HLSC-EV treatment, even in a short-duration model, was feasible and effectively reduced liver injury during hypoxic NMP.

Background: While growing evidence supports the use of hypothermic oxygenated machine perfusion (HOPE) in liver transplantation, its effects on liver metabolism are still incompletely understood. Methods: To assess liver metabolism during HOPE using microdialysis (MD), we conducted an open-label, observational pilot study on 10 consecutive grafts treated with dual-HOPE (D-HOPE). Microdialysate and perfusate levels of glucose, lactate, pyruvate, glutamate, and flavin mononucleotide (FMN) were measured during back table preparation and D-HOPE and correlated to graft function and patient outcome. Results: Median (IQR) MD and D-HOPE time was 228 (210, 245) and 116 (103, 143) min. Three grafts developed early allograft dysfunction (EAD), with one requiring retransplantation. During D-HOPE, MD glucose and lactate levels increased (ANOVA = 9.88 [p = 0.01] and 3.71 [p = 0.08]). Their 2nd-hour levels were higher in EAD group and positively correlated with L-GrAFT score. 2ndhour MD glucose and lactate were also positively correlated with cold ischemia time, macrovesicular steatosis, weight gain during D-HOPE, and perfusate FMN.These correlations were not apparent when perfusate levels were considered.In contrast, MD FMN levels invariably dropped steeply after D-HOPE start, whereas perfusate FMN was higher in dysfunctioning grafts. Conclusion:MD glucose and lactate during D-HOPE are markers of hepatocellular injury and could represent additional elements of the viability assessment.

Summary Livers from donors after circulatory death (DCD) are a promising option to increase the donor pool, but their use is associated with higher complication rate and inferior graft survival. Normothermic machine perfusion (NMP) keeps the graft at 37°C, providing nutrients and oxygen supply. Human liver stem cell‐derived extracellular vesicles (HLSC‐EVs) are able to reduce liver injury and promote regeneration. We investigated the efficacy of a reconditioning strategy with HLSC‐EVs in an experimental model of NMP. Following total hepatectomy, rat livers were divided into 4 groups: (i) healthy livers, (ii) warm ischemic livers (60 min of warm ischemia), (iii) warm ischemic livers treated with 5 × 108 HLSC‐EVs/g‐liver, and (iv) warm ischemic livers treated with a 25 × 108 HLSC‐EVs/g‐liver. NMP lasted 6 h and HLSC‐EVs (Unicyte AG, Germany) were administered within the first 15 min. Compared to controls, HLSC‐EV treatment significantly reduced transaminases release. Moreover, HLSC‐EVs enhanced liver metabolism by promoting phosphate utilization and pH self‐regulation. As compared to controls, the higher dose of HLSC‐EV was associated with significantly higher bile production and lower intrahepatic resistance. Histologically, this group showed reduced necrosis and enhanced proliferation. In conclusion, HLSC‐EV treatment during NMP was feasible and effective in reducing injury in a DCD model with prolonged warm ischemia.

Although machine perfusion (MP) is being increasingly adopted in liver transplantation, indications, timing, and modality are debated. To investigate current indications for MP a web-based Google Forms survey was launched in January 2021 and addressed to 127 experts in the field, identified among first and corresponding Authors of MP literature in the last 10 years. The survey presented 10 real-life cases of donor-recipient matching, asking whether the liver would be accepted (Q1), whether MP would be used in that particular setting (Q2) and, if so, by which MP modality (Q3) and at what timing during preservation (Q4).Respondents could also comment on each case. The agreement was evaluated using Krippendorff's alpha coefficient. Answers from 39 (30.1%) participants disclosed significant heterogeneity in graft acceptance, MP indications, technique, and timing. Agreement between respondents was generally poor (Q1, α = 0.11; Q2, α = 0.14; Q3, α = 0.12, Q4, α = 0.11). Overall, respondents preferred hypothermic MP and an end-ischemic approach in 56.3% and 81.1% of cases, respectively. A total of 18 (46.2%) participants considered only one MP approach, whereas 17 (43.6%) and 3 (7.7%) considered using alternatively 2 or 3 different techniques. Of 38 comments, 17 (44.7%) were about the use of MP for graft viability assessment before implantation. This survey shows considerable variability in MP indications, emphasizing the need to identify scenarios of optimal utilization for each technique. Viability assessment emerges as a fundamental need of transplant professionals when considering the use of MP.

Hepatic ischemia-reperfusion injury (IRI) is observed in liver transplantation and hepato-biliary surgery and is associated with an inflammatory response. Human liver stem cell-derived extracellular vesicles (HLSC-EV) have been demonstrated to reduce liver damage in different experimental settings by accelerating regeneration and by modulating inflammation. The aim of the present study was to investigate whether HLSC-EV may protect liver from IRI in a mouse experimental model. Segmental IRI was obtained by selective clamping of intrahepatic pedicles for 90 min followed by 6 h of reperfusion. HLSC-EV were administered intravenously at the end of the ischemic period and histopathological and biochemical alterations were evaluated in comparison with controls injected with vehicle alone. Intra liver localization of labeled HLSC-EV was assessed by in in vivo Imaging System (IVIS) and the internalization into hepatocytes was confirmed by fluorescence analyses. As compared to the control group, administration of 3 × 109 particles (EV1 group) significantly reduced alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) release, necrosis extension and cytokines expression (TNF-α, CCL-2 and CXCL-10). However, the administration of an increased dose of HLSC-EV (7.5 × 109 particles, EV2 group) showed no significant improvement in respect to controls at enzyme and histology levels, despite a significantly lower cytokine expression. In conclusion, this study demonstrated that 3 × 109 HLSC-EV were able to modulate hepatic IRI by preserving tissue integrity and by reducing transaminases release and inflammatory cytokines expression. By contrast, a higher dose was ineffective suggesting a restricted window of biological activity.