Available online at www.sciencedirect.com

Transplantation Reviews 26 (2012) 156–162www.elsevier.com/locate/trre

The role of normothermic extracorporeal perfusion in minimizingischemia reperfusion injury

Thomas Vogela, Jens G. Brockmannb, Constantin Coussiosc, Peter J. Frienda,⁎aNuffield Department of Surgical Sciences, University of Oxford, The Churchill Hospital, OX3 7LJ, United Kingdom

bDivision of Visceral and Transplant Surgery, University Hospital Zurich, United KingdomcDepartment of Engineering Science, University of Oxford, OX3 7LJ, United Kingdom

Abstract

The primary objective of organ preservation is to deliver a viable graft with minimal risk of impaired postoperative graft function. Incurrent clinical practice, preservation of transplanted organs is based on hypothermia. Organs are flushed and stored using specificpreservation solutions to reduce cellular metabolism and prevent cell swelling. However, the ongoing organ donor shortage and consequentexpansion of donor criteria to include the use of grafts that would once have been discarded as unsuitable have underlined the need for atechnique that prevents any further damage during the preservation period. The principle of normothermic machine perfusion preservation isthe maintenance of cellular metabolism in a physiological environment throughout the preservation period. Normothermic preservation, atleast in theory, thereby overcomes the 3 major weaknesses inherent in traditional static cold storage by (1) avoiding ischemia/reperfusioninjury, (2) avoiding cold injury, and (3) allowing viability assessment. Furthermore, normothermic machine perfusion might transpire to bethe ideal vehicle to deliver other therapeutic interventions during preservation to modulate and optimize the graft before transplantation. Byrestoring function in marginal donor organs and enabling the clinician to appraise its viability, the donor pool might be greatly expanded.© 2012 Elsevier Inc. All rights reserved.

1. Introduction

Providing a viable graft for transplantation is theoverriding objective of organ preservation. Optimal organscan be transplanted with a minimum of risk of significantpostoperative graft dysfunction; but the increasing demandfor transplantation has created a need for additional organsources, and acceptance criteria have been widened. Theseso-called marginal organs, or organs from “extended criteriadonors,” include organs from elderly donors, organs procuredfrom donors after cardiac death, or organs from donors withother medical conditions. These organs are known to be moreprone to primary nonfunction or delayed graft function,which can be devastating after transplantation of immediatelylife-preserving organs such as heart or liver, but which alsohas severe consequences after transplantation of other organssuch as the kidney or pancreas. Expansion of donor criteria,

⁎ Corresponding author. Tel.: +44 01865 223872; fax: +44 01865226196.

E-mail address: peter.friend@nds.ox.ac.uk (P.J. Friend).

0955-470X/$ – see front matter © 2012 Elsevier Inc. All rights reserved.doi:10.1016/j.trre.2011.02.004

therefore, necessitates new approaches to the prevention offurther organ damage during the preservation period tomaintain acceptable outcomes of transplantation surgery.

The characteristics of an “ideal” preservation method aresummarized in Table 1. It is clear that current organpreservation methodology lacks several of these criteria.

The current clinical practice of organ preservation ingeneral is based on hypothermia. Organs are flushed andcooled with specific preservation solutions at ice tempera-ture; the 2 main principles are reduction of metabolic activityof the biological tissue (by cooling) and prevention of cellularswelling (as a consequence of the components of thesolution). At the moment flow of oxygenated blood isterminated, the supply of oxygen, cofactors, and nutrientsstops concomitantly. However, cell metabolic activity is nothalted at ice temperature, but merely slowed 1.5- to 2-fold forevery 10°C drop in temperature. Anaerobic metabolismcontinues, with resultant depletion of energy stores andaccumulation of metabolic products including breakdownproducts of adenosine triphosphate (ATP) [1]. Depletion ofATP causes loss of transcellular electrolyte gradients, influxof free calcium, and subsequent activation of phospholipases;

Table 1Characteristics of an ideal preservation technology

Comment

Preservation time Longer preservation times offer several advantages; best recipient matching, longer travel distances,optimal recipient preparation, surgery with rested teams, etc

Reduction of ischemic injury Injury sustained before or during organ retrieval must be limited and reversed, if possible; organ resuscitationGraft assessment/viability assessment Appraisal of organ function before transplantation to minimize organs unnecessarily discarded for transplantation

because of uncertainties of posttransplantation graft function; remove “subjectivity” from organ assessmentIntervention Interventions carried out during the preservation time might include drug therapy to improve postoperative graft

function as well as organ and or recipient targeted gene therapy and immunomodulationAvailability The technology should be readily available to organ retrieval teams; preparation time should be minimal to be used

with unstable donors or in organ donation after cardiac deathManageability The technology or device should be easy to handle, mobile, and preferably used during the entire preservation period

from donor hospital until transplantation at the transplant centerSafety Risks inherent in the preservation technique or associated with the use of a technical device should be minimalCost-effectiveness Increased costs would have to be offset against the benefits of increased transplant numbers and improved outcomes

157T. Vogel et al. / Transplantation Reviews 26 (2012) 156–162

this is a main contributor to cell swelling and lysis [2].In addition, accumulation of metabolic products duringischemia forms the basis for production of toxic moleculesafter reperfusion, which promote downstream pathways ofischemia/reperfusion injury [3]. In organs retrieved from non–heart-beating donors—with an inevitable period of oxygendeprivation between cardiac arrest and organ perfusion—thedeleterious effects of cold ischemia are superimposed on theinjury sustained during this warm ischemic phase.

Current organ preservation by static cold storage thus fallsshort in several aspects that are of particular importance forsuccessful transplantation of extended criteria organs:

Injury sustained during donor death and organ procure-ment is not reversed.Cold preservation causes itself time-dependent injurybeyond that induced by ischemia.Viability of the organ cannot be assessed beforetransplantation.Preservation times are very limited.

2. Normothermic perfusion

Normothermic machine perfusion of isolated organs isnot a novel technology. As early as in the first half of the20th century, Alexis Carrel [4] and his coworker CharlesLindbergh perfused abdominal organs with oxygenatedserum at normothermia and demonstrated viability forseveral days. Indeed, the first successful human livertransplants, carried out by Starzl et al [5] in 1967, weretransplanted after graft pretreatment with machine perfusionof diluted oxygenated blood. In the following decades,however, further development and clinical use of machineperfusion technology were hampered by several factors:

Introduction of improved cold preservation solutions inthe 1980s with substantial improvement in transplantationoutcomeComplexity of the warm perfusion technology, necessi-tating bulky devices and trained staff to operateHigh costs

At that time, research into extracorporeal machineperfusions largely centered on liver support [6-8]. In thelast decade, however, driven by the need to use marginaldonor organs and enabled by improvements in cardiopul-monary bypass technology, there has been a resurgence ofinterest in machine perfusion as a means of optimizing organpreservation for the purpose of transplantation.

Machine preservation of an organ at body temperaturerepresents a complete reversal of current thinking in terms oforgan preservation. In contrast to cold storage, theunderlying principle of normothermic machine perfusionpreservation is to maintain cellular metabolism in aphysiologic environment throughout the preservation period.Ischemia is prevented by perfusing the organ with oxygen-rich medium; continuous circulation of metabolic substratesand removal of waste products provide the basis for ATPproduction. Experimentally, this has been shown to reverseATP loss following ischemia (of the liver) and thus toreverse injury sustained [9-11]. Besides restoration ofenergy charges, normothermic perfusion was shown topromote upregulation of protective proteins including hemeoxygenase–1 (HO-1) and other members of the heat shockfamily [12,13].

Knowledge of the basic mechanisms of ischemia-reperfusion injury is in large part based on experimentalwork in rodents. To address more clinical issues, however,porcine transplantation has emerged as the preferred modelof liver preservation and transplantation experiments[14,15]. In a landmark article, Schön et al [16] demonstrateda substantial survival benefit for liver grafts after normo-thermic preservation when compared with livers after coldstorage preservation. In these experiments, pig livers weretransplanted successfully after 1 hour of warm ischemiafollowed by 4 hours of normothermic machine perfusion.This contrasted with the group of cold preserved organs, inwhich there were no survivors. Recently, Brockmann et al[17] described results of transplantation experiments in aporcine model of heart-beating and non–heart-beating donorscenarios. Perfusion was commenced either at the time ofcessation of circulation (as in a heart-beating organ

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donation) or after a period of warm ischemia (simulatingnon–heart-beating organ donation). Following a 5-hourpreservation period, there was no difference in outcome inthe heart-beating transplantation setting. However, after 20hours of preservation, there were significant advantages fornormothermic preservation when compared with coldpreservation in both groups of donor scenarios. Theseadvantages applied to enzyme release, histology, and animalsurvival. It was notable that there was no difference insurvival of recipients of heart-beating compared with non–heart-beating donor organs in the 20-hour warm-preservedgroups (86% vs 83%).

In the kidney, Hosgood et al [18] have reported goodresults of a period of normothermic preservation at the endof renal preservation in a porcine autotransplant model.Published data on perfusion preservation of thoracic organsfor transplantation are more scarce, although some reportssuggest that it may be very beneficial [19,20]; and in thecase of the heart, several normothermic approaches arecurrently pursued [21-23]. The heart was indeed probablythe first organ to be perfused at normothermia in a clinicaltransplant setting, although no data on these perfusionshave been published in a scientific journal to date [24]. The“Organ Care System” developed by the Transmedicscompany was used for these trials; it was demonstratedthat it permits ex vivo donor heart assessment, includingidentification of occult pathologic condition such as donorcoronary disease [25]. In a recent publication by Lindstedtet al [26], results of a small series of lung transplants afternormothermic reconditioning have been reported; theauthors applied a brief period of normothermic extracor-poreal perfusion under ventilator support to donor lungsthat had previously been rejected for transplantation.Preliminary data showed acceptable outcome after trans-plantation of these marginal grafts. Also in the liver,available data demonstrate clear evidence of the potential ofthis technology and provide a powerful argument toproceed to clinical trials. The first step will be todemonstrate that the perfusion characteristics shown forthe pig liver are valid in human livers.

2.1. Normothermic perfusion devices

The perfusion circuits used by most research groups areassembled from standard cardiopulmonary bypass compo-nents and tubing. Principle constituents are a blood reservoir,pump(s) (in some instances embodying 2 pumps because ofthe dual blood supply to the liver), oxygenator, and a heatexchanging device [16,27-29]. In contrast to hypothermicmachine perfusion, organ perfusion at normothermia isperformed at physiological pressures [16,18,30]. There issome preference for the use of centrifugal rather than rollerpumps, mainly to reduce the trauma to red blood cells. In ourown system, arterial perfusion is pumped directly, whereasthe portal vein is perfused via gravity making use of theautoregulative abilities of the liver [31-33].

Because oxygen is consumed at a high rate by functioningtissues at normothermia, the perfusion solution requires aspecialized oxygen carrier; and blood is most widely used forthis purpose. Other critical components of the perfusate(though not all in every device) include nutrition (glucose,insulin, amino acids); drugs to prevent thrombosis ormicrocirculatory failure (heparin, prostacyclin); and agentsto reduce cellular edema, cholestasis, and free radical injury[30]. There has been ongoing debate about the desirabilityfor fluctuating ambient pressure (to mimic the effect ofrespiration). This adds considerable complexity; in theperfusion circuit designed by the Berlin group for normo-thermic preservation of the liver, the organ is exposed tooscillating pressures to improve homogeneity of microcir-culation, a technique initially described by Velasquez et al in1970 [34,35]. Furthermore, this circuit includes a dialysissystem to regulate the pH and electrolyte concentrations.Other groups have focussed on varying aspects of thetechnology, for example, modulating the flow characteristicsof the perfusate with pulsatile perfusion [22].

Portability is probably an important criterion; experi-mental data suggest that prolonged cold ischemia of theorgan before attachment to the preservation circuit ishighly detrimental [36,37]. For a perfusion device to beintroduced into the clinical arena, it will need to be mobileto allow transportation to the donor hospital and back tothe transplant center.

3. Benefits of normothermic perfusion

3.1. Viability assessment

One of the most important advantages of normothermicperfusion over static cold organ preservation is the ability toassess viability. Because the organ is functioning during theperiod of preservation, it is possible to measure keyparameters before transplantation to assess function beforecommitting the recipient to transplantation. Viability mea-sures have been defined and validated in animal transplantstudies [17]. Several parameters (bile output, base excess,aspartate aminotransferase/alanine aminotransferase, hya-luronic acid, portal pressure, and portal venous resistance)reliably predicted those livers that would fail after trans-plantation—and did so within 4 hours of the start ofperfusion. If the ability to predict posttransplant viabilitywere confirmed in clinical practice, a major limitation of coldstorage in the era of extended criteria donor organs would beovercome. In this scenario, once the surgeon retrieves amarginal organ, viability parameters measured on theperfusion device would indicate a nonfunctioning graftbefore transplantation, enabling the organ to be discardedwithout jeopardizing a recipient. This would not onlydecrease the risk of primary nonfunction after transplantationbut also minimize the number of viable extended criteriaorgans that are discarded, thereby expanding the potentialdonor pool.

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3.2. Extended preservation time

Complete replication of the physiological environment ofthe organ by means of normothermic perfusion would,theoretically, allow preservation for an unlimited period. Incurrent practice, however, preservation times are limited byorgan-specific safe cold ischemia times; and transplantationoften has to be carried out outside routine working hourswith only limited availability of theater staff, anesthetists,and interdisciplinary support. It is well known, however, thatout-of-hours surgery is associated with increased risks andcosts [38,39]. Prolonging the maximum preservation timewould improve the logistics of transplantation substantially,eventually converting organ transplantation from an emer-gency into an elective procedure. Elective transplant surgeryalso entails the chance of optimized recipient selection,evaluation, and preparation. In case of organs specificallysensitive to ischemic times, longer preservation may enablelong-distance sharing of these organs between centers. Lastbut not least, extended preservation time allows for optimaltissue matching, which may lead to improved outcomes inorgans currently not being matched because of timeconstraints, for example, the heart and liver [40,41].

3.3. Preservation time interventions

If long-term perfusion preservation at normothermiabecomes feasible, this opens new perspectives for furthertherapeutic interventions before transplantation. Thesemight include simple drug therapies [42,43], but thetechnique might turn out to be the ideal vehicle to reduceor alter the immunological response after transplantation bymeans of immunomodulation or other interventional grafttreatment [44,45]. Organs that remain unsuitable for solidorgan transplantation might still be of therapeutic value forhepatic support procedures [46] or hepatocyte transplanta-tion [47] using the core technology of normothermicmachine perfusion.

3.3.1. Drug interventionAttempts to influence organ quality and graft survival after

transplantation by additives to storage or perfusion solutionsare as old as organ preservation itself. These attempts did notstop with the introduction of University of Wisconsinsolution and histidine-tryptophan-ketoglutarate solution inthe late 1980s. Numerous compounds have been tested eitherin the organ donor or during preservation, with variablesuccess in experimental models and clinical practice. Severalof these compounds might be more effective whenadministered in a physiological environment.

As outlined above, reperfusion injury is mediated by acascade of deleterious cellular responses leading to aninflammatory response. Recently, it has been shown that byinduction of protective genes and prevention of apoptosis,reperfusion injury can be substantially diminished[13,48,49]. In particular, HO-1 and other inducible membersof the heat shock protein family have been reported to

provide protection against ischemia reperfusion injury.Expression of genes and induction of new protein synthesisare active cellular processes that are severely hampered atlow temperature [50]. Furthermore, the short time scale ofcurrent organ preservation is a handicap for the induction ofgene expression. Experimental data on HO-1 expressionsuggest that induction in vivo needs to be initiated 24 hoursbefore organ retrieval, a time frame not available in theclinical setting [13]. Furthermore, the induction of protectivestrategies during preservation (rather than in the donor beforeretrieval) enable organ-specific therapies to be delivered.

3.4. Gene delivery

The benefits of normothermic perfusion preservation interms of organ-specific targeted drug intervention might beeven more crucial in gene therapy approaches. Duringpreservation, whole organs are uniquely accessible formodification. Gene therapy in transplantation thus far hasbeen confined to animal models, mostly rodents. However,potential targets for gene therapy in transplantation aremanifold [51]. The range includes modulation of costimulatoryand apoptosis pathways and specific manipulation of cytokineexpression and leukocyte recruitment. In addition, immuno-modulatory and cytoprotective enzymes including HO-1(as described above) can also be upregulated by gene transfer.

Gene therapy is also an attractive way to ameliorate orprevent acute cellular rejection. The advantage normother-mic organ preservation offers over hypothermic preservationin this model is again the physiologic environment, which ismuch more likely to promote both gene transfection andfunction. Gene delivery to the isolated organ preventssystemic exposure to potentially harmful or toxic effects ofthe gene vector system [52]. Similarly, different organs havevarying tropism for different vectors and promoter systems.In the isolated perfused organ, the most ideal vector systemcan be used for each tissue [53].

3.5. Combination with normothermic recirculation

The method of normothermic recirculation was firstdescribed in 1994. Hoshino et al [54,55] reported thatdonor in situ oxygenation by means of a cardiopulmonarybypass circuit gave substantial benefit to graft quality fromnon–heart-beating liver donors. In brief, the donor iscannulated immediately after cardiac arrest and certificationof death. Perfusion is achieved using a cardiopulmonarybypass pump, extracorporeal membrane oxygenation, and aheat exchange device. The advantage of this technique lies inits rapid application after cardiac death—in particular whencardiac arrest is unpredicted as is the case in donors fromMaastricht categories I, II, or IV. The rationale ofnormothermic recirculation is to reverse the depleted cellularenergy status that follows a period of cardiac arrest. It isbelieved that the same molecular mechanism may apply as inischemic preconditioning [56], a phenomenon widelyrecognized to reduce postischemic injury in different organs,

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including the liver. The first clinical report of this technique,recently published, has demonstrated feasibility. Ten of 40potential donation after circulatory death livers weretransplanted [57]; one graft was lost to primary nonfunction,and another was lost to hepatic artery thrombosis.

In donation after cardiac death organ donors, thecombination of normothermic recirculation with normother-mic machine perfusion preservation might optimize cellularenergetics and provide conditions for cellular recovery. In aseries of pig experiments using normothermic recirculationin an DCD model with prolonged warm ischemia, it wasshown that normothermic recirculation leads to substantialincrease in energy charge of livers when compared withlivers retrieved without normothermic recirculation. It iswell known that the energy charge of liver grafts issignificantly correlated with graft viability [58,59]. Therepair of injury sustained during warm ischemia andsubsequent avoidance of cold ischemia may allow organsfrom donors with much longer warm ischemic times than arecurrently possible to be transplanted successfully.

4. Conclusion

At a time when the discrepancy between donor organdemand and supply is increasing and patients are dying inincreasing numbers on the waiting list, there is a pressingneed to expand the donor pool and use all available organs.For many years, hypothermic, static preservation has beenthe universal standard. Although limited in terms ofpreservation duration, it has had the major advantages ofsimplicity, portability, and affordability. In the “less-than-optimal” organ, however, the deleterious effect of simplecold storage on organ viability becomes the critical factor;and in this regard, it is apparent that cold preservation hasreached its limits. Normothermic preservation overcomes 3main weaknesses inherent in hypothermic preservation byavoiding ischemia/reperfusion injury, avoiding cold injury,and allowing viability assessment [60].

Experimental data suggest that the quality of organpreservation can be improved substantially by normothermicmachine perfusion with the maintenance of a supply ofoxygen and nutrition and the avoidance of cooling[30,33,36,61-63]. However, these data are based on organpreservation and transplantation studies in animal models;and proof in the clinical setting is still awaited. Nevertheless,available evidence provides a powerful argument to proceedto clinical trials.

Inherently, the technology of normothermic organ perfu-sion is complex, much more than in hypothermic machineperfusion. A continuous supply of warm, oxygenatedperfusate is vital—any technical failure during preservationof a human organmay have a disastrous impact, rendering theorgan nonviable. As well as absolute reliability, any deviceused in clinical organ preservation has to fulfill furtherrequirements: First, the size must be small enough to be

moved in standard ambulance cars (and small planes orhelicopters) between donor hospitals and transplant centres.It has been shown experimentally that even quite briefperiods of cold ischemia are detrimental to the organquality, making stationary machine perfusion devices—based at the recipient transplant center—not a feasibleoption. The device must have appropriate battery capacityto enable at least some period of transport independent ofmains electricity.

The technique of normothermic preservation and its clinicalimplementation will be expensive. As well as the costs of thedevice and trained staff, there will be expenses for disposablesand maintenance. However, these costs will be balancedagainst the benefit of the new preservation technology. Ifnormothermic perfusion enables longer preservation times andthe use of more extended criteria organs, fatty livers, and liversfrom non–heart-beating donors, then the greater cost andcomplexity may well be justified.

The authors declare a financial interest in OrganOx, aspin-out company from the University of Oxford that isdeveloping normothermic preservation for clinical use.

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