Marginal grafts, such as fatty livers, grafts from older donors, or livers retrieved after cardiac death (DCD) tolerate conventional, cold static storage only poorly. We developed a novel model of subnormothermic ex vivo liver perfusion for preservation, assessment, and repair of marginal liver grafts prior to transplantation.
The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression. One strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (DCD). The current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. In addition, cold static organ storage does not allow graft assessment or repair prior to transplantation.
These shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. The optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.
In spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. Recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.
This paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. We will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs.
Liver transplantation is the only treatment option for patients with end stage liver disease or advanced hepatocellular carcinoma. For the last 25 years, the number of waiting list candidates has gradually increased and exceeded the number of available grafts. The number of heart beating donors has decreased over the last decade. At the same time, numbers of marginal grafts, such as donation after cardiac death (DCD), as well as old and fatty livers have increased1,2.
Marginal grafts are often declined for liver transplantation because of the higher chance of primary graft non- or delayed function. In DCD grafts, development of ischemic type biliary strictures (ITBS) is of special concern. With the conventional static cold preservation technique, ITBS occur in about 10-40% of DCD grafts. In the majority of patients, ITBS leads to re-transplantation or patient death. Especially prolonged warm and cold ischemic times are risk factors for ITBS3-7. Donor age, genetic predispositions (such as CCR5 delta 32), and the choice of preservation solution have also been discussed as additional risk factors7-10. Partial microthrombosis of the peribiliary vessels has been suggested as potential mechanism for ITBS after liver transplantation with DCD grafts11.
Prior to the clinical introduction of liver transplantation, ex vivo liver perfusions have been used to study hepatic metabolism and physiology12,13. After liver transplantation found its way into the clinical setting in the 1960s, innumerable attempts have been made to use ex vivo liver perfusion as a preservation method by mimicking physiological nutrition and oxygenation conditions. Its utility for preservation of marginal grafts has been investigated in the last decade, but it did not reach standard clinical care. We recently described a reduction in bile duct injury in DCD liver transplantation by ex vivo perfused preservation14. Different approaches regarding the perfusion solution were made. The selection ranges from cellular solutions like whole blood from the donor animal or packed red cells in combination with human plasma, to acellular approaches like machine University of Wisconsin solution, IGL solution, or Steen solution14-19.
The temperature ranges from 4-37 °C20. The nomenclature in hypothermic, subnormothermic, and normothermic is very variable and inconsistent. All different techniques, solutions, and temperature settings aim at 1) stable perfusion conditions, 2) sufficient oxygenation, and 3) re-establishment of organ function. An enhanced preservation capacity as well as the ability of organ assessment and treatment during normothermic and subnormothermic perfusion faces higher technical complexity and costs compared to hypothermic perfusion20,21.
We have developed a subnormothermic ex vivo liver perfusion system over the last 4 years. The system can be used to 1) “recharge” the hepatic energy content, 2) to assess the graft’s quality, and to 3) repair marginal livers prior to transplantation. The following protocol contains all information for a stable hepatic perfusion.
A schematic overview of the protocol is presented in Figure 1.
Figure 1. Study protocol. The porcine study design of liver injury is based on a donation after cardiac death (DCD) model. After dissection of all liver vessels, cardiac death is induced followed by 45 min of warm graft ischemia. To simulate a graft transport in between the donor and recipient hospitals in a clinical setting, the graft is stored on ice for 4 hr after cold, dual flush. After cold storage, the organ is subnormothermic perfused for 6 hr in order to assess the perfusion stability. In a transplant model, the perfusion time could be shorter in order to recharge energy storage and to assess the organ viability. Please click here to view a larger version of this figure.
1. Animals
NOTE: Male Yorkshire pigs, 30-35 kg, were utilized for this study. All animals received humane care in compliance with the ‘‘Principles of Laboratory Animal Care’’ formulated by the National Society for Medical Research and the ‘‘Guide for the Care of Laboratory Animals’’ published by the National Institutes of Health. The Animal Care Committee of the Toronto General Research Institute approved all studies.
2. Organ Retrieval
3. Ex vivo Liver Perfusion
Below, we present the results of 5 perfusion experiments with DCD-grafts after 45 min warm- and 4 hr cold ischemia prior to the start of the subnormothermic ex vivo perfusion.
The main goal for an ex vivo liver perfusion is to ensure a sufficient oxygen supply to the organ. Ischemia causes vasoconstriction, thus increasing the perfusion resistance. Achieving constant vascular flows with stable pressures is a good indicator of adequate oxygenation. During an induction period of 1-2 hr the perfusion solution and the organ are warmed up to 33 °C, which deceases the vascular resistance of the liver. Once the target temperature of 33 °C is achieved, flow values level at a constant, nearly physiological range for the rest of the 6 hr perfusion time (Figures 3A-3D).
At the same time, the organ becomes metabolically active. Figure 4A shows the venous pO2, a marker of oxygen consumption. Within the initial 2 hr the venous pO2 declines to a constant plateau. At this metabolically active state, the liver starts producing bile (Figure 4B). The dialyzer provides a balanced electrolyte homeostasis (Figures 4C-4D). An initial hyperkalemia is quickly leveled out. Online AST measurement serves as monitoring of hepatocellular damage. Figure 5 displays only a shallow linear AST increase over the entire perfusion period. H&E staining after 6 hr of perfusion reveals hepatocyte necrosis <5 % with an intact lobular and sinusoidal structure (Figure 6). PAS staining at the same time point shows replenished cellular glycogen storage compared to exhausted storage in cold preserved DCD-grafts (Figure 7).
Figure 3. Perfusion flows and pressure (n = 5, error bars show standard deviation). (A,B) Hepatic artery (HA) flow and pressure: During the warming phase in the first 1-2 hr, the flow increases at stable pressures and is constant afterwards. Looking at the decreasing portal venous pressure (C), the increase of HA flow towards the end of the perfusion might be an autoregulatory reaction of the liver. (C,D) The portal venous (PV) flow increases corresponding to the HA flow during the first 2 hr of warming. The pressures remain relatively stable.
Figure 4. Monitoring parameters (n = 5, error bars show standard deviation). (A) The venous pO2 as a marker of oxygen demand and metabolic activity decreases within the initial phase of warming due to activated cellular metabolism; it remains stable afterwards. (B) Bile production as a marker of metabolic activity starts at temperatures around 30 °C and, thus, between the first and second hour of perfusion. (C,D) The dialyzer assures electrolyte homeostasis; an initial hyperkalemia is quickly balanced.
Figure 5. AST (n = 5, error bars show standard deviation). AST is a sensitive marker of hepatocellular injury; the shallow increase suggests no significant injury during ex vivo perfusion.
Figure 6. H&E staining (20X magnification). (A) Sham liver sample before warm ischemia, one representative liver lobule with intact architecture. (B) Liver sample after 45 min of warm ischemia, 4 hr of cold ischemia, and 6 hr of subnormothermic perfusion, the lobular architecture is intact without necrosis and only minimal cell swelling, the sinusoidal spaces are mildly dilated in comparison to the sham sample.
In a pig model that mimics DCD liver transplantation, we demonstrated that subnormothermic liver perfusion with a cellular perfusion solution results in stable perfusion parameters, minimal hepatocyte injury, and active hepatic metabolism. Our subnormothermic perfusion set up has proven to recover a hepatocellular homeostasis and metabolism. Glycogen storage is restored and metabolites are discarded.
Ex vivo liver perfusion as preservation technique offers for the first time the opportunity to assess markers of graft function and injury during organ preservation and prior to transplantation. Beside the macroscopic evaluation of the graft perfusion homogeneity, flow values provide a good indicator of the graft’s viability and the extent of the ischemic injury it had suffered earlier29. Oxygen consumption and bile production are markers of metabolic function. Levels of hepatic enzymes like AST can be used to assess the degree and dynamics of hepatocellular injury30. This thorough graft assessment may allow a reliable discrimination between transplantable and non-transplantable marginal organs.
We chose a subnormothermic temperature of 33 °C in our perfusion system because the temperature is sufficient to allow metabolism as well as ATP and glycogen synthesis. At the same time, it provides a decreased oxygen demand in comparison to normothermic perfusion settings which provides additional safety against ischemic injury. In general, perfusion temperatures above 30 °C have shown to minimize cold ischemic injury and provide sufficient metabolic activity31.
Contrary to other groups, we did not use whole blood as perfusate, but a normo-osmotic albumin solution (Steen) with washed and filtered red blood cells. By excluding the plasma components as well as thrombocytes and leukocytes, the perfusion solution is designed to minimize pro-inflammatory signaling during the ex vivo perfusion.
In addition to the graft assessment, stable perfusion conditions over several hours allow graft treatment. Numerous molecules have shown to attenuate reperfusion injury under experimental conditions32. However, almost no treatment regime has made its way into clinical practice, yet. One reason seems to be the lack of opportunity to apply those treatments during cold storage. A metabolically active liver on an ex vivo perfusion system is optimal for applying any kind of treatment. In this regard, not only treatments to ameliorate reperfusion conditions like attenuation of Kupffer cell activity or scavenging of reactive oxygen species are conceivable but also treatments like gene therapy to condition the graft, e.g., against Hepatitis C recurrence. Other potential strategies could include reduction on steatosis during the ex vivo perfusion period33.
In summary, ex vivo liver perfusion is a novel strategy to minimize cold ischemic injury and to assess marginal liver grafts prior to liver transplantation. The ex vivo perfusion setting provides unique opportunity to repair and condition grafts prior to transplantation.
The authors have nothing to disclose.
The study was supported by research grants of the Roche Organ Transplant Research Foundation (ROTRF) and Astellas. Markus Selzner was supported by an ASTS Career Development Award. Matthias Knaak was supported by the Astellas Research Scholarship. We thank Uwe Mummenhoff and the Birmingham family for their generous support.
circuit | Maquet (Hirrlingen, GER) | custom made | main reservoir (3L, 3/8" outflow) |
– | portal reservoir (1.5L, 1/4", outflow) | ||
– | centrifugal pump | ||
– | oxygenator | ||
– | leukocyte filter | ||
tubing (1/4" x 1/16") | Raumedic (Helmbrechts, GER) | MED7506 | |
tubing (3/8" x 3/32") | Raumedic (Helmbrechts, GER) | MED7536 | |
tubing connectors | Raumedic (Helmbrechts, GER) | various sizes | |
dialysis filter, Optiflux F160NR | Fresenius Medical Care (Waltham, MA) | F160NR | |
STEEN solution | XVIVO (Göteborg, SWE) | 19004 | 2L |
dialysis acid concentrate A | Baxter (Mississauga, ON) | D12188M | 45ml |
amino acid, Travasol 10% | Baxter (Mississauga, ON) | JB6760 | 100ml |
Sodium Pyruvate | Sigma-Aldrich (St. Louis, MO) | P2256 | 1.1g |
Heparin | Sandoz Canada Inc (Toronto, ON) | 10750 | 40000 iU |
Calcium Gluconate | Pharmaceutical Partners of Canada (Richmond Hill, ON) | C31110 | 10mg |
fast acting Insulin | various vendors | 1000 iU | |
Cefazoline | various vendors | 1g | |
Metronidazole | Baxter (Mississauga, ON) | JB3401 | 500mg |