Normothermic 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.

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.

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.

Normothermic extracorporeal human liver perfusion following donation after cardiac death

Normothermic extracorporeal human liver perfusion following donation after cardiac death

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.

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.

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.

A comparison of bile composition from heart-beating and non–heart-beating rabbit organ donors during normothermic extracorporeal liver perfusion: Experimental evaluation using proton magentic resonance spectroscopy

Background Organs retrieved from marginal and non–heart-beating donors (NHBDs) have sustained variable degrees of preretrieval damage that result in an increased incidence of complications. Normothermic extracorporeal liver perfusion (NELP) provides an opportunity to evaluate and resuscitate such organs. The aim of this study was to identify markers of ischemic injury in bile during perfusion of livers from heart-beating donors (HBDs) and NHBDs. Methods Livers were retrieved from New Zealand white rabbits. The HBD group ( n = 4) had no in situ warm ischemia before retrieval and the NHBD group ( n = 4), 45 minutes of in situ warm ischemia before liver retrieval. After 40 minutes of postretrieval cold ischemia, all livers were dual vessel reperfused, normothermically with oxygenated buffer solution supplemented with rabbit red blood cells, for 6 hours. Bile was collected and examined with 1HMRS. Results Perfusion bile from HBD group showed an increased concentration of bile acids, lactate, glucose, and phosphatidylcholine, but a decreased concentration of acetate as compared to retrieval bile. This trend was further enhanced in NHBD group. The mean ± SD (in μmol/L) were bile acids (10.48 ± 2.8 vs 26.05 ± 12.1 vs 44.5 ± 44.5), lactate (10.66 ± 4.5 vs 14.66 ± 5.2 vs 13.22 ± 1.8), glucose (5.37 ± 2 vs 21.2 ± 5.0 vs 29.09 ± 15.3), phosphatidylcholine (0.21 ± 0.02 vs 5.57 ± 1.7 vs 6.42 ± 0.3), and acetate (1.8 ± 0.5 vs 0.39 ± 0.1 vs 0.38 ± 0.09) for retrieval bile, HBD perfusion bile, and NHBD perfusion bile, respectively. One animal from each group did not produce any bile during perfusion. Conclusions 1HMRS of biliary constituents revealed differences with the type of ischemia. These indices may be potential markers of the extent of warm ischemic injury and the functional activity of an extracorporeally perfused liver.

Near-infrared spectroscopic assessment of mitochondrial oxygenation status—comparison during normothermic extracorporeal liver perfusion by buffer only or buffer fortified with washed red blood cells: an experimental study

Background Use of marginal and non–heart-beating donors leads to an increased incidence of complications after clinical liver transplantation. Normothermic extracorporeal liver perfusion (NELP) may allow resuscitation and evaluation of such organs. Despite recent success in long-term liver preservation by NELP, no methods of organ evaluation have been defined. Mitochondrial cytochrome oxidase (Cyt Ox) levels reflect oxygen and substrate delivery, and hence ATP production at the cellular level. This study used near-infrared spectroscopy (NIRS) to measure Cyt Ox levels during NELP. Methods Livers retrieved from New Zealand white rabbits were immediately perfused in an extracorporeal circuit with oxygenated buffer (group A, n = 4) or red blood cell (RBC)–fortified buffer (group B, n = 4). Perfusion was continued for 3 hours at 37°C pH 7.4, and perfusate was gassed with 95%O 2/5%CO 2 at 1 liter per minute. Cyt Ox levels were monitored continuously by NIRS and bile output was measured. Results Cyt Ox was reduced at the start of perfusion in both groups, but even more rapidly in the buffer-perfused group. After initial deterioration, Cyt Ox levels improved significantly ( P < .05) with perfusion in the RBG-perfused group, but remained impaired in the buffer group 5.74 ± 1.51 Δμmol/L and −25.77 ± 21.94 Δμmol/L for groups B and A, respectively, at 180 minutes. Differences in bile output were not significant (19.33 ± 9.50 and 25.00 ± 16.81 μmol/min/100 g for groups B and A respectively). Conclusions Cyt Ox levels may offer better viability markers than bile output. NIRS is a practical method to measure tissue oxygenation, and RBC-based perfusion provided better oxygenation during NELP.

Liver transplantation after organ preservation with normothermic extracorporeal perfusion.

To study normothermic extracorporeal liver perfusion (NELP) as a means to preserve livers for transplantation and to reverse warm ischemic injury. The authors provide experimental evidence that successful transplantation after 4 hours of normothermic extracorporeal liver perfusion is possible and as reliable as 4 hours of cold preservation in University of Wisconsin solution. NELP preserves liver function completely and can reverse 60 minutes of warm ischemic injury in non-heart-beating donors. Thirty-six German Landrace pigs received transplants in six groups. Group 1 animals received direct transplantation. Group 2 received transplants after 4 hours of cold preservation with University of Wisconsin solution and Group 3 animals after 4 hours of NELP. Group 4 animals sustained 1 hour of warm ischemia before transplantation. Group 5 animals received transplants after 1 hour of warm ischemia and 4 hours of cold preservation and Group 6 animals after 1 hour of warm ischemia and 4 hours of NELP. All animals receiving livers treated by NELP survived more than 7 days after the transplant (Groups 3 and 6). In contrast, all animals in Group 5 developed primary graft nonfunction within 24 hours after transplantation. The technique of NELP holds the potential to keep a mammalian liver outside the body completely functional, possibly for more than 4 hours. NELP can be used for liver preservation before transplantation or for the use of organs from non-heart-beating donors.

Xenogeneic, Extracorporeal Liver Perfusion in Primates Improves the Ratio of Branched-Chain Amino Acids to Aromatic Amino Acids (Fischer’s Ratio)

In fulminant hepatic failure (FHF), the development of hepatic encephalopathy is associated with grossly abnormal concentrations of plasma amino acids (PAA). Normalization of the ratio of branched-chain amino acids to aromatic amino acids (Fischer’s ratio) correlates with clinical improvement. This study evaluated changes in PAA metabolism during 4 h of isolated, normothermic extracorporeal liver perfusion using a newly designed system containing human blood and a rhesus monkey liver. Bile and urea production were within the physiological range. Release of the transaminases AST, ALT and LDH were minimal. The ratio of branched (valine, leucine, isoleucine) to aromatic (tyrosine, phenylalanine) amino acids increased significantly. These results indicate that a xenogeneic extracorporeal liver perfusion system is capable of significantly increasing Fischer’s ratio and may play a role in treating and bridging patients in FHF in the future.