Donor Liver Preservation Research Articles

Prolonged hypothermic machine perfusion enables daytime liver transplantation – an IDEAL stage 2 prospective clinical trial

Liver transplantation is traditionally performed around the clock to minimize organ ischemic time. However, the prospect of prolonging preservation times holds the potential to streamline logistics and transform liver transplantation into a semi-elective procedure, reducing the need for nighttime surgeries. Dual hypothermic oxygenated machine perfusion (DHOPE) of donor livers for 1-2h mitigates ischemia-reperfusion injury and improves transplant outcomes. Preclinical studies have shown that DHOPE can safely extend the preservation of donor livers for up to 24h. We conducted an IDEAL stage 2 prospective clinical trial comparing prolonged (≥4h) DHOPE to conventional (1-2h) DHOPE for brain-dead donor livers, enabling transplantation the following morning. Liver allocation to each group was based on donor hepatectomy end times. The primary safety endpoint was a composite of all serious adverse events (SAE) within 30 days after transplantation. The primary feasibility endpoint was defined as the number of patients assigned and successfully receiving a prolonged DHOPE-perfused liver graft. Trial registration at: WHO International Clinical Trial Registry Platform, number NL8740. Between November 1, 2020 and July 16, 2022, 24 patients were enrolled. The median preservation time was 14.5h (interquartile range [IQR], 13.9-15.5) for the prolonged group (n=12) and 7.9h (IQR, 7.6-8.6) for the control group (n=12; p=0.01). In each group, three patients (25%; 95% CI 3.9-46%, p=1) experienced a SAE. Markers of ischemia-reperfusion injury and oxidative stress in both perfusate and recipients were consistently low and showed no notable discrepancies between the two groups. All patients assigned to either the prolonged group or control group successfully received a liver graft perfused with either prolonged DHOPE or control DHOPE, respectively. This first-in-human clinical trial demonstrates the safety and feasibility of DHOPE in prolonging the preservation time of donor livers to enable daytime transplantation. The ability to extend the preservation window to up to 20h using hypothermic oxygenated machine preservation at a 10°C temperature has the potential to reshape the landscape of liver transplantation. University Medical Center Groningen, the Netherlands.

Development of a novel artificial normothermic organ perfusion preservation solution and its application in DCD donor liver preservation

Development of a novel artificial normothermic organ perfusion preservation solution and its application in DCD donor liver preservation

The role of normothermic machine perfusion (NMP) in the preservation of ex-vivo liver before transplantation: A review.

The discrepancy between the number of patients awaiting liver transplantation and the number of available donors has become a key issue in the transplant setting. There is a limited access to liver transplantation, as a result, it is increasingly dependent on the use of extended criteria donors (ECD) to increase the organ donor pool and address rising demand. However, there are still many unknown risks associated with the use of ECD, among which preservation before liver transplantation is important in determining whether patients would experience complications survive after liver transplantation. In contrast to traditional static cold preservation of donor livers, normothermic machine perfusion (NMP) may reduce preservation injury, improve graft viability, and potentially ex vivo assessment of graft viability before transplantation. Data seem to suggest that NMP can enhance the preservation of liver transplantation to some extent and improve the early outcome after transplantation. In this review, we provided an overview of NMP and its application in ex vivo liver preservation and pre-transplantation, and we summarized the data from current clinical trials of normothermic liver perfusion.

A Meta-Analysis and Systematic Review of Normothermic and Hypothermic Machine Perfusion in Liver Transplantation

The gap between the demand and supply of donor livers is still a considerable challenge. Since static cold storage is not sufficient in marginal livers, machine perfusion is being explored as an alternative. The objective of this study was to assess (dual) hypothermic oxygenated machine perfusion (HOPE/D-HOPE) and normothermic machine perfusion (NMP) in contrast to static cold storage (SCS). Three databases were searched to identify studies about machine perfusion. Graft and patient survival and postoperative complications were evaluated using the random effects model. the incidence of biliary complications was lower in HOPE vs. SCS (OR: 0.59, 95% CI: 0.36-0.98, p = 0.04, I2: 0%). There was no significant difference in biliary complications between NMP and SCS (OR: 0.76, 95% CI: 0.41-1.40, p = 0.38, I2: 55%). Graft and patient survival were significantly better in HOPE than in SCS (HR: 0.40, 95% CI: 0.23-0.71, p = 0.002, I2: 0%) and (pooled HR: 0.43, 95% CI: 0.20-0.93, p = 0.03, I2: 0%). Graft and patient survival were not significantly different between NMP and SCS. HOPE/D-HOPE and NMP are promising alternatives to SCS for donor liver preservation. They may help address the widening gap between the demand for and availability of donor livers by enabling the rescue and transplantation of marginal livers.

Application of polymerized porcine hemoglobin in the ex vivo normothermic machine perfusion of rat livers.

Background: In contrast to traditional static cold preservation of donor livers, normothermic machine perfusion (NMP) may reduce preservation injury, improve graft viability and potentially allows ex vivo assessment of graft viability before transplantation. The polymerized porcine hemoglobin is a kind of hemoglobin oxygen carrier prepared by crosslinking porcine hemoglobin by glutaraldehyde to form a polymer. The pPolyHb has been proved to have the ability of transporting oxygen which could repair the organ ischemia-reperfusion injury in rats. Objective: In order to evaluate the effectiveness of rat liver perfusion in vitro based on pPolyHb, we established the NMP system, optimized the perfusate basic formula and explored the optimal proportion of pPolyHb and basal perfusate. Methods: The liver was removed and perfused for 6h at 37°C. We compared the efficacy of liver perfusion with different ratios of pPolyHb. Subsequently, compared the perfusion effect using Krebs Henseleit solution and pPolyHb perfusate of the optimal proportion, and compared with the liver preserved with UW solution. At 0h, 1h, 3h and 6h after perfusion, appropriate samples were collected for blood gas analysis and liver injury indexes detection. Some tissue samples were collected for H&E staining and TUNEL staining to observe the morphology and detect the apoptosis rate of liver cells. And we used Western Blot test to detect the expression of Bcl-2 and Bax in the tissues. Results: According to the final results, the optimal addition ratio of pPolyHb was 24%. By comparing the values of Bcl-2/Bax, the apoptosis rate of pPolyHb group was significantly reduced. Under this ratio, the results of H&E staining and TUNEL staining showed that the liver morphology was well preserved without additional signs of hepatocyte ischemia, biliary tract injury, or hepatic sinusoid injury, and hepatocyte apoptosis was relatively mild. Conclusion: Through the above-mentioned study we show that within 6h of perfusion based on pPolyHb, liver physiological and biochemical activities may essentially be maintained in vitro. This study demonstrates that a pPolyHb-based perfusate is feasible for NMP of rat livers. This opens up a prospect for further research on NMP.

Machine perfusion preservation of donor livers and their target temperature

Machine perfusion preservation of donor livers and their target temperature

Ex situ machine preservation of donor livers for transplantation: HOPE for all?

Hypothermic oxygenated machine perfusion (HOPE) reduces ischaemia–reperfusion injury of donor livers and thereby improves outcomes after transplantation. End-ischaemic normothermic machine perfusion (NMP) enables assessment of hepatobiliary viability and selection of livers that would otherwise have been declined for transplantation. We advocate the combined use of (dual) HOPE and NMP for livers that are considered high risk, but may still be transplanted safely after ex situ resuscitation and assessment of hepatobiliary viability. Combined dual HOPE–NMP has the potential to substantially decrease the high rates of deceased donor liver discard.

High Mobility Group Box 1 Contributes to the Acute Rejection of Liver Allografts by Activating Dendritic Cells.

Acute rejection induced by the recognition of donor alloantigens by recipient T cells leads to graft failure in liver transplant recipients. The role of high mobility group box 1 (HMGB1), an inflammatory mediator, in the acute allograft rejection of liver transplants is unknown. Here, rat orthotopic liver transplantation was successfully established to analyze the expression pattern of HMGB1 in liver allografts and its potential role in promoting the maturation of dendritic cells (DCs) to promote T cell proliferation and differentiation. Five and 10 days after transplantation, allografts showed a marked upregulation of HMGB1 expression accompanied by elevated levels of serum transaminase and CD3+ and CD86+ inflammatory cell infiltration. Furthermore, in vitro experiments showed HMGB1 increased the expressions of co-stimulatory molecules (CD80, CD83, and MHC class II) on bone marrow-derived DCs. HMGB1-pulsed DCs induced naive CD4+ T cells to differentiate to Th1 and Th17 subsets secreting IFN-γ and IL-17, respectively. Further in vivo experiments confirmed the administration of glycyrrhizic acid, a natural HMGB1 inhibitor, during donor liver preservation had therapeutic effects by reducing inflammation and hepatocyte damage reflected by a decline in serum transaminase and prolonged allograft survival time. These results suggest the involvement of HMBG1 in acute liver allograft rejection and that it might be a candidate therapeutic target to avoid acute rejection after liver transplantation.

Advances in supercooling liver preservation

Liver transplantation is currently the only effective treatment for end-stage liver disease. The preservation of donor liver before transplantation is important. But both traditional static cold storage and machine perfusion are limited by the preservation time, so that the allotment space of donor liver is limited, which inevitably leads to the abandonment of part of donor liver.At present, to find a preservation technology that not only guarantees the quality of donor liver but also has a longer effective preservation time is the direction of joint efforts of all clinicians. Supercooling liver preservation(SLP) to find a preservation technology that not only guarantees the quality of donor liver but also has a longer effective preservation time is the direction of joint efforts of all clinicians. SLP, a new method based on using cryoprotectants to keep donor liver under -6 ℃ and recovering the graft with subnormothermic machine perfusion that enables long-term transplantation survival following 4 days of liver preservation, made a revolutionary breakthrough in the field of liver preservation, carved out a new field for the research of liver preservation. This article reviews the latest experimental research progress of SLP in the field of liver transplantation.

Progress in research on donor liver preservation technology in vitro

Liver transplantation is the most effective therapeutic options for the patients at the advanced stage, but with the amount of transplant surgery increasing, margin donors are used for transplantation in the case of severe donor organ deficiency. However, the commonly used cold storage technique has poor preservation effect on margin donors, resulting in an increase in the incidence of complications after transplantation. The donated liver quality is one the most important factors for the patients long term survival, so there is an urgent need for a new type of organ preservation technology to preserve the margin donors in vitro. This paper summarized the current research on the ex-vivo preservation methods of liver and the new mechanical perfusion preservation methods. Key words: Liver transplantation; Normothermic machine perfusion; Hypothermic machine perfusion; Subormothermic machine perfusion; Static cold storage

Hypothermic oxygenated machine perfusion enables 24-hour Ex situ preservation of porcine donation after circulatory death livers

Background: End-ischemic hypothermic oxygenated machine perfusion reduces ischemia-reperfusion injury of donor livers during storage and transplantation. However, it is unknown whether this technique also supports an extension of the ex situ preservation time. We aimed to determine whether liver grafts from donation after circulatory death (DCD) porcine donors can be effectively preserved up to 24-hours using dual hypothermic oxygenated machine perfusion (DHOPE). Methods Porcine DCD livers were subjected to 2h static cold storage (SCS), followed by 2, 6, or 24 hours DHOPE (n=6 per group), using Belzer UW solution. Hepatocellular and bile duct viability were tested during 4h normothermic ex situ reperfusion with autologous blood. DCD livers preserved by 24h SCS (n=2) served as controls. Results: In all three study groups, portal venous and arterial flows remained stable during DHOPE. After normothermic reperfusion, there were no significant differences in lactate clearance, blood pH, glucose, and alanine aminotransferase levels among the DHOPE-preserved levels. All DHOPE-preserved livers produced bile and there were no significant differences in biliary pH, HCO3-, and LDH between the groups at 4h after reperfusion. Moreover, levels of malondialdehyde and HMGB-1 (markers for oxidative stress) in serum and liver parenchyma were similar for all DHOPE-preserved livers. Levels of cell-free DNA, a marker of cell death, were similar between the groups. Histological analysis of bile ducts and liver parenchyma also revealed no differences. In contrast, liver preserved by 24h SCS did not produce bile after reperfusion and turned bluish with reducing portal and arterial flows, representative for a non-viable liver. Histology revealed massive cellular necrosis. Conclusion: While 24h SCS preservation of DCD liver grafts lead to non-viable livers after warm reperfusion, DHOPE enabled successful ex situ preservation of donor livers up to 24h. If confirmed with human liver grafts, this technique can facilitate logistics of allocation and transplantation. Illustrative example of a porcine liver at the end of 24-hour DHOPE (A), and at the end of reperfusion after 24-hour DHOPE (B) or 24-hourSCS (C)

Does Ischemia Free Liver Procurement Under Normothermic Perfusion Benefit the Outcome of Liver Transplantation?

BackgroundIn contrast to conventional static cold preservation, normothermic machine perfusion (NMP) provides a beneficial alternative preservation of donor livers. However, the liver still suffered cold ischemic injury before attaching to the perfusion device.Material/MethodsTo prevent cold ischemic injury during procurement, we describe a novel procedure called ischemia-free liver procurement (IFLP) under NMP. Two liver grafts were procured from brain death donor under NMP and underwent 2-hour ex vivo NMP followed by 3 and 6 hours of static cold preservation. From procurement to post-transplantation course, evidence was collected to prove that IFLP is safe and benefits recipients.ResultsThe post-transplantation course was uneventful, and the liver function tests and histological study revealed minimal hepatocyte and biliary epithelium injury during the preservation.ConclusionsThis preliminary experience demonstrates the clinical feasibility and safety of IFLP under NMP which offering opportunities to increase the number of donor livers and to improve the organ function.

Development and Assessment of Normothermic Machine Perfusion Preservation for Extracorporeal Splitting of Pig Liver.

BACKGROUND Studies have shown that normothermic machine perfusion (NMP) exerts a significant protective role for donations after cardiac death (DCD) livers and verified the effects of NMP in preservation, repair, and preoperative assessment of human donor livers. The objective of this study was to verify the effectiveness and stability of NMP system by splitting pig livers in perfusion preservation. MATERIAL AND METHODS Four healthy Ba-Ma miniature pigs were used. The livers were harvested, and then connected to the NMP system to split the livers. The indexes during the splitting were recorded, the serological indices were measured, and routine pathological examination of liver tissue were observed. RESULTS With splitting, the portal vein pressure and the hepatic artery pressure were not significantly changed. The flow in the portal vein and hepatic artery reduced respectively from 455±107.55 mmHg, 180.75±59.46 mmHg before splitting to 392.5±125.27 mmHg and 126.25±6.99 mmHg after splitting. The liver preservation temperature was controlled to 38.83±0.24 to 38.93±0.43°C during splitting. At the beginning of perfusion preservation, the alanine aminotransferase(ALT), lactate dehydrogenase(LDH), and alkaline phosphatase (ALP) were lower than the baseline level; ALT and LDH gradually increased with splitting, whereas ALP was maintained in a lower level in the splitting. At the beginning of perfusion preservation, aspartate aminotransferase(AST) was comparable with the baseline value, and it was gradually increased with splitting. The cell structure in the liver before NMP preservation and after splitting did not show significant changes. CONCLUSIONS The NMP device developed in this study can be used for donor liver preservation and splitting, without causing significant liver damage.

Ex Situ Normothermic Machine Perfusion of Donor Livers.

In contrast to conventional static cold preservation (0-4 °C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 °C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 °C and 37 °C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.

<em>Ex Situ</em> Normothermic Machine Perfusion of Donor Livers

In contrast to conventional static cold preservation (0-4 °C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 °C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 °C and 37 °C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.

Analysis of the curative effect of ABO-incompatible liver transplantation in the treatment in patients with acute severe liver disease

To analyze and evaluate the clinical effect of ABO-incompatible liver transplantation in the treatment of acute severe liver disease. A retrospective clinical study was conducted. The clinical data of 4 136 patients undergoing orthotopic liver transplantation in Organ Transplantation Center of Tianjin First Center Hospital from September 1999 to December 2013 were analyzed. The criteria of patients enrolled were as following: model for end-stage liver disease (MELD) score ≥ 20, the donor's and recipient's blood types were different, age 18-70 years, and undergone primary non-bypass orthotopic liver transplantation. According to the rate of compliance with the principles of blood transfusion, the cases were divided into two groups: ABO-compatible group (ABO-C group, n=41), ABO-incompatible group (ABO-I group, n=22). The patients in ABO-I group received basiliximab + methylprednisolone for immune induction therapy during operation, basiliximab + tacrolimus + mycophenolate + cortisol as quadruple immunosuppressive regimen after operation. They also received subcutaneous injection of low molecular heparin for anticoagulant therapy after operation, and oral warfarin or aspirin and clopidogrel bisulfate instead after 7 days. They also received routine alprostadil after operation. The remaining treatment was the same as that of ABO-C group. The clinical data, postoperative complications, rejection and survival rates of two groups were statistically analyzed. There were no significant differences in gender, age, MELD score, complicated with tumor, quality of donor liver, length of cold preservation of donor liver, duration of operation, and blood loss during operation between ABO-C and ABO-I groups. Number of splenectomy during operation was significantly higher in ABO-I group than that in ABO-C group (5 cases vs. 1 case, χ² = 4.687, P=0.030). The 3-month, 6-month, 1-year, 3-year and 5-year survival rates of ABO-C group were 89.5%, 78.3%, 72.5%, 69.1% and 61.8%, respectively, while those of ABO-I group were 78.9%, 72.9%, 65.6%, 56.2% and 46.8%, respectively. There was no significant difference in the cumulative survival rate between two groups (Log Rank, χ² = 0.647, df=1, P=0.421). The postoperative infection rate in ABO-I group was significantly higher than that of ABO-C group [63.6% (14/22) vs. 31.7% (13/41), χ² = 5.960, P=0.015]. There were no significant difference in postoperative complications of biliary tract [22.7% (5/22) vs. 12.2% (5/41), χ² = 0.531, P=0.466], vascular complications [31.8% (7/22) vs. 12.2% (5/41), χ² = 2.416, P=0.120], or rejection as diagnosed by pathology [22.7% (5/22) vs. 9.8% (4/41), χ² = 1.051, P=0.305] between ABO-I and ABO-C groups. Although ABO-incompatible liver transplantation was followed by higher postoperative infection rate and perioperative mortality, ABO-incompatible liver transplantation can still be used to save the patient with acute severe liver disease as there is a shortage of compatible donor at present.

Ex vivo Normothermic Machine Perfusion and Viability Testing of Discarded Human Donor Livers

In contrast to traditional static cold preservation of donor livers, normothermic machine perfusion may reduce preservation injury, improve graft viability and potentially allows ex vivo assessment of graft viability before transplantation. We have studied the feasibility of normothermic machine perfusion in four discarded human donor livers. Normothermic machine perfusion consisted of pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous portal perfusion for 6 h. Two hollow fiber membrane oxygenators provided oxygenation of the perfusion fluid. Biochemical markers in the perfusion fluid reflected minimal hepatic injury and improving function. Lactate levels decreased to normal values, reflecting active metabolism by the liver (mean lactate 10.0 ± 2.3 mmol/L at 30 min to 2.3 ± 1.2 mmol/L at 6 h). Bile production was observed throughout the 6 h perfusion period (mean rate 8.16 ± 0.65 g/h after the first hour). Histological examination before and after 6 h of perfusion showed well-preserved liver morphology without signs of additional hepatocellular ischemia, biliary injury or sinusoidal damage. In conclusion, this study shows that normothermic machine perfusion of human donor livers is technically feasible. It allows assessment of graft viability before transplantation, which opens new avenues for organ selection, therapeutic interventions and preconditioning.

Role of Cholangiocyte Bile Acid Transporters in Large Bile Duct Injury After Rat Liver Transplantation

The pathogenesis of nonanastomotic strictures with a patent hepatic artery remains to be investigated. This study focuses on the role of cholangiocyte bile acid transporters in bile duct injury after liver transplantation. Sprague-Dawley rats were divided into three groups (n=20 for each): the sham-operated group (Sham), the transplant group with 1-hr donor liver cold preservation (CP-1h), and the transplant group with 12-hr donor liver cold preservation (CP-12h). Bile was collected for biochemical analysis. The histopathologic evaluation of bile duct injury was performed and the cholangiocyte bile acid transporters apical sodium-dependent bile acid transporter (ASBT), ileal lipid binding protein (ILBP), and Ostalpha/Ostbeta were investigated. RESULTS.: The immunohistochemical assay suggested that ASBT and ILBP were expressed exclusively on large bile duct epithelial cells, whereas Ostalpha and Ostbeta were expressed on both small and large bile ducts. Western blot and quantitative polymerase chain reaction analysis showed that the expression levels of these transporters dramatically decreased after transplantation. It took seven to 14 days for ILBP, Ostalpha, and Ostbeta to recover, whereas ASBT recovered within 3 days and even reached a peak above the normal level seven days after operation. In the CP-12h group, the ratios of the ASBT/ILBP, ASBT/Ostalpha and ASBT/Ostbeta expression levels were correlated with the injury severity scores of large but not small bile ducts. The results suggest that the unparallel alteration of cholangiocyte bile acid transporters may play a potential role in large bile duct injury after liver transplantation with prolonged donor liver preservation.

Endogenous Bile Salts Are Associated With Bile Duct Injury in the Rat Liver Transplantation Model

Nonanastomotic biliary strictures are a serious complication after orthotopic liver transplantation (OLT) and are difficult to cure. We examined the role of endogenous bile salt toxicity in the pathogenesis of bile duct injury after OLT. A total of 90 Sprague-Dawley rats were divided into three groups: Sham-operated group (Sham, n=30), OLT group with 1 hr donor liver preservation (OLT-1h, n=30), and OLT group with 12 hr donor liver preservation (OLT-12h, n=30). Bile was collected and analyzed biochemically. The histopathologic study was performed to determine the intrahepatic bile duct morphologic changes. Hepatic expressions of bile transporters Ntcp, Bsep, Mdr2, and Oatps were detected. During the first 2 weeks after transplantation, bile salt secretion was not parallel with phospholipid secretion, resulting in high biliary bile salt-to-phospholipid (BS:PL) ratio. The expression of bile transporters was consistent with the change of bile composition. Bile duct injury correlated significantly with bile salt secretion and BS:PL ratio. Moreover, OLT group with longer donor liver preservation time (OLT-12h) had significantly lower Mdr2 messenger RNA/protein level, higher BS:PL ratio, and better correlation between BS:PL ratio and bile duct injury compared with those of OLT-1h. The unparallel secretion of bile salts and phospholipids results in cytotoxic bile formation with high BS:PL ratio after liver transplantation. Longer donor liver preservation time will increase graft bile cytotoxicity. The results of this study suggest that endogenous bile salts play a role in the pathogenesis of bile duct injury after OLT.

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.