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Keywords:

  • Discarded human liver;
  • liver transplantation;
  • machine perfusion;
  • normothermic;
  • preservation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

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.


Abbreviations
ALT

alanine aminotransferase

DBD

donation after brain death

DCD

donation after cardiac death

ECD

extended criteria donors

gamma-GT

gamma-glutamyl transferase

LDH

lactate dehydrogenase

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Although the traditional organ preservation method of static cold storage is sufficient for preserving most livers for transplantation, it fails to maintain viability in part of the grafts of suboptimal quality, or so-called extended criteria donor (ECD) grafts. Higher rates of delayed graft function, primary nonfunction and cholangiopathies are seen in recipients of donation after cardiac death (DCD), older and steatotic grafts [1, 2]. As the discrepancy between available donor organs and the number of patients waiting for transplantation increases, more ECD grafts will be used, necessitating the development of new and better preservation and selection methods.

Machine perfusion of donor livers offers potential to meet the requirements to preserve ECD grafts. A relatively simple option is hypothermic machine perfusion (0–4°C), offering oxygen supply to the liver and the removal of waste products. However, although hypothermic perfusion has achieved adequate results in near-clinical and clinical setting [3, 4], its use seems to offer too little protection in suboptimal grafts [5, 6]. An important advantage of normothermic perfusion (37°C) over static cold preservation and hypothermic perfusion is the full metabolic support and the possibility to assess graft viability before transplantation. In addition, liver grafts could potentially be optimized by pharmacological conditioning during normothermic perfusion. However, although normothermic machine perfusion may provide better viability testing and resuscitation, it requires challenging, near-physiological conditions [7]. To date, successful normothermic perfusion of livers has been reported only in animal models [8-12]. Although these results are promising, feasibility of normothermic machine perfusion in human livers remains to be demonstrated. We studied the feasibility of normothermic, oxygenated machine preservation and ex vivo viability testing of discarded human ECD livers, using a newly developed liver perfusion machine.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Donor livers

Between May 2012 and August 2012, four human livers derived from DCD donors that were declined for transplantation by all three transplant centers in the Netherlands were included in this study. Characteristics of these four livers are presented in Table 1. In all cases permission to use the liver for this study was obtained from the relatives. The study was approved by the medical ethical committee of the University Medical Center Groningen and the Nederlandse Transplantatie Stichting, the competent authority for organ donation in the Netherlands.

Table 1. Characteristics of the discarded human donor livers used for normothermic machine perfusion
 Liver no.
Donor characteristics1234
  1. The grade of steatosis was based on the grading system established by a leading group of expert liver pathologists (Kleiner DE, Brunt EM, Van Natta M, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005; 41: 1313–1321). Grade I translates to 5–33% of macrovesicular steatosis. DBD = donation after brain death; DCD = donation after cardiac death; HTK = histidine-tryptophan-ketoglutarate; UW = University of Wisconsin.

DCD/DBDDCDDCDDCDDCD
Age (years)66443864
SexMaleFemaleFemaleMale
Height (m)1.851.651.701.80
Weight (kg)105926575
Cause of brain injurySubdural hematomaSubarachnoid hemorrhageDrowningTraumatic brain injury
Weight of the liver (kg)2.062.372.061.75
Reason for rejection for transplantationDCD, age 60+ and steatosis35 min of O2-saturation <30% before deathTumor detected, malignancy could not be excludedDCD, age 60+ and alcohol abuse in medical history.
Time between withdrawal of mechanical, ventilated or organ-perfusion support and cardiac death17 min40 min11 min24 min
Time between cardiac death and cold perfusion17 min16 min12 min17 min
Cold ischemic preservation4 h 46 min6 h 29 min9 h 28 min6 h 56 min
Preservation solution during initial cold preservationHTKHTKHTKUW
Normothermic perfusion time6 h6 h6 h6 h
Histological grade of steatosisGrade 1Grade 0Grade 0Grade 0

Normothermic machine perfusion

For normothermic perfusion of the donor livers we used a CE marked (European Union certification of safety, health and environmental requirements) device that enables dual perfusion via both the hepatic artery and the portal vein in a closed circuit (Liver Assist®, Organ Assist, Groningen, The Netherlands; Figure 1). Two rotary pumps provided a pulsatile flow to the hepatic artery and a continuous flow to the portal vein. Two hollow fiber membrane oxygenators provided oxygenation of the perfusion solution, as well as removal of CO2. The system was both pressure and temperature controlled, which allowed auto regulation of the blood flow through the liver, with constant pressure at variable flow rates. Flow, pressure and temperature were displayed on the device in real-time. Pressure was limited to a mean of 50 mmHg in the hepatic artery and 11 mmHg in the portal vein. The temperature was set to 37°C and a new sterile disposable set of tubing, reservoir and oxygenators was used for each liver.

image

Figure 1. A schematic drawing (A) and a photo (B) of the perfusion machine used for normothermic perfusion of human donor livers (Liver Assist®, Organ Assist, Groningen, The Netherlands).

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Composition of perfusion fluid

We aimed for a perfusion fluid containing all nutrients, oxygen and protective substances required by a metabolically active liver. Detailed information on the various components and the biochemical characteristics of the perfusion fluid are provided in Tables 2 and 3. The base of our perfusion fluid was human packed red blood cells and fresh frozen plasma; blood group and Rhesus factor identical to the donor liver. These blood products were obtained from the local blood bank and derived from third party blood donors. Because of the relatively low hematocrit of the perfusion solution (0.23 ± 0.01 at the start of perfusion) the pO2 was targeted at supernormal values (60 kPa) in order to provide sufficient oxygen to the liver. Heparin was added for anticoagulation, and vitamins, glucose, amino acids and trace elements were added to provide sufficient nutrients for the liver. Broad-spectrum antibiotics cefazolin and metronidazole were added to prevent bacterial growth and graft infection under normothermic conditions. To prevent the development of interstitial edema or intracellular contraction, the oncotic and osmotic pressure of the perfusion fluid was adjusted by adding sterile H2O, saline and human albumin. The targeted osmolarity of the perfusion fluid was 302 mosmol/L. Sodium bicarbonate was added for buffering capacity.

Table 2. Composition of the perfusion fluid used for normothermic machine perfusion of human donor livers
ComponentQuantity
Red blood cell concentrate (hematocrit 60%)750 mL
Fresh frozen plasma900 mL
Human albumin 200g/L (Albuman®, Sanquin)100 mL
Modified parenteral nutrition (Clinimix® N17G35E, Baxter international Inc.)7.35 mL
Multivitamins for infusion (Cernevit®, Baxter international Inc.)7 μL
Concentrated trace elements for infusion (Nutritrace®, B. Braun Melsungen AG)7.35 mL
Metronidazol for i.v. administration (5 mg/mL) (Flagyl®, Sanofi-Aventis)40 mL
Cefazolin 1000 mg flask 5 mL powder for i.v. administration (Servazolin®, Sandoz )2 mL
Fast-acting insulin (100 IU/mL) (Actrapid®, Novo Nordisk)20 mL
Calcium glubionate, intravenous solution 10%, 137.5 mg/mL (Sandoz®)40 mL
Sterile H2O51.3 mL
NaCl 0.9% solution160 mL
Sodium bicarbonate 8.4% solution31 mL
Heparin 5000 IE/mL for i.v. administration4 mL
Total volume2120 mL
Table 3. Biochemical characteristics of the perfusion fluid before connecting the liver, after 30 min and 6 h of normothermic perfusion
Chemical variableBefore start perfusionAt 30 minAt 6 hReference values in blood
  1. To convert values for glucose to mg/dL, multiply by 18.02. To convert values for lactate to mg/dL, multiply by 9.01. To convert values for hemoglobin to g/dL, multiply by 1.650. Data are expressed as mean ± SEM.

pH7.59 ± 0.117.53 ± 0.097.36 ± 0.017.35 – 7.45
pCO2 (kPa)3.5 ± 0.41.9 ± 0.85.0 ± 1.04.6 – 6.0
pO2 (kPa)74.8 ± 2.469.7 ± 3.653.1 ± 8.09.5 – 13.5
sO2 (%)100 ± 0.1100 ± 0.2100 ± 0.296 – 99
HCO3 (mmol/L)28 ± 1011 ± 321 ± 321 – 25
Base excess (mmol/L)4.9 ± 10.5-12.6 ± 4.4-5.1 ± 2.8– 3.0 to 3.0
Na+ (mmol/L)150 ± 7139 ± 5144 ± 4135 – 145
K+ (mmol/L)5.3 ± 0.64.8 ± 0.96.5 ± 1.03.5 – 5.0
Free Ca2+ (mmol/L)0.62 ± 0.031.47 ± 0.151.32 ± 0.141.15 – 1.29
Glucose (mmol/L)13.7 ± 0.627.8 ± 6.011.3 ± 5.54 – 9
Lactate (mmol/L)6.3 ± 0.510.0 ± 2.32.3 ± 1.20.5 – 2.2
Hemoglobin (mmol/L)4.7 ± 0.13.8 ± 0.13.9 ± 0.28.7 – 10.6
Hematocrit0.23 ± 0.010.19 ± 0.010.20 ± 0.010.42 – 0.52

Procurement and preparation of donor livers

All liver procurement procedures were performed by one of the regional multi-organ recovery teams, in the Netherlands, using a standard technique of in situ cooling and flush-out with ice cold preservation fluid (University of Wisconsin [UW] or histidine–tryptophan–ketoglutarate [HTK] solution). Care was taken to leave a segment of the supratruncal aorta attached. Livers were packed and stored in standard sterile donor organ bags and a box with crushed ice, and subsequently transported to our center. Immediately upon arrival of the donor liver in the operating room, the back table procedure was performed by an experienced transplant surgeon. Arteries and the portal vein were dissected free and the arterial cannula was inserted and secured in the proximal end of the supratruncal aorta segment. The distal end of the aorta was closed below the origin of the celiac trunc, using a prolene 4–0 suture. A second cannula was inserted and secured in the distal end of the portal vein. The cannulas for the hepatic artery and the portal vein are part of the sterile disposables provided by Organ Assist. The cystic duct was ligated to prevent bile flow from the gallbladder into the common bile duct. The gallbladder was not removed to avoid continuous bleeding from the gallbladder fossa in a fully heparinized system. A Meredith silicon catheter no. 8 was inserted and secured in the distal common bile duct. After preparation on the back table, the liver was weighed. Immediately before connecting the liver to the perfusion machine, the liver was rapidly flushed with 1 L cold NaCl 0.9% solution (only when UW solution was used for preservation), followed by 1 L warm NaCl 0.9% solution.

Liver function assessment

During machine perfusion, samples were taken from the perfusion fluid every 30 min and analyzed immediately for blood gas parameters (pO2, pCO2, sO2, HCO3 and pH) and for biochemical parameters (glucose, calcium, lactate, potassium and sodium) by an ABL800 FLEX analyzer (Radiometer, Brønshøj, Denmark). In addition, plasma from the perfusion fluid was collected (after 5 min centrifugation 2700 rpm at 4°C), frozen and stored at –80°C for determination of alkaline phosphatase, gamma-glutamyl transferase (gamma-GT), alanine aminotransferase (ALT), urea and total bilirubin, using standard biochemical methods. Bile production was measured at 30 min intervals by weighing the Eppendorf tubes in which bile was collected from the biliary drain. Biliary epithelial cell function was assessed by measuring biliary bicarbonate concentration. For this purpose, bile samples were collected under mineral oil and analyzed immediately using an ABL800 FLEX analyzer (Radiometer). Biliary concentration of gamma-GT and lactate dehydrogenase (LDH) were measured as biomarkers of biliary epithelial cell injury [13], and biliary bilirubin concentration was measured as biomarker of hepatocellular secretory function, using standard biochemical methods.

Histological evaluation

Biopsies were obtained from the liver parenchyma as well as the extrahepatic bile ducts before and after machine perfusion and stored in formalin for histological evaluation. Paraffin-embedded slides of liver biopsies were prepared for hematoxylin and eosin (H&E) staining and complementary liver staining with periodic acid-Schiff after diastase digestion, Masson's trichrome, iron and reticulin. Additional slides were prepared for immunohistochemical detection of activated caspase-3 (Asp175, Cell Signaling #9661; 1:100 dilution), a marker for apoptosis. Paraffin-embedded slides of extrahepatic bile ducts were prepared for H&E staining and complementary staining with periodic acid-Schiff after diastase digestion. Slides of the extrahepatic bile ducts were graded according to a systematic scoring system of bile duct injury as first described by Hansen et al. [14]. All liver and bile duct slides were examined by an experienced liver pathologist (ASH Gouw) using light microscopy.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Perfusion characteristics

Immediately after arrival in our center, donor livers were prepared on the back table and connected to the normothermic perfusion system, resulting in a mean cold storage period of 415 ± 58 min. Both the arterial and portal flow increased during the first 30 min of normothermic perfusion and remained stable thereafter, with a mean arterial flow of 283 ± 29 mL/min and a mean portal flow of 686 ± 25 mL/min at 6 h (Figure 2A). The perfusion machine provided adequate oxygenation of the perfusion fluid and extraction of carbon dioxide (Figure 2B). Macroscopically all livers seemed well perfused (Figure 3).

image

Figure 2. Functional parameters of the perfusion machine and markers of liver viability and injury during 6 h of normothermic machine perfusion (n = 4). (A) and (B) Perfusion characteristics and gas exchange parameters. (C) and (D) Concentrations of hepatobiliary injury and function markers in the perfusion fluid. (E) Bile production during perfusion (n = 3). (F) Eppendorf tubes containing bile from graft #1; demonstrating a gradual change to a darker shade over time, in parallel with increasing biliary concentration of bilirubin and bicarbonate. (G) Evolution of biliary concentrations of gamma-GT and LDH as markers of biliary injury. Data are expressed as mean ± SEM.

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image

Figure 3. Photos of liver #1 before and during normothermic machine perfusion. (A) Just before connecting the liver to the perfusion machine. (B) One minute after connecting the liver. (C) Twenty minutes after connecting the liver. (D) After 6 h of normothermic machine perfusion. During operation the organ chamber is covered by a transparent cover to maintain a moist environment for the liver (not shown in these pictures).

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Assessment of hepatic injury and function

Changes in the concentration of biochemical markers in the perfusion fluid reflected minimal hepatic injury and improving function (Figures 2C and D). After a temporary rise in ALT directly after the start of perfusion, ALT concentrations remained stable. Other markers of cell injury also remained stable (gamma-GT and potassium) or decreased (alkaline phosphatase; Figure 2C). Lactate concentrations initially increased but subsequently 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; Figure 2D). Similarly, glucose levels decreased to near-normal values (mean 11 ± 5 mmol/L at 6 h). Bicarbonate levels increased during perfusion to physiological levels (mean 20.7 ± 2.8 mmol/L at 6 h). Overall, the pH at the start of perfusion was slightly alkalotic, but in all procedures it returned to normal values within the first hour (mean pH 7.40 ± 0.06 at 1 h; Figure 2B). Urea, produced by the liver, increased from 5.9 ± 1.0 mmol/L at 30 min to 12.9 ± 2.3 mmol/L at 6 h (Figure 2D).

Bile production was observed throughout the entire perfusion period (Figure 2E). The mean rate of bile production after the first hour was 8.16 ± 0.65 g/h. The quality of bile improved during perfusion as indicated by a gradual change in color to a darker shade and increasing concentrations of biliary bilirubin and bicarbonate (Figure 2F). Biliary gamma-GT and LDH concentrations decreased after an initial peak at 90 min of perfusion (Figure 2G).

Liver and extrahepatic bile duct histology

Histological examination of H&E stained liver biopsies showed no major differences between biopsies taken before and after 6 h of perfusion (Figure 4). In general, hepatocytes seemed viable without additional ischemic changes and no signs of biliary injury or sinusoidal damage were present in the liver biopsies after machine perfusion. Additional liver staining with Masson's trichrome, reticulin, iron, periodic acid-Schiff after diastase digestion and immunohistochemistry for caspase-3 confirmed normal liver morphology and provided no evidence of additional apoptotic cell death after machine perfusion (data not shown).

image

Figure 4. Hematoxylin and eosin staining of liver biopsies before (A–D 0 h) and after (A–D 6 h) 6 h of normothermic machine perfusion, showing well-preserved microscopic architecture in all four perfused livers.

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Histological examination of biopsies from the distal end of the extrahepatic bile duct taken before machine preservation revealed partial loss of the biliary epithelial cell layer similar to what has been described by Hansen et al. [14] in human liver transplantation (Figure 5). Further histological assessment of bile duct wall injury revealed no evidence of major intramural bleeding and no microthrombi in the vasculature. The amount of arteriolonecrosis remained stable in graft #3 and #4, but increased with one degree in grafts #1 and #2. After perfusion, normal morphology of the bile duct wall stroma with presence of cell nuclei was seen in graft #3, but a decrease in the number of cell nuclei in the stroma was noted in the other grafts.

image

Figure 5. Hematoxylin and eosin stainings of biopsies from the distal end of the extrahepatic bile duct (graft #3), taken before and after 6 h of normothermic machine perfusion. Panel (A) shows an overview at 100× magnification, the lumen of the bile duct is marked by the asterisk. Similar to what has been described in over 80% of human livers before clinical transplantation [14], biopsies taken before machine preservation revealed partial loss of the biliary epithelial cell layer. The amount of biliary epithelial cell loss did not increase during 6 h of machine perfusion. Further histological assessment at higher magnification (panel B and C) revealed no evidence of major intramural bleeding and no microthrombi in the vasculature. Well-preserved bile duct wall stroma is marked by # in panel (B). Arrows in panel (C) indicate arteries/arterioles of the peribiliary plexus with normal morphology.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

In this study, we demonstrated feasibility of normothermic machine perfusion of four discarded human donor livers. There were no technical failures of the perfusion machine and all livers were well perfused and oxygenated. The livers, although discarded for varying reasons, functioned well ex vivo. Macroscopically well-perfused livers, continuous bile production (possibly the most important outcome parameter for a well-functioning liver [Ref. [9]]) and normalization of various biochemical parameters in the perfusion fluid were noted over time in all procedures. Histological examinations confirmed adequate preservation of liver morphology.

This is the first description of successful normothermic machine perfusion of human livers, ever since the first unsuccessful attempts of hypothermic perfusion of human livers by Starzl et al. in 1967, after which the clinical use and development of human liver perfusion devices stagnated [15]. Only recently, renewed clinical application of machine preservation has been reported by Guarrera et al. [4]. The device used by these investigators, however, was based on hypothermic perfusion (at 4°C) without oxygenation of the perfusion fluid. In contrast to oxygenated, normothermic machine perfusion, hypothermic perfusion does not allow an adequate functional assessment of liver viability before transplantation. Especially in an era of donor organ shortage, normothermic machine preservation has great potential as a tool to increase the number of donor livers by enabling pretransplant assessment of ECD livers that would otherwise not have been accepted for transplantation because of the presumed too high risk of primary nonfunction.

Based on a porcine model of normothermic perfusion, bile output has been suggested as the most significant parameter of liver viability [9]. We observed increasing bile output in all four livers tested during 6 h of normothermic machine preservation. Assessment of bile composition showed a gradual increase in biliary bilirubin and bicarbonate concentration, reflecting recovery of secretory function of hepatocytes and biliary epithelial cells, respectively. In parallel with this, biomarkers of biliary epithelial cell injury such as biliary concentrations of gamma-GGT and LDH decreased, indicating that there was no ongoing biliary epithelial cell injury during the 6 h of perfusion.

In a recent clinical study in 93 liver transplants, Hansen et al. [14] have observed a >50% loss of biliary epithelium in 82.8% of extrahepatic bile duct biopsies taken before transplantation. Therefore, it is not surprising that we observed similar changes in our biopsies before machine perfusion. Although some signs of bile duct wall injury as described by Hansen et al., such as intramural bleeding and microthrombi in the vasculature, could not be detected, other signs such as arteriolonecrosis and loss of cell nuclei in the bile duct wall stroma were observed after perfusion. Collectively, these histological findings indicate that normothermic machine perfusion alone is not sufficient to completely avoid ischemia/reperfusion injury of the extrahepatic bile ducts. It needs to be awaited whether modifications of the perfusion fluid, for example by adding cytoprotective hydrophilic bile salts, will result in a reduction of the degree of biliary ischemia/reperfusion injury and a subsequent lower rate of biliary complications after transplantation. Clearly this is an area that still requires intensive further research.

Whether ex vivo bile output and assessment of other viability parameters can be used as reliable parameters to discriminate viable from nonviable human donor livers will also require confirmation in a larger cohort. This work is in progress in our center and the ultimate goal will be to perform a clinical trial. One option would be to apply normothermic machine perfusion in ECD livers that have been rejected for transplantation and will otherwise be discarded. This is a strategy followed by Guarrera et al. in a clinical trial on hypothermic liver machine perfusion [4]. Alternatively, one could think of a randomized clinical trial of ECD livers, such as livers obtained from DCD donors, using biliary strictures as the primary endpoint.

Transplantation of ECD livers, such as steatotic or old livers, results in an increased risk of primary nonfunction or delayed graft function [1]. In the case of DCD livers, the most important complication is the development of cholangiopathies [2]. Although we have been able to assess overall viability of the perfused livers by measurement of bile production, serial analysis of biochemical markers in the perfusion fluid and bile, as well as histological examination, accurate functional assessment of biliary epithelium viability is more difficult. We have used biliary secretion of bicarbonate as a biomarker of biliary epithelial function. Biliary epithelial cells contribute significantly to bile volume and flow by active secretion of bicarbonate and we have observed a recovery of biliary bicarbonate secretion during machine perfusion. The amount of bicarbonate secretion in the bile, however, is the result of all biliary epithelial cells present in the intra- and extrahepatic bile ducts and it does not necessarily reflect function of the epithelium of the larger bile ducts, which are most prone for the development of stricture after transplantation. More research in this area is clearly needed [16].

In conclusion, this study shows that normothermic machine perfusion of human donor livers is technically feasible. Normothermic perfusion of ECD livers allows assessment of graft viability before transplantation and opens new avenues for donor organ selection, therapeutic interventions and preconditioning. This may not only improve organ quality and function, but will also lead to a considerable expansion of the number of organs available for transplantation.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

This research work was financially supported by grants provided by Innovatief Actieprogramma Groningen (IAG-3), Jan Kornelis de Cock Stichting and Tekke Huizingafonds, all in The Netherlands. We are grateful to Arjan van der Plaats and Martin Kuizenga (Organ Assist, Groningen, Netherlands) for their technical support and assistance during the perfusion experiments. Furthermore, we are appreciative to all the Dutch transplantation coordinators, Ernst Buiter and Cees Brugman in particular, for identifying the potential discarded livers and obtaining informed consent.

Disclosure

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References