Ex-situ Perfusion Research Articles

Circulating factors, in both donor and ex-vivo heart perfusion, correlate with heart recovery in a pig model of DCD

BackgroundHeart transplantation with donation after circulatory death and ex-situ heart perfusion offers excellent outcomes and increased transplantation rates. However, improved graft evaluation techniques are required to ensure effective utilization of grafts. Therefore, we investigated circulating factors, both in-situ and ex-situ, as potential biomarkers for cardiac graft quality. MethodsCirculatory death was simulated in anesthetized male pigs with warm ischemic durations of 0, 10, 20, or 30 min. Hearts were explanted and underwent ex-situ perfusion for 3h in an unloaded mode, followed by left ventricular loading for 1h, to evaluate cardiac recovery (outcomes). Multiple donor blood and ex-situ perfusate samples were used for biomarker evaluation with either standard biochemical techniques or nuclear magnetic resonance spectroscopy. ResultsCirculating adrenaline, both in the donor and at 10 min ex-situ heart perfusion, negatively correlated with cardiac recovery (p <0.05 for all). We identified several new potential biomarkers for cardiac graft quality that can be measured rapidly and simultaneously with nuclear magnetic resonance spectroscopy. At multiple timepoints during unloaded ex-situ heart perfusion, perfusate levels of acetone, betaine, creatine, creatinine, fumarate, hypoxanthine, lactate, pyruvate and succinate (p <0.05 for all) significantly correlated with outcomes; the optimal timepoint being 60 min. ConclusionsIn heart donation after circulatory death, circulating adrenaline levels are valuable for cardiac graft evaluation. Nuclear magnetic resonance spectroscopy is of particular interest, as it measures multiple metabolites in a short timeframe. Improved biomarkers may allow more precision and therefore better support clinical decisions about transplantation suitability.

Should advanced perfusion be the standard of care for donation after circulatory death liver transplant?

As an alternative to static cold storage (SCS), advanced perfusion techniques such as normothermic regional perfusion and ex-situ perfusion (normothermic or hypothermic) have emerged as a way to improve the ischemic injury suffered by donation after circulatory death (DCD) livers. Multiple studies have been published that have demonstrated superior post-DCD liver transplant outcomes when using advanced perfusion compared with SCS. In particular, these studies have shown lower rates of ischemic cholangiopathy with advanced perfusion. In addition to the improved post-liver transplant outcomes, studies have also demonstrated higher rates of liver utilization from DCD donors when advanced perfusion is used compared with SCS. Given the high rates of graft loss in patients who develop ischemic cholangiopathy, the significant reduction seen in DCD donor livers that have undergone advanced perfusion represents a key step in more broad utilization of these livers. With such compelling evidence from multiple trials, it seems reasonable to ask the question: should advanced perfusion be the standard of care for DCD liver transplant?

Metabolomic profiling during ex situ normothermic perfusion before heart transplantation defines patterns of substrate utilization and correlates with markers of allograft injury

PURPOSECardiac metabolism is altered in heart failure and ischemia-reperfusion injury states. We hypothesized that metabolomic profiling during ex-situ normothermic perfusion before heart transplantation (HT) would lend insight into myocardial substrate utilization and report on subclinical and clinical allograft dysfunction risk. METHODSMetabolomic profiling was performed on serial samples of ex-situ normothermic perfusate assaying biomarkers of myocardial injury in lactate and cardiac troponin I (TnI) as well as metabolites (66 acylcarnitines, 15 amino acids, non-esterified fatty acids (NEFA), ketones, and 3-hydroxybutyrate). We tested for change over time in injury biomarkers and metabolites, along with differential changes by recovery strategy (donation after circulatory death (DCD) v. donation after brain death (DBD)). We examined associations between metabolites, injury biomarkers, and primary graft dysfunction (PGD). Analyses were performed using linear mixed models adjusted for recovery strategy, assay batch, donor predicted heart mass, and time. RESULTSA total of 176 samples from 92 ex-situ perfusion runs were taken from donors with a mean age of 35 years (SD 11.3) years and a median total ex-situ perfusion time of 234 (IQR 84) minutes. Lactate trends over time differed significantly by recovery strategy, while TnI increased during ex-situ perfusion regardless of DCD v. DBD status. We found fuel substrates were rapidly depleted during ex-situ perfusion, most notably the branched-chain amino acids leucine/isoleucine, as well as ketones, 3-hydroxybutyrate, and NEFA (least squares (LS) mean difference from the first to last timepoint -1.7 - -4.5, FDR q<0.001). Several long-chain acylcarnitines (LCAC), including C16, C18, C18:1, C18:2, C18:3, C20:3, and C20:4 increased during the perfusion run (LS mean difference 0.42 – 0.67, q<0.001). Many LCACs were strongly associated with lactate and TnI. The change over time of many LCACs was significantly different for DCD vs. DBD, suggesting differential trends in fuel substrate utilization by ischemic injury pattern. Changes in leucine/isoleucine, arginine, C12:1-OH/C10:1-DC, and C16-OH/C14-DC were associated with increased odds of moderate-severe PGD. Neither end-of-run nor change in lactate or TnI was associated with PGD. CONCLUSIONMetabolomic profiling of ex-situ normothermic perfusion solution reveals a pattern of fuel substrate utilization that correlates with subclinical and clinical allograft dysfunction. This study highlights a potential role for interventions focused on fuel substrate modification in allograft conditioning during ex-situ perfusion to improve allograft outcomes.

(190) Superior Cardiac Protection in Combined Ex-Situ Perfusion of Heart and Liver: Lessons Learned

(190) Superior Cardiac Protection in Combined Ex-Situ Perfusion of Heart and Liver: Lessons Learned

ORGAN THERAPEUTICS DURING EX-SITU DYNAMIC PRESERVATION. A LOOK INTO THE FUTURE

Machine perfusion has transformed the field of organ preservation permitting longer preservation and open a new avenue for graft treatment. As we are entering an era of “precision medicine”, the organ transplant field is becoming equipped with the tools necessary to personalize and optimize organs designed specifically to withstand injurious pathways that occur during transplantation. Here we highlight recent progress using different treatment strategies during ex-situ perfusion. In the future, customized graft therapy will create a reality where organs will be optimized, personalized, and likely be available on demand.

P2.21: Protection of Normal Metabolic Function of Healthy Pig Livers Following Prolonged Normothermic Ex-situ Perfusion

P2.21: Protection of Normal Metabolic Function of Healthy Pig Livers Following Prolonged Normothermic Ex-situ Perfusion

The role of ex-situ perfusion for thoracic organs.

Ex-situ machine perfusion for both heart (HTx) and lung transplantation (LuTx) reduces ischemia-reperfusion injury (IRI), allows for greater flexibility in geographical donor management, continuous monitoring, organ assessment for extended evaluation, and potential reconditioning of marginal organs. In this review, we will delineate the impact of machine perfusion, characterize novel opportunities, and outline potential challenges lying ahead to improve further implementation. Due to the success of several randomized controlled trials (RCT), comparing cold storage to machine perfusion in HTx and LuTx, implementation and innovation continues. Indeed, it represents a promising interface for organ-specific therapies targeting IRI, allo-immune responses, and graft reconditioning. These mostly experimental efforts range from genetic approaches and nanotechnology to cellular therapies, involving mesenchymal stem cell application. Despite tremendous potential, prior to clinical transition, more data is needed. Collectively, machine perfusion constitutes the vanguard in thoracic organ transplantation research with extensive potential for expanding the donor pool, enhancing transplant outcomes as well as developing novel therapy approaches.

Pediatric heart transplantation following donation after circulatory death, distant procurement, and ex-situ perfusion.

Limited availability of suitable donor hearts remains a challenge to pediatric heart transplantation, contributing to waitlist mortality. Controlled donation after circulatory death (DCD) has demonstrated success in adults. Early series of pediatric DCD heart transplantation using cold storage alone reported significant early mortality. We report a collaboration between 2 centers in the United Kingdom, combining expertise in adult DCD organ retrieval and pediatric transplantation. This retrospective series comprises 6 children (4 male, all >20 kg) undergoing DCD heart transplantation at Great Ormond Street Hospital between 1 February and 30 September 2020, following retrieval with direct procurement and perfusion using portable normothermic machine perfusion by the Royal Papworth Hospital service. Baseline characteristics and 1-year follow-up were compared to 9 children who underwent donation after brain death (DBD) transplants contemporaneously. Mean DCD donor age was 24.67 years and mean DCD recipient age was 13.83 years. Mean functional warm ischemic time was 28.5 minutes and ex-situ heart perfusion time was 280 minutes. Median ICU and hospital stay were 9 and 17 days, respectively. All children survived to 1-year post-transplant. Survival and ICU and hospital stay were similar between the DCD and DBD cohorts. Performing DCD transplants resulted in a 66.7% increase in transplants for children >20 kg at GOSH during the study. This series demonstrates that DCD heart transplant can be performed safely with excellent short-term survival in children. Although the cohort is small, there was no significant difference in major outcomes compared to a DBD cohort.

93. The Effects of Subnormothermic Plasma and Red Blood Cell Based Perfusion on the Function and Viability of Limb Allografts

Purpose: Vascularized composite allotranplantation (VCA) is an evolving area of modern transplantation medicine to reconstruct composite tissue defects such as abdominal wall, face, and limb. One of the challenges in VCA is the narrow window of tolerable ischemia time which limits the number of available donors and imposes a single center transplantation approach. Current standard of care, static cold storage, is only effective for short periods and may result in severe tissue damage and acute rejection when prolonged. Ex-situ perfusion has the potential to overcome these challenges by extending allowable pre-transplantation ischemia time. However, optimum perfusate composition, preservation temperature, and hemodynamic parameters of perfusion are still debated. Over the past decade, the benefits of addition of fresh plasma in hemorrhagic shock resuscitation and attenuation of subsequent reperfusion injury have been recognized. Whether these observations are due to high or low molecular weight (HMW and LMW, respectively) plasma proteins require further investigation. This study investigated the effects of the addition of plasma on the viability of perfused limb allografts with prolonged ischemia time. Methods: All limb transplantations were performed orthotopically between male Lewis rats (200-250 g). In group 1, amputated limbs were transplanted immediately to determine the effects of surgical trauma. In group 2, amputated limbs were preserved at 4°C simulating the clinical standard of care. In other groups, amputated limbs were perfused for 6 hours at 20° to 22°C using one of the following perfusates groups and transplanted to another recipient (in less than 90 minutes): RBC only (G3), RBC + whole plasma (G4), RBC + LMW plasma (G5), RBC + HMW plasma (G6). At 3 months post-transplantation, surviving recipients had end-point assessment using electromyography where compound muscle action potentials (CMAP) and extensor digitorum longus (EDL) muscle force generation were compared to contralateral healthy limb. Muscle and nerve histology samples were collected at the end of EMG testing. Results: Recipients in the group perfused with RBC and HMW plasma proteins had severe reperfusion injury and did not survive beyond 3 days. Among surviving recipients, the addition of whole plasma did not appear to improve amplitude and latency on gastrocnemius and EDL muscles compared to immediately transplanted limbs. Groups perfused with RBC only and RBC with LMW plasma demonstrated excellent amplitude and latencies on both muscle groups, with no significant difference compared to immediately transplanted limbs (p>0.05). The EDL muscle in these groups also generated similar tetanic and twitch forces compared to the immediate transplantation group (p>0.05). Conclusion: Ex-situ perfusion at 20° to 22°C for 6 hours using RBC and LMW plasma appears to protect both fast and slow twitch muscles, and improve axonal regeneration and myelin sheath thickness to a degree comparable with the immediately transplanted group.

Macrophage-Derived Extracellular Vesicles (EVs) Have Functional and Metabolic Effects on Cardiac Grafts in a Preclinical Model of Donation After Circulatory Death (DCD)

<h3>Purpose</h3> Donation after circulatory death (DCD) could substantially increase cardiac graft availability. Cardiac ischemia and reperfusion, as occurs during DCD, can trigger the recruitment of macrophages as well as their polarization from the M0 to M1 state, which may occur in parallel with increased glycogen turnover and pentose phosphate flux. Extracellular vesicles (EVs) can transmit the metabolic status of their parent cell to recipient cells. As post-ischemic glucose metabolism is recognized to affect the cardiac recovery, we investigated the effects of M0 and M1 macrophage-derived EV administration during reperfusion on post-ischemic functional recovery and glucose metabolism using an isolated rat heart model of DCD. <h3>Methods</h3> Hearts isolated from adult, male rats were aerobically perfused for 20 minutes, followed by 27 minutes continued perfusion or 27 minutes of warm, global ischemia, and 60 minutes aerobic reperfusion with or without the intravascular administration of EVs. Four experimental groups were examined: 1) continued perfusion, no EVs (No ISCH); 2) ischemia, no EVs (ISCH); 3) ischemia with EVs from M0 macrophages (ISCH M0); 4) ischemia with EVs from M1 macrophages (ISCH M1). Contractile and metabolic post-ischemic recovery were evaluated. Data are reported as mean ± standard deviation. <h3>Results</h3> Left ventricular (LV) work (heart rate-developed pressure product) at 60 minutes reperfusion was significantly decreased in ISCH (17 ± 3) and ISCH M1 (19 ± 3), but not in ISCH M0 (22 ± 3), compared to No ISCH (26 ± 4 mmHg*beats*min^{−1 *}10³; p<0.05 for both). Rates of glycolysis during reperfusion were not different among groups; however, glycogen content was significantly lower after 60 min reperfusion in ISCH M1 (8 ± 2) compared to ISCH M0 (17 ± 3 μmol/g dry; p<0.05). <h3>Conclusion</h3> The ischemia-induced reduction in left ventricular work was improved with M0-, but not M1-, derived EVs. M0 and M1-derived EVs also differentially affected glucose metabolism; M1-macrophage derived EVs limited glycogen content, possibly by increasing glycogen use or turnover and stimulating recipient cells to mimic parent cell metabolic phenotypes. Circulating EVs in the ex-situ perfusion of DCD hearts may be possible targets for modulating metabolic function and optimizing graft recovery following warm ischemia.

Pediatric Heart Transplantation Following Donation after Circulatory Death, Distant Procurement and Ex-Situ Perfusion

Purpose There remains a significant shortage of organs for children listed for heart transplant. This may be ameliorated by controlled donation after circulatory death (DCD), which has proven success in adults. However, there are numerous challenges in retrieval, assessment and transportation of DCD hearts. We report a unique collaboration between 2 centres, combining expertise in DCD organ retrieval and paediatric transplantation. Methods All families of children over 20 kg listed for heart transplantation were approached for DCD listing; all consented (n = 20). DCD hearts were procured by direct retrieval and perfusion, and then mounted, perfused and assessed on an ex-situ cardiac perfusion machine (Organ Care System (OCS), TransMedics, Inc., Andover, MA), before transfer to the implanting unit. Results Between 1 Feb and 30 Jun 2020, 5 children received a DCD heart (aged 12 to 16 years; two female). Two had previous cardiac surgery, and none had transpulmonary gradient > 8 mmHg. Donor median age was 19.5 years (15-43); three were male. Donor heart mean functional warm ischaemic time was 25 minutes (22 - 28). Mean travel distance was 150 miles (40 - 220) and mean ex-situ perfusion time was 266 minutes (192 - 325). Initial arterial lactate on the OCS was 8.8 mmol/l (4.7-11.7) and venous 8.0 mmol/l (4.7 - 10.1); these fell to 4.7 mmol/l (2.2 - 7.4) and 4.4 mmol/L (2 - 7.1) respectively during transport. No recipients required post-operative mechanical support. Median ITU stay was 8 days (7 - 11) and total hospital stay was 16 days (12 - 21). Pre-discharge echocardiography showed good biventricular function in all recipients except one with mild left and moderate right ventricular dysfunction; all now have excellent graft function. In the same period, 6 DBD transplants were performed in those listed for DCD; although not reaching statistical significance, ventilation, inotrope use, ITU and hospital stay were all shorter in the DCD group. Conclusion The use of previously unavailable DCD hearts has increased transplant activity by 83% in children > 20 kg in this early series. By combining the expertise of one unit in organ retrieval using novel technologies with another in paediatric transplantation, all patients had excellent short-term outcomes. Development of the DCD program to include the use of smaller organs is now a priority for both units.

Primary graft nonfunction in liver transplantation: can we predict? A single-center experience

Background Primary nonfunction (PNF) after liver transplantation is a lethal condition, requiring immediate retransplantation. The precise cause is not well known yet. The aim of this study is to determine the incidence of PNF in our liver recipients, potential risk factors, and outcome. Patients and methods A total of 248 adult liver transplant recipients from 2014 till 2017 at our Transplant Unit were included after excluding nine patients for missing data. Of 248 patients, five (2%) had PNF; two of these patients have been excluded for the purpose of data analysis, as they had machine perfusion (One Liver Assist, and the other Organox). Of the non-PNF 243 recipients, 36 patients receiving livers from donors undergoing in-situ normothermic regional perfusion or ex-situ normothermic perfusion were not included, leaving 207 patients, so the total number was 210 patients. Donor and graft variables studied including age, BMI, serum sodium, cold ischemia time, warm ischemia time, operative time, graft type, and severity of steatosis. Recipient variables included primary liver disease; United Kingdom Model for End-Stage Liver Disease (UKELD) score; posttransplant biochemistry; potential risk factors, including dialysis, inotropes, mechanical ventilation, and pretransplant portal vein thrombosis; hospital and ICU stay; and patient survival. Results UKELD score was the only significant recipient variable (P=0.044). Among donor and graft variables, notably all PNF patients received donation after circulatory death grafts. Posttransplant laboratory values were strikingly worse, clearly indicating more pronounced hepatic and renal impairment in PNF group. Creatinine levels on days 1, 3, and 5 were significantly worse. Hospital and ICU stays were longer for PNF group, with ICU stay significantly longer [median of 7 vs. 2 days (P=0.014)], with no death in PNF group. Conclusion The main risk factors for PNF in our practice were donation after circulatory death grafts and more sick (higher UKELD) recipients.

Transplantation of a heart donated after circulatory death via thoraco-abdominal normothermic regional perfusion and results from the first Spanish case

BackgroundControlled donation after circulatory death (cDCD) has emerged as one of the main strategies for increasing the organ donor pool. Because of the ischemic injury that follows the withdrawal of life-sustaining therapies, hearts from cDCD donors have not been considered for transplantation until recently. The ex-situ perfusion of hearts directly procured from cDCD donors has been used to allow the continuous perfusion of the organ and the assessment of myocardial viability prior to transplantation. Based on our experience with abdominal normothermic regional perfusion in cDCD, we designed a protocol to recover and validate hearts from cDCD donors using thoraco-abdominal normothermic regional perfusion without the utilization of an ex-situ device.Case presentationWe describe the first case of a cDCD heart transplant performed with this approach in Spain. The donor was a 43-year-old asthmatic female diagnosed with severe hypoxic encephalopathy. She was considered a potential cDCD donor and a suitable candidate for multiorgan procurement including the heart via thoraco-abdominal normothermic regional perfusion. The heart recipient was a 60-year-old male diagnosed with amyloid cardiomyopathy. Cold ischemia time was 55 min. The surgery was uneventful.ConclusionsThis case report, the first of its kind in Spain, supports the feasibility of evaluating and successfully transplanting cDCD hearts without the need for ex-situ perfusion based on the use of thoraco-abdominal normothermic regional perfusion opening the way for multiorgan donation in cDCD.

A SINGLE-CENTRE, FIVE-YEAR EXPERIENCE WITH DCD HEART TRANSPLANTATION

Introduction: In 2014 the world first DCD heart transplant with distant procurement (DPP) was performed in Sydney, Australia. Programs are now established in the UK and Europe, as well as a recently commenced multi-centre trial in the USA, with >150 performed worldwide. In established centres, DCD donors account for >20% of heart transplants with excellent early- and mid-term outcomes. Despite these encouraging results, there is still much to be learnt about DCD heart transplantation, and with increasing numbers of hospitals wishing to start DCD heart programs this paper will describe the protocols, lessons learnt and current outcomes from our experience. Materials and Methods: Forty-one DCD heart transplants were performed at our institution between July 2014 and February 2020, utilising a DPP and ex-situ perfusion protocol. Male or female DCD donors under the age of 55 years with no known history of cardiac disease were accepted for retrieval. Recipient selection was based on blood group and cross-matching outcomes, heart mass, and clinical urgency. The donor hearts were perfused on the TransMedics OCS device as outlined in Table 1.For analysis, donor details were collected using electronic donor records (Donate Life, Australia) with recipient data collected from clinical notes and electronic databases at our institution. Results: Patients receiving DCD allografts had excellent outcomes with 97.6 and 95.1% 1- and 5-year survival, respectively.In spite of the similar donor and recipient profiles and a similar rate of utilization of allografts reperfused on the OCS device (74% and 72%, early and late cohort, respectively), the rate of delayed graft function requiring ECMO support has dropped dramatically, from 35% to 14%.Interestingly, prolongation of the functional warm ischemic time was not associated with an increased rate of ECMO, however prolongation of the asystolic warm ischemic time was. A longer agonal period from withdrawal to circulatory arrest was in fact associated with a lower rate of ECMO.Discussion: Over our DCD program experience we have made several changes likely to have contributed to the reduced perioperative ECMO requirement. Primarily, we have adopted a more ‘protective’ approach to the management of the heart on the OCS device, targeting in particular lower coronary flows, with a view to reducing endothelial dysfunction and myocardial oedema. In addition, a design change to the OCS module now allows for a controlled cooling of the heart to 16°C prior to delivery of cardioplegia, proving superior protection to the heart during implantation. Conclusion: DCD heart transplantation clearly provides an additional source of donor allografts with excellent recipient outcomes. Our experiences have reinforced the benefit of a ‘protective’ perfusion strategy on the OCS device, and also raise questions about the true tolerable warm ischemic time, which requires further investigation. Curran Foundation.

Mitochondrial Transplantation for Myocardial Protection in Ex-Situ Perfused Hearts Donated after Cardio-Circulatory Death

Donation after circulatory death (DCD) offers an additional source of cardiac allografts, potentially allowing expansion of the donor pool. In this study, we investigated the efficacy of mitochondrial transplantation to improve myocardial function in DCD hearts. Cardiocirculatory death was induced in Yorkshire pigs (40-50 kg, n=22) by cessation of mechanical ventilation. After 20 minutes of warm ischemia, cardioplegia was administered. The hearts were then reperfused on an ex-situ blood perfusion system for 240 minutes. Following 15 minutes of ex-situ perfusion, mitochondria (5 × 109 in respiration buffer, Mitochondria, n=8) or respiration buffer alone (Vehicle, n=8) were delivered as a single bolus to the coronary arteries. A control group (Sham, n=6) was not subjected to warm ischemia. Hearts were perfused in a resting mode for 120 minutes before converting the system to a continuous isovolumic loading mode. Left ventricular (LV) global function, myocardial oxygen consumption, and arterial lactates were compared by two-way repeated-measures analysis of variance (ANOVA) with Bonferroni adjustment. At the end of 240 minutes of reperfusion, LV peak developed pressure (LVdevP) and dP/dt max were significantly higher in the Sham group than in Vehicle (p<0.001 both) and Mitochondria (p=0.002 and p=0.022, respectively). Hearts treated with mitochondria showed a significantly greater LVdevP (p=0.002), dP/dt max (p<0.001) and myocardial oxygen consumption (p=0.004) compared to Vehicle (Figure A-C). There was no significant difference in myocardial oxygen consumption between Sham and Mitochondria (p>0.05), which was significantly lower in Vehicle (p=0.004). No differences were found in arterial lactate levels (Figure D) (p> 0.05). Mitochondrial transplantation significantly preserves myocardial function and oxygen consumption in DCD hearts, thus providing an additional possible benefit for expanding heart donor pool.

CONTRACTILITY VERSUS METABOLIC CARDIAC ASSESSMENT DURING EX SITU HEAR PERFUSION: A PRE-CLINICAL TRANSPLANT STUDY

Assessment of DCD hearts during ex situ perfusion (ESP) is required to determine suitability of these organs for transplant. To date, metabolic and contractility parameters have not been compared using a full transplant model. We aimed to identify which parameters measured during ESP could predict myocardial performance post-transplantation. Porcine hearts were procured from beating-heart donations (n=6) or following hypoxic circulatory arrest and 15min of warm ischemia (n=6). They underwent 4h of ESP, transplantation, and reperfusion for 3h. Cardiac function was assessed during ESP using left (LV) and right (RV) ventricular pressure-volume loops, and metabolic parameters. We performed linear regressions to identify significant correlations. Several parameters correlated with recovery of cardiac function (Table 1). LV developed pressure was the best predictor of LV Max dP/dt (R2=0.523; p=0.018) while myocardial oxygen consumption (MVO2) at 30min of ESP better predicted LV stroke work (R2=0.625; p=0.006). RV Tau during ESP was the best predictor of post-transplant RV function (R2=0.615; p=0.021). Several metabolic parameters predicted cardiac index. LV Preload Recruitable Stroke Work was the only contractile measurement to predict cardiac index (R2=0.467; p=0.021), while MVO2 at 30min was the best predictor (R2=0.578; p=0.007). Both contractility and metabolic markers measured during ESP were able to predict myocardial performance following transplantation. Assessment of metabolic parameters demonstrated a more significant number of correlations and provided a better evaluation of cardiac index. This is contradictory with previous reports showing contractile evaluation would provide a more accurate estimation of early transplant function. However, contractility parameters were better to evaluate isolated LV and RV function post-transplant. Further studies are needed to clarify this question.

Contractility versus Metabolic Cardiac Assessment during Ex Situ Heart Perfusion: A Pre-Clinical Transplant Study

Purpose Assessment of DCD hearts during ex situ perfusion (ESP) is required to determine suitability of these organs for transplant. To date, metabolic and contractility parameters have not been compared using a full transplant model. We aimed to identify which parameters measured during ESP could predict myocardial performance post-transplantation Methods Porcine hearts were procured from beating-heart donations (n=6) or following hypoxic circulatory arrest and 15min of warm ischemia (n=6). They underwent 4h of ESP, transplantation, and reperfusion for 3h. Cardiac function was assessed during ESP using left (LV) and right (RV) ventricular pressure-volume loops, and metabolic parameters. We performed linear regressions to identify significant correlations Results Several parameters correlated with recovery of cardiac function (Table 1). LV developed pressure was the best predictor of LV Max dP/dt (R2=0.523; p=0.018) while myocardial oxygen consumption (MVO2) at 30min of ESP better predicted LV stroke work (R2=0.625; p=0.006). RV Tau during ESP was the best predictor of post-transplant RV function (R2=0.615; p=0.021). Several metabolic parameters predicted cardiac index. LV Preload Recruitable Stroke Work was the only contractile measurement to predict cardiac index (R2=0.467; p=0.021), while MVO2 at 30min was the best predictor (R2=0.578; p=0.007) Conclusion Both contractility and metabolic markers measured during ESP were able to predict myocardial performance following transplantation. Assessment of metabolic parameters demonstrated a more significant number of correlations and provided a better evaluation of cardiac index. This is contradictory with previous reports showing contractile evaluation would provide a more accurate estimation of early transplant function. However, contractility parameters were better to evaluate isolated LV and RV function post-transplant. Further studies are needed to clarify this question.

Development of an Ex-Situ Limb Perfusion System for a Rodent Model.

Ex-situ perfusion (ESP) is a promising method in preserving vascularized composite tissue allografts (VCAs) with potential to widen donor procurement to larger geographic areas. To optimize the method of preservation, we developed a small animal model to conduct biomolecular investigations. Twenty rat hind limbs (18.2 ± 1.3 g) were procured and connected to our custom-made ESP system. Perfusion pressure and flow parameters were measured with hourly blood gas analysis under near-normothermic (30-35˚C) conditions. Perfusate was prepared with swine hemoglobin (6-9 g/dL) and STEEN Solution. After 6 hours of perfusion, gastrocnemius muscles were evaluated for their histology and metabolomic profiling. Following 3 sets of experiments, perfusion was maintained at an average flow of 0.9 ± 0.24 mL/min and resulted in lactate levels of 3.78 ± 1.02 mmol/L. Metabolomic analysis revealed maintained cellular energy stores (total adenylates perfusion 0.698 ± 0.052 versus baseline 0.685 ± 0.091 umols/ug, p = 0.831), and histologic analysis revealed no evidence of barotrauma or myodegeneration. Rat hind limbs were viable after 6 hours of ESP on our miniaturized ESP system. This study is the first to document the ex-situ hind limb perfusion platform on a rodent model. These experimental findings have potential to guide future research to extend the viable duration of VCA preservation.

Procurement and ex-situ perfusion of isolated slaughterhouse-derived livers as a model of donors after circulatory death.

Ex-situ machine perfusion (MP) techniques are increasingly used in clinical settings, especially on grafts derived from donors after cardiac death (DCD). However, comprehension of biological effects elicited during MP are largely unknown and a substantial number of animal studies are presently focused on this topic. The aim of the present study was to describe a model of DCD based on ex-situ perfusion of liver grafts derived from animals dedicated to food production. Procurement took place within a slaughterhouse facility. A clinically fashioned closed circuit normothermic MP (NMP) was built up. Autologous blood-enriched perfusion fluid was adopted. Perfusate and tissue samples were collected to asses NMP functionality. Grafts were classified as transplantable (LT-G) or not (n-LT) according to clinical criteria, while histopathological analysis was used to confirm graft viability. After cold storage, the liver grafts were connected to the NMP. During the rewarming phase, temperature and flows were progressively increased to reach target values. At the end of NMP, 4 grafts were classified as LT-G and 3 nLT-G. Histology confirmed absence of major damage in LT-G, while diffuse necrosis appeared in nLT-G. Interestingly, in nLT-G an early impairment of hepatocyte respiratory chain, leading to cell necrosis and graft non-viability, was documented for the first time. These parameters, together with indocyanine-green dye and citrate clearance could contribute to graft evaluation in clinical settings. In conclusion, this model provides a promising and reproducible method to replace dedicated experimental animals in DCD and MP research, in line with the 3Rs principles.

Procurement and ex-situ perfusion of isolated slaughterhouse-derived livers as a model of donors after circulatory death_suppl

Ex-situ machine perfusion (MP) techniques are increasingly used in clinical settings, especially on grafts from donors after cardiac death (DCD). However, the biological effects elicited by machine perfusion are largely unknown, and a sub­stantial number of animal studies are presently focused on this topic. The aim of the present study was to describe a model of DCD based on ex-situ perfusion of liver grafts derived from animals used for food production.Procurement took place within a slaughterhouse facility. After cold storage, the liver grafts were perfused with autologous blood-enriched perfusion fluid in a clinically fashioned closed circuit normothermic MP (NMP). During the rewarming phase, temperature and flows were progressively increased to reach target values. Perfusate and tissue samples were collected to assess NMP functionality. Grafts were classified as transplantable (LT-G) or not (nLT-G) according to clinical criteria, and viability was confirmed by histopathological analysis.Four grafts were classified as LT-G and three as nLT-G at the end of NMP. Histology confirmed the absence of major damage in LT-G, while nLT-G presented diffuse necrosis. Interestingly, an early impairment of the hepatocyte respiratory chain, leading to cell necrosis and graft non-viability, was documented in nLT-G for the first time. These parameters, together with indocyanine-green dye and citrate clearance could contribute to graft evaluation in clinical settings. The model provides a promising and reproducible method to replace dedicated experimental animals in research on machine perfusion of DCD grafts in line with the 3Rs principles