Ex-vivo Perfusion Research Articles

Connexin43 Inhibition Prevents Human Vein Grafts Intimal Hyperplasia.

Venous bypass grafts often fail following arterial implantation due to excessive smooth muscle cells (VSMC) proliferation and consequent intimal hyperplasia (IH). Intercellular communication mediated by Connexins (Cx) regulates differentiation, growth and proliferation in various cell types. Microarray analysis of vein grafts in a model of bilateral rabbit jugular vein graft revealed Cx43 as an early upregulated gene. Additional experiments conducted using an ex-vivo human saphenous veins perfusion system (EVPS) confirmed that Cx43 was rapidly increased in human veins subjected ex-vivo to arterial hemodynamics. Cx43 knock-down by RNA interference, or adenoviral-mediated overexpression, respectively inhibited or stimulated the proliferation of primary human VSMC in vitro. Furthermore, Cx blockade with carbenoxolone or the specific Cx43 inhibitory peptide 43gap26 prevented the burst in myointimal proliferation and IH formation in human saphenous veins. Our data demonstrated that Cx43 controls proliferation and the formation of IH after arterial engraftment.

Carbonic Anhydrase Protects Fatty Liver Grafts against Ischemic Reperfusion Damage.

Carbonic anhydrases (CAs) are ubiquitous metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and a proton. CAs are involved in numerous physiological and pathological processes, including acid-base homeostasis, electrolyte balance, oxygen delivery to tissues and nitric oxide generation. Given that these processes are found to be dysregulated during ischemia reperfusion injury (IRI), and taking into account the high vulnerability of steatotic livers to preservation injury, we hypothesized a new role for CA as a pharmacological agent able to protect against ischemic damage. Two different aspects of the role of CA II in fatty liver grafts preservation were evaluated: 1) the effect of its addition to Institut Georges Lopez (IGL-1) storage solution after cold ischemia; 2) and after 24h of cold storage followed by two hours of normothermic ex-vivo perfusion. In all cases, liver injury, CA II protein concentration, CA II mRNA levels and CA II activity were determined. In case of the ex-vivo perfusion, we further assessed liver function (bile production, bromosulfophthalein clearance) and Western blot analysis of phosphorylated adenosine monophosphate activated protein kinase (AMPK), mitogen activated protein kinases family (MAPKs) and endoplasmic reticulum stress (ERS) parameters (GRP78, PERK, IRE, eIF2α and ATF6). We found that CA II was downregulated after cold ischemia. The addition of bovine CA II to IGL-1 preservation solution efficiently protected steatotic liver against cold IRI. In the case of reperfusion, CA II protection was associated with better function, AMPK activation and the prevention of ERS and MAPKs activation. Interestingly, CA II supplementation was not associated with enhanced CO2 hydration. The results suggest that CA II modulation may be a promising target for fatty liver graft preservation.

2. Challenge of organ shortage is enormous, but we’re heading towards a future where we have time and ability to improve and augment organs before more and better transplants

Over the past four decades, Lung transplant has evolved to a successful modality for treating end stage lung disease in selected patients. However, lung transplant is not problem-free. Primary graft dysfunction is the most severe form of post transplant lung ischemia and reperfusion injury and is correlated with early and late mortality after lung transplantation. Many studies have been performed to optimize the donor lung preservation in order to minimize ischemic injury to the donor lung. However, no major breakthrough has been made in donor lung preservation. Cold flush and storage has been accepted as a standard preservation of donor lungs in clinical lung transplantation. With the concept of living organ transplant, that is, heart beating, lung breathing, kidney making urine and liver producing bile, Transmedics developed a portable ex-vivo warm perfusion system that allows the donor lungs functioning and “breathing” in a near-physiologic state outside the body during transport, thus minimizing the donor lung ischemic time. Gregor Warnecke and his colleagues reported first-in-man experience of the portable OCS lung device for concomitant preservation, assessment and transport of donor lungs. It demonstrated that lungs can be safely preserved with the OCS lung, resulting in complete organ use and successful transplantation in their high-risk recipients. As one of the major lung transplant centers participating in the INSPIRE trial, the lung transplant team at Ronald Reagan UCLA medical center successfully performed the nation’s first “breathing lung” transplant in November, 2012. The INSPIRE trial has been conducted in leading lung transplant centers in Europe, the United States, Australia, and Canada. The interim results from the INSPIRE trial are suggesting significant improvement in post lung transplant clinical outcomes using the TransMedics OCS lung portable perfusion and ventilation system. In addition, other important clinical parameters such as in-hospital mortality, 6-month survival, rate of lung-related complications, time on mechanical ventilation and ICU stay may be better in the OCS group as compared to cold storage. With OCS, surgeons may accept suboptimal organs or those obtained from non-heart-beating donors through assessment of donor lung function at donor site, thus expending the donor pool without compromising patient outcome. The OCS lung enables surgeons to continuously monitor and assess the lung function and quality until transplantation. By doing so, it makes the recipient transplant team coordinate with the procure team easier, the timing of recipient incision more flexible. It allows two separate single lung transplantation that share lungs from one donor proceeding one after the other. OCS lung also allows surgeons to procure lungs over longer distance, leading to development of an international organ sharing system. We believe that this OCS lung technology has a potential of bringing human lung transplantation to a new cornerstone.

Rivaroxaban transfer across the dually perfused isolated human placental cotyledon

The purpose of this study was to determine the rate and extent of rivaroxaban transfer across the term human placenta and determine whether passive diffusion was the primary mechanism involved in this transfer. The transplacental pharmacokinetics of rivaroxaban was determined with the ex-vivo placenta perfusion model. Rivaroxaban was added to the maternal or fetal circulation only (250 ng/mL). Additional experiments were conducted under equilibrative conditions with the addition of rivaroxaban to both the maternal and fetal circulations (250 ng/mL). Rivaroxaban concentrations were measured with the use of liquid chromatography-tandem mass spectrometry. There was rapid transfer of rivaroxaban across the human placenta in both the maternal-to-fetal and fetal-to-maternal directions, as evidenced by transfer ratios of 0.69 (interquartile range, 0.58-0.73; n = 5) and 0.69 (interquartile range, 0.67-0.71; n = 2), respectively, after 3 hours. Under equilibrative conditions (n = 2), rivaroxaban concentrations remained relatively constant, which suggests that rivaroxaban crosses the placenta down a concentration gradient. This is the first direct evidence of rivaroxaban transfer across the term human placenta from both the mother-to-fetus and fetus-to-mother directions. Our results document that unbound rivaroxaban rapidly crosses the placental barrier via passive diffusion. However, because rivaroxaban is highly bound to plasma proteins (up to 95%), this suggests that the amount of unbound drug that may reach the fetus is likely much lower. Additional studies will need to explore its safety before administering rivaroxaban to a pregnant woman.

Adult heart transplantation with distant procurement and ex-vivo preservation of donor hearts after circulatory death: a case series

Orthotopic heart transplantation is the gold-standard long-term treatment for medically refractive end-stage heart failure. However, suitable cardiac donors are scarce. Although donation after circulatory death has been used for kidney, liver, and lung transplantation, it is not used for heart transplantation. We report a case series of heart transplantations from donors after circulatory death. The recipients were patients at St Vincent's Hospital, Sydney, Australia. They received Maastricht category III controlled hearts donated after circulatory death from people younger than 40 years and with a maximum warm ischaemic time of 30 min. We retrieved four hearts through initial myocardial protection with supplemented cardioplegia and transferred to an Organ Care System (Transmedics) for preservation, resuscitation, and transportation to the recipient hospital. Three recipients (two men, one woman; mean age 52 years) with low transpulmonary gradients (<8 mm Hg) and without previous cardiac surgery received the transplants. Donor heart warm ischaemic times were 28 min, 25 min, and 22 min, with ex-vivo Organ Care System perfusion times of 257 min, 260 min, and 245 min. Arteriovenous lactate values at the start of perfusion were 8·3-8·1 mmol/L for patient 1, 6·79-6·48 mmol/L for patient 2, and 7·6-7·4 mmol/L for patient 3. End of perfusion lactate values were 3·6-3·6 mmol/L, 2·8-2·3 mmol/L, and 2·69-2·54 mmol/L, respectively, showing favourable lactate uptake. Two patients needed temporary mechanical support. All three recipients had normal cardiac function within a week of transplantation and are making a good recovery at 176, 91, and 77 days after transplantation. Strict limitations on donor eligibility, optimised myocardial protection, and use of a portable ex-vivo organ perfusion platform can enable successful, distantly procured orthotopic transplantation of hearts donated after circulatory death. NHMRC, John T Reid Charitable Trust, EVOS Trust Fund, Harry Windsor Trust Fund.

Ex-vivo perfusion of donor hearts for human heart transplantation (PROCEED II): a prospective, open-label, multicentre, randomised non-inferiority trial

The Organ Care System is the only clinical platform for ex-vivo perfusion of human donor hearts. The system preserves the donor heart in a warm beating state during transport from the donor hospital to the recipient hospital. We aimed to assess the clinical outcomes of the Organ Care System compared with standard cold storage of human donor hearts for transplantation. We did this prospective, open-label, multicentre, randomised non-inferiority trial at ten heart-transplant centres in the USA and Europe. Eligible heart-transplant candidates (aged >18 years) were randomly assigned (1:1) to receive donor hearts preserved with either the Organ Care System or standard cold storage. Participants, investigators, and medical staff were not masked to group assignment. The primary endpoint was 30 day patient and graft survival, with a 10% non-inferiority margin. We did analyses in the intention-to-treat, as-treated, and per-protocol populations. This trial is registered with ClinicalTrials.gov, number NCT00855712. Between June 29, 2010, and Sept 16, 2013, we randomly assigned 130 patients to the Organ Care System group (n=67) or the standard cold storage group (n=63). 30 day patient and graft survival rates were 94% (n=63) in the Organ Care System group and 97% (n=61) in the standard cold storage group (difference 2·8%, one-sided 95% upper confidence bound 8·8; p=0·45). Eight (13%) patients in the Organ Care System group and nine (14%) patients in the standard cold storage group had cardiac-related serious adverse events. Heart transplantation using donor hearts adequately preserved with the Organ Care System or with standard cold storage yield similar short-term clinical outcomes. The metabolic assessment capability of the Organ Care System needs further study. TransMedics.

Changing paradigms in organ preservation and resuscitation.

Shortage of donor organs has increased consideration for use of historically excluded grafts. Ex-vivo machine perfusion is an emerging technology that holds the potential for organ resuscitation and reconditioning, potentially increasing the quality and number of organs available for transplantation. This article aims to review the recent advances in machine perfusion and organ preservation solutions. Flow and pressure-based machine perfusion has shown improved kidney graft function and survival, especially among expanded criteria donors. Pressure-based machine perfusion is demonstrating promising results in preservation and resuscitation of liver, pancreas, heart, and also lung grafts. August 2014 marked Food and Drug Administration approval of XPS XVIVO Perfusion System (XVIVO Perfusion Inc., Englewood, Colorado, USA), a device for preserving and resuscitating lung allografts initially considered unsuitable for transplantation. Although there is no consensus among physicians about the optimal preservation solution, adding antiapoptotic and cell protective agents to preservation solutions is an interesting research area that offers potential to improve preservation. Ex-vivo machine perfusion of solid organs is a promising method that provides the opportunity for resuscitation and reconditioning of suboptimal grafts, expanding the number and quality of donor organs.

(752) - Ex-Vivo Heart Perfusion Using the Transmedics Organ Care System - A Single Center Experience

Single flush antegrade perfusion and cold storage is a simple and economic way for donor heart preservation. However, the ischemic tolerance is limited by cellular energy resources. Due to the increasing organ shortage an increasing number of marginal donor hearts with an increased risk of primary graft failure (PGF) is used. Ex-vivo perfusion using oxygenated warm blood perfusion of the heart during transport might offer a possible solution to increase the utility of donor organs and decrease the risk of PGF.

(244) - Metabolic Assessment of Marginal Donor Lungs During Ex-Vivo Perfusion (EVLP): New Parameters for Decision Making

EVLP has become a widespread method to evaluate marginal donor lungs. Nevertheless, strong evidence on accurate judgment of graft quality by individual functional parameters is still missing. Anaerobic glucose metabolism might be dependent on graft quality and serve as a parameter for decision making during EVLP. This is an analysis of our prospective EVLP database. Since 2010, 43 EVLP were performed with initially rejected marginal donor lungs. 28 (65%) of these lungs were accepted after EVLP and transplanted with results comparable to standard donor lungs. Absolute glucose consumption and lactate production were calculated and corrected for hourly changes of perfusate and additional glucose administration. These values were compared between accepted (group A) and rejected lungs (group B). Linear regression was performed and the slope values were indexed for donor pTLC to correct for graft size. Mean EVLP Duration was 275±87 min. (group A, n=28) vs. 297 min. (group B, n=15: 238 min; p=0.039). Subsequently accepted lungs showed a significantly lower total glucose consumption (T1h:-899 vs.-1258 mg; p=0.29/ T2h:-1625 vs.-2293 mg; p=0.058/ T3h: 2432 vs.-3380 mg; p=0.015/ T4h:-2812 vs.-4771 mg; p<0.001) and lower lactate production (T1h:7.5 vs.9.5mmol; p=0.44/ T2h: 15 vs. 19.6 mmol; p=0.09/ T3h: 22.3 vs. 29 mmol; p=0.02/ T4h:27 vs. 42.7mmol; p<0.001). In linear regression analysis glucose consumption and lactate production slopes were significantly different between the groups (glucose:-2.03 ±1.03 vs.-3.06 ±1.41 mg/min/LTLCp;p=0.021/ lactate: 0.019 ±0.008 vs. 0.028 ±0.013 mmol/min/LTLCp; p=0.038. Lactate and glucose slopes were significantly correlated(r=-0.798; p<0.001) During EVLP, lactate and glucose changes are linear and highly correlated over time. Glucose consumption and lactate production are significantly higher in unacceptable donor lungs and should be considered as important parameters for decision making during EVLP.

Upregulation of Krüppel-Like Factor 6 is associated with acute liver injury in mice and humans

Background: Krüppel-like factor 6 (KLF6) is a ubiquitously expressed, multifunctional transcription factor and tumor suppressor gene. In previous studies, we identified KLF6 as an important transcription factor in hepatocyte glucose and lipid homeostasis, and downregulation of KLF6 was associated with accelerated tumor-growth of hepatocellular cancer. So far, no data is available on the role of KLF6 in acute liver injury and regeneration. Here, we aimed to investigate the expression pattern of hepatocyte KLF6 in patients with acute liver failure (ALF), in a human ex-vivo perfusion model of acetaminophen induced liver injury and to study the effects of hepatocyte specific KLF6 depletion in mice undergoing partial hepatectomy (PHx). Methods: Liver samples from 10 patients with drug-induced ALF were obtained (either liver biopsy or explanted liver in patients who underwent liver transplantation) and KLF6 expression was quantified via immunohistochemistry (IHC) and compared to liver samples from 10 non-cirrhotic NAFLD patients with simple steatosis as controls. In another setting, non-cirrhotic liver tissue was obtained from partial liver resection for metastatic surgery in 8 patients. In an established ex-vivo perfusion model, these samples were treated with acetaminophen (APAP) up to 30 hours. KLF6 and HGF (hepatocyte growth factor) mRNA expression was quantified before and after APAP treatment. In a murine model of PHx (n= 6 mice/group), we assessed KLF6 expression before and at different timepoints after PHx. Also, hepatocyte specific KLF6 knockout mice underwent PHx and we performed PCNA staining at different timepoints to assess hepatocyte proliferation, compared to controls (n= 6 mice/group). Results: IHC in ALF patients revealed significant upregulation of KLF6 protein within hepatocytes compared to controls. APAP perfusion of non-cirrhotic liver tissue significantly induced the expression of KLF6 (4.7-fold, p = 0.003) as well as the regeneration-associated molecule HGF (3.9-fold, p = 0.02). In mice, PHx also led to significant induction of KLF6 expression at different timepoints (3.8-fold, p = 0.03). In hepatocyte specific KLF6 knockouts, hepatocyte proliferation, as assessed with PCNA staining was significantly induced at early timepoints (p < 0.05). Conclusion: Here, we were the first to study KLF6 expression in ALF. Our findings suggest an important role for KLF6 in liver regeneration, as KLF6 expression is upregulated in different models of acute liver injury and ALF patients. Hepatocyte proliferation following PHx was induced in mice with KLF6 knockdown, compared to wildtype controls, suggesting a role for KLF6 in hepatic regeneration. Further studies and data analysis will be needed to identify the individual mechanisms for KLF6 mediated effects in acute liver injury.

Investigations on acetaminophen-induced liver injury using an ex-vivo perfusion model with human liver sections

Introduction: A perfusion system for human liver sections able to keep hepatic functionality for up to 30 hours was applied to study liver damage mediated by acetaminophen (APAP) in a human tissue based model. Methods: Perfusions were performed for 30 hours and hourly taken samples from the perfusate were analyzed for markers of general metabolism, hepatic synthesis capacity and liver cell damage. APAP was added after 8h with amounts relative to the liver weight (6.5 mg/g). N-Acetylcysteine was added after 20h to reduce liver damage in the course of perfusion. Liver function was determined by clearance of indocyanine green (ICG) after 4, 20 and 28h and normalized to the value at 4h. Results: ICG clearance in three liver sections perfused without APAP to obtain reference values was 98 ± 12% after 20h and 77 ± 18% after 28h. Liver function in two smaller liver sections (38 g) incubated with APAP was reduced to 41 and 71% after 20h and 22% after 28h, respectively. Liver function in two larger liver pieces (57 g) was dropped to 25 and 36% after 20h and to 18 and 10% after 28h, respectively, exhibiting nearly complete cell death at the end of experiment. Conclusion: The hepatotoxic sensitivity of the model with patient-relevant amounts of APAP was shown within the perfusion period. Further studies are required to investigate the optimal conditions to gain damaged, but not deceased tissue after 30h for subsequent analysis. Thus, this model is useful as human-based artificial liver system for the study of new drug candidates and pathologies of the liver.

Transplacental transfer of 2-naphthol in human placenta

ObjectiveTo determine the transfer of 2-naphthol (2-NPH) in fullterm human placental tissues. MethodsSix placentas were studied. The ex-vivo dual closed-loop human placental cotyledon perfusion model was used. 2-NPH was added to the perfusate in the maternal compartment. Samples were obtained from the maternal and fetal up to 360min measuring. ResultsThe mean fetal weight was 2880±304.2g. Mean perfused cotyledon weight was 26.3 (±5.5)g. All unperfused placental tissue samples contained NPH with a mean level of 7.98 (±1.73)μg\\g compared to a mean of 15.58 (±4.53)μg\\g after 360min perfusion. A rapid drop in maternal 2-NPH concentration was observed; from 5.54μg\\g in the first 15min and 13.8μg\\g in 360min. The fetal side increased from 0.65μg\\g in the initial 15min to 1.5μg\\g in 360min. The transfer rate of NPH was much lower than that of antipyrine. Conclusion2-NPH has the ability to rapidly across the placenta from the maternal to the fetal compartment within 15min. The placenta seems to play a role in limiting the passage of 2-NPH in the fetal compartment.

LPS from bovine serum albumin drives TNF-α release during ex-vivo placenta perfusion experiments, contaminates the perfusion system but can be effectively removed by oxidative cleaning

IntroductionThe dual ex-vivo perfusion of human placental tissue is useful to study inflammatory pathways. We found significant TNF-α release in negative controls similar in concentration to lipopolysaccharide (LPS) stimulated placentas. The aim of the current study was to (i) identify sources driving TNF-α release and (ii) develop an approach to control for it. Method(i) To determine sources leading to TNF-α release, solutions frequently circulated through the perfusion system and perfusion media with different bovine serum albumin (BSA) quality were exposed to mouse macrophage cell lines (RAW264.7) and subsequently measured for TNF-α expression. (ii) To assess memory effects and validate cleaning procedures, sham perfusion experiments were conducted either in the presence or absence of exogenous LPS, in new tubing that was contaminated, cleaned and analyzed for the effectiveness of LPS removal. Oxidative and acid–base cleaning were tested for their effectiveness to reduce LPS contamination. ResultsTNF-α release, observed in negative control experiments, was attributed to the use of LPS-contaminated BSA as well as inadequate cleaning of the perfusion system. Once introduced in the perfusion system, LPS accumulated and created a memory effect. Oxidative but not acid–base depyrogenation effectively reduced LPS levels to concentrations that were in accordance with FDA guidelines (<0.5 EU/mL) for medical equipment redeemed appropriate for re-use. DiscussionLPS contamination of the placenta perfusion model could have confounding effects on experimental outcomes leading to misinterpretation of data. To circumvent LPS contamination LPS-free BSA and oxidative depyrogenation cleaning techniques should be implemented in future placental perfusion studies.

Criteria for viability assessment of discarded human donor livers during ex-vivo normothermic machine perfusion

Criteria for viability assessment of discarded human donor livers during ex-vivo normothermic machine perfusion

High-risk donors: extending our criteria in times of organ shortage.

Increasing waiting lists and declining transplant numbers due to organ shortage are a global problem that needs a multimodal approach to overcome this situation. Extending the criteria for transplantation may be one part of the solution. There are political efforts to increase the donor rate and change the listing criteria and the allocation process. Recently, the cardiac allocation score was introduced enhancing the factor urgency to the allocation process. Marginal donor organs can be accepted using ex-vivo perfusion strategies. Experimental approaches, such as donation after circulatory death and xenotransplantation, need to be further developed to be applied to humans and increase the pool of available transplant organs. Organ shortage needs new approaches to overcome the discrepancy between the number of patients on the wait list and performed heart transplantations, reduce wait list mortality and improve long-term outcomes after transplantation.

Dual closed-loop ex-vivo placental perfusion model: Evidence for relevant LPS contamination and development of a procedure for depyrogenation

Dual closed-loop ex-vivo placental perfusion model: Evidence for relevant LPS contamination and development of a procedure for depyrogenation

The bile duct in donation after cardiac death donor liver transplant.

This review considers the biliary complications associated with liver transplantation using donation after cardiac death (DCD) donor grafts. The increasing use of DCD liver grafts with their increased incidence of biliary complications is discussed. The ethics of this greater use is briefly analysed. Recent animal and human study evidence to support the peribiliary vascular plexus' role in ischaemic cholangiopathy is reviewed. Recent advances in in-vivo and ex-vivo perfusion are explored. In particular, the latest theories regarding perfusion's peribiliary plexus preserving effects and the mechanism by which biliary regeneration may be promoted as a consequence are discussed. This article explores the need for DCD liver graft use and the associated biliary complications. The current theories regarding the cause of DCD biliary complications are reviewed, as are the current strategies to reduce them.

The marginal kidney donor.

The current era of organ shortage has necessitated a widening of criteria for donation, considering donors who would have been considered unsuitable before. This review summarizes the recent advances in strategies to maximize the use of marginal kidneys without compromising the outcomes. Various strategies have been studied and implemented to optimize procurement and allocation of marginal kidneys, and to preserve their function. In particular, a growing number of transplant centers are using donors after circulatory death. Whereas normothermic ex-vivo and postmortem perfusion are promising procedures to improve the outcomes of marginal grafts in the future, dual-kidney transplantation is a viable approach which is at present potentially underutilized. Despite active research on new strategies to evaluate organ quality, pretransplant biopsy assessment currently remains the most reliable method. The practice of using living donors with advanced age is supported by available evidence, whereas the use of young living donors with minor medical abnormalities needs further investigation. Progress has been made in the recent years, clarifying the best criteria for evaluating, recovering, and allocating marginal kidney donors. However, further research is needed, with special regards to the criteria for using marginal living-kidney donors.

Criteria for Viability Assessment of Discarded Human Donor Livers During Ex-Vivo Normothermic Machine Perfusion.

Criteria for Viability Assessment of Discarded Human Donor Livers During Ex-Vivo Normothermic Machine Perfusion.

Establishing an Ex-Vivo Pancreas Perfusion Model On Discarded Human Grafts.

Establishing an Ex-Vivo Pancreas Perfusion Model On Discarded Human Grafts.