Extracorporeal Liver Perfusion Research Articles

Normothermic liver machine perfusion as a dynamic platform for regenerative purposes: What does the future have in store for us?

Liver transplantation has become an immense success; nevertheless, far more recipients are registered on waiting lists than there are available donor livers for transplantation. High-risk, extended criteria donor livers are increasingly used to reduce the discrepancy between organ demand and supply. Especially for high-risk livers, dynamic preservation using machine perfusion can decrease post-transplantation complications and may increase donor liver utilisation by improving graft quality and enabling viability testing before transplantation. To further increase the availability of donor livers suitable for transplantation, new strategies are required that make it possible to use organs that are initially too damaged to be transplanted. With the current progress in experimental liver transplantation research, (long-term) normothermic machine perfusion may be used in the future as a dynamic platform for regenerative medicine approaches, enabling repair and regeneration of injured donor livers. Currently explored therapeutics such as defatting cocktails, RNA interference, senolytics, and stem cell therapy may assist in the repair and/or regeneration of injured livers before transplantation. This review will provide a forecast of the future utility of normothermic machine perfusion in decreasing the imbalance between donor liver demand and supply by enabling the repair and regeneration of damaged donor livers.

Bridging to Allotransplantation-Is Pig Liver Xenotransplantation the Best Option?

In the past 20 y, the number of patients in the United States who died while waiting for a human donor liver totaled >52 000. The median national wait time for patients with acute liver failure and the most urgent liver transplant listing was 7 d in 2018. The need for a clinical "bridge" to allotransplantation is clear. Current options for supporting patients with acute liver failure include artificial liver support devices, extracorporeal liver perfusion, and hepatocyte transplantation, all of which have shown mixed results with regard to survival benefit and are largely experimental. Progress in the transplantation of genetically engineered pig liver grafts in nonhuman primates has grown steadily, with survival of the pig graft extended to almost 1 mo in 2017. Further advances may justify consideration of a pig liver transplant as a clinical bridge to allotransplantation. We provide a brief history of pig liver xenotransplantation, summarize the most recent progress in pig-to-nonhuman primate liver transplantation models, and suggest criteria that may be considered for patient selection for a clinical trial of bridging by genetically engineered pig liver xenotransplantation to liver allotransplantation.

Improving Liver Graft Function Using CD47 Blockade in the Setting of Normothermic Machine Perfusion

Toward the goal of using more livers for transplantation, transplant centers are looking to increase the use of organs from "marginal" donors. Livers from these donors, however, have been shown to be more susceptible to preservation and reperfusion injury. Using a porcine model of donation after circulatory death, we studied the use of antibody-mediated CD47 blockade to further improve liver graft function undergoing normothermic machine perfusion. Livers from 20 pigs (5 per group) were brought under either 30 or 60 min of warm ischemia time followed by the administration of CD47 monoclonal antibody (CD47mAb) treatment or immunoglobulin G control antibodies and 6 h of normothermic extracorporeal liver perfusion. After 6 h of normothermic extracorporeal liver perfusion, CD47mAb-treated livers with 30 or 60 min warm ischemia time had significantly lower alanine transaminase levels and higher bile production compared with their respective control groups. Blockade of the CD47 signaling pathway resulted in significantly lower thrombospondin-1 protein levels, lower expression of caspase-3, and higher expression of phosphorylated extracellular signal-regulated kinase. These findings suggested that CD47mAb treatment decreases ischemia/reperfusion injury through CD47/thrombospondin-1 signaling downregulation and the presence of necrosis/apoptosis after reperfusion and could increase liver regeneration during normothermic perfusion of the liver.

Donation after Circulatory Death in Paediatric Liver Transplantation: Current Status and Future Perspectives in the Machine Perfusion Era.

Efforts have been made by the transplant community to expand the deceased donor pool in paediatric liver transplantation (LT). The growing experience on donation after circulatory death (DCD) for adult LT has encouraged its use also in children, albeit in selective cases, opening new perspectives for paediatric patients. Even though there has recently been a slight increase in the number of DCD livers transplanted in children, with satisfactory graft and patient outcomes, the use of DCD grafts in paediatric recipients is still controversial due to morbid outcomes associated with DCD grafts. In this context, recent advances in the optimization of donor support by extracorporeal membrane oxygenation and in the graft preservation by liver machine perfusion could find application in order to expand the donor pool in paediatric LT. In the present study we review the current literature on DCD liver grafts transplanted in children and on the use of extracorporeal donor support and liver perfusion machines in paediatrics, with the aim of defining the current status and future perspectives of paediatric LT.

Development of a normothermic extracorporeal liver perfusion system toward improving viability and function of human extended criteria donor livers.

Donor organ shortages have led to an increased interest in finding new approaches to recover organs from extended criteria donors (ECD). Normothermic extracorporeal liver perfusion (NELP) has been proposed as a superior preservation method to reduce ischemia/reperfusion injury (IRI), precondition suboptimal grafts, and treat ECD livers so that they can be successfully used for transplantation. The aim of this study was to investigate the beneficial effects of a modified NELP circuit on discarded human livers. Seven human livers that were rejected for transplantation were placed on a modified NELP circuit for 8 hours. Perfusate samples and needle core biopsies were obtained at hourly intervals. A defatting solution that contained exendin-4 (50 nM) and L-carnitine (10 mM) was added to the perfusate for 2 steatotic livers. NELP provided normal temperature, electrolytes, and pH and glucose levels in the perfusate along with physiological vascular flows and pressures. Functional, biochemical, and microscopic evaluation revealed no additional injuries to the grafts during NELP with an improved oxygen extraction ratio (>0.5) and stabilized markers of hepatic injury. All livers synthesized adequate amounts of bile and coagulation factors. We also demonstrated a mild reduction (10%) of macroglobular steatosis with the use of the defatting solution. Histology demonstrated normal parenchymal architecture and a minimal to complete lack of IRI at the end of NELP. In conclusion, a modified NELP circuit preserved hepatocyte architecture, recovered synthetic functions, and hepatobiliary parameters of ECD livers without additional injuries to the grafts. This approach has the potential to increase the donor pool for clinical transplantation. Liver Transplantation 22 979-993 2016 AASLD.

Normothermic extracorporeal liver perfusion for donation after cardiac death (DCD) livers

The susceptibility of extended criteria livers to ischemia reperfusion injury is a major obstacle in organ cold preservation. Normothermic extracorporeal liver perfusion (NELP) has been investigated to reduce ischemic damage, restore physiologic function, and assess viability of the liver prior to transplant. The goal of this study is to compare physiological parameters of livers maintained continuously on NELP to those preserved in cold solution. Livers from 9 female landrace pigs were subjected to either 20 minutes (W20-NELP), 40 minutes (W40-NELP), or 60 minutes (W60-NELP) of warm ischemia followed by 6 hours of NELP followed by a 2-hour NELP evaluation phase. This was compared with 3 livers subjected to 40 minutes of warm ischemia time followed by 6 hours of cold storage (W40-Cold) and a 2-hour NELP evaluation phase. Groups were compared with the 2-way analysis of variance test. NELP stabilized transaminases accompanied by significant improvement in bile production and decline in lactate and INR values in all W-NELP groups. Histologic analysis demonstrated significant improvement from 0 hour (mild-to-moderate sinusoidal dilation and zone 3 necrosis) to the end of the NELP run (minimal necrosis and mild IRI). Comparison of W40-NELP and W40-Cold revealed greater bile production and oxygen extraction ratio in W40-NELP. In contrast, markers of cellular and functional damage were increased in the W40-Cold group. NELP improves metabolic and functional parameters of livers with either short or extended warm ischemia times compared with livers subjected to comparable cold ischemia times.

A Human-Specific Mutation Limits Nonhuman Primate Efficacy in Preclinical Xenotransplantation Studies

Patients diagnosed with fulminant hepatic failure face high mortality rates. A potential therapeutic approach for these patients is the use of extracorporeal porcine liver perfusion, to serve as a form of "liver dialysis." Previously, our laboratory has shown that, during a 72-hour extracorporeal perfusion with human blood, porcine Kupffer cells bind to and phagocytose human erythrocytes causing the hematocrit to fall to 2.5% of the original value. Subsequently, erythrocyte binding has been shown to involve N-acetylneuraminic acid (Neu5Ac) on the surface of human erythrocytes and sialoadhesin on the surface of the porcine Kupffer cells. Given that no primate other than the human is known to express the majority of its sialic acid as Neu5Ac, we evaluated whether nonhuman primates would provide adequate evaluation of the loss of erythrocytes that might be expected in a clinical trial of extracorporeal porcine liver perfusion. We found that while porcine macrophages readily bound human erythrocytes, binding of nonhuman primate erythrocytes was significantly reduced (P<0.001). This study suggests that nonhuman primates may fail to serve as an adequate model for studying extracorporeal porcine liver perfusion because of the fact that porcine macrophages do not bind nonhuman primate erythrocytes.

Normothermic extracorporeal human liver perfusion following donation after cardiac death

Normothermic extracorporeal human liver perfusion following donation after cardiac death

Porcine Sialoadhesin: A Newly Identified Xenogeneic Innate Immune Receptor

Extracorporeal porcine liver perfusion is being developed as a bridge to liver allotransplantation for patients with fulminant hepatic failure. This strategy is limited by porcine Kupffer cell destruction of human erythrocytes, mediated by lectin binding of a sialic acid motif in the absence of antibody and complement. Sialoadhesin, a macrophage restricted lectin that binds sialic acid, was originally described as a sheep erythrocyte binding receptor. Given similarities between sialoadhesin and the unidentified macrophage lectin in our model, we hypothesized porcine sialoadhesin contributed to recognition of human erythrocytes. Two additional types of macrophages were identified to bind human erythrocytes-spleen and alveolar. Expression of sialoadhesin was confirmed by immunofluorescence in porcine tissues and by flow cytometry on primary macrophages. A stable transgenic cell line expressing porcine sialoadhesin (pSn CHO) bound human erythrocytes, while a sialoadhesin mutant cell line did not. Porcine macrophage and pSn CHO recognition of human erythrocytes was inhibited approximately 90% by an antiporcine sialoadhesin monoclonal antibody and by human erythrocyte glycoproteins. Furthermore, this binding was substantially reduced by sialidase treatment of erythrocytes. These data support the hypothesis that porcine sialoadhesin is a xenogeneic receptor that mediates porcine macrophage binding of human erythrocytes in a sialic acid-dependent manner.

Histological changes during extracorporeal perfusions of the porcine liver: implications for temporary support during acute liver failures

Ex vivo perfused porcine livers have been used for temporary support during acute liver failure. The aim of this study was to assess both the histological changes and temporal pattern of the changes that occur during extracorporeal liver perfusions and to correlate these with factors that may influence them. Five porcine livers were harvested, preserved in cold ice and reperfused for 6h in an extracorporeal circuit using autologous normothermic blood. Tissue biopsies were collected hourly. The Ishak score was used to quantify hepatic necrosis, and immunohistochemistry was used to evaluate apoptosis and regeneration. Liver weight, perfusion parameters, arterial blood gases and blood samples were also collected. The Ishak score peaked immediately before and 4 h after the start of reperfusion. Scattered necrosis, microvesicular steatotic vacuolization, sinusoidal dilatation and red cell extravasation were present. Anion gap acidosis was associated with the Ishak score. An inverse correlation was present between liver regeneration and necrosis, and between liver weight and regeneration. No changes were observed for apoptosis. Among the inflammatory cytokines evaluated, interleukin-6 and -8 levels increased significantly during the perfusions. Hepatic necrosis was always present during the extracorporeal perfusions, followed a definite pattern and was inversely correlated with regeneration. Apoptosis did not increase over baseline levels. The meaning of these findings and their correlation with clinical outcomes during acute hepatic failures deserve further investigation.

Blocking Porcine Sialoadhesin Improves Extracorporeal Porcine Liver Xenoperfusion

Background: Patients in fulminant hepatic failure (FHF) currently do not have a temporary means of support while awaiting liver transplantation. A potential therapeutic approach for patients in FHF is the use of extracorporeal perfusion with porcine livers as a form of “liver dialysis”. During a 72 hour extracorporeal perfusion of porcine livers with human blood, porcine Kupffer cells (KC) bind to and phagocytose human red blood cells (hRBC) causing the hematocrit to decrease to 2.5% of the original value. Our laboratory has identified sialoadhesin (Sn) on the surface of porcine KC as the lectin responsible for binding N-acetylneuraminic acid (Neu5Ac) on the surface of the hRBC. In order for extracorporeal porcine liver perfusion to be used as a therapy for patients in liver failure, all forms of anti-human rejection must be resolved. We evaluated whether targeting Sn prevents the recognition and subsequent destruction of hRBCs seen during extracorporeal porcine liver xenoperfusion. Methods:In vitro experiments were performed wherein cultured porcine macrophages (pMΦ) were pretreated with the anti-porcine Sn monoclonal antibody, 1F1. pMΦ were subsequently assayed for their ability to bind hRBC. Next, ex vivo experiments were performed wherein six wild type pig livers were perfused with isolated hRBCs for 72 hours. In one group, three livers were treated with 1F1. As a control group, three livers were treated with an antibody of the same isotype but with no known specificity for Sn. Results: We have shown that treatment of cultured pMΦ with 10ug/ml of 1F1 abolishes the ability of the pMΦ to bind hRBC (p < 0.001). Additionally, we have shown that the addition of 1F1 to an extracorporeal porcine liver xenoperfusion model significantly reduces the loss of hRBC over a 72 hour period (p < 0.01). Sustained liver function was demonstrated by continued bile production and other markers. Conclusions: This study suggests that the destruction of human erythrocytes in an extracorporeal porcine liver xenoperfusion model is, in part, mediated by Sn expressed on the surface of porcine KC. Furthermore, this data illustrates that the addition of anti-Sn antibody to the circulation of a pig-to-human xenoperfusion may inhibit the destruction of human erythrocytes by porcine KC. Future studies should be conducted utilizing livers derived from Sn deficient pigs.

Extracorporeal Liver Perfusion System for Artificial Liver Support Across a Membrane

An extracorporeal porcine liver perfusion (ECPLP) system circumvents the limitations of hepatocyte based bio-artificial liver, but its clinical application has been limited so far due to the potential risk of transmission of porcine endogenous retroviruses. The aim of this study was to develop an ECPLP model that can provide artificial hepatic support across a semi-permeable membrane, which has the potential to block porcine viruses due to its pore size. Livers from white landrace pigs were perfused with normothermic oxygenated blood using Medtronic BP560 centrifugal pump (Medtronic, Inc., Minneapolis. MN). This ECPLP system was used to support a "surrogate" patient across the filter Evaclio-EC4A. Function of liver was measured by indocyanine green retention at 15 min (ICGR15). Clearance of galactose, ammonia, and para-aminobenzoic acid infused into the "surrogate" patient circulation was calculated to assess liver support across the membrane. The study was designed as test (n = 15) versus control (n = 5), with control experiments having no liver in the circuit. For the test experiments, we perfused 15 livers with mean hepatic artery pressure of 87 mm Hg and flows of 1.2 L/min. ICGR15 in test experiments was 11%. Ammonia clearance was 945 mg/min/kg, galactose metabolic rate was 111.7 mg/min/Kg, and the hippurate ratio was 91% in the test. In contrast, the control experiments did not show any significant change in the concentration of any of these compounds. Our ECPLP model was able to provide hepatic support in an experimental setting across a hollow fiber filter. Further work on an anhepatic animal is needed prior to application in human trials.

Subnormothermic Machine Perfusion at Both 20°C and 30°C Recovers Ischemic Rat Livers for Successful Transplantation

Utilizing livers from donors after cardiac death could significantly expand the donor pool. We have previously shown that normothermic (37°C) extracorporeal liver perfusion significantly improves transplantation outcomes of ischemic rat livers. Here we investigate whether recovery of ischemic livers is possible using sub-normothermic machine perfusion at 20°C and 30°C. Livers from male Lewis rats were divided into five groups after 1 h of warm ischemia (WI): (1) WI only, (2) 5 h of static cold storage (SCS), or 5 h of MP at (3) 20°C, (4) 30°C, and (5) 37°C. Long-term graft performance was evaluated for 28 d post-transplantation. Acute graft performance was evaluated during a 2 h normothermic sanguineous reperfusion ex vivo. Fresh livers with 5 h of SCS were positive transplant controls while fresh livers were positive reperfusion controls. Following machine perfusion (MP) (Groups 3, 4, and 5), ischemically damaged livers could be orthotopically transplanted into syngeneic recipients with 100% survival (N ≥ 4) after 4 wk. On the other hand, animals from WI only, or WI + SCS groups all died within 24 h of transplantation. Fresh livers preserved using SCS had the highest alanine aminotransferase (ALT), aspartate aminotransferase (AST), and the lowest bile production during reperfusion, while at 28 d post-transplantation, livers preserved at 20°C and 30°C had the highest total bilirubin values. MP at both 20°C and 30°C eliminated temperature control in perfusion systems and recovered ischemically damaged rat livers. Postoperatively, low transaminases suggest a beneficial effect of sub-normothermic perfusion, while rising total bilirubin levels suggest inadequate prevention of ischemia- or hypothermia-induced biliary damage.

Action of quercetin-5’-sulfonic acid sodium salt (NaQSA) on SOD activity and liver function in extracorporeal liver perfusion (ELP) in rats

Action of quercetin-5’-sulfonic acid sodium salt (NaQSA) on SOD activity and liver function in extracorporeal liver perfusion (ELP) in rats

Action of quercetin-5’-sulfonic acid sodium salt (NaQSA) on ADMA/DDAH pathway in extracorporeal liver perfusion (ELP) in rats

Action of quercetin-5’-sulfonic acid sodium salt (NaQSA) on ADMA/DDAH pathway in extracorporeal liver perfusion (ELP) in rats

Pig liver xenotransplantation as a bridge to allotransplantation: which patients might benefit?

Acute liver failure is a potentially devastating clinical syndrome that, without liver transplantation (Tx), is associated with high mortality. Rapid deterioration in clinical status and a shortage of deceased human organs prohibits liver Tx in many patients. Bridging to liver Tx has been attempted by various approaches, for example, bioartificial liver support, extracorporeal pig liver perfusion, and hepatocyte Tx, but none of these approaches has convincingly improved patient survival. The orthotopic Tx of a genetically engineered pig liver could theoretically provide successful bridging. Immediate availability, perfect metabolic condition, adequate size-match and hepatocyte mass, and freedom from potentially pathogenic microorganisms could be assured. The advantages and disadvantages of bridging by pig liver Tx compared with other approaches are discussed. The selection of patients for an initial clinical trial of pig liver Tx would be similar to that of various prior trials in patients experiencing rapid and severe deterioration in liver function. The ability to give truly informed consent for a pig bridging procedure at the time of listing for liver Tx renders the patient with acute-on-chronic liver failure or primary allograft failure is a preferable candidate for this procedure than a patient who is admitted urgently with acute (fulminant) liver failure in whom consent may not be possible. Although several barriers to successful pig organ xenoTx remain, for example, coagulation dysfunction between pig and primate, if these can be resolved by further genetic engineering of the organ-source pigs, a pig liver may prove life saving to patients dying rapidly of liver failure.

Indocyanine green R15 ratio depends directly on liver perfusion flow rate

Indocyanine green (ICG) is a synthetic dye that is widely used to evaluate liver function in critically ill patients, before liver resection or after liver transplantation. Controversy still exists about the impact exerted on the ICG ratio after 15 min (ICG R15) by differences in liver perfusion rates, hyperdynamic states, or patient cardiac output. We studied the role of different liver perfusion rates on the ICG R15 ratio in a normothermic extracorporeal liver perfusion system under standardized conditions. Livers from landrace pigs (40-50 kg) were perfused with fresh porcine blood. Normal and high perfusion rates were defined as 1 ml and 2 ml/g liver/min, respectively. Perfusate pressure of the hepatic artery and portal vein were within the physiological range in both groups. According to manufacturer's instructions, 0.5 mg of ICG per kg was applied and the ICG R15 was calculated. Calculations were based on fifteen experiments in five liver perfusions. Bile production, liver function and histology were analyzed. All perfusions were characterized by physiological bile production, lack of hepatocellular damage and normal histology. ICG R15 ratio in group I, perfused with 1 ml/g liver, was 18.9 +/- 6%. In group II, perfused with 2 ml/g liver, the ICG R15 ratio was 7.2 +/- 3%. The difference between groups 1 and 2 was statistically significant (p < 0.05). ICG R15 is reliable within one group at defined perfusion rates. Doubled perfusion rates contribute to higher ICG clearance. For clinical application we would like to suggest considering cardiac output of the patient for interpretation of ICG ratios.

Recovery of warm ischemic rat liver grafts by normothermic extracorporeal perfusion.

Liver transplantation is currently the only established treatment of end-stage liver disease, but it is limited by a severe shortage of viable donor livers. Donors after cardiac death (DCD) are an untapped source that could significantly increase the pool of available livers. Preservation of these DCD livers by conventional static cold storage (SCS) is associated with an unacceptable risk of primary nonfunction and delayed graft failure. Normothermic extracorporeal liver perfusion (NELP) has been suggested as an improvement over SCS. Livers recovered from male Lewis rats were subjected to 1 hr of warm ischemia and preserved with 5 hr of SCS or NELP, and transplanted into syngeneic recipients. As additional controls, non-ischemic livers preserved with 6 hr of SCS or NELP and unpreserved ischemic livers were transplanted. After NELP, ischemically damaged livers could be orthotopically transplanted into syngeneic recipients with 92% survival (n=13) after 4 weeks, which was comparable with control animals that received healthy livers preserved by SCS (n=9) or NELP (n=11) for 6 hr. On the other hand, animals from ischemia/SCS control group all died within 12 hr postoperatively (n=6). Similarly, animals that received ischemic livers without preservation all died within 24 hr after transplantation (n=6). These results suggest that NELP has the potential to reclaim warm ischemic livers that would not be transplantable otherwise. The rat model in this study is a useful platform to further optimize NELP as a method of recovery and preservation of DCD livers.

Preservation of non-heart-beating donor livers in extracorporeal liver perfusion and histidine-trytophan-ketoglutarate solution

To compare the preservation of non-heart-beating donor (NHBD) livers in cold histidine-trytophan-ketoglutarate (HTK) solution and extracorporeal liver perfusion (ECLP). Livers harvested from healthy pigs were stored for 10 h in cold HTK solution (group A, n = 4) or perfused with oxygenated autologous blood at body temperature (group B, n = 4). Both groups were then tested on the circuit for 4 h. Bile production, hemodynamic parameters, hepatocyte markers and reperfusion injury of extracorporeal livers were tested in each group. Liver tissues from each group were examined at the end of reperfusion. At 1, 2, 3 and 4 h after reperfusion, bile production, hemodynamic parameters, hepatocyte markers and reperfusion injury of livers in group A were statistically different from those in group B (P < 0.05 or P < 0.01). ECLP is better than HTK solution to preserve NHBD livers. ECLP can assess the graft viability before liver transplantation.

A Model for Normothermic Preservation of the Rat Liver

Current techniques for the preservation of donor livers typically rely on cold temperatures (approximately 0-4 degrees C) to slow down metabolic processes. Recently, normothermic extracorporeal liver perfusion (NELP) has regained interest as a potentially promising approach for long-term liver preservation. Unlike cold-storage techniques, NELP attempts to maintain the liver in a near physiological environment, thus enabling normal metabolic and tissue repair processes to take place, which may help in the recovery of ischemically damaged and fatty donor livers, both of which represent significant untapped sources of donor livers. However, NELP is technically more complex than cold-storage techniques, and the lack of standardized small animal models limits its development. Here we describe a rat NELP system that is simple and cost-effective to run. We show that rat livers that underwent NELP for 6 h could be routinely transplanted into syngeneic recipient rats with excellent 1-month survival. During perfusion, the release of cytosolic enzymes, bile and urea production, and oxygen uptake rate could be readily monitored, thus providing a comprehensive picture of hepatic function before transplantation. This system will help in the optimization of NELP in several ways, such as for the improvement of perfusion conditions and the development of quantitative metabolic criteria for hepatic viability.