menghini and wedge biopsies were collected at time 0, 2 and 6 hours (**) and immediately fixed in formalin or snap-frozen in liquid nitrogen. Blood gas analysis was carried out and perfusate was sampled at 30 min time intervals throughout. In addition, bile production was measured at time 4 and 6 hours (#) and analysed at 6 hours.</p

Viability criteria achievement by the livers in each group.

Viability criteria achievement by the livers in each group.</p

Immunohistochemical assessment of oxidative tissue injury and activation of the inflammatory cascade.

Panel A shows the tissue expression of the uncoupling protein 2, a marker for reactive oxygen species (ROS) generation; the left-hand picture shows a moderate diffuse reaction of the staining at time 0, mainly localised at peri-portal areas, which changed to a mild reaction at the end of the 6 hours perfusion. The graph on the right shows the variation in the two groups at these time points. This downregulation in ROS production is associated with a reduction in tissue expression of 4-hydroxynonenal (4-HNE) (Panel B), an established marker for lipid peroxidation during oxidative stress. The lowered oxidative injury decreases the tissue expression of the cluster of differentiation (CD)14 in macrophages (Panel C). CD14 is part of the signalosome of the toll-like receptor 4, which in turn leads to the activation of inflammatory and endothelial cells, perpetuating and aggravating tissue injury. In accordance, a downward trend can be seen during the perfusion in the presence of activated leukocytes, identified by the expression of the CD11b (Panel D) and activated endothelial cells, as represented by the expression of the vascular cell adhesion molecule 1 (VCAM-1) (Panel E). Therefore, both combined protocols exhibited a similar decreasing trend in the expression of markers of ischaemia-reperfusion injury during the perfusion. In the graphs, dots represent individual organs at the time points, and the line is the median of the values for each group.</p

Donor demographics, liver characteristics and machine perfusion parameters.

Donor demographics, liver characteristics and machine perfusion parameters.</p

Stability of HBOC-based perfusate at different temperatures and its impact on mitochondrial function at hypothermic temperatures.

Panel A shows on the left-hand graph that haemoglobin levels were stable during the D-HOPE phase, and after rewarming, they were similar to the HOPE+NMP group that received fresh HBOC-based perfusate. Methaemoglobin levels (middle graph) were stable during D-HOPE and increased slowly during the rewarming, reaching comparable levels to the HOPE+NMP group during the normothermic phase. Despite the increase in the levels of methaemoglobin, arterial oxygen saturation was constant throughout the perfusion. Panel B compares markers of mitochondrial respiration between the study groups. Both groups presented a downward trend in oxygen requirements (oxygen uptake—HOPE+NMP group and oxygen consumption—cold-to-warm group), with a similar drop in the release of carbon dioxide (CO2) in the perfusate. Reflecting an efficient mitochondrial oxidative function, both groups replenished adenosine trisphosphate (ATP) stores during the hypothermic phase. In the graphs, dots represent individual organs at the time points, and the line is the median of the values for each group. For the bar graph, the median and interquartile range are represented.</p