SRS191 - Metabolomic profiling of pancreas grafts preserved by oxygenated hypothermic machine perfusion

All human islets used in research and for the clinical treatment of diabetes are subject to ischemic damage during pancreas procurement, preservation, and islet isolation. A major factor influencing islet function is exposure of pancreata to cold ischemia during unavoidable windows of preservation by static cold storage (SCS). Improved preservation methods may prevent this functional deterioration. In the present study, we investigated whether pancreas preservation by gaseous oxygen perfusion (persufflation) better preserved islet function versus SCS. Human pancreata were preserved by SCS or by persufflation in combination with SCS. Islets were subsequently isolated, and preparations in each group matched for SCS or total preservation time were compared using dynamic glucose-stimulated insulin secretion as a measure of β-cell function and RNA sequencing to elucidate transcriptomic changes. Persufflated pancreata had reduced SCS time, which resulted in islets with higher glucose-stimulated insulin secretion compared to islets from SCS only pancreata. RNA sequencing of islets from persufflated pancreata identified reduced inflammatory and greater metabolic gene expression, consistent with expectations of reducing cold ischemic exposure. Portions of these transcriptional responses were not associated with time spent in SCS and were attributable to pancreatic reoxygenation. Furthermore, persufflation extended the total preservation time by 50% without any detectable decline in islet function or viability. These data demonstrate that pancreas preservation by persufflation rather than SCS before islet isolation reduces inflammatory responses and promotes metabolic pathways in human islets, which results in improved β cell function.

Donor Heart Preservation by Hypothermic Ex Vivo Perfusion - Improved Recipient Survival and Successful Prolongation of Ischemic Time

IntroductionPost-ischaemic hypothermic machine perfusion (HMP) may be beneficial in recovery of marginal kidney grafts. The full capacity of conventional HMP (with passive oxygenation) to recondition an organ has not been realised. We investigated whether HMP can ameliorate ischemic damage caused by extremely prolonged static cold storage (SCS). MethodsPorcine kidneys underwent 4-h (SCS4,n = 4) or 52-h (SCS52,n = 4) SCS, followed by 10 h of HMP and were then subjected to 2 h of isolated normothermic reperfusion (NRP). ResultsThere was a post-SCS graft weight loss in SCS52 vs SCS4 kidneys. SCS52 kidneys showed viable perfusion dynamics during HMP, with significantly shorter times to reach viable parameters vs SCS4 kidneys (p < 0.027). During NRP SCS52 kidneys demonstrated similar trends in perfusion dynamics, renal function, oxygen consumptions, lactate production, and tubular injury to SCS4 kidneys. ConclusionGraft weight loss after SCS, reducing resistance to perfusion, may facilitate better HMP dynamics and graft reconditioning. Clinicians utilising HMP should be aware of this phenomenon when using HMP in kidneys exposed to extreme periods of SCS. HMP after an extended period of SCS can resuscitate kidneys to a level equitable of viability as those after a short period of SCS. Utilising passive oxygenation however may be limiting such recovery and interventions utilising active oxygenation may provide benefit in such organs.

Better preservation strategies for the storage of donation after circulatory death grafts are essential to improve graft function and to increase the kidney donor pool. We compared continuous normothermic ex vivo kidney perfusion (NEVKP) with hypothermic anoxic machine perfusion (HAMP) and static cold storage (SCS) in a porcine kidney autotransplantation model. Porcine kidneys were exposed to 30 minutes of warm ischemia and then reimplanted following either 16 hours of either SCS, HAMP (LifePort 1.0), or NEVKP before autotransplantation (n = 5 per group). The contralateral kidney was removed. Animals were followed for 8 days. Grafts preserved by NEVKP demonstrated improved function with more rapid recovery compared with HAMP and SCS (mean peak serum creatinine: 3.66 ± 1.33 mg/dL [postoperative d 1 [(POD1)], 8.82 ± 3.17 mg/dL [POD2], and 12.90 ± 2.19 mg/dL [POD3], respectively). The NEVKP group demonstrated significantly increased creatinine clearance calculated on POD3 (63.6 ± 19.0 mL/min) compared with HAMP (13.5 ± 10.3 mL/min, P = 0.001) and SCS (4.0 ± 2.6 mL/min, P = 0.001). Histopathologic injury scores on POD8 were lower in both perfused groups (NEVKP and HAMP, score: 1-1.5) compared with SCS (score: 1-3, P = 0.3), without reaching statistical significance. NEVKP storage significantly improved early kidney function compared with both cold preservation strategies, although HAMP also demonstrates improvement over SCS. NEVKP may represent a novel, superior preservation option for donation after circulatory death renal grafts compared with conventional hypothermic methods.

Machine perfusion is an organ preservation strategy used to improve function over simple storage in a cold environment. This article presents an updated systematic review and meta-analysis of machine perfusion in deceased donor kidneys. RCTs from November 2018 to July 2023 comparing machine perfusion versus static cold storage in kidney transplantation were evaluated for systematic review. The primary outcome in meta-analysis was delayed graft function. A total 19 studies were included, and 16 comparing hypothermic machine perfusion with static cold storage were analysed. The risk of delayed graft function was lower with hypothermic machine perfusion (risk ratio (RR) 0.77, 95% c.i. 0.69 to 0.86), even in kidneys after circulatory death (RR 0.78, 0.68 to 0.90) or brain death (RR 0.73, 0.63 to 0.84). Full hypothermic machine perfusion decreased the risk of delayed graft function (RR 0.69, 0.60 to 0.79), whereas partial hypothermic machine perfusion did not (RR 0.92, 0.69 to 1.22). Normothermic machine perfusion or short-term oxygenated hypothermic machine perfusion preservation after static cold storage was equivalent to static cold storage in terms of delayed graft function and 1-year graft survival. Hypothermic machine perfusion reduces delayed graft function risks and normothermic approaches show promise.

Background and Aims:In liver transplantation, cold preservation induces ischemia, resulting in significant reperfusion injury. Hypothermic oxygenated machine perfusion (HMP-O2) has shown benefits compared to static cold storage (SCS) by limiting ischemia-reperfusion injury. This study reports outcomes using a novel portable HMP-O2 device in the first US randomized control trial.Approach and Results:The PILOT trial (NCT03484455) was a multicenter, randomized, open-label, noninferiority trial, with participants randomized to HMP-O2 or SCS. HMP-O2 livers were preserved using the Lifeport Liver Transporter and Vasosol perfusion solution. The primary outcome was early allograft dysfunction. Noninferiority margin was 7.5%. From April 3, 2019, to July 12, 2022, 179 patients were randomized to HMP-O2 (n=90) or SCS (n=89). The per-protocol cohort included 63 HMP-O2 and 73 SCS. Early allograft dysfunction occurred in 11.1% HMP-O2 (N=7) and 16.4% SCS (N=12). The risk difference between HMP-O2 and SCS was −5.33% (one-sided 95% upper confidence limit of 5.81%), establishing noninferiority. The risk of graft failure as predicted by Liver Graft Assessment Following Transplant score at seven days (L-GrAFT7) was lower with HMP-O2 [median (IQR) 3.4% (2.4–6.5) vs. 4.5% (2.9–9.4), p=0.024]. Primary nonfunction occurred in 2.2% of all SCS (n=3, p=0.10). Biliary strictures occurred in 16.4% SCS (n=12) and 6.3% (n=4) HMP-O2 (p=0.18). Nonanastomotic biliary strictures occurred only in SCS (n=4).Conclusions:HMP-O2 demonstrates safety and noninferior efficacy for liver graft preservation in comparison to SCS. Early allograft failure by L-GrAFT7 was lower in HMP-O2, suggesting improved early clinical function. Recipients of HMP-O2 livers also demonstrated a lower incidence of primary nonfunction and biliary strictures, although this difference did not reach significance.

To underscore the advantages of hypothermic machine perfusion (HMP) in light of the recent discoveries. The phase I clinical trial of liver HMP has suggested superiority over static cold storage (SCS) preservation. The liver transplant arena has gained more sophisticated insights into the molecular mechanism and biomechanical understanding behind the benefits of HMP. Its ability to attenuate ischemia-reperfusion injury translates to reduction in ischemia-reperfusion injury with improved early allograft function. HMP has the potential to predict graft function as well as enable resuscitation of grafts from extended criteria donors. Targeted therapeutic interventions via HMP will further contribute to optimal preservation and reconditioning of the allografts. HMP's advantages do not merely derive from its mechanical forte in maintaining the microvasculature patent. It is also a versatile clinical tool with the ability to deliver metabolic substrates, antioxidants and therapeutic agents to the ex-vivo graft, dilution of waste products generated by inefficient or anaerobic respiration, intraoperative ex-vivo assessment and prediction of the graft's future performance posttransplantation. With demonstrated superiority over SCS, HMP holds promise for expanding the donor pool and becoming the gold standard for liver preservation.

In transplantation, hypothermic machine perfusion (HMP) has been shown to be superior to static cold storage (SCS) in terms of functional outcomes. Ex vivo machine perfusion offers the possibility to deliver drugs or other active substances, such as Mesenchymal Stem Cells (MSCs), directly into an organ without affecting the recipient. MSCs are multipotent, self-renewing cells with tissue-repair capacities, and their application to ameliorate ischemia- reperfusion injury (IRI) is being investigated in several preclinical and clinical studies. The aim of this study was to introduce MSCs into a translational model of hypothermic machine perfusion and to test the efficiency and feasibility of this method. Methods: three rodent kidneys, six porcine kidneys and three human kidneys underwent HMP with 1-5 × 106 labelled MSCs within respective perfusates. Only porcine kidneys were compared to a control group of 6 kidneys undergoing HMP without MSCs, followed by mimicked reperfusion with whole blood at 37 °C for 2 h for all 12 kidneys. Reperfusion perfusate samples were analyzed for levels of NGAL and IL-β by ELISA. Functional parameters, including urinary output, oxygen consumption and creatinine clearance, were compared and found to be similar between the MSC treatment group and the control group in the porcine model. IL-1β levels were higher in perfusate and urine samples in the MSC group, with a median of 285.3 ng/mL (IQR 224.3-407.8 ng/mL) vs. 209.2 ng/mL (IQR 174.9-220.1), p = 0.51 and 105.3 ng/mL (IQR 71.03-164.7 ng/mL) vs. 307.7 ng/mL (IQR 190.9-349.6 ng/mL), p = 0.16, respectively. MSCs could be traced within the kidneys in all models using widefield microscopy after HMP. The application of Mesenchymal Stem Cells in an ex vivo hypothermic machine perfusion setting is feasible, and MSCs can be delivered into the kidney grafts during HMP. Functional parameters during mimicked reperfusion were not altered in treated kidney grafts. Changes in levels of IL-1β suggest that MSCs might have an effect on the kidney grafts, and whether this leads to a positive or a negative outcome on IRI in transplantation needs to be determined in further experiments.

One of the main factors limiting potential uptake of pancreas transplantation, particularly in the United Kingdom, is the shortage of grafts. There has therefore been a recent expansion, particularly in the United Kingdom, in the utilization of grafts from donation after cardiac death (DCD) donors. These grafts are subjected to a greater ischemic insult and are arguably at higher risk of poor functional outcome. Although conventional preservation techniques may be adequate for donation after brain death (DBD) and low-risk DCD pancreases, as the number of DCD pancreas transplants increase and the threshold for rejecting organs decreases, the importance of optimal preservation techniques is going to increase. Over recent years, there have been significant advances in preservation techniques for DCD kidneys, improving the outcome of these marginal grafts. However, the use of such techniques for pancreas preservation is extremely limited and mainly historical. This overview describes the background and results of the established method of pancreas preservation for DBD, namely, cold static storage, and describes the use of the two-layer method. It also reviews pulsatile machine perfusion and normothermic perfusion for pancreas preservation techniques, which have shown promise in the preservation of DCD kidney grafts. The use of these techniques in pancreas preservation is predominantly historical but warrants reevaluation as to the feasibility of applying these techniques to DCD pancreas grafts not only for preservation but also for viability assessment. Further areas for development of pancreas preservation are discussed.

Graft optimization is a necessity in order to develop uterus transplantation from brain-dead donors, as a complement to living donors, as these grafts are rare and the last organs retrieved in multiple organ donation. The aim of this study was to assess the feasibility and interest of hypothermic machine perfusion (HMP) in uterus transplantation using a porcine model; secondary outcomes were the evaluation of the graft's tolerance to a prolonged cold ischaemia time and to find new biomarkers of uterus viability. Fifteen uterus allotransplantations were performed in a porcine model, after 18 h of cold ischaemia, divided in three groups: Static cold storage in a HTK solution, HMP (with the VitaSmart (™) machine Bridge to Life Ltd.) with a UW-MP solution, and static cold storage in a UW solution. The main outcome was macroscopic: uterine arteries pulsatility, recoloration, and bleeding at the cut. Secondary outcomes were histological analyses (Zitkute and inflammation scores), caspase3 immunohistochemistry and plasmatic dosage of biomarkers. 14/15 allotransplantations were performed according to the protocol and met the criteria of macroscopic vitality. Grafts treated with HMP (MP did not show significantly more tissue) damage than the recipient's uterus, contrary to grafts in static cold storage, independently of the solution used. This difference disappeared one and 3 h after uterus transplantation. Plasma dosages before and after uterus transplantation did not allow to identify a new biomarker of uterus viability. HMP is feasible in a porcine model, without inflicting damage on the grafts during cold ischaemia time. Grafts exposed to HMP seemed to better endure reperfusion phenomena, but this advantage did not last over time.

Outcomes of donation after circulatory death kidneys undergoing hypothermic machine perfusion following static cold storage: A UK population-based cohort study.

Ex situ machine perfusion is increasingly used to preserve and assess donor livers before transplantation. Compared with traditional static cold storage (SCS), machine perfusion exposes livers to an additional risk of microbial contamination. However, information on the risk of microbial transmission during machine perfusion is lacking. All livers that underwent either hypothermic oxygenated machine perfusion (HOPE) or normothermic machine perfusion (NMP) in our center between September 2021 and September 2023, and during which samples were taken from SCS fluid and/or machine perfusion solution for microbiological examination, were included in this retrospective, observational clinical study. Microbial transmission was examined from SCS fluid to machine perfusion solution fluid and, subsequently, to recipients of these livers. A total of 90 cases of liver machine perfusion were included: 59 HOPE and 31 NMP. SCS preservation fluid cultures before HOPE or NMP were positive for at least 1 microorganism in 52% of the cases. After HOPE, there were no cases of positive machine perfusion fluid or evidence of microbial transmission to the recipients. After NMP, in 1 (3%) patient Escherichia coli was grown from abdominal drain fluid, the same bacterial strain that was also grown from the SCS preservation fluid before NMP. This E coli was resistant to the antibiotics that are routinely added to the NMP perfusion fluid. The risk of microbial transmission after machine perfusion is very low but not absent. We recommend routine sampling of machine perfusion fluid at the end of the procedure for microbiological analysis.

Liver transplantation (LT) remains the definitive treatment for end-stage liver disease, but it continues to face two major challenges: ischemia-reperfusion injury (IRI), which compromises graft function, and a critical shortage of suitable donor organs. To address the latter, the use of marginal grafts from extended criteria donors (ECD) and donation after circulatory death (DCD) has increased, although these organs are more susceptible to IRI. This review aims to evaluate the current landscape of machine perfusion (MP) technologies-hypothermic, subnormothermic, and normothermic-and their role in improving graft preservation, function, and utilization in LT. MP has emerged as a promising alternative to static cold storage (SCS), offering the potential to assess graft viability and reduce IRI. Hypothermic machine perfusion (HMP), particularly when oxygenated (HOPE), shows protective effects on the biliary system and may reduce ischemic injury, though data on improved graft survival remain limited. Subnormothermic perfusion is associated with enhanced ATP restoration and reduced cold-induced injury in preclinical models but lacks robust clinical validation. Normothermic machine perfusion (NMP) allows real-time functional assessment and supports active metabolism, with clinical trials demonstrating reduced early allograft dysfunction and increased use of marginal grafts. Recent studies suggest that combining techniques, such as HOPE followed by NMP, may offer synergistic benefits, although optimal protocols remain under investigation. Machine perfusion technologies represent a significant advancement in liver transplantation by improving preservation strategies and enabling the use of suboptimal grafts. While NMP appears most promising for functional assessment and extended preservation, HOPE shows particular value in end-ischemic reconditioning. Although MP is not yet a complete replacement for SCS, its potential to improve outcomes and expand the donor pool is increasingly supported by emerging clinical evidence. Further large-scale, randomized trials are essential to determine best practices and establish MP as a standard component of LT protocols.

Impact of static cold storage VS hypothermic machine preservation on ischemic kidney graft: inflammatory cytokines and adhesion molecules as markers of ischemia/reperfusion tissue damage. Our preliminary results

Hypothermic Machine Perfusion Results in a Marginal Kidney Transplant Programme