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Background: Ex vivo normothermic machine liver perfusion (NMLP) involves artificial cannulation of vessels and generation of flow pressures. This could lead to shear stress-induced endothelial damage, predisposing to vascular complications, or improved preservation of donor artery quality. This study aims to assess the spatial donor hepatic artery (HA) endothelial quality downstream of the cannulation site after end-ischaemic NMLP., Methods: Remnant HA segments from the coeliac trunk up to the gastroduodenal artery branching were obtained after NMLP (n = 15) and after static cold storage (SCS) preservation (n = 15). Specimens were fixed in 10% neutral buffered formalin and sectioned at pre-determined anatomical sites downstream of the coeliac trunk. CD31 immunohistostaining was used to assess endothelial integrity by a 5-point ordinal scale (grade 0: intact endothelial lining, grade 5: complete denudation). Endothelial integrity after SCS was used as a control for the state of the endothelium at commencement of NMP., Results: In the SCS specimens, regardless of the anatomical site, near complete endothelial denudation was present throughout the HA (median scores 4.5-5). After NMLP, significantly less endothelial loss in the distal HA was present compared to SCS grafts (NMLP vs. SCS: median grade 3 vs. 4.5; p = 0.042). In NMLP specimens, near complete endothelial denudation was present at the cannulation site in all cases (median grade: 5), with significantly less loss of the endothelial lining the further from the cannulation site (proximal vs. distal, median grade 5 vs. 3; p = 0.005)., Conclusion: Loss of endothelial lining throughout the HA after SCS and at the cannulation site after NMLP suggests extensive damage related to surgical handling and preservation injury. Gradual improved endothelial lining along more distal sites of the HA after NMLP indicates potential for re-endothelialisation. The regenerative effect of NMLP on artery quality seems to occur to a greater extent further from the cannulation site. Therefore, arterial cannulation for machine perfusion of liver grafts should ideally be as proximal as possible on the coeliac trunk or aortic patch, while the site of anastomosis should preferentially be attempted distal on the common HA., (© 2021. Crown.)

The increasing disparity between the number of patients listed for transplantation and the number of suitable organs has led to the increasing use of extended criteria donors (ECDs). ECDs are at increased risk of developing ischaemia reperfusion injury and greater risk of post-transplant complications. Ischaemia reperfusion injury is a major complication of organ transplantation defined as the inflammatory changes seen following the disruption and restoration of blood flow to an organ-it is a multifactorial process with the potential to cause both local and systemic organ failure. The utilisation of machine perfusion under normothermic (37 degrees Celsius) and hypothermic (4-10 degrees Celsius) has proven to be a significant advancement in organ preservation and restoration. One of the key benefits is its ability to optimise suboptimal organs for successful transplantation. This review is focused on examining ischaemia reperfusion injury and how machine perfusion ameliorates the graft's response to this.

Purpose of Review: To summarize key studies in liver preservation published over the last 3 years and evaluate benefits and limitations of the different perfusion techniques. Selected experimental applications that may be translated to the clinical use will be also discussed., Recent Findings: Normothermic machine perfusion (NMP) has transitioned into clinical practice. Viability assessment is a reliable tool for clinical decision-making, and safety of the back-to-base approach has facilitated adoption of the technology. Data supporting well tolerated use of declined livers after NMP and new protocols selecting complex recipients aim to improve access to suitable organs. Hypothermic machine perfusion (HMP) is showing promising clinical results by decreasing biliary complications in recipients' receiving organs donated after circulatory death (DCD) and improving early graft function in extended criteria organs. Long-term data of HMP on DCD livers shows improved graft survival over standard SCS. Novel approaches utilizing sequential HMP--NMP or ischaemia-free preservation aim to improve outcomes of extended criteria organs., Summary: Machine perfusion for organ transplantation has become an established technique but the field is rapidly evolving. Ongoing research focuses on evaluation of the intervention efficacy and finding optimal indications to use each perfusion strategy according to graft type and clinical scenario., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

This review article focuses on current clinical outcomes with novel perfusion strategies in organ transplantation. Broadly, these approaches can be divided into in situ regional perfusion in the donor and ex situ machine perfusion of individual organs. In both settings, hypothermic and normothermic techniques are in clinical use. Evidence from full text articles, abstracts, and data presented at scientific meetings has been considered. Animal studies have been excluded. The review focuses on kidney, liver, pancreas, heart, and lungs. The level of evidence ranges from quasi-experimental work in human pancreas to multiple meta-analyses of Randomized Controlled Trials for hypothermic machine perfusion of kidneys. The data in this review were presented to experts in organ perfusion and preservation at the National Health Service Blood and Transplant Preservation and Perfusion Future Strategy Summit in London in October 2018. The outcomes of the meeting are discussed in the review after due consideration of the available evidence base.

There is a limited access to liver transplantation, however, many organs are discarded based on subjective assessment only. Here we report the VITTAL clinical trial (ClinicalTrials.gov number NCT02740608) outcomes, using normothermic machine perfusion (NMP) to objectively assess livers discarded by all UK centres meeting specific high-risk criteria. Thirty-one livers were enroled and assessed by viability criteria based on the lactate clearance to levels ≤2.5 mmol/L within 4 h. The viability was achieved by 22 (71%) organs, that were transplanted after a median preservation time of 18 h, with 100% 90-day survival. During the median follow up of 542 days, 4 (18%) patients developed biliary strictures requiring re-transplantation. This trial demonstrates that viability testing with NMP is feasible and in this study enabled successful transplantation of 71% of discarded livers, with 100% 90-day patient and graft survival; it does not seem to prevent non-anastomotic biliary strictures in livers donated after circulatory death with prolonged warm ischaemia.

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., Competing Interests: The machine perfusion research was funded by the Queen Elizabeth Hospital Birmingham Charity’s Liver Foundation UK. YLB is funded by the NIHR Wellcome Trust. Hemopure® was provided free of charge by Hemoglobin Oxygen Therapeutics LLC. The company has not had any role in the study design, data collection, analysis, interpretation or manuscript preparation. The authors are employees of the University Hospital Birmingham or the University of Birmingham and none of them received any payment or have any conflict of interest related to this manuscript. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

Strategies to increase the use of steatotic donor livers are required to tackle the mortality on the transplant waiting list. We aimed to test the efficacy of pharmacological enhancement of the lipid metabolism of human livers during ex situ normothermic machine perfusion to promote defatting and improve the functional recovery of the organs. Because of steatosis, 10 livers were discarded and were allocated either to a defatting group that had the perfusate supplemented with a combination of drugs to enhance lipid metabolism or to a control group that received perfusion fluid with vehicle only. Steatosis was assessed using tissue homogenate and histological analyses. Markers for lipid oxidation and solubilization, oxidative injury, inflammation, and biliary function were evaluated by enzyme-linked immunosorbent assay, immunohistochemistry, and in-gel protein detection. Treatment reduced tissue triglycerides by 38% and macrovesicular steatosis by 40% over 6 hours. This effect was driven by increased solubility of the triglycerides (P = 0.04), and mitochondrial oxidation as assessed by increased ketogenesis (P = 0.008) and adenosine triphosphate synthesis (P = 0.01) were associated with increased levels of the enzymes acyl-coenzyme A oxidase 1, carnitine palmitoyltransferase 1A, and acetyl-coenzyme A synthetase. Concomitantly, defatted livers exhibited enhanced metabolic functional parameters such as urea production (P = 0.03), lower vascular resistance, lower release of alanine aminotransferase (P = 0.049), and higher bile production (P = 0.008) with a higher bile pH (P = 0.03). The treatment down-regulated the expression of markers for oxidative injury as well as activation of immune cells (CD14; CD11b) and reduced the release of inflammatory cytokines in the perfusate (tumor necrosis factor α; interleukin 1β). In conclusion, pharmacological enhancement of intracellular lipid metabolism during normothermic machine perfusion decreased the lipid content of human livers within 6 hours. It also improved the intracellular metabolic support to the organs, leading to successful functional recovery and decreased expression of markers of reperfusion injury., (Copyright © 2019 The Authors. Liver Transplantation published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.)

Hypothermic oxygenated perfusion (HOPE) and normothermic perfusion are seen as distinct techniques of ex situ machine perfusion of the liver. We aimed to demonstrate the feasibility of combining both techniques and whether it would improve functional parameters of donor livers into transplant standards. Ten discarded human donor livers had either 6 hours of normothermic perfusion (n = 5) or 2 hours of HOPE followed by 4 hours of normothermic perfusion (n = 5). Liver function was assessed according to our viability criteria; markers of tissue injury and hepatic metabolic activity were compared between groups. Donor characteristics were comparable. During the hypothermic perfusion phase, livers down-regulated mitochondrial respiration (oxygen uptake, P = 0.04; partial pressure of carbon dioxide perfusate, P = 0.04) and increased adenosine triphosphate levels 1.8-fold. Following normothermic perfusion, those organs achieved lower tissue expression of markers of oxidative injury (4-hydroxynonenal, P = 0.008; CD14 expression, P = 0.008) and inflammation (CD11b, P = 0.02; vascular cell adhesion molecule 1, P = 0.05) compared with livers that had normothermic perfusion alone. All livers in the combined group achieved viability criteria, whereas 40% (2/5) in the normothermic group failed (P = 0.22). In conclusion, this study suggests that a combined protocol of hypothermic oxygenated and normothermic perfusions might attenuate oxidative stress, tissue inflammation, and improve metabolic recovery of the highest-risk donor livers compared with normothermic perfusion alone., (Copyright © 2018 The Authors. Liver Transplantation published by Wiley Periodicals, Inc. on behalf of American Association for the Study of Liver Diseases.)

Long-standing research has shown that increased lipid content in donor livers is associated with inferior graft outcomes posttransplant. The global epidemic that is obesity has increased the prevalence of steatosis in organ donors, to the extent that it has become one of the main reasons for declining livers for transplantation. Consequently, it is one of the major culprits behind the discrepancy between the number of donor livers offered for transplantation and those that go on to be transplanted. Steatotic livers are characterized by poor microcirculation, depleted energy stores because of an impaired capacity for mitochondrial recovery, and a propensity for an exaggerated inflammatory response following reperfusion injury culminating in poorer graft function postoperatively. Ex situ machine perfusion, currently a novel method in graft preservation, is showing great promise in providing a tool for the recovery and reconditioning of marginal livers. Hence, reconditioning these steatotic livers using machine perfusion has the potential to increase the number of liver transplants performed. In this review, we consider the problematic issues associated with fatty livers in the realm of transplantation and discuss pharmacological and nonpharmacological options that are being developed to enhance recovery of these organs using machine perfusion and defatting strategies., (© 2018 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Increased use of high-risk allografts is critical to meet the demand for liver transplantation. We aimed to identify criteria predicting viability of organs, currently declined for clinical transplantation, using functional assessment during normothermic machine perfusion (NMP). Twelve discarded human livers were subjected to NMP following static cold storage. Livers were perfused with a packed red cell-based fluid at 37°C for 6 hours. Multilevel statistical models for repeated measures were employed to investigate the trend of perfusate blood gas profiles and vascular flow characteristics over time and the effect of lactate-clearing (LC) and non-lactate-clearing (non-LC) ability of the livers. The relationship of lactate clearance capability with bile production and histological and molecular findings were also examined. After 2 hours of perfusion, median lactate concentrations were 3.0 and 14.6 mmol/L in the LC and non-LC groups, respectively. LC livers produced more bile and maintained a stable perfusate pH and vascular flow >150 and 500 mL/minute through the hepatic artery and portal vein, respectively. Histology revealed discrepancies between subjectively discarded livers compared with objective findings. There were minimal morphological changes in the LC group, whereas non-LC livers often showed hepatocellular injury and reduced glycogen deposition. Adenosine triphosphate levels in the LC group increased compared with the non-LC livers. We propose composite viability criteria consisting of lactate clearance, pH maintenance, bile production, vascular flow patterns, and liver macroscopic appearance. These have been tested successfully in clinical transplantation. In conclusion, NMP allows an objective assessment of liver function that may reduce the risk and permit use of currently unused high-risk livers., (© 2018 by the American Association for the Study of Liver Diseases.)

While majority of liver transplants worldwide continue to be performed using deceased donor organs, the demands for donor livers continues to exceed the current supply. In an attempt to maximize the number of potentially usable donor livers and expand the current donor pool, there is intense global research by various groups exploring the role of machine perfusion in the liver transplantation, particularly with respect to the machine perfusion of extended-criteria liver donors. In this review, the authors summarize the current field of machine perfusion strategies as applied to deceased donor liver transplantation and how therapeutic targeting of the liver sinusoidal endothelial cell may improve the quality of donor livers., Competing Interests: Disclosure The authors report no conflicts of interest in this work., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Introduction: The use of marginal or extended criteria donor livers is increasing. These organs carry a greater risk of initial dysfunction and early failure, as well as inferior long-term outcomes. As such, many are rejected due to a perceived risk of use and use varies widely between centres. Ex situ normothermic machine perfusion of the liver (NMP-L) may enable the safe transplantation of organs that meet defined objective criteria denoting their high-risk status and are currently being declined for use by all the UK transplant centres., Methods and Analysis: Viability testing and transplantation of marginal livers is an open-label, non-randomised, prospective, single-arm trial designed to determine whether currently unused donor livers can be salvaged and safely transplanted with equivalent outcomes in terms of patient survival. The procured rejected livers must meet predefined criteria that objectively denote their marginal condition. The liver is subjected to NMP-L following a period of static cold storage. Organs metabolising lactate to ≤2.5 mmol/L within 4 hours of the perfusion commencing in combination with two or more of the following parameters-bile production, metabolism of glucose, a hepatic arterial flow rate ≥150 mL/min and a portal venous flow rate ≥500 mL/min, a pH ≥7.30 and/or maintain a homogeneous perfusion-will be considered viable and transplanted into a suitable consented recipient. The coprimary outcome measures are the success rate of NMP-L to produce a transplantable organ and 90-day patient post-transplant survival., Ethics and Dissemination: The protocol was approved by the National Research Ethics Service (London-Dulwich Research Ethics Committee, 16/LO/1056), the Medicines and Healthcare Products Regulatory Agency and is endorsed by the National Health Service Blood and Transplant Research, Innovation and Novel Technologies Advisory Group. The findings of this trial will be disseminated through national and international presentations and peer-reviewed publications., Trial Registration Number: NCT02740608; Pre-results., Competing Interests: Competing interests: RWL and YB receive salary as Wellcome Trust research fellows., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Background: Normothermic machine perfusion of the liver (NMP-L) is a novel technique that preserves liver grafts under near-physiological conditions while maintaining their normal metabolic activity. This process requires an adequate oxygen supply, typically delivered by packed red blood cells (RBC). We present the first experience using an acellular hemoglobin-based oxygen carrier (HBOC) Hemopure in a human model of NMP-L., Methods: Five discarded high-risk human livers were perfused with HBOC-based perfusion fluid and matched to 5 RBC-perfused livers. Perfusion parameters, oxygen extraction, metabolic activity, and histological features were compared during 6 hours of NMP-L. The cytotoxicity of Hemopure was also tested on human hepatic primary cell line cultures using an in vitro model of ischemia reperfusion injury., Results: The vascular flow parameters and the perfusate lactate clearance were similar in both groups. The HBOC-perfused livers extracted more oxygen than those perfused with RBCs (O2 extraction ratio 13.75 vs 9.43 % ×10 per gram of tissue, P = 0.001). In vitro exposure to Hemopure did not alter intracellular levels of reactive oxygen species, and there was no increase in apoptosis or necrosis observed in any of the tested cell lines. Histological findings were comparable between groups. There was no evidence of histological damage caused by Hemopure., Conclusions: Hemopure can be used as an alternative oxygen carrier to packed red cells in NMP-L perfusion fluid.

Purpose of Review: Normothermic machine perfusion of the liver (NMP-L) is a novel technology recently introduced into the practice of liver transplantation. This review recapitulates benefits of normothermic perfusion over conventional static cold storage and summarizes recent publications in this area., Recent Findings: The first clinical trials have demonstrated both safety and feasibility of NMP-L. They have shown that machine perfusion can entirely replace cold storage or be commenced following a period of cold ischaemia. The technology currently allows transplant teams to extend the period of organ preservation for up to 24 h. Results from the first randomized control trial comparing NMP-L with static cold storage will be available soon. One major advantage of NMP-L technology over other parallel technologies is the potential to assess liver function during NMP-L. Several case series have suggested parameters usable for liver viability testing during NMP-L including bile production and clearance of lactic acidosis. NMP-L allows viability testing of high-risk livers. It has shown the potential to increase utilization of donor organs and improve transplant procedure logistics., Summary: NMP-L is likely to become an important technology that will improve organ preservation as well as have the potential to improve utilization of extended criteria donor livers.

The demand for liver transplantation (LT) exceeds supply, with rising waiting list mortality. Utilization of high-risk organs is low and a substantial number of procured livers are discarded. We report the first series of five transplants with rejected livers following viability assessment by normothermic machine perfusion of the liver (NMP-L). The evaluation protocol consisted of perfusate lactate, bile production, vascular flows, and liver appearance. All livers were exposed to a variable period of static cold storage prior to commencing NMP-L. Four organs were recovered from donors after circulatory death and rejected due to prolonged donor warm ischemic times; one liver from a brain-death donor was declined for high liver function tests (LFTs). The median (range) total graft preservation time was 798 (range 724-951) min. The transplant procedure was uneventful in every recipient, with immediate function in all grafts. The median in-hospital stay was 10 (range 6-14) days. At present, all recipients are well, with normalized LFTs at median follow-up of 7 (range 6-19) months. Viability assessment of high-risk grafts using NMP-L provides specific information on liver function and can permit their transplantation while minimizing the recipient risk of primary graft nonfunction. This novel approach may increase organ availability for LT., (© Copyright 2016 The Authors. American Journal of Transplantation published by Wiley Periodicals, Inc. on behalf of American Society of Transplant Surgeons.)

Background: Donation after circulatory death (DCD) liver grafts have supplemented the donor organ pool, but certain adverse outcomes have prevented exploration of the full potential of such organs. The aim of this study was to determine key differences in basic energy metabolism between DCD and donation after brainstem death (DBD) grafts., Methods: Microdialysis samples from DCD and DBD allograft parenchyma from cold storage to 48 h after reperfusion were analysed by colorimetric methods. Interstitial lactate, pyruvate and glycerol levels were measured and the lactate/pyruvate ratio was calculated to estimate energy depletion of the grafts. Histological features of ischaemia and reperfusion injury were assessed., Results: Donor age, extent of steatosis and cold ischaemia time were comparable between ten DCD and 20 DBD organs. DCD grafts had higher levels of interstitial lactate (median 11.6 versus 1.2 mmol/l; P = 0.015) and increased lactate/pyruvate ratio (792 versus 38; P = 0.001) during cold storage. There was no significant difference in glycerol levels between DCD and DBD grafts (225.1 versus 127.5 µmol/l respectively; P = 0.700). Rapid restoration of energy levels with lactate clearance, increased pyruvate levels and reduced lactate/pyruvate ratio was seen following reperfusion of functioning DCD grafts, parallel with levels in DBD grafts. Histology revealed more pronounced glycogen depletion in DCD grafts. Three allografts that failed owing to primary non-function showed energy exhaustion with severe glycogen depletion., Conclusion: Liver grafts from DCD donors exhibited depletion of intracellular energy reserves during cold storage. Failed allografts showed severe energy depletion. Modified organ preservation techniques to minimize organ injury related to altered energy metabolism may enable better utilization of donor organs after circulatory death., (© 2014 BJS Society Ltd. Published by John Wiley & Sons Ltd.)

Background: The impact of the process of liver transplantation on glucose metabolism in the graft was studied using microdialysis., Methods: Microdialysis catheters were inserted into 15 human livers to monitor metabolic changes that took place during organ harvest, the process of backtable preparation of the graft, and following implantation in the recipient where it remained in situ for 48 hours. The cannula was perfused with isotonic solution and hourly samples of perfusate were collected and analyzed., Results: Six livers showed serum biochemical evidence of ischemia/reperfusion (IR) injury with 24 hours aspartate transaminase (AST) levels >2000 IU/L (Group A) whereas the remaining patients showed little evidence of IR injury (Group B). In Group A, lactate levels in the donor microdialysate rose to >6 mM (P < 0.05), were significantly higher during backtable preparation of the liver (>15 mM; P < 0.03), and took longer to normalize in the recipient following implantation (18 vs. 8 hours, P < 0.03) than lactate levels of the livers of patients in Group B who did not develop ischemia reperfusion injury. No significant differences were observed in glucose, pyruvate, or glycerol concentrations between the two groups., Conclusions: Interstitial lactic acidosis in the donor allograft is associated with significant reperfusion injury on implantation.

The outcome of 228 liver grafts in 208 elective adult recipients was assessed to identify any adverse effects of extending the length of cold preservation with UW solution beyond 12 hr. A total of 114 grafts had been preserved < 12 hr--median 9.5 (group I) and 114 > 12 hr--median 14.5 (group II). Intraoperative blood and blood product usage, graft function, hospital stay, and graft and patient survival were identical in the two groups. Biliary strictures occurred in 5.7% of grafts (8 anastomotic (3 group I, 5 group II; 5 hilar/nonanastomotic: 3 group I, 2 group II). There was no graft or patient loss due to the hilar strictures but 1 patient died following reconstruction of an anastomotic stricture (0.4%). It is concluded that extending the cold preservation to approximately 15 hr does not adversely affect outcome after liver transplantation.