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Abstract The partial understanding of the biological events that occur during normothermic machine perfusion (NMP) and particularly during prolonged perfusion might hinder its deployment in clinical transplantation. The aim of our study was to implement a rat model of prolonged NMP to characterize the bio-molecular phenotype and metabolism of the perfused organs. Livers (n = 5/group) were procured and underwent 4 h (NMP4h) or 12 h (NMP12h) NMP, respectively, using a perfusion fluid supplemented with an acellular oxygen carrier. Organs that were not exposed to any procedure served as controls (Native). All perfused organs met clinically derived viability criteria at the end of NMP. Factors related to stress-response and survival were increased after prolonged perfusion. No signs of oxidative damage were detected in both NMP groups. Evaluation of metabolite profiles showed preserved mitochondrial function, activation of Cori cycle, induction of lipolysis, acetogenesis and ketogenesis in livers exposed to 12 h-NMP. Increased concentrations of metabolites involved in glycogen synthesis, glucuronidation, bile acid conjugation, and antioxidant response were likewise observed. In conclusion, our NMP12h model was able to sustain liver viability and function, thereby deeply changing cell homeostasis to maintain a newly developed equilibrium. Our findings provide valuable information for the implementation of optimized protocols for prolonged NMP.

Liver transplantation is known to generate significant inflammation in the entire organ based on the metabolic profile and the tissue’s ability to recover from the ischemia-reperfusion injury (IRI). This cascade contributes to post-transplant complications, affecting both the synthetic liver function (immediate) and the scar development in the biliary tree. The new occurrence of biliary strictures, and the recurrence of malignant and benign liver diseases, such as cholangiocarcinoma (CCA) and primary sclerosing cholangitis (PSC), are direct consequences linked to this inflammation. The accumulation of toxic metabolites, such as succinate, causes undirected electron flows, triggering the releases of reactive oxygen species (ROS) from a severely dysfunctional mitochondrial complex 1. This initiates the inflammatory IRI cascade, with subsequent ischemic biliary stricturing, and the upregulation of pro-tumorigenic signaling. Such inflammation is both local and systemic, promoting an immunocompromised status that can lead to the recurrence of underlying liver disease, both malignant and benign in nature. The traditional treatment for CCA was resection, when possible, followed by cytotoxic chemotherapy. Liver transplant oncology is increasingly recognized as a potentially curative approach for patients with intrahepatic (iCCA) and perihilar (pCCA) cholangiocarcinoma. The link between IRI and disease recurrence is increasingly recognized in transplant oncology for hepatocellular carcinoma. However, smaller numbers have prevented similar analyses for CCA. The mechanistic link may be even more critical in this disease, as IRI causes the most profound damage to the intrahepatic bile ducts. This article reviews the underlying mechanisms associated with biliary inflammation and biliary pathology after liver transplantation. One main focus is on the link between transplant-related IRI-associated inflammation and the recurrence of cholangiocarcinoma and benign liver diseases of the biliary tree. Risk factors and protective strategies are highlighted.

Background. Although hypothermic oxygenated perfusion (HOPE) improves posttransplant outcomes, setting up machine perfusion programs may be subjected to specific obstacles under different conditions. This study aims to describe the establishment of HOPE in a real-life setting in Brazil. Methods. Extended criteria donors in donation after brain death organs preserved by HOPE were accepted for higher-risk candidates needing expedited transplantation, perceived as those who would benefit most from the technique because of its limited availability. Extended criteria donors was defined by the Eurotransplant criteria. High-risk transplant candidates were characterized by suboptimal surgical conditions related to the recipient or the procedure. Results. Six HOPE-preserved grafts were transplanted from February 2022 to August 2022. The mean donor risk index was 1.7 (SD 0.5). One organ was severely steatotic, and 3 had an anticipated cold ischemia time above 12 h. Recipients’ mean model for end-stage liver disease was 28.67 (SD 6.79), with 1 case of retransplant, 1 of refractory ascites, and 1 of acute-on-chronic liver failure. The mean cold ischemia time was 5 h 42 min (SD 82 min), HOPE 6 h 3 min (SD 150 min), and total preservation time 11 h 46 min (SD 184 min). No case had early allograft dysfunction. The mean length of hospital stay was 10 d with 100% graft and patient survival and no ischemic cholangiopathies at a median follow-up of 15 mo (min 12, max 18). Costs and country-specific legal regulations for device utilization were the major hurdles to implementing the program. Conclusion. We presented a pathway to introduce and rationalize the use of HOPE in a scenario of challenging donor-recipient matching with good results. These findings may aid in implementing machine perfusion programs, especially in settings with limited resources or complex transplant logistics.

Summary: Background: To report on a concept of liver assessment during ex situ hypothermic oxygenated perfusion (HOPE) and its significant impact on liver utilization. Methods: An analysis of prospectively collected data on donation after circulatory death (DCD) livers, treated by HOPE at our institution, during a 11-year period between January 2012 and December 2022. Findings: Four hundred and fifteen DCD Maastricht III livers were offered during the study period in Switzerland, resulting in 249 liver transplants. Of those, we performed 158 DCD III liver transplants at our institution, with 1-year patient survival and death censored graft survival (death with functioning graft) of 87 and 89%, respectively, thus comparable to benchmark graft survivals of ideal DBD and DCD liver transplants (89% and 86%). Correspondingly, graft loss for primary non-function or cholangiopathy was overall low, i.e., 7/158 (4.4%) and 11/158 (6.9%), despite more than 82% of DCD liver grafts ranked high (6–10 points) or futile risk (>10 points) according to the UK-DCD score. Consistently, death censored graft survival was not different between low-, high-risk or futile DCD III livers. The key behind these achievements was the careful development and implementation of a routine perfusate assessment of mitochondrial biomarkers for injury and function, i.e., release of flavin mononucleotide from complex I, perfusate NADH, and mitochondrial CO2 production during HOPE, allowing a more objective interpretation of liver quality on a subcellular level, compared to donor derived data. Interpretation: HOPE after cold storage is a highly suitable and easy to perform perfusion approach, which allows reliable liver graft assessment, enabling surgeons to make a fact based decision on whether or not to implant the organ. HOPE-treatment should be combined with viability assessment particularly when used for high-risk organs, including DCD livers or organs with relevant steatosis. Funding: This study was supported by the Swiss National Foundation (SNF) grant 320030_189055/1 to PD.

Summary: The risk of cancer recurrence after liver surgery mainly depends on tumour biology, but preclinical and clinical evidence suggests that the degree of perioperative liver injury plays a role in creating a favourable microenvironment for tumour cell engraftment or proliferation of dormant micro-metastases. Understanding the contribution of perioperative liver injury to tumour recurrence is imperative, as these pathways are potentially actionable. In this review, we examine the key mechanisms of perioperative liver injury, which comprise mechanical handling and surgical stress, ischaemia-reperfusion injury, and parenchymal loss leading to liver regeneration. We explore how these processes can trigger downstream cascades leading to the activation of the immune system and the pro-inflammatory response, cellular proliferation, angiogenesis, anti-apoptotic signals, and release of circulating tumour cells. Finally, we discuss the novel therapies under investigation to decrease ischaemia-reperfusion injury and increase regeneration after liver surgery, including pharmaceutical agents, inflow modulation, and machine perfusion.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death and the sixth most diagnosed malignancy worldwide. Serum alpha-fetoprotein (AFP) is the traditional, ubiquitous biomarker for HCC. However, there has been an increasing call for the use of multiple biomarkers to optimize care for these patients. AFP, AFP-L3, and prothrombin induced by vitamin K absence II (DCP) have described clinical utility for HCC, but unfortunately, they also have well established and significant limitations. Circulating tumor DNA (ctDNA), genomic glycosylation, and even totally non-invasive salivary metabolomics and/or micro-RNAS demonstrate great promise for early detection and long-term surveillance, but still require large-scale prospective validation to definitively validate their clinical validity. This review aims to provide an update on clinically available and emerging biomarkers for HCC, focusing on their respective clinical strengths and weaknesses.

Introduction: Circulating tumor DNA (ctDNA) is emerging as a promising, non-invasive diagnostic and surveillance biomarker in solid organ malignancy. However, its utility before and after liver transplant (LT) for patients with primary and secondary liver cancers is still underexplored. Methods: Patients undergoing LT for hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), and colorectal liver metastases (CRLM) with ctDNA testing were included. CtDNA testing was conducted pre-transplant, post-transplant, or both (sequential) from 11/2019 to 09/2023 using Guardant360, Guardant Reveal, and Guardant360 CDx. Results: 21 patients with HCC (n = 9, 43%), CRLM (n = 8, 38%), CCA (n = 3, 14%), and mixed HCC/CCA (n = 1, 5%) were included in the study. The median follow-up time was 15 months (range: 1–124). The median time from pre-operative testing to surgery was 3 months (IQR: 1–4; range: 0–5), and from surgery to post-operative testing, it was 9 months (IQR: 2–22; range: 0.4–112). A total of 13 (62%) patients had pre-transplant testing, with 8 (62%) having ctDNA detected (ctDNA+) and 5 (32%) not having ctDNA detected (ctDNA-). A total of 18 (86%) patients had post-transplant testing, 11 (61%) of whom were ctDNA+ and 7 (33%) of whom were ctDNA-. The absolute recurrence rates were 50% (n = 5) in those who were ctDNA+ vs. 25% (n = 1) in those who were ctDNA- in the post-transplant setting, though this difference was not statistically significant (p = 0.367). Six (29%) patients (HCC = 3, CCA = 1, CRLM = 2) experienced recurrence with a median recurrence-free survival of 14 (IQR: 6–40) months. Four of these patients had positive post-transplant ctDNA collected following diagnosis of recurrence, while one patient had positive post-transplant ctDNA collected preceding recurrence. A total of 10 (48%) patients had sequential ctDNA testing, of whom n = 5 (50%) achieved ctDNA clearance (+/−). The remainder were ctDNA+/+ (n = 3, 30%), ctDNA−/− (n = 1, 10%), and ctDNA−/+ (n = 1, 11%). Three (30%) patients showed the acquisition of new genomic alterations following transplant, all without recurrence. Overall, the median tumor mutation burden (TMB) decreased from 1.23 mut/Mb pre-transplant to 0.00 mut/Mb post-transplant. Conclusions: Patients with ctDNA positivity experienced recurrence at a higher rate than the ctDNA- patients, indicating the potential role of ctDNA in predicting recurrence after curative-intent transplant. Based on sequential testing, LT has the potential to clear ctDNA, demonstrating the capability of LT in the treatment of systemic disease. Transplant providers should be aware of the potential of donor-derived cell-free DNA and improved approaches are necessary to address such concerns.

Hydrodynamic tail vein injection (HTV) is the “gold standard” for delivering naked DNA vectors to mouse liver, thereby transfecting predominately perivenous hepatocytes. While HTV corrects metabolic liver defects such as phenylketonuria or cystathionine β-synthase deficiency, correction of spfash mice with ornithine transcarbamylase (OTC) deficiency was not possible despite overexpression in the liver, as the OTC enzyme is primarily expressed in periportal hepatocytes. To target periportal hepatocytes, we established hydrodynamic retrograde intrabiliary injection (HRII) in mice and optimized minicircle (MC) vector delivery using luciferase as a marker gene. HRII resulted in a transfection efficiency below 1%, 100-fold lower than HTV. While HRII induced minimal liver toxicity compared with HTV, overexpression of luciferase by both methods, but not of a natural liver-specific enzyme, elicited an immune response that led to the elimination of luciferase expression. Further testing of MC vectors delivered via HRII in spfash mice did not result in sufficient therapeutic efficacy and needs further optimization and/or selection of the corrected cells. This study reveals that luciferase expression is toxic for the liver. Furthermore, physical delivery of MC vectors via the bile duct has the potential to treat defects restricted to periportal hepatocytes, which opens new doors for non-viral liver-directed gene therapy.

Waiting list mortality together, with limited availability of organs, are one of the major challenges in liver transplantation (LT). Especially in the paediatric population, another limiting factor is the scarcity of transplantable liver grafts due to additional concerns regarding graft size matching. In adults, donation after circulatory death (DCD) liver grafts have been used to expand the donor pool with satisfactory results. Although several studies suggest that DCD livers could also be used in paediatric recipients with good outcomes, their utilisation in children is still limited to a small number of reports. Novel organ perfusion strategies could be used to improve organ quality and help to increase the number of DCD grafts utilised for children. With the current manuscript, we present the available literature of LT using DCD grafts in paediatric recipients, discussing current challenges with the use of these livers in children and how machine perfusion technologies could be of impact in the future.

Building the inner layer of our blood vessels, the endothelium forms an important line communicating with deeper parenchymal cells in our organs. Previously considered passive, endothelial cells are increasingly recognized as key players in intercellular crosstalk, vascular homeostasis, and blood fluidity. Comparable to other cells, their metabolic function strongly depends on mitochondrial health, and the response to flow changes observed in endothelial cells is linked to their mitochondrial metabolism. Despite the direct impact of new dynamic preservation concepts in organ transplantation, the impact of different perfusion conditions on sinusoidal endothelial cells is not yet explored well enough. This article therefore describes the key role of liver sinusoidal endothelial cells (LSECs) together with their mitochondrial function in the context of liver transplantation. The currently available ex situ machine perfusion strategies are described with their effect on LSEC health. Specific perfusion conditions, including perfusion pressure, duration, and perfusate oxygenation are critically discussed considering the metabolic function and integrity of liver endothelial cells and their mitochondria.

Short bubble and subsequent surface oxygenation is an innovative oxygenation technique and alternative for membrane oxygenation during hypothermic machine perfusion (HMP). The metabolic effect of the interruption of surface oxygenation for 4 h (mimicking organ transport) during HMP was compared to continuous surface and membrane oxygenation in a pig kidney ex situ preservation model. After 30 min of warm ischemia by vascular clamping, a kidney of a ±40 kg pig was procured and subsequently preserved according to one of the following groups: (1) 22-h HMP + intermittent surface oxygenation (n = 12); (2) 22-h HMP + continuous membrane oxygenation (n = 6); and (3) 22-h HMP + continuous surface oxygenation (n = 7). Brief perfusate O2 uploading before kidney perfusion was either obtained by direct bubble (groups 1, 3) or by membrane (group 2) oxygenation. Bubble oxygenation during minimum 15 min was as efficient as membrane oxygenation in achieving supraphysiological perfusate pO2 levels before kidney perfusion. Metabolic tissue analysis (i.e., lactate, succinate, ATP, NADH, and FMN) during and at the end of the preservation period demonstrated similar mitochondrial protection between all study groups. Short bubble and subsequent intermittent surface oxygenation of the perfusate of an HMP-kidney might be an effective and cheap preservation strategy to protect mitochondria, eliminating the need/costs of a membrane oxygenator and oxygen source during transport.

Background: Organ perfusion technology is increasingly used in many countries, with a focus, however, on the Western world. This study investigates the current international trends and obstacles to the broader routine implementation of dynamic perfusion concepts in liver transplantation. Methods: A web-based anonymous survey was launched in 2021. Experts of all involved specializations from 70 centers in 34 countries were contacted, based on the published literature and experience in the field of abdominal organ perfusion. Results: Overall, 143 participants from 23 countries completed the survey. Most respondents were male (67.8%) and transplant surgeons (64.3%) working at university hospitals (67.9%). The majority had experience with organ perfusion (82%), applying mainly hypothermic machine perfusion (HMP; 38%) and other concepts. While most (94.4%) expect a higher utilization of marginal organs with machine perfusion, the majority considers HMP the best technique to reduce liver discard-rates. While most respondents (90%) believed machine perfusion should be fully commissioned, the lack of funding (34%) and knowledge (16%) as well as limited staff (19%) were the three main obstacles to a routine clinical implementation. Conclusion: Although dynamic preservation concepts are increasingly used in clinical practice, significant challenges remain. Specific financial pathways, uniform regulations, and tight collaborations among involved experts are needed to achieve wider global clinical use.

Although machine perfusion is a hot topic today, we are just at the beginning of understanding the underlying mechanisms of protection. Recently, the first randomized controlled trial reported a significant reduction of ischemic cholangiopathies after transplantation of livers donated after circulatory death, provided the grafts were treated with an endischemic hypothermic oxygenated perfusion (HOPE). This approach has been known for more than fifty years, and was initially mainly used to preserve kidneys before implantation. Today there is an increasing interest in this and other dynamic preservation technologies and various centers have tested different approaches in clinical trials and cohort studies. Based on this, there is a need for uniform perfusion settings (perfusion route and duration), and the development of general guidelines regarding the duration of cold storage in context of the overall donor risk is also required to better compare various trial results. This article will highlight how cold perfusion protects organs mechanistically, and target such technical challenges with the perfusion setting. Finally, the options for viability testing during hypothermic perfusion will be discussed.

The frequent use of marginal livers forces transplant centres to explore novel technologies to improve organ quality and outcomes after implantation. Organ perfusion techniques are therefore frequently discussed with an ever-increasing number of experimental and clinical studies. Two main approaches, hypothermic and normothermic perfusion, are the leading strategies to be introduced in clinical practice in many western countries today. Despite this success, the number of studies, which provide robust data on the underlying mechanisms of protection conveyed through this technology remains scarce, particularly in context of different stages of ischemia-reperfusion-injury (IRI). Prior to a successful clinical implementation of machine perfusion, the concept of IRI and potential key molecules, which should be addressed to reduce IRI-associated inflammation, requires a better exploration. During ischemia, Krebs cycle metabolites, including succinate play a crucial role with their direct impact on the production of reactive oxygen species (ROS) at mitochondrial complex I upon reperfusion. Such features are even more pronounced under normothermic conditions and lead to even higher levels of downstream inflammation. The direct consequence appears with an activation of the innate immune system. The number of articles, which focus on the impact of machine perfusion with and without the use of specific perfusate additives to modulate the inflammatory cascade after transplantation is very small. This review describes first, the subcellular processes found in mitochondria, which instigate the IRI cascade together with proinflammatory downstream effects and their link to the innate immune system. Next, the impact of currently established machine perfusion strategies is described with a focus on protective mechanisms known for the different perfusion approaches. Finally, the role of such dynamic preservation techniques to deliver specific agents, which appear currently of interest to modulate this posttransplant inflammation, is discussed together with future aspects in this field.

The aim of this work was to assess the association between graft-to-recipient weight ratio (GRWR) in adult-to-adult living donor liver transplantation (LDLT) and hepatocellular carcinoma (HCC) recurrence. A search of the MEDLINE and EMBASE databases was performed until December 2022 for studies comparing different GRWRs in the prognosis of HCC recipients in LDLT. Data were pooled to evaluate 1- and 3-year survival rates. We identified three studies, including a total of 782 patients (168 GRWR < 0.8 vs. 614 GRWR ≥ 0.8%). The pooled overall survival was 85% and 77% at one year and 90% and 83% at three years for GRWR < 0.8 and GRWR ≥ 0.8, respectively. The largest series found that, in patients within Milan criteria, the GRWR was not associated with lower oncological outcomes. However, patients with HCC outside the Milan criteria with a GRWR < 0.8% had lower survival and higher tumor recurrence rates. The GRWR < 0.8% appears to be associated with lower survival rates in HCC recipients, particularly for candidates with tumors outside established HCC criteria. Although the data are scarce, the results of this study suggest that considering the individual GRWR not only as risk factor for small-for-size-syndrome but also as contributor to HCC recurrence in patients undergoing LDLT would be beneficial. Novel perfusion technologies and pharmacological interventions may contribute to improving outcomes.

The shortage of viable donated livers limits patient access to liver transplantation. Here the authors report the use of normothermic machine perfusion to help identify viable organs from livers discarded based on current clinical criteria, which are then transplanted to recipients in a single-arm clinical trial.

Based on the renaissance of dynamic preservation techniques, extended criteria donor (ECD) livers reclaimed a valuable eligibility in the transplantable organ pool. Being more vulnerable to ischemia, ECD livers carry an increased risk of early allograft dysfunction, primary non-function and biliary complications and, hence, unveiled the limitations of static cold storage (SCS). There is growing evidence that dynamic preservation techniques—dissimilar to SCS—mitigate reperfusion injury by reconditioning organs prior transplantation and therefore represent a useful platform to assess viability. Yet, a debate is ongoing about the advantages and disadvantages of different perfusion strategies and their best possible applications for specific categories of marginal livers, including organs from donors after circulatory death (DCD) and brain death (DBD) with extended criteria, split livers and steatotic grafts. This review critically discusses the current clinical spectrum of livers from ECD donors together with the various challenges and posttransplant outcomes in the context of standard cold storage preservation. Based on this, the potential role of machine perfusion techniques is highlighted next. Finally, future perspectives focusing on how to achieve higher utilization rates of the available donor pool are highlighted.

Tumor recurrence after liver transplantation has been linked to multiple factors, including the recipient’s tumor burden, donor factors, and ischemia-reperfusion injury (IRI). The increasing number of livers accepted from extended criteria donors has forced the transplant community to push the development of dynamic perfusion strategies. The reason behind this progress is the urgent need to reduce the clinical consequences of IRI. Two concepts appear most beneficial and include either the avoidance of ischemia, e.g., the replacement of cold storage by machine perfusion, or secondly, an endischemic organ improvement through perfusion in the recipient center prior to implantation. While several concepts, including normothermic perfusion, were found to reduce recipient transaminase levels and early allograft dysfunction, hypothermic oxygenated perfusion also reduced IRI-associated post-transplant complications and costs. With the impact on mitochondrial injury and subsequent less IRI-inflammation, this endischemic perfusion was also found to reduce the recurrence of hepatocellular carcinoma after liver transplantation. Firstly, this article highlights the contributing factors to tumor recurrence, including the surgical and medical tissue trauma and underlying mechanisms of IRI-associated inflammation. Secondly, it focuses on the role of mitochondria and associated interventions to reduce cancer recurrence. Finally, the role of machine perfusion technology as a delivery tool and as an individual treatment is discussed together with the currently available clinical studies.

Machine perfusion (MP) allows the maintenance of liver cells in a metabolically active state ex vivo and can potentially revert metabolic perturbations caused by donor warm ischemia, procurement, and static cold storage (SCS). The present preclinical research investigated the metabolic outcome of the MP procedure by analyzing rat liver tissue, bile, and perfusate samples by means of high-field (600 MHz) nuclear magnetic resonance (NMR) spectroscopy. An established rat model of normothermic MP (NMP) was used. Experiments were carried out with the addition of an oxygen carrier (OxC) to the perfusion fluid (OxC-NMP, n = 5) or without (h-NMP, n = 5). Bile and perfusate samples were collected throughout the procedure, while biopsies were only taken at the end of NMP. Two additional groups were: (1) Native, in which tissue or bile specimens were collected from rats in resting conditions; and (2) SCS, in which biopsies were taken from cold-stored livers. Generally, NMP groups showed a distinctive metabolomic signature in all the analyzed biological matrices. In particular, many of the differentially expressed metabolites were involved in mitochondrial biochemical pathways. Succinate, acetate, 3-hydroxybutyrate, creatine, and O-phosphocholine were deeply modulated in ex vivo perfused livers compared to both the Native and SCS groups. These novel results demonstrate a broad modulation of mitochondrial metabolism during NMP that exceeds energy production and redox balance maintenance.

Background: Mitochondrial succinate accumulation has been suggested as key event for ischemia reperfusion injury in mice. No specific data are however available on behavior of liver mitochondria during ex situ machine perfusion in clinical transplant models. Methods: We investigated mitochondrial metabolism of isolated perfused rat livers before transplantation. Livers were exposed to warm and cold ischemia to simulate donation after circulatory death (DCD) and organ transport. Subsequently, livers were perfused with oxygenated Belzer-MPS for 1h, at hypothermic or normothermic conditions. Various experiments were performed with supplemented succinate and/or mitochondrial inhibitors. The perfusate, liver tissues, and isolated mitochondria were analyzed by mass-spectroscopy and fluorimetry. Additionally, rat DCD livers were transplanted after 1h hypothermic or normothermic oxygenated perfusion. In parallel, perfusate samples were analysed during HOPE-treatment of human DCD livers before transplantation. Findings: Succinate exposure during rat liver perfusion triggered a dose-dependent release of mitochondrial Flavin-Mononucleotide (FMN) and NADH in perfusates under normothermic conditions. In contrast, perfusate FMN was 3-8 fold lower under hypothermic conditions, suggesting less mitochondrial injury during cold re-oxygenation compared to normothermic conditions. HOPE-treatment induced a mitochondrial reprogramming with uploading of the nucleotide pool and effective succinate metabolism. This resulted in a clear superiority after liver transplantation compared to normothermic perfusion. Finally, the degree of mitochondrial injury during HOPE of human DCD livers, quantified by perfusate FMN and NADH, was predictive for liver function. Interpretation: Mitochondrial injury determines outcome of transplanted rodent and human livers. Hypothermic oxygenated perfusion improves mitochondrial function, and allows viability assessment of liver grafts before implantation. Funding: detailed information can be found in Acknowledgments.

Background: Pre-clinical research with multi-potent adult progenitor cells (MAPC® cells, Multistem, Athersys Inc., Cleveland, Ohio) suggests their potential as an anti-inflammatory and immunomodulatory therapy in organ transplantation. Normothermic machine perfusion of the liver (NMP-L) has been proposed as a way of introducing therapeutic agents into the donor organ. Delivery of cellular therapy to human donor livers using this technique has not yet been described in the literature. The primary objectives of this study were to develop a technique for delivering cellular therapy to human donor livers using NMP-L and demonstrate engraftment.Methods: Six discarded human livers were perfused for 6 h at 37°C using the Liver Assist (Organ Assist, Groningen). 50 × 106 CMPTX-labeled MAPC cells were infused directly into the right lobe via the hepatic artery (HA, n = 3) or portal vein (PV, n = 3) over 20 min at different time points during the perfusion. Perfusion parameters were recorded and central and peripheral biopsies were taken at multiple time-points from both lobes and subjected to standard histological stains and confocal microscopy. Perfusate was analyzed using a 35-plex multiplex assay and proteomic analysis.Results: There was no detrimental effect on perfusion flow parameters on infusion of MAPC cells by either route. Three out of six livers met established criteria for organ viability. Confocal microscopy demonstrated engraftment of MAPC cells across vascular endothelium when perfused via the artery. 35-plex multiplex analysis of perfusate yielded 13 positive targets, 9 of which appeared to be related to the infusion of MAPC cells (including Interleukin's 1b, 4, 5, 6, 8, 10, MCP-1, GM-CSF, SDF-1a). Proteomic analysis revealed 295 unique proteins in the perfusate from time-points following the infusion of cellular therapy, many of which have strong links to MAPC cells and mesenchymal stem cells in the literature. Functional enrichment analysis demonstrated their immunomodulatory potential.Conclusion: We have demonstrated that cells can be delivered directly to the target organ, prior to host immune cell population exposure and without compromising the perfusion. Transendothelial migration occurs following arterial infusion. MAPC cells appear to secrete a host of soluble factors that would have anti-inflammatory and immunomodulatory benefits in a human model of liver transplantation.

[This corrects the article DOI: 10.1371/journal.pone.0224066.].

Hypothermic-oxygenated-machine-perfusion (HOPE) allows assessment/reconditioning of livers procured from high-risk donors before transplantation. Graft referral to HOPE mostly depends on surgeons’ subjective judgment, as objective criteria are still insufficient. We investigated whether analysis of effluent fluids collected upon organ flush during static-cold-storage can improve selection criteria for HOPE utilization. Effluents were analyzed to determine cytolysis enzymes, metabolites, inflammation-related mediators, and damage-associated-molecular-patterns. Molecular profiles were assessed by unsupervised cluster analysis. Differences between “machine perfusion (MP)-yes” vs. “MP-no”; “brain-death (DBD) vs. donation-after-circulatory-death (DCD)”; “early-allograft-dysfunction (EAD)-yes” vs. “EAD-no” groups, as well as correlation between effluent variables and transplantation outcome, were investigated. Livers assigned to HOPE (n = 18) showed a different molecular profile relative to grafts transplanted without this procedure (n = 21, p = 0.021). Increases in the inflammatory mediators PTX3 (p = 0.048), CXCL8/IL-8 (p = 0.017), TNF-α (p = 0.038), and ANGPTL4 (p = 0.010) were observed, whereas the anti-inflammatory cytokine IL-10 was reduced (p = 0.007). Peculiar inflammation, cell death, and coagulation signatures were observed in fluids collected from DCD livers compared to those from DBD grafts. AST (p = 0.034), ALT (p = 0.047), and LDH (p = 0.047) were higher in the “EAD-yes” compared to the “EAD-no” group. Cytolysis markers and hyaluronan correlated with recipient creatinine, AST, and ICU stay. The study demonstrates that effluent molecular analysis can provide directions about the use of HOPE.

Limited duration of transgene expression, insertional mutagenesis, and size limitations for transgene cassettes pose challenges and risk factors for many gene therapy vectors. Here, we report on physiological expression of liver phenylalanine hydroxylase (PAH) by delivery of naked DNA/minicircle (MC)-based vectors for correction of homozygous enu2 mice, a model of human phenylketonuria (PKU). Because MC vectors lack a defined size limit, we constructed a MC vector expressing a codon-optimized murine Pah cDNA that includes a truncated intron and is under the transcriptional control of a 3.6-kb native Pah promoter/enhancer sequence. This vector, delivered via hydrodynamic injection, yielded therapeutic liver PAH activity and sustained correction of blood phenylalanine comparable to viral or synthetic liver promoters. Therapeutic efficacy was seen with vector copy numbers of 95% loss of vector genomes and PAH activity in liver, demonstrating that MC vectors had not integrated into the liver genome. In conclusion, MC vectors, which do not have a defined size-limitation, offer a favorable safety profile for hepatic gene therapy due to their non-integration in combination with native promoters.

INTRODUCTION:The combination of hypothermic and normothermic machine perfusion (HMP+NMP) of the liver provides individual benefits of both techniques, improving the rescue of marginal organs. The aim of this study was to investigate the effect on the bioenergetic status and the oxidative-mediated tissue injury of an uninterrupted combined protocol of HMP+NMP using a single haemoglobin-based oxygen carrier (HBOC)-based perfusate. METHODS:Ten discarded human donor livers had either 2 hours of dual hypothermic oxygenated perfusion (D-HOPE) with sequential controlled rewarming (COR) and then NMP using the HBOC-based perfusate uninterruptedly (cold-to-warm group); or 2 hours of hypothermic oxygenated perfusion (HOPE) with an oxygen carrier-free perfusate, followed by perfusate exchange and then NMP with an HBOC-based perfusate. Markers of liver function, tissue adenosine triphosphate (ATP) levels and tissue injury were systematically assessed. RESULTS:The hypothermic phase downregulated mitochondrial respiration and increased ATP levels in both groups. The cold-to-warm group presented higher arterial vascular resistance during rewarming/NMP (p = 0.03) with a trend of lower arterial flow (p = 0.09). At the end of NMP tissue expression of markers of reactive oxygen species production, oxidative injury and inflammation were comparable between the groups. CONCLUSION:The uninterrupted combined protocol of HMP+NMP using an HBOC-based perfusate-cold-to-warm MP-mitigated the oxidative-mediated tissue injury and enhanced hepatic energy stores, similarly to an interrupted combined protocol; however, it simplified the logistics of this combination and may favour its clinical applicability.

Based on the continuous increase of donor risk, with a majority of organs classified as marginal, quality assessment and prediction of liver function is of utmost importance. This is also caused by the notoriously lack of effective replacement of a failing liver by a device or intensive care treatment. While various parameters of liver function and injury are well-known from clinical practice, the majority of specific tests require prolonged diagnostic time and are more difficult to assess ex situ. In addition, viability assessment of procured organs needs time, because the development of the full picture of cellular injury and the initiation of repair processes depends on metabolic active tissue and reoxygenation with full blood over several hours or days. Measuring injury during cold storage preservation is therefore unlikely to predict the viability after transplantation. In contrast, dynamic organ preservation strategies offer a great opportunity to assess organs before implantation through analysis of recirculating perfusates, bile and perfused liver tissue. Accordingly, several parameters targeting hepatocyte or cholangiocyte function or metabolism have been recently suggested as potential viability tests before organ transplantation. We summarize here a current status of respective machine perfusion tests, and report their clinical relevance.

Tumour recurrence is currently a hot topic in liver transplantation. The basic mechanisms are increasingly discussed, and, for example, recurrence of hepatocellular carcinoma is often described in pre-injured donor livers, which frequently suffer from significant ischemia/reperfusion injury. This review article highlights the underlying mechanisms and describes the specific tissue milieu required to promote tumour recurrence after liver transplantation. We summarise the current literature in this field and show risk factors that contribute to a pro-tumour-recurrent environment. Finally, the potential role of new machine perfusion technology is discussed, including the most recent data, which demonstrate a protective effect of hypothermic oxygenated perfusion before liver transplantation.

Ischemia-reperfusion injury (IRI) constitutes a significant source of morbidity and mortality after orthotopic liver transplantation (OLT). The allograft is metabolically impaired during warm and cold ischemia and is further damaged by a paradox reperfusion injury after revascularization and reoxygenation. Short-term and long-term complications including post-reperfusion syndrome, delayed graft function, and immune activation have been associated with IRI. Due to the current critical organ shortage, extended criteria grafts are increasingly considered for transplantation, however, with an elevated risk to develop significant features of IRI. In recent years, ex vivo machine perfusion (MP) of the donor liver has witnessed significant advancements. Here, we describe the concept of hypothermic (oxygenated) machine perfusion (HMP/HOPE) approaches and highlight which allografts may benefit from this technology. This review also summarizes clinical applications and the main aspects of ongoing randomized controlled trials on hypothermic perfusion. The mechanistic aspects of IRI and hypothermic MP—which include tissue energy replenishment, optimization of mitochondrial function, and the reduction of oxidative and inflammatory damage following reperfusion—will be comprehensively discussed within the context of current preclinical and clinical evidence. Finally, we highlight novel trends and future perspectives in the field of hypothermic MP in the context of recent findings of basic and translational research.

We describe in this review the different types of injuries caused to the biliary tree after liver transplantation. Furthermore, we explain underlying mechanisms and why oxygenated perfusion concepts could not only protect livers, but also repair high-risk grafts to prevent severe biliary complications and graft loss. Accordingly, we summarize experimental studies and clinical applications of machine liver perfusion with a focus on biliary complications after liver transplantation. Key points: (1) Acute inflammation with subsequent chronic ongoing liver inflammation and injury are the main triggers for cholangiocyte injury and biliary tree transformation, including non-anastomotic strictures; (2) Hypothermic oxygenated perfusion (HOPE) protects livers from initial oxidative injury at normothermic reperfusion after liver transplantation. This is a unique feature of a cold oxygenation approach, which is effective also end-ischemically, e.g., after cold storage, due to mitochondrial repair mechanisms. In contrast, normothermic oxygenated perfusion concepts protect by reducing cold ischemia, and are therefore most beneficial when applied instead of cold storage; (3) Due to less downstream activation of cholangiocytes, hypothermic oxygenated perfusion also significantly reduces the development of biliary strictures after liver transplantation.