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ABSTRACT Background Methods Results Conclusions Machine perfusion is a promising strategy for safeguarding liver transplants donated after cardiac death (DCD). In this study, we developed and validated a novel machine perfusion approach for mitigating risk factors and salvaging severe DCD livers.A novel hypothermic oxygenated perfusion (HOPE) system was developed, incorporating two pumps and an elastic water sac to emulate the functionality of the cardiac cycle. Compared to conventional systems (HOPE S1 and S2), the novel HOPE system (HOPE S3) was evaluated in rats, utilizing healthy livers perfused with methylene blue diluted using Histidine‐tryptophan‐ketoglutarate (HTK) solution or DCD livers subjected to 60 min of warm ischemia without heparin administration. Liver perfusion outcomes were assessed through macroscopic and microscopic evaluations, molecular analyses, and orthotopic liver transplantation (OLT).DCD livers subjected to HOPE systems' perfusion exhibited decreased injury and enhanced survival rates compared to static cold storage following 60 min of warm ischemia (DCD + SCS). The 4‐week post‐transplantation survival rates were 0%, 20%, and 33% in the DCD + SCS, HOPE S1, and HOPE S2 groups, respectively. HOPE S3 conferred protection against hepatocyte and non‐parenchymal cell injury, resulting in a 67% animal survival rate following 60 min of warm donor ischemia (HOPE S3). Assessments of hepatic sinusoidal microcirculation, morphological changes, and molecular alterations in preserved livers further confirmed these findings.The newly devised machine perfusion system can enhance and uniform liver perfusion and may become a promising tool for revitalizing DCD liver grafts afflicted with severe warm ischemic injuries. [ABSTRACT FROM AUTHOR]

Background Cardiac donation after circulatory death is a promising option to increase graft availability. Graft preservation with 30 minutes of hypothermic oxygenated perfusion (HOPE) before normothermic machine perfusion may improve cardiac recovery as compared with cold static storage, the current clinical standard. We investigated the role of preserved nitric oxide synthase activity during HOPE on its beneficial effects. Methods and Results Using a rat model of donation after circulatory death, hearts underwent in situ ischemia (21 minutes), were explanted for a cold storage period (30 minutes), and then reperfused under normothermic conditions (60 minutes) with left ventricular loading. Three cold storage conditions were compared: cold static storage, HOPE, and HOPE with Nω‐nitro‐L‐arginine methyl ester (nitric oxide synthase inhibitor). To evaluate potential confounding effects of high coronary flow during early reperfusion in HOPE hearts, bradykinin was administered to normalize coronary flow to HOPE levels in 2 additional groups (cold static storage and HOPE with Nω‐nitro‐L‐arginine methyl ester). Cardiac recovery was significantly improved in HOPE versus cold static storage hearts, as determined by cardiac output, left ventricular work, contraction and relaxation rates, and coronary flow (P

Machine perfusion is increasingly being tested in clinical transplantation. Despite this, the number of large prospective clinical trials remains limited. The aim of this study was to compare the impact of machine perfusion vs. static cold storage (SCS) on outcomes after liver transplantation. A systematic search of MEDLINE, EMBASE, CINAHL and the Cochrane Central Register of Controlled Trials (CENTRAL) was conducted to identify randomized-controlled trials (RCTs) comparing "post-transplant" outcomes following machine perfusion vs. SCS. Data were pooled using random effect models. Risk ratios (RRs) were calculated for relevant outcomes. The quality of evidence was rated using the GRADE-framework. Seven RCTs were identified (four on hypothermic oxygenated [HOPE] and three on normothermic machine perfusion [NMP]), including a total number of 1,017 patients. Both techniques were associated with significantly lower rates of early allograft dysfunction (NMP: n = 41/282, SCS: n = 74/253, RR 0.50, 95% CI 0.30-0.86, p = 0.01, I2 = 39%; HOPE: n = 45/241, SCS: n = 97/241, RR 0.48, 95% CI 0.35-0.65, p < 0.00001, I2 = 5%). The HOPE approach led to a significant reduction in major complications (Clavien Grade ≥IIIb; HOPE: n = 90/241; SCS: n = 117/241, RR 0.76, 95% CI 0.63-0.93, p = 0.006, I2 = 0%), "re-transplantation" (HOPE: n = 1/163; SCS: n = 11/163; RR 0.21, 95% CI 0.04-0.96, p = 0.04; I2 = 0%) and graft loss (HOPE: n = 7/163; SCS: n = 19/163; RR 0.40, 95% CI 0.17-0.95, p = 0.04; I2 = 0%). Both perfusion techniques were found to 'likely' reduce overall biliary complications and non-anastomotic strictures. Although this study provides the highest current evidence on the role of machine perfusion, outcomes remain limited to a 1-year follow-up after liver transplantation. Comparative RCTs and large real-world cohort studies with longer follow-up are required to enhance the robustness of the data further, thereby supporting the introduction of perfusion technologies into routine clinical practice. CRD42022355252. For a decade, two dynamic perfusion concepts have increasingly been tested in several transplant centres worldwide. We undertook the first systematic review and meta-analysis and identified seven published RCTs, including 1,017 patients, evaluating the effect of machine perfusion (hypothermic and normothermic perfusion techniques) compared to static cold storage in liver transplantation. Both perfusion techniques were associated with lower rates of early allograft dysfunction in the first week after liver transplantation. Hypothermic oxygenated perfusion led to a reduction in major complications, lower "re-transplantation" rates and better graft survival. Both perfusion strategies were found to 'likely' reduce overall biliary complications and non-anastomotic biliary strictures. This study provides the highest current evidence on the role of machine perfusion. Outcomes remain limited to a 1-year post-transplant follow-up. Larger cohort studies with longer follow-up and clinical trials comparing the perfusion techniques are required. This is especially relevant to provide clarity and optimise implementation processes further to support the commissioning of this technology worldwide. [Display omitted] • First systematic review and meta-analysis evaluating the effect of HOPE and NMP compared to SCS in liver transplantation. • HOPE and NMP were associated with lower rates of early allograft dysfunction in the first week after liver transplantation. • HOPE led to a significant reduction in major complications, lower "re-transplantation" rates and better graft survival. • Despite being the highest level of evidence, outcome data on machine perfusion remain limited to 1-year post-transplant follow-up. [ABSTRACT FROM AUTHOR]

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.

Simple Summary: Little is known about the functions and intracellular mechanisms of the endothelial cells in liver grafts. In particular, we still know little about the effect of the most recent machine perfusion techniques currently applied to improve liver transplant outcomes. In this study we analyzed different endothelial markers of both immunohistochemical and gene expression in two different biopsies (for each donor). We observed a severe depression of endothelial trophism in liver grafts, which is restored after reperfusion. This is interesting for further studies on liver grafts, especially considering that the execution of HOPE seems to improve this functional recovery. We propose that our results may help improve the knowledge on graft tissues in order to customize the perfusion techniques prior to transplant and, therefore, improve liver transplant outcomes. The aim of the present study was to evaluate the homeostasis and trophism of liver sinusoidal endothelial cells (LSECs) in vivo in different stages of liver graft donation, in order to understand the effects of graft ischemia and perfusion on LSEC activity in liver grafts. Special attention was paid to grafts that underwent hypothermic oxygenated perfusion (HOPE). Forty-seven donors were prospectively enrolled, and two distinct biopsies were performed in each case: one allocation biopsy (at the stage of organ allocation) and one post-perfusion biopsy, performed after graft implant in the recipients. In all biopsies, immunohistochemistry and RT-PCR analyses were carried out for the endothelial markers CD34, ERG, Nestin, and VEGFR-2. We observed an increase in CD34 immunoreactivity in LSEC during the whole preservation/perfusion period (p < 0.001). Nestin and ERG expression was low in allocation biopsies, but increased in post-perfusion biopsies, in both immunohistochemistry and RT-PCR (p < 0.001). An inverse correlation was observed between ERG positivity and donor age. Our results indicate that LSEC trophism is severely depressed in liver grafts, but it is restored after reperfusion in standard conditions. The execution of HOPE seems to improve this recovery, confirming the effectiveness of this machine perfusion technique in restoring endothelial functions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 development of cholangiopathies after liver transplantation impacts on the quality and duration of graft and patient survival, contributing to higher costs as numerous interventions are required to treat strictures and infections at the biliary tree. Prolonged donor warm ischaemia time in combination with additional cold storage are key risk factors for the development of biliary strictures. Based on this, the clinical implementation of dynamic preservation strategies is a current hot topic in the field of donation after circulatory death (DCD) liver transplantation. Despite various retrospective studies reporting promising results, also regarding biliary complications, there are only a few randomised-controlled trials on machine perfusion. Recently, the group from Groningen has published the first randomised-controlled trial on hypothermic oxygenated perfusion (HOPE), demonstrating a significant reduction of symptomatic ischaemic cholangiopathies with the use of a short period of HOPE before DCD liver implantation. The most likely mechanism for this important effect, also shown in several experimental studies, is based on mitochondrial reprogramming under hypothermic aerobic conditions, e.g. exposure to oxygen in the cold, with a controlled and slow metabolism of ischaemically accumulated succinate and simultaneous ATP replenishment. This unique feature prevents mitochondrial oxidative injury and further downstream tissue inflammation. HOPE treatment therefore supports livers by protecting them from ischaemia-reperfusion injury (IRI), and thereby also prevents the development of post-transplant biliary injury. With reduced IRI-associated inflammation, recipients are also protected from activation of the innate immune system, with less acute rejections seen after HOPE. [ABSTRACT FROM AUTHOR]

Background: Normothermic regional perfusion (NRP) and machine perfusion (MP) are variously used in many European centers to improve the outcomes after liver transplantation from donation after circulatory death (DCD). In Italy, a combination of NRP and subsequent MP has been used since the start of the activity. While NRP is mandatory for every DCD recovery, the subsequent use of MP is left to each center. Methods: We have designed a national survey to investigate practices and policies of these techniques. The questionnaire included 46 questions and was distributed to all the 21 Italian centers using an online form between June and July 2021. Results: The overall response rate was 100%. A local NRP program for controlled Maastricht type 3 DCD was active in 11/21 (52.4%) centers. Organization and availability of personnel were perceived as the main difficulties in starting such a program. Between 2015 and 2020, 119 DCD livers were transplanted, with an overall utilization rate of 69.2%. Pump flow and gross aspect were considered the most reliable parameters in liver selection during NRP. Eight (72.7%) centers adopted subsequent hypothermic MP, 1 (9.1%) center normothermic MP, and the remaining 2 (18.2%) used both MP types. Conclusion: This first snapshot survey shows that NRP with subsequent MP is the most used protocol in Italy for DCD livers, although some heterogeneity exists in the type and purpose of MP between centers. Overall, this policy ensures a high utilization rate, considering the high risk of the DCD donor population in Italy. [ABSTRACT FROM AUTHOR]

Background: After cold storage (CS) and subsequent transplantation, fatty liver is more inclined to develop liver dysfunction and serious postoperative complications in contrast to healthy liver. Hypothermic oxygenated perfusion (HOPE) is a safe and efficacious system, which can repair fatty liver and reduce ischemia‐reperfusion injury. The aim of this research is to investigate the function of Brg1/Nrf2/HO‐1 signaling pathway in the protective effect of HOPE on ischemia‐reperfusion injury of fatty liver. Methods: The mouse fatty liver model was successfully established and verified by hematoxylin‐eosin (HE) staining and oil red O staining. The animals were divided into Control group, CS group and HOPE group. The levels of liver enzyme and lactate in the perfusate were used to measure liver function and cellular metabolism. HE staining and TUNEL staining were utilized to assess the tissue structure and apoptosis, respectively. The levels of superoxide dismutase, malondialdehyde and reactive oxygen species in liver tissue were measured to quantitatively analyze the degree of oxidative stress, and the expressions of protein Brg1, Nrf2 and HO‐1 were detected by means of the western blot. Double‐labeling immunofluorescence was to explore the colocalization of Brg1 and Nrf2. Results: The injury of the liver in the CS group was more serious than that in the control group. However, HOPE could significantly reduce the injury, which was manifested by the improvement of liver function and cellular metabolism, and the lower degrees of apoptosis, necrosis and oxidative stress. Furthermore, the expressions of Brg1, Nrf2 and HO‐1 in the HOPE group were significantly increased than those in the CS group. Conclusions: One‐hour HOPE treatment before reperfusion can obviously improve the injury of fatty liver in mice. The underlying mechanism may be that the interaction of Brg1 and Nrf2 can selectively activate the transcription of HO‐1. [ABSTRACT FROM AUTHOR]

Prolonged warm ischemic is the main cause discarding donated organs after cardiac death. Here, we identified that prolonged warm ischemic time induced disseminated intravascular coagulation and severe capillary vasospasm after cardiac death of rat kidneys. Additionally, we found a significant accumulation of fibrinogen in a hypoxic cell culture of human umbilical vein epithelial cells and in isolated kidneys exposed to prolonged warm ischemic following flushing out of blood. However, pre-flushing the kidney with snake venom plasmin in a 90-minute warm ischemic model maximized removal of micro thrombi and facilitated the delivery of oxygen and therapeutic agents. Application of carbon monoxide-releasing CORM-401 during ex vivo hypothermic oxygenated perfusion achieved multipath protective effects in prolonged warm ischemic kidneys. This led to significant improvements in perfusion parameters, restoration of the microcirculation, amelioration of mitochondrial injury, oxidative stress, and apoptosis. This benefit resulted in significantly prolonged warm ischemic kidney recipient survival rates of 70%, compared with none in those receiving ex vivo hypothermic oxygenated perfusion alone. Significantly, ex vivo hypothermic oxygenated perfusion combined with cytoprotective carbon monoxide releasing CORM-401 treatment meaningfully protected the donated kidney after cardiac death from ischemia-reperfusion injury by reducing inflammation, oxidative stress, apoptosis, and pathological damage. Thus, our study suggests a new combination treatment strategy to potentially expand the donor pool by increasing use of organs after cardiac death and salvaging prolonged warm ischemic kidneys., (Copyright © 2024 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

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.

Objective: The aim of this study was to investigate tumor recurrence after liver transplantation for hepatocellular carcinoma (HCC), with and without hypothermic oxygenated liver perfusion (HOPE) before transplantation. Patients and Methods: We analyzed all liver recipients with HCC, transplanted between January 2012 and September 2019 with donation after circulatory death (DCD) livers after previous end-ischemic HOPE-treatment (n = 70, Center A). Tumor parameters and key confounders were compared to consecutive recipients with HCC, transplanted during the same observation period with an unperfused DBD liver (n = 70). In a next step, we analyzed unperfused DCD (n = 70) and DBD liver recipients (n = 70), transplanted for HCC at an external center (Center B). Results: Tumor parameters were not significantly different between HOPE-treated DCD and unperfused DBD liver recipients at Center A. One-third of patients were outside established tumor thresholds, for example, Milan criteria, in both groups. Despite no difference in tumor load, we found a 4-fold higher tumor recurrence rate in unperfused DBD livers (25.7%, 18/70), compared to only 5.7% (n = 4/70) recipients with tumor recurrence in the HOPE-treated DCD cohort (P = 0.002) in Center A. The tumor recurrence rate was also twice higher in unperfused DCD and DBD recipients at the external Center B, despite significant less cases outside Milan. HOPE-treatment of DCD livers resulted therefore in a 5-year tumor-free survival of 92% in HCC recipients, compared to 73%, 82.7%, and 81.2% in patients receiving unperfused DBD or DCD livers, from both centers. Conclusion: We suggest that a simple machine liver perfusion approach appears advantageous to protect from HCC recurrence after liver transplantation, despite extended tumor criteria. [ABSTRACT FROM AUTHOR]

Objective: The aim of this study was to determine the predictive value of machine perfusate analysis on graft outcome. Background: Ex situ machine perfusion (MP) is gaining increasing interest to potentially repair injured organs and to assess organ function. In the field of liver transplantation, however, no studies exist on reliable prediction of graft function during MP. Methods: We have used hypothermic oxygenated perfusion (HOPE) for donation after circulatory death (DCD) or extended criteria donation after brain death (DBD) human liver grafts during the last 7 years. Our series includes 100 HOPE-treated liver-transplanted patients with an overall tumor-censored 5-year graft survival of 89%. We monitored 54 livers during HOPE in terms of fluorometric analysis of released mitochondrial flavin (flavin mononucleotide, FMN) in the machine perfusate. Results: Real-time optical measurement of mitochondrial FMN release in machine perfusates of livers disclosed a strong correlation with lactate clearance and coagulation factors at day 1 and 2 after transplantation. Receiver-operating characteristic curve analysis revealed an area under the curve (AUROC) of 0.79 [95% confidence interval (CI), 0.62-0.97] for severe allograft dysfunction and for early graft loss (AUROC 0.93, 95% CI, 0.84-1.0). Conclusions: Assessment of flavin, a marker of mitochondrial complex I injury, in the perfusate provides a fast prediction of liver graft function and loss during ex situ MP before implantation. This finding may have high clinical relevance, as liver grafts from extended DBD or DCD donors carry considerable risks for recipients. On-line estimation of outcome before implantation would therefore substantially increase safe utilization of liver grafts. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • End-ischemic HOPE protected against arterial and biliary complications, resulting in significantly less graft loss. • Equivalent outcomes were achieved with HOPE as with primary DBD liver transplants. • HOPE after cold storage is a simple and effective method to treat high-risk DCD livers prior to implantation. Background & Aims Donation after circulatory death (DCD) liver transplantation is known for potentially worse outcomes because of higher rates of graft non-function or irreversible cholangiopathy. The impact of machine liver perfusion techniques on these complications remains elusive. We aimed to provide data on 5-year outcomes in patients receiving DCD liver transplants, after donor organs had been treated by hypothermic oxygenated perfusion (HOPE). Methods Fifty HOPE-treated DCD liver transplants performed in Zurich between 2012 and 3/2017 were matched with 50 primary donation after brain death (DBD) liver transplants, and with 50 untreated DCD liver transplants in Birmingham. Match factors focussed on short cold ischaemia, comparable recipient age and low recipient laboratory model for end-stage liver disease scores. Primary endpoints were post-transplant complications, and non-tumour-related patient death or graft loss. Results Despite extended donor warm ischaemia, HOPE-treated DCD liver transplants achieved similar overall graft survival, compared to standard DBD liver transplants. Particularly, graft loss due to any non-tumour-related causes occurred in 8% (4/50) of cases. In contrast, untreated DCD livers resulted in non-tumour-related graft failure in one-third (16/50) of cases (p = 0.005), despite significantly (p <0.001) shorter functional donor warm ischaemia. Five-year graft survival, censored for tumour death, was 94% for HOPE-treated DCD liver transplants vs. 78% in untreated DCD liver transplants (p = 0.024). Conclusions The 5-year outcomes of HOPE-treated DCD liver transplants were similar to those of DBD primary transplants and superior to those of untreated DCD liver transplants, despite much higher risk. These results suggest that a simple end-ischaemic perfusion approach is very effective and may open the field for safe utilisation of extended DCD liver grafts. Lay summary Machine perfusion techniques are currently being introduced into the clinic, with the aim of optimising injured grafts prior to implantation. While short-term effects of machine liver perfusion have been frequently reported in terms of hepatocellular enzyme release and early graft function, the long-term benefit on irreversible graft loss has been unclear. Herein, we report on 5-year graft survival in donation after cardiac death livers, treated either by conventional cold storage, or by 1–2 h of hypothermic oxygenated perfusion (HOPE) after cold storage. Graft loss was significantly less in HOPE-treated livers, despite longer donor warm ischaemia times. Therefore, HOPE after cold storage appears to be a simple and effective method to treat high-risk livers before implantation. [ABSTRACT FROM AUTHOR]

Background & Aims Pretreatment of marginal organs by perfusion is a promising opportunity to make more organs available for transplantation. Protection of human donation after cardiac death (DCD) livers by a novel machine perfusion technique, hypothermic oxygenated perfusion (HOPE), was recently established. Herein, we tested whether HOPE is also useful for fatty liver grafts, using a rodent transplant model. Methods Rats were fed over three weeks with a special methionine-choline-deficient diet (MCDD) to induce severe hepatic macrosteatosis (≥60%). Afterwards, livers were transplanted with either minimal or 12 h cold storage. Additional liver grafts were treated after 12 h cold storage with 1 h HOPE before transplantation. Graft injury after orthotopic liver transplantation (OLT) was assessed in terms of oxidative stress, damage-associated molecular patterns release, toll-like receptor-4 activation, cytokine release, endothelial activation, and the development of necrosis and fibrosis. Results Implantation of cold stored macrosteatotic liver grafts induced massive reperfusion injury after OLT, compared to controls (non-fatty livers). HOPE treatment after cold storage failed to change the degree of steatosis itself, but markedly decreased reperfusion injury after OLT, as detected by less oxidative stress, less nuclear injury, less Kupffer- and endothelial cell activation, as well as less fibrosis within one week after OLT. Protective effects were lost in the absence of oxygen in the HOPE perfusate. Conclusion HOPE after cold storage of fatty livers prevents significant reperfusion injury and improves graft function, comparable to the effects of HOPE in DCD livers and DCD kidneys. HOPE treatment is easy and may become a universal concept to further expand the donor pool. Lay summary An increasing number of donor livers contain fat. It is important to harness marginal livers, which may contain fat, as the stock of donor livers is limited. Hypothermic oxygenated perfusion (HOPE) prevents reperfusion injury and improves liver graft function. HOPE offers a simple and low-cost option for treating liver grafts in transplant centers, even after cold storage, instead of transporting machines to the place of procurement. HOPE could be used globally to expand the donor pool. [ABSTRACT FROM AUTHOR]

Background: Normothermic regional perfusion (NRP) and hypothermic-oxygenated-perfusion (HOPE), were both shown to improve outcomes after liver transplantation from donors after circulatory death (DCD). Comparative clinical and mechanistical studies are however lacking., Methods: A rodent model of NRP and HOPE, both in the donor, was developed. Following asystolic donor warm ischemia time (DWIT), the abdominal compartment was perfused either with a donor-blood-based-perfusate at 37 °C (NRP) or with oxygenated Belzer-MPS at 10 °C (donor-HOPE) for 2 h. Livers were then procured and underwent 5 h static cold storage (CS), followed by transplantation. Un-perfused and HOPE-treated DCD-livers (after CS) and healthy livers (DBD) with direct implantation after NRP served as controls. Endpoints included the entire spectrum of ischemia-reperfusion-injury., Findings: Healthy control livers (DBD) showed minimal signs of inflammation during 2 h NRP and achieved 100% posttransplant recipient survival. In contrast, DCD livers with 30 and 60 min DWIT suffered from greater mitochondrial injury and inflammation as measured by increased perfusate Lactate, FMN- and HMGB-1-levels with subsequent Toll-like-receptor activation during NRP. In contrast, donor-HOPE (instead of NRP) led to significantly less mitochondrial-complex-I-injury and inflammation. Results after donor-HOPE were comparable to ex-situ HOPE after CS. Most DCD-liver recipients survived when treated with one HOPE-technique (86%), compared to only 40% after NRP (p = 0.0053). Following a reduction of DWIT (15 min), DCD liver recipients achieved comparable survivals with NRP (80%)., Interpretation: High-risk DCD livers benefit more from HOPE-treatment, either immediately in the donor or after cold storage. Comparative prospective clinical studies are required to translate the results., Funding: Funding was provided by the Swiss National Science Foundation (grant no: 32003B-140776/1, 3200B-153012/1, 320030-189055/1, and 31IC30-166909) and supported by University Careggi (grant no 32003B-140776/1) and the OTT (grant No.: DRGT641/2019, cod.prog. 19CT03) and the Max Planck Society. Work in the A.G. laboratory was partially supported by the NIH R01NS112381 and R21NS125466 grants., Competing Interests: Declaration of interests The authors have no conflicts of interest to disclose related to this study. The experiments and analysis in the study consortium were funded by the listed sources, including the Swiss National Science Foundation, the University Careggi, the OTT, the Max Planck Society and the NIH., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: The aim of this study was to investigate novel and easily applicable preservation perfusion techniques in kidney grafts obtained from donors after circulatory death (DCD). Background: A novel perfusion approach, hypothermic oxygenated perfusion (HOPE), used for DCD liver grafts, is based on cold perfusion for 1 hour by an oxygenated solution before implantation. Here, we aimed to test HOPE in a rodent model of kidney grafts associated with substantial warm ischemia. Methods: Rat kidneys were exposed to 30 minutes in situ warm ischemia, without application of heparin. Kidneys were removed and cold stored for 4 and 18 hours, mimicking DCD organ procurement and conventional preservation. In additional experiments, kidneys were normothermically perfused with oxygenated blood for 1 hour after cold storage. In a third group, kidneys were perfused by HOPE for 1 hour after cold storage. In each group, orthotopic kidney transplantation was performed after recipient nephrectomy. Results: HOPE-treated DCD kidneys showed dramatically better function after transplantation, than cold-stored grafts in terms of nuclear injury, macrophage activation, endothelium activation, tubulus damage, and graft function. A short period of warm oxygenated perfusion before implantation improved graft quality as compared with cold storage, but was significantly less effective in all endpoints compared with HOPE. The effect of HOPE was dependent on perfusate oxygenation in the cold. Conclusions: HOPE of DCD kidneys was superior to other clinically used preservation approaches, consistent to earlier results in livers. On the basis of this, we assume a strong and generalized effect on solid organ viability by HOPE before transplantation. These results justify a clinical trial. [ABSTRACT FROM AUTHOR]

Hypothermic oxygenated perfusion (HOPE) has become widespread for the preservation of liver grafts, making tangled the relationship among the use of extended criteria donors (ECD), graft histology and transplant outcome. To prospectively validate the impact of the graft histology on transplant outcome in recipient receiving liver grafts from ECD after HOPE. Ninety-three ECD grafts were prospectively enrolled; 49 (52.7 %) were perfused with HOPE according to our protocols. All clinical, histological and follow-up data were collected. Grafts with portal fibrosis stage ≥ 3 according to Ishak's (evaluated with Reticulin stain) had a significantly higher incidence of early allograft dysfunction (EAD) and 6-month-dysfunction (p = 0.026 and p = 0.049), with more days in Intensive Care Unit (p = 0.050). Lobular fibrosis correlated with post-liver transplant kidney function (p = 0.019). Moderate-to-severe chronic portal inflammation was correlated with graft survival on both multivariate and univariate analyses (p < 0.001), but this risk factor is sensibly reduced by the execution of HOPE. The use of liver grafts with portal fibrosis stage ≥ 3 implies a higher risk of post-transplant complications. Portal inflammation represents an important prognostic factor as well, but the execution of HOPE represents a valid tool to improve graft survival. [ABSTRACT FROM AUTHOR]

Background: Exposure of donor liver grafts to prolonged periods of warm ischemia before procurement causes injuries including intrahepatic cholangiopathy, which may lead to graft loss. Due to unavoidable prolonged ischemic time before procurement in donation after cardiac death (DCD) donation in 1 participating center, each liver graft of this center was pretreated with the new machine perfusion "Hypothermic Oxygenated PErfusion" (HOPE) in an attempt to improve graft quality before implantation. Methods: HOPE-treated DCD livers (n=25) were matched and compared with normally preserved (static cold preservation) DCD liver grafts (n=50) from 2 well-established European programs. Criteria for matching included duration of warm ischemia and key confounders summarized in the balance of risk score. In a second step, perfused and unperfused DCD livers were compared with liver grafts from standard brain dead donors (n=50), also matched to the balance of risk score, serving as baseline controls. Results: HOPE treatment of DCD livers significantly decreased graft injury compared with matched cold-stored DCD livers regarding peak alanineaminotransferase (1239 vs 2065 U/L, P=0.02), intrahepatic cholangiopathy (0% vs 22%, P=0.015), biliary complications (20% vs 46%, P=0.042), and 1-year graft survival (90% vs 69%, P=0.035). No graft failure due to intrahepatic cholangiopathy or nonfunction occurred in HOPE-treated livers, whereas 18% of unperfused DCD livers needed retransplantation. In addition, HOPE-perfused DCD livers achieved similar results as control donation after brain death livers in all investigated endpoints. Conclusions: HOPE seems to offer important benefits in preserving higherrisk DCD liver grafts. [ABSTRACT FROM AUTHOR]

Background & Aims A variety of liver perfusion techniques have been proposed to protect liver grafts prior to implantation. We compared hypothermic and normothermic oxygenated perfusion techniques in a rat liver transplant model, using higher risk grafts obtained after cardiac arrest (DCD). Methods Rat livers were subjected to 30 or 60 min in situ warm ischemia, without application of heparin. Livers were excised and stored for 4 h at 4 °C, mimicking DCD organ procurement, followed by conventional organ transport. In experimental groups, DCD liver grafts received a 4 h normothermic oxygenated perfusion through the portal vein and the hepatic artery instead of cold storage. The perfusate consisted of either full blood or leukocyte-depleted blood (normothermic groups). Other livers underwent hypothermic oxygenated perfusion (HOPE) for 1 h after warm ischemia and 4 h cold storage (HOPE group). Liver injury was assessed during machine perfusion and after isolated liver reperfusion, and by orthotopic liver transplantation (OLT). Results DCD livers, subjected to normothermic perfusion, disclosed reduced injury and improved survival compared to cold storage after limited warm ischemia of 30 min (70%; 7/10), but failed to protect from lethal injury in grafts exposed to 60 min warm ischemia (0%; 0/10). This finding was consistent with Kupffer and endothelial cell activation in cold stored and normothermic perfused livers. In contrast, HOPE protected from hepatocyte and non-parenchymal cell injury and led to 90% (9/10) and 63% (5/8) animal survival after 30 and 60 min of donor warm ischemia, respectively. Conclusions This is the first evidence that HOPE is superior to normothermic oxygenated perfusion in a clinically relevant model through modulation of the innate immunity and endothelial cell activation. [ABSTRACT FROM AUTHOR]

Background & Aims: Due to ethical rules in most countries, long ischemia times are unavoidable prior to organ procurement of donors without a heartbeat, which can cause early graft failure after liver transplantation or late biliary strictures. Hypothermic oxygenated machine perfusion, used prior to graft implantation, may rescue these high risk organs. Methods: Eight patients with end stage liver diseases received human livers, obtained after controlled cardiac death (Maastricht category III), with a median donor warm ischemia time of 38min, followed by a standard cold flush and static storage at 4°C. Hypothermic oxygenated perfusion (HOPE) was applied for 1–2h prior to implantation through the portal vein. The HOPE-perfusate was cooled at 10°C and oxygenated (pO2 60kPa) using an ECOPS device (Organ Assist®). Perfusion pressure was maintained below 3mmHg. Results: Each machine perfused liver graft disclosed excellent early function after transplantation. The release of liver enzymes and kidney function, as well as ICU and hospital stays were comparable or better than in matched liver grafts from brain death donors. No evidence of intrahepatic biliary complications could be documented within a median follow up of 8.5months. Conclusions: This is the first report on cold machine perfusion of human liver grafts obtained after cardiac arrest and subsequent transplantation. Application of HOPE appears well tolerated, easy-to-use, and protective against early and later injuries. [ABSTRACT FROM AUTHOR]

Background & Aims: The use of livers from donors after cardiac arrest (DCD) is increasing in many countries to overcome organ shortage. Due to additional warm ischemia before preservation, those grafts are at higher risk of failure and bile duct injury. Several competing rescue strategies by machine perfusion techniques have been developed with, however, unclear effects on biliary injury. We analyze the impact of an end-ischemic Hypothermic Oxygenated PErfusion (HOPE) approach applied only through the portal vein for 1h before graft implantation. Methods: Rat livers were subjected to 30-min in situ warm ischemia, followed by subsequent 4-h cold storage, mimicking DCD-organ procurement and conventional organ transport. Livers in the HOPE group underwent also passive cold storage for 4h, but were subsequently machine perfused for 1h before implantation. Outcome was tested by liver transplantation (LT) at 12h after implantation (n=10 each group) and after 4weeks (n=10 each group), focusing on early reperfusion injury, immune response, and later intrahepatic biliary injury. Results: All animals survived after LT. However, reperfusion injury was significantly decreased by HOPE treatment as tested by hepatocyte injury, Kupffer cell activation, and endothelial cell activation. Recipients receiving non-perfused DCD livers disclosed less body weight gain, increased bilirubin, and severe intrahepatic biliary fibrosis. In contrast, HOPE treated DCD livers were protected from biliary injury, as detected by cholestasis parameter and histology. Conclusions: We demonstrate in a DCD liver transplant model that end-ischemic hypothermic oxygenated perfusion is a powerful strategy for protection against biliary injury. [Copyright &y& Elsevier]

• HOPE combined with TJ-5 ameliorates DCD liver IRI. • HOPE combined with TJ-5 reduces oxidative stress, inflammation and apoptosis. • HOPE combined with TJ-5 inhibits TLR/MyD88 signaling during DCD liver IRI. Ischemia-reperfusion injury (IRI) is an inevitable pathological process during donation after circulatory death (DCD) liver transplantation, which contributes to serious damage to the graft. Oxidative stress, inflammation and apoptosis are all fatal causes of IRI of the liver. Hypothermic oxygenated perfusion (HOPE), as an emerging dynamic preservation technology, has a more significant effect on reducing DCD liver IRI than static cold storage (CS) mainly by regulating oxidative stress and inflammation. To further enhance the effect of HOPE and reveal its underlying mechanisms, investigators have recently combined HOPE with various methods. Excessive activation of the TLR/MyD88 signaling pathway can lead to severe immune inflammatory response. TJ-M2010-5 (TJ-5), a novel thiazaol-aminoramification MyD88 inhibitor, plays an essential role in the treatment of various diseases or pathological injuries in mice, such as hepatocellular carcinoma, acute liver injury and myocardial IRI. However, little is known about the role of TJ-5 in HOPE alleviating DCD liver IRI. Herein, we sought to investigate the role of HOPE combined with TJ-5 in reducing DCD liver IRI. We found that HOPE combined with TJ-5 significantly reduced oxidative stress, lessened inflammation, and decreased apoptosis during DCD liver IRI. Furthermore, HOPE combined with TJ-5 exerted their effects by inhibiting the TLR/MyD88 signaling pathway. Overall, these results demonstrated that HOPE combined with TJ-5 has a significant effect on alleviating DCD liver IRI. Therefore, the combined application of HOPE and TJ-5 may be an available and valid treatment option for DCD liver IRI. [ABSTRACT FROM AUTHOR]