BACKGROUND: The increasing demand for transplantable organs results in rising utilization of extended criteria donor organs (ECD). Normothermic machine perfusion (NMP) has emerged as a promising method to evaluate liver grafts under near-physiological conditions prior to transplantation. Despite these advances, there is a lack of easily assessable and non-invasive biomarkers to reliably predict graft viability and postoperative function. This study aims to assess the predictive value of selected perfusate-derived miRNAs during NMP for graft viability and the occurrence of early allograft dysfunction (EAD) following liver transplantation.

METHODS: In this study, a total of 33 liver grafts undergoing NMP between 2021 and 2024 at the Medical University of Vienna were analyzed. Perfusate samples were collected at three predefined time points: baseline (0 minutes), after 5 minutes, and after 60 minutes of perfusion. Expression levels of miRNA 122-5p, 151a-5p, and 192-5p were quantified using the commercially available hepatomiR^®Kit. For transplanted grafts, the incidence of EAD was assessed according to the criteria established by Olthoff et al. In addition, conventional perfusate markers including aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and lactate were measured for comparative analysis.

RESULTS: Out of the 33 livers evaluated, 22 grafts (66.7%) were transplanted, while 11 livers (33.3%) were discarded due to not meeting the required criteria that were previously established. 15 grafts (68.18%) out of the 22 transplanted livers developed EAD. No significant differences in miRNA expression were observed between transplanted and discarded livers. In contrast, miRNA levels were significantly elevated in grafts that subsequently developed EAD. Specifically, miRNA-122-5p levels were higher after 60 minutes of perfusion (p = 0.007), miRNA-151a-5p was elevated after 5 minutes (p = 0.026) and 60 minutes (p = 0.005), while miRNA-192-5p levels increased after 5 minutes (p = 0.021) and 60 minutes (p = 0.003). Receiver operating characteristic (ROC) analysis demonstrated strong predictive accuracy for EAD after 60 minutes of NMP, with an AUC of 0.857 for miRNA-122-5p, 0.867 for miRNA-151a-5p, and 0.886 for miRNA-192-5p. These values surpassed those of conventional markers such as AST (AUC 0.833) and LDH (AUC 0.786), whereas lactate did not show any predictive potential with an AUC of 0.478 after 60 minutes.

CONCLUSIONS: Perfusate-derived miRNAs during NMP serve as non-invasive, sensitive predictors of early allograft dysfunction and outperform established biochemical parameters. Integration of HepatomiR levels into clinical viability assessment could improve organ selection when utilizing marginal grafts and therefore optimize graft survival after transplantation.

Introduction:

Normothermic machine perfusion (NMP) has emerged as a promising platform to preserve and evaluate donor livers prior to transplantation. However, the molecular processes underlying graft behavior during NMP remain poorly understood, particularly in marginal grafts that do not meet criteria for transplantation. To better characterize transcriptional changes during perfusion, we analyzed paired liver biopsies obtained before and after 12 hours of NMP in grafts not utilized for transplantation.

Methods:

Eighteen marginal donor livers that did not meet criteria for transplantation were subjected to NMP. Paired biopsies were collected prior to initiation of perfusion and after 12 hours. High-throughput sequencing of messenger RNA (mRNA) and microRNA (miRNA) was performed, followed by differential expression analysis. Regulatory interactions were predicted through in silico network modeling, and functional relevance was explored using Gene Ontology Biological Process (GO-BP) enrichment.

Results:

We identified 1,740 mRNA transcripts and 245 miRNAs with significant differential regulation during NMP. Network analysis revealed 29 mRNA targets associated with the regulation of eight miRNAs. The transcriptional profile was consistent with stress-biased regeneration, metabolic inflexibility, and endothelial activation. Specifically, strong downregulation of pyruvate dehydrogenase kinase 4 (PDK4) and acyl-CoA oxidase 1 (ACOX1) indicated suppression of fatty acid oxidation and increased glucose dependence, a maladaptive metabolic switch previously linked to ischemia–reperfusion and steatosis. Reduced growth hormone receptor expression suggested blunted growth hormone/insulin-like growth factor signaling, potentially impairing regenerative capacity. Endothelial destabilization was reflected by decreased expression of junctional regulators (MYLK, TMEM47, PCDH9, TENM2), which are involved in maintaining sinusoidal barrier integrity. Conversely, pro-stress and pro-apoptotic mediators including HSPA1B and BBC3/PUMA were upregulated, as were SERPINE1/PAI-1, RIPK2, ZC3H12A/MCPIP1, and CHKA, implicating proteotoxic stress, inflammatory signaling, thrombo-fibrotic priming, and enhanced phosphatidylcholine biosynthesis. Together, these transcriptional shifts suggest that livers failing viability testing enter a state of energetic strain, endothelial activation, and pro-inflammatory stress. GO-BP enrichment analysis corroborated these findings, with vascular regulation and metabolic pathways emerging as the most significantly modulated processes.

Conclusion:

Transcriptomic profiling of marginal liver grafts during NMP reveals a phenotype marked by impaired metabolic flexibility, blunted regenerative signaling, and endothelial activation, accompanied by heightened stress, apoptotic, and inflammatory pathways. These features likely contribute to the inability of such grafts to recover viability during perfusion. Understanding these maladaptive responses at the molecular level may guide targeted interventions to improve graft reconditioning, refine viability assessment, and expand the pool of usable donor livers.

The global demand for transplantable organs continues to rise, driving exploration of methods to extend preservation times and facilitate longer-distance transport. In this context, subzero preservation strategies may offer potential advantages by slowing metabolic processes and reducing tissue degradation. This study aimed to evaluate the feasibility of utilizing a novel organ preservation solution containing a nature-inspired anti-icing agent, XT-ViVo®, alongside the organ transporter TimeSeal®, for transport aboard a commercial transatlantic flight. To this end, porcine kidneys were harvested in Baltimore, USA, flushed with XT-ViVo®, stored at -5°C, and transported via a commercial aircraft to Innsbruck, Europe, using the TimeSeal® device designed to maintain exact target temperatures. After storage durations ranging from about 19.5 to 72.5 hours, graft function was assessed during six hours of Normothermic Machine Perfusion (NMP) using the Kidney Assist platform. Biopsies were collected before, during, and after perfusion to assess organ viability and mitochondrial activity via high-resolution respirometry. Additionally, biochemical analyses of perfusate and urine samples were performed, and microperfusion was evaluated noninvasively by hyperspectral imaging. The organ transporter appeared to maintain the temperature close to the target -5.0 °C with moderate variation, suggesting effective thermal stability during transport. During NMP, the kidneys showed stable hemodynamics post subzero storage with consistent flow rates, urine production, and a renal resistance index within expected physiological ranges. Oxygen consumption peaked after around two hours of reperfusion but appeared to taper off toward the end of the perfusion period. pH and lactate levels in perfusate samples remained within physiological range. Measurements of mitochondrial function indicated that while succinate was the dominant substrate, there was a gradual decline in oxidative phosphorylation capacity and ATP production efficiency as perfusion progressed, coupled with indications of increasing outer mitochondrial membrane impairment. Hyperspectral imaging demonstrated consistent microvascular perfusion throughout the six-hour perfusion. Collectively, these findings provide first preliminary evidence that the combined use of ice-free subzero preservation with XT-ViVo® and subsequent NMP may offer a clinically feasible approach for organ preservation and global organ transport.

Background: Hypothermic oxygenated machine perfusion (HOPE) improves outcomes after liver transplantation (LTx), yet standardized biomarkers for graft viability assessment during perfusion are lacking, as no readily available and easily assessable monitoring parameters exist currently.

Methods: This study included all patients who underwent LTx with grafts perfused via HOPE at the Medical University of Vienna between 2018 and 2023. HOPE was performed according to institutional standard operating procedures using University of Wisconsin machine perfusion solution. Blood gas analyses were obtained every 30 minutes for up to 120 minutes during perfusion. Hospitalization data was prospectively collected and retrospectively analyzed. Early allograft dysfunction (EAD) was the primary outcome parameter and defined according to Olthoff et al-.

Results: A total of 158 patients were included in the analysis, of whom 44 (27.8%) developed EAD. Grafts that developed EAD exhibited significantly lower sodium concentrations at all measured time points, whereas lactate levels were elevated in EAD grafts at 60 and 90 minutes during HOPE. Sodium and lactate measurements at 90 minutes demonstrated the highest predictive value for EAD, with areas under the receiver operating characteristic curve of 0.741 and 0.633, respectively. Optimal cut-off values were identified as 85 mmol/L for sodium and 4.3 mmol/L for lactate at 90 minutes. Based on these thresholds, a low-risk cohort (high sodium, low lactate) exhibited an EAD incidence of 17.6%, whereas a high-risk cohort (low sodium, high lactate) showed a markedly higher incidence of 69.2% (p ≤0,001).

Conclusion: Assessment of sodium and lactate levels at 90 minutes during HOPE effectively identifies high-risk donor grafts prior to LTx. Accordingly, the evaluation of these readily available perfusate biomarkers prior to decision for transplantation might improve patient outcome.

Introduction: Kidney transplantation remains the gold standard treatment for end-stage renal dysfunction. However, the prolonged shortage of suitable donors further compounded by the increasing utilization of extended-criteria donors emphasizes the urgent need for advancements in organ preservation techniques. While static cold storage (SCS) is widely adopted due to its cost-effectiveness and simplicity, it still requires further optimization. Preclinical evidence suggests that methane (CH₄) has potent anti-inflammatory, antioxidative, and mitochondrial-stabilizing properties, indicating potential utility as a bioactive additive in preservation solutions. This study aimed to develop a clinically relevant porcine kidney transplantation model to investigate the efficacy of methane-enriched Custodiol (HTK) solution in improving SCS outcomes.

Methods: Under general anaesthesia and mechanical ventilation, pigs underwent left nephrectomy followed by recovery (approval no. V/3262/2022). Retrieved kidneys were perfused and stored for 16 hours at 4°C in either standard HTK or methane-enriched HTK solution (HTK+CH₄; enriched with 25 L of 2.1% CH₄-air mixture; n=5/group). Kidneys were then reimplanted, and contralateral nephrectomy performed following reinduction of anesthesia. Following transplantation, animals were monitored for 24 hours, assessing renal artery flow (RAF), urine output (UO), serum creatinine levels, systemic haemodynamics, diuretic requirements, renal myeloperoxidase (MPO) enzyme activity and kidney mitochondrial function.

Results: There was no significant difference in systemic haemodynamics between the two groups. However, kidneys preserved in HTK+CH₄ demonstrated superior perfusion, as evidenced by consistently higher RAF and lower renal vascular resistance throughout the post-transplantation period. Furthermore, the HTK+CH₄ group required less diuretic support and exhibited improved UO and urine specific gravity, along with lower serum creatinine levels, all indicating enhanced renal function. Renal inflammation, as reflected by MPO activity, was significantly reduced, and mitochondrial outer membrane integrity was better preserved in kidneys stored in HTK+CH₄.

Conclusions: This translational porcine model effectively facilitates the evaluation of organ preservation strategies. Our findings suggest that methane-enriched HTK solution confers significant renoprotective effects, enhancing graft function and perfusion in the context of kidney transplantation.