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Normothermic ex situ liver perfusion (NEsLP) offers the opportunity to assess biomarkers of graft function and injury. We investigated NEsLP parameters (biomarkers and markers) for the assessment of liver viability in a porcine transplantation model. Grafts from heart-beating donors (HBD), and from donors with 30 minutes (donation after cardiac death [DCD]30'), 70 minutes (DCD70'), and 120 minutes (DCD120') of warm ischemia were studied. The HBD, DCD30', and DCD70'-groups had 100% survival. In contrast, 70% developed primary nonfunction (PNF) and died in the DCD120'-group. Hepatocellular function during NEsLP showed low lactate (≤1.1 mmol/L) in all the groups except the DCD120'-group (>2 mmol/L) at 4 hours of perfusion (P = .04). The fold-urea increase was significantly lower in the DCD120'-group (≤0.4) compared to the other groups (≥0.65) (P = .01). As for cholangiocyte function, bile/perfusate glucose ratio was significantly lower (<0.6) in all the groups except the DCD120'-group (≥0.9) after 3 hours of perfusion (<0.01). Bile/perfusate Na+ ratio was significantly higher (≥1.2) after 3 hours of perfusion in all the groups except for the DCD120'-group (≤1) (P < .01). Three hours after transplantation, the DCD120'-group had a significantly higher international normalized ratio (>5) compared to the rest of the groups (≤1.9) (P = .02). Rocuronium levels were higher at all the time-points in the animals that developed PNF during NEsLP and after transplantation. This study demonstrates that biomarkers and markers of hepatocellular and cholangiocyte function during NEsLP correlate with the degree of ischemic injury and posttransplant function., (© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: PPAR-gamma (γ) is highly expressed in macrophages and its activation affects their polarization. The effect of PPAR-γ activation on Kupffer cells (KCs) and liver ischemia-reperfusion injury (IRI) has not yet been evaluated. We investigated the effect of PPAR-γ activation on KC-polarization and IRI., Materials and Methods: Seventy percent (70%) liver ischemia was induced for 60mins. PPAR-γ-agonist or vehicle was administrated before reperfusion. PPAR-γ-antagonist was used to block PPAR-γ activation. Liver injury, necrosis, and apoptosis were assessed post-reperfusion. Flow-cytometry determined KC-phenotypes (pro-inflammatory Nitric Oxide +, anti-inflammatory CD206+ and anti-inflammatory IL-10+)., Results: Liver injury assessed by serum AST was significantly decreased in PPAR-γ-agonist versus control group at all time points post reperfusion (1hr: 3092±105 vs 4469±551; p = 0.042; 6hr: 7041±1160 vs 12193±1143; p = 0.015; 12hr: 5746±328 vs 8608±1259; p = 0.049). Furthermore, liver apoptosis measured by TUNEL-staining was significantly reduced in PPAR-γ-agonist versus control group post reperfusion (1hr:2.46±0.49 vs 6.90±0.85%;p = 0.001; 6hr:26.40±2.93 vs 50.13±8.29%; p = 0.048). H&E staining demonstrated less necrosis in PPAR-γ-agonist versus control group (24hr:26.66±4.78 vs 45.62±4.57%; p = 0.032). The percentage of pro-inflammatory NO+ KCs was significantly lower at all post reperfusion time points in the PPAR-γ-agonist versus control group (1hr:28.49±4.99 vs 53.54±9.15%; p = 0.040; 6hr:5.51±0.54 vs 31.12±9.58%; p = 0.009; 24hr:4.15±1.50 vs 17.10±4.77%; p = 0.043). In contrast, percentage of anti-inflammatory CD206+ KCs was significantly higher in PPAR-γ-agonist versus control group prior to IRI (8.62±0.96 vs 4.88 ±0.50%; p = 0.04). Administration of PPAR-γ-antagonist reversed the beneficial effects on AST, apoptosis, and pro-inflammatory NO+ KCs., Conclusion: PPAR-γ activation reduces IRI and decreases the pro-inflammatory NO+ Kupffer cells. PPAR-γ activation can become an important tool to improve outcomes in liver surgery through decreasing the pro-inflammatory phenotype of KCs and IRI.

Background: The optimal vasodilator to avoid hepatic artery vasospasm during normothermic ex vivo liver perfusion (NEVLP) is yet to be determined. We compared safety and efficacy of BQ123 (endothelin1 antagonist), epoprostenol (prostacyclin analogue), and verapamil (calcium channel antagonist)., Methods: Livers from porcine heart beating donors were perfused for 3 hours and transplanted into recipient pigs. Four groups were compared: group 1, livers perfused with a dose of 1.25 mg of BQ123 at baseline and at 2 hours of perfusion; group 2, epoprostenol at a continuous infusion of 4 mg/h; group 3, verapamil 2.5 mg at baseline and at 2 hours of perfusion; group 4, no vasodilator used during ex vivo perfusion. Liver injury and function were assessed during perfusion, and daily posttransplantation until postoperative day (POD) 3. All groups were compared with a cold storage group for postoperative graft function., Results: Hepatic artery flow during NEVLP was significantly higher in BQ123 compared with verapamil, epoprostenol, and no vasodilator-treated livers. Aspartate aminotransferase levels were significantly lower with BQ123 and verapamil compared with epoprostenol and control group during perfusion. Peak aspartate aminotransferase levels were lower in pigs receiving BQ123 and verapamil perfused grafts compared with epoprostenol and control group. International Normalized Ratio, alkaline phosphatase, and total bilirubin levels were lower in the BQ123 and verapamil groups compared to epoprostenol group. Cold storage group had increased markers of ischemia reperfusion injury and slower graft function recovery compared to machine perfused grafts., Conclusion: The use of BQ123, epoprostenol, and verapamil during NEVLP is safe. Livers perfused with BQ123 and verapamil have higher hepatic artery flow and reduced hepatocyte injury during perfusion compared with epoprostenol. Hepatic artery flow is significantly reduced in the absence of vasodilators during NEVLP.

Normothermic ex vivo liver perfusion (NEVLP) improves graft preservation by avoiding cold ischemia injury. We investigated whether the protective effects of NEVLP can be further improved by applying strategies targeted on reducing the activation of proinflammatory cytokines during perfusion. Livers retrieved under heart-beating conditions were perfused for 4 hours. Following the preservation period, a pig liver transplantation was performed. In group 1 (n = 5), anti-inflammatory strategies (alprostadil, n-acetylcysteine, carbon monoxide, sevoflurane, and subnormothermic temperature [33°C]) were applied. This was compared with a perfused control group (group 2) where livers (n = 5) were perfused at 37°C without anti-inflammatory agents, similar to the setup used in current European clinical trials, and to a control group preserved with static cold storage (group 3). During 3-day follow-up, markers of reperfusion injury, bile duct injury, and liver function were examined. Aspartate aminotransferase (AST) levels during perfusion were significantly lower in the study versus control group at 1 hour (52 ± 6 versus 162 ± 86 U/L; P = 0.01), 2 hours (43 ± 5 versus 191 ± 111 U/L; P = 0.008), and 3 hours (24 ± 16 versus 218 ± 121 U/L; P = 0.009). During perfusion, group 1 versus group 2 had reduced interleukin (IL) 6, tumor necrosis factor α, and galactosidase levels and increased IL10 levels. After transplantation, group 1 had lower AST peak levels compared with group 2 and group 3 (1400 ± 653 versus 2097 ± 1071 versus 1747 ± 842 U/L; P = 0.47) without reaching significance. Bilirubin levels were significantly lower in group 1 versus group 2 at day 1 (3.6 ± 1.5 versus 6.60 ± 1.5 μmol/L; P = 0.02) and 3 (2 ± 1.1 versus 9.7 ± 7.6 μmol/L; P = 0.01). A trend toward decreased hyaluronic acid, as a marker of improved endothelial cell function, was observed at 1, 3, and 5 hours after reperfusion in group 1 versus group 2. Only 1 early death occurred in each group (80% survival). In conclusion, addition of anti-inflammatory strategies further improves warm perfused preservation. Liver Transplantation 22 1573-1583 2016 AASLD., (© 2016 by the American Association for the Study of Liver Diseases.)

The liver and spleen are major biological barriers to translating nanomedicines because they sequester the majority of administered nanomaterials and prevent delivery to diseased tissue. Here we examined the blood clearance mechanism of administered hard nanomaterials in relation to blood flow dynamics, organ microarchitecture and cellular phenotype. We found that nanomaterial velocity reduces 1,000-fold as they enter and traverse the liver, leading to 7.5 times more nanomaterial interaction with hepatic cells relative to peripheral cells. In the liver, Kupffer cells (84.8 ± 6.4%), hepatic B cells (81.5 ± 9.3%) and liver sinusoidal endothelial cells (64.6 ± 13.7%) interacted with administered PEGylated quantum dots, but splenic macrophages took up less material (25.4 ± 10.1%) due to differences in phenotype. The uptake patterns were similar for two other nanomaterial types and five different surface chemistries. Potential new strategies to overcome off-target nanomaterial accumulation may involve manipulating intra-organ flow dynamics and modulating the cellular phenotype to alter hepatic cell interactions.

The European trial investigating normothermic ex vivo liver perfusion (NEVLP) as a preservation technique for liver transplantation (LT) uses gelofusine, a non-US Food and Drug Administration-approved, bovine-derived, gelatin-based perfusion solution. We report a safety and feasibility clinical NEVLP trial with human albumin-based Steen solution. Transplant outcomes of 10 human liver grafts that were perfused on the Metra device at 37 °C with Steen solution, plus 3 units of erythrocytes were compared with a matched historical control group of 30 grafts using cold storage (CS) as the preservation technique. Ten liver grafts were perfused for 480 minutes (340-580 minutes). All livers cleared lactate (final lactate 1.46 mmol/L; 0.56-1.74 mmol/L) and produced bile (61 mL; 14-146 mL) during perfusion. No technical problems occurred during perfusion, and all NEVLP-preserved grafts functioned well after LT. NEVLP versus CS had lower aspartate aminotransferase and alanine aminotransferase values on postoperative days 1-3 without reaching significance. No difference in postoperative graft function between NEVLP and CS grafts was detected as measured by day 7 international normalized ratio (1.1 [1-1.56] versus 1.1 [1-1.3]; P = 0.5) and bilirubin (1.5; 1-7.7 mg/dL versus 2.78; 0.4-15 mg/dL; P = 0.5). No difference was found in the duration of intensive care unit stay (median, 1 versus 2 days; range, 0-8 versus 0-23 days; P = 0.5) and posttransplant hospital stay (median, 11 versus 13 days; range, 8-17 versus 7-89 days; P = 0.23). Major complications (Dindo-Clavien ≥ 3b) occurred in 1 patient in the NEVLP group (10%) compared with 7 (23%) patients in the CS group (P = 0.5). No graft loss or patient death was observed in either group. Liver preservation with normothermic ex vivo perfusion with the Metra device using Steen solution is safe and results in comparable outcomes to CS after LT. Using US Food and Drug Administration-approved Steen solution will avoid a potential regulatory barrier in North America. Liver Transplantation 22 1501-1508 2016 AASLD., (© 2016 American Association for the Study of Liver Diseases.)