Study design.

<p>Series 1: Isolated primary human hepatocytes (PHH) were left in standard media for 2 days and then received media supplemented with fatty acids. After 2 days of fat loading the fatty PHH were allocated to the defatting treatment group where the media was supplemented with the defatting cocktail of drugs, and the control groups, the standard control group and the vehicle control group that received vehicle only. Lean hepatocytes were kept in standard culture conditions throughout the experimental period. The experimentation period lasted for two days thereafter. Series 2: Human intra-hepatic endothelial cells (HIEC) and cholangiocytes were immuno-magnetically separated with Dynabeads conjugated with cell-specific monoclonal antibody. The cells were left in culture for 2 days in standard media to reach confluence and then were allocated to the intervention group that received the defatting cocktail and the control groups, the standard control group and the vehicle control group that had the media supplemented with the vehicle only. The experimentation period lasts for two days thereafter.</p

Defatting of fat loaded primary human hepatocytes (PHH).

<p>Panel A: The positive are of oil red O of the defatting treatment group was reduced by 28% in comparison with the vehicle control group over 24 hours and 54% over 48 hours. Panel B: Intracellular triglyceride levels of the defatting treatment group were reduced by 32% within 24 hours of treatment and 35% within 48 hours, in comparison with the fatty vehicle control group. Panel C: Oil red O staining picture of PHH of the defatting group at the end of the 48 hours of treatment. There is a predominance of small lipid droplets in the cytoplasm of the cells and the nucleus is in its usual position. Series 2: shows a series of oil red O staining pictures from PHH in culture at different time points of the experiments. Panel D shows lean cells in culture, after the incubation with fatty acids they become loaded with fat (panel E). Those fat loaded PHH were then incubated with only the vehicle of the drugs for 48 hours and the lipid content decreased over time (panel F) or had the defatting treatment that showed the significant higher decrease in the area of oil red O (panel G). Data report the median of three separate experiments performed in quadruplicate and errors bars the interquartile range. Comparisons performed using two-tailed t-test. * = p<0.05.</p

Release of total ketones in the supernatants.

<p>Fat loaded primary human hepatocytes that had the defatting treatment showed an increase in cell culture supernatant levels of total ketone bodies of 1.22-fold over 24 hours and 1.40-fold over 48 hours. Data reports the median of three separate experiments performed in quadruplicate and errors bars the interquartile range. Comparisons performed using two-tailed t-test. * = p<0.05.</p

Assessment of the cytotoxicity of the defatting cocktail to human cells of the liver via MTT assay.

<p>Panel A: the toxicity of the defatting cocktail was tested in primary human hepatocytes and results showed a significant improvement of 11% in viability of the defatting treatment group compared with the fatty vehicle control group. Panel B: Treatment of human intra-hepatic endothelial cells (HIEC) with the drugs had no effect on cell viability compared with the control groups. Panel C: treatment of cholangiocytes with the defatting cocktail did not demonstrate any cytotoxic effect to the cells and indicated a slight improvement in viability compared to the control groups. Panels D and E: Phase contrast microscopy showing representative images of HIEC (Panel D) and cholangiocytes (Panel E) at different time points. No gross modifications in cell integrity were observed in either cell type which was consistent and supportive of the MTT data. Data report the median of three separate experiments performed in quadruplicate and errors bars the interquartile range. Comparisons performed using two-tailed t-test. * = p<0.05.</p

Results of fat loading of primary human hepatocytes.

<p>Panel A: The supplementation of the media with the combination of fatty acids resulted in a cell viability rate of 81% after 48 hours of incubation. Panel B: Oil red O staining image of PHH at the end of the fat loading period. There is predominance of large lipid droplets displacing the nucleus of the cells to the periphery (black arrow). Panel C: At the end of 48 hours of fatting load there was a significant increase of 14-fold of the positive area of oil red O. Panel D: Intracellular triglycerides increased 8-fold within 48 hours of incubation with fatty acids. Data report the median of three separate experiments performed in quadruplicate and errors bars the interquartile range. Comparisons performed using two-tailed t-test. * = p<0.05.</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

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

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