New strategies and concepts in organ preservation.

Eur Surg Res 2015;54:114–126 DOI: 10.1159/000369455 Received: July 29, 2014 Accepted after revision: October 28, 2014 Published online: December 3, 2014

© 2014 S. Karger AG, Basel 0014–312X/14/0544–0114$39.50/0 www.karger.com/esr

Invited Review

New Strategies and Concepts in Organ Preservation Tanja Hoffmann

Thomas Minor

Surgical Research Division, Clinic of Surgery, University of Bonn, Bonn, Germany

Key Words Epigenetics · Extended criteria donor · Krüppel-like factor 2 · Machine perfusion · Organ preservation · Oxygen persufflation Abstract Organ transplantation is still affected by a notable degree of preservation-associated ischemia and reperfusion injury, which can seriously hamper early graft function. The increasing extension of the criteria for donor organ acceptance, especially for organs that have suffered from periods of warm ischemic injury prior to graft retrieval, results in even higher demands on preserving these ischemia-sensitive grafts. Growing attention is thus directed towards more dynamic preservation methods instead of simple static storage. Particularly in grafts that are retrieved after cardiac standstill of the donor, provision of oxygen to enable some kind of regenerative metabolism appears to be desirable, although the optimal temperature for oxygenated preservation/revitalization is still under debate. Hybrid solutions, comprising conventional cold storage for ease of graft procurement and transportation together with more sophisticated ‘in-house’ reconditioning protocols after arrival at the implantation clinic, might help to minimize graft injury during the critical transition from preservation to © 2014 S. Karger AG, Basel reperfusion.

Introduction

T. Minor Surgical Research Division, Clinic of Surgery University of Bonn DE–53127 Bonn (Germany) E-Mail minor @ uni-bonn.de

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The basic method for organ preservation involves vascular flush-out with a specifically designed preservation solution at hypothermia and subsequent static storage. Simplicity, cost-effectiveness and ease of transport make this technique the most widely used method

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Hoffmann and Minor: New Strategies and Concepts in Organ Preservation

for the preservation of organs. In view of the increasing shortage of donor organs, the donor acceptance criteria for graft retrieval have been extended, and organs that would have been discarded in the past are increasingly used in current routine. More and more transplantation centers nowadays accept less-than-optimal organs and grafts from donors whose hearts stopped beating before donation [donation after cardiac death (DCD)]; depending on the criteria applied, it may be estimated that approximately one third to half of the donors can be defined as marginal donors. With the ever-growing concerns about lifestyle in Western society, this problem will become more important in the coming years. It is obvious that the reduced organ quality will have an impact on graft function after transplantation. Especially with organs that suffered from prolonged ischemic periods, early function can be seriously decreased, as indicated by the fact that 60–80% of the kidneys retrieved from DCD donors do not function properly after transplantation and need dialysis support. Despite all recent improvement in donor treatment and harvesting protocols, preservation-associated ischemia and reperfusion injury prevail as pertinent factors responsible for primary dysfunction after the activation of the grafted organ on cellular, molecular and epigenetic levels.

The detrimental anaerobic metabolism could be retarded by lowering the temperature to approximately 4 ° C (hypothermia), which reduces the cellular energy demand to little more than 10% of normothermic values. Further improvement is obtained by the use of specifically designed preservation solutions for flush-out and storage of the organ, a procedure pioneered by G. Collins with the first description of the ‘Collins Solution’, using specific compounds to counteract acidosis and intra- or extracellular edema. While little improvement could be achieved by further modulations of the temperature, a lot of research effort has been put into the refinement of the preservation solution, along with the development of new and more effective formulations. The University of Wisconsin solution has been considered the gold standard for the preservation of abdominal organs for several decades, although its high viscosity, mainly due to the colloid hydroxyethyl starch (HES), may be considered a drawback in some instances. Moreover, HES has been shown to favor erythrocyte aggregation, which might be counterproductive in flushing organs, especially when procured after cardiac arrest of the donor [1, 2]. Another solution increasingly used at present is the histidine-tryptophan-ketoglutarate (HTK) solution originally developed for cardioplegia. Later experience with this colloid-free solution has shown HTK to also be equivalent to University of Wisconsin solution for abdominal organ preservation in clinical practice [3]. Recent developments involve the preservation solution Polysol, which contains 60 components including impermeants, antioxidants, vitamins, energy substrates and amino acids. For colloidal support, Polysol is supplemented with polyethylene glycol (35 kDa), which does not increase viscosity as seen with HES-containing solutions. However, after several promising results in animal studies, the first clinical trial was prematurely terminated due to increased rejection rates in the treatment arm [4]. Custodiol-N, a derivative of the classic HTK solution, represents another attempt to implement modern pathophysiological insights into the composition of preservation solutions [5]. Newly added compounds (glycine, alanine, N-acetylhistidine and iron chelators) inhibited hypoxic and cold-induced cell injury, resulting in a clear reduction of preservation injury in experimental models [6, 7]. Clinical trials of the use of Custodiol-N in organ preservation are currently under way.


Cold Storage Solutions

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AQIX is a solution mimicking physiological serum conditions with an N,N-bis(2hydroxyethyl)-2-aminoethanesulfonic acid (BES)-hydrogen carbonate pH buffering system which shows a buffering capacity over a large range of temperatures from hypo- to normothermia and can thus also be used in quite a warm environment [8]. A further promising approach to improving graft viability during cold storage has been seen in the preequilibration of the preservation medium with gaseous compounds acting on cellular signal transduction. Thus, rat kidneys that were preserved in a 70% xenon-saturated medium showed significantly better function after transplantation than controls, which was associated with a net antiapoptotic effect of the gas [9]. Other investigations have shown protective properties of nitric oxide [10] or carbon monoxide [11], mitigating inflammation, apoptosis or vascular resistance. Interestingly, carbon monoxide could also be applied through the nongaseous administration of so-called CO-releasing molecules, which consistently release CO in biological tissues [12]. Dynamic Preservation

However, in order to make optimum use of critical organs, dynamic preservation techniques were proposed, aiming to reverse the deleterious priming of the graft primarily by means of continuous ex vivo tissue perfusion and/or oxygenation immediately after harvest.

The benefit of aerobiosis during extracorporeal preservation of the organ has been related to the fact that even at low temperatures of about 4 ° C, a residual metabolism is operative in the tissue which provides restorative capacities if oxygen is provided as energetic fuel. While machine perfusion of the kidney may be considered an established clinical procedure in the hands of experienced transplant specialists, this is not at all the case with regard to the pancreas or the liver. Nonetheless, interest is also growing in machine perfusion of liver grafts, and first encouraging results in humans have been published [13]. The group of Guarrera [14] reported a significant reduction in serum injury markers such as peak transaminase levels 1 week after transplantation following liver preservation by machine perfusion, along with a 30% shorter hospital stay, as compared to cold storage. Additionally, they could confirm previous experimental data on the hypothermic machine perfusion (HMP)-induced reduction of inflammatory cytokines and adhesion molecules upon reperfusion. Adequate hemodynamic perfusion in those organs, however, is technically much more demanding than it is in the kidney. An elegant alternative to machine perfusion for aerobic organ preservation, circumventing the technical demands required for pancreas or liver perfusion, consists in using the technique of gaseous oxygen persufflation, that is, insufflation of medical-grade oxygen via the vascular system of the otherwise cold-stored organ [15]. Being substantially less cumbersome than the machine perfusion approach, but appearing to be equally effective in preclinical studies [16], this technique has recently found its way from the experimental stage to clinical testing [17, 18]. Moreover, this method represents an attractive study tool to isolate the effect of tissue oxygenation during ischemia from any interfering effect inherent in continuous organ perfusion such as better equilibration of the tissue with the preservation solution, provision or washout of metabolites or pulsatile shear stress to the vasculature. The energetic homeostasis provided by steady persufflation with oxygen could be documented in the rat liver by an overall elevation of tissue ATP concentrations in line with a homogenously


Aerobiosis

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normalized redox status as visualized by NADH autofluorescence experiments [19]. The ultrastructure of mitochondria as well as sinusoidal endothelial cells could be impressively maintained by gaseous oxygen persufflation, especially in fatty livers [20]. Oxygen seems to be important also for pancreas preservation. Adaptation of the oxygen gas persufflation technique to the long-term storage of pancreata has led to superior yields in poststorage islet cell isolation in the hands of several groups [21, 22]. Aiming in the same direction, attempts have been undertaken to preoxygenate the initial flush-out solution [23] in order to improve pancreas tissue integrity during storage conditions. Although increased partial pressures of oxygen could be measured [23], no attempt has yet been made to document the association with functional outcome measures. Future studies are needed to evaluate the graft-protecting potential of this approach Adequate tissue energetics is thought to improve cellular ion and signal homeostasis during preservation, to mitigate the graft’s proclivity for suffering from further tissue damage during the period of a second warm ischemia upon implantation surgery and to fuel initial function upon reperfusion [24, 25]. Since the fate of an organ depends on whether cell death or regeneration prevails, autophagic processes, which seem to influence regeneration after ischemic insult, should come into the focus of organ preservation research. Depending on the amount of injured mitochondria and the energetic status of the cell, the prevailing cellular reaction to an ischemic insult will be necrosis, apoptosis or autophagy [26]. Autophagy normally occurs at low rates in cells to perform homeostatic functions and is regulated by a number of proteins like the autophagy-related proteins (Atg) or beclin-1 [27]. Autophagy allows the cell not only to recycle amino acids but also to remove damaged organelles, thereby eliminating oxidative stress and allowing cellular remodeling for survival [28, 29]. Indeed, autophagic proteins appear to decrease during and after warm anoxia in isolated hepatocytes [27] and intact liver grafts [30]. Defective autophagy, in turn, culminates in the onset of mitochondrial membrane permeability transition and cellular death after warm reperfusion [27, 31]. Gaseous oxygen persufflation of the graft during ischemic preservation, however, was found to mitigate the decline in cellular autophagic clearance and consecutive graft necrosis [30]. After all, little is known about the usefulness and optimal concentration of oxygen during HMP, and systematic investigations into this topic have been missing for a long time. Equilibration with room air was thought to be sufficient in some studies, and no oxygenation was described in others. Only recently, in a liver preservation model, has it been shown that HMP with an air-equilibrated perfusate seemed to adequately preserve livers during machine preservation but eventually produced results upon warm reperfusion inferior to those in grafts which were treated by HMP with 100% oxygenation [32]. Similar data were reported for a porcine renal transplant model, which demonstrated a significantly superior preservation after HMP if the perfusate was equilibrated with 100% oxygen [33]. Both studies, however, were conducted on predamaged grafts retrieved only after cardiac arrest of the donor. In contrast, a pivotal role of active oxygenation during hypothermic perfusion has been questioned for kidneys from donors with intact circulation [34]. Obviously, the need for oxygen delivery is somehow inversely correlated with the severity of the ischemic insult or the quality of the graft and might become mandatory if regenerative processes are desirable, as after preceding warm ischemic insult in the donor. Although most current analyses of the clinical use of HMP in renal preservation have actually found benefits over simple cold storage in terms of reduced primary dys- or nonfunction [35–37], one recent review draws a more skeptical conclusion [38]. This report, however, places a primary focus on graft mortality, which, putatively for statistical reasons, has not been the primary endpoint in most of the controlled studies and, hence, might not come up directly with significant results. Nonetheless, in the latest randomized controlled



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study on kidneys donated after brain death, the 3-year study graft survival after machine perfusion was superior to that after cold storage, with differences being most pronounced for kidneys recovered from expanded criteria donors [39]. As far as delayed graft function and organ quality might be taken as clinically relevant outcome characteristics, the advantages of HMP over cold storage seem to be widely accepted, and renal preservation by machine perfusion has become an established alternative in many countries. Additionally, recent investigations into cost/benefit relations have come up with the clear conclusion that a reduction of delayed graft function and the ensuing requirement for dialysis would be a pertinent denominator in favor of machine perfusion preservation. Moreover, recent discoveries in the area of perfusion mechanistics, such as low-pressure perfusion to avoid mechanical stress to the organ vasculature [40] or the need for active oxygenation in grafts from DCD suggested in the mean time [24, 32, 41], should be taken into account when evaluating negative reports from clinical trials undertaken with less appropriate technologies. Apart from its possible impact on immediate parenchymal cell structure and function, tissue hypoxia could also induce rapid epigenetic modifications. One important example hereof is the alteration of the methylation state in gene promoter regions that can modulate gene expression, normally repressed by methylation of the CpG promoter region which reduces transcription factor access to gene-regulatory binding sites [42]. Dysregulation of this process might be heritable upon mitosis and thus be pertinent to long-term graft function. A first example of this mechanism has been given by Parker et al. [43], who found a persistent demethylation at the complement 3 promoter after ischemic preservation of rat kidneys. Other investigators, however, conversely report global DNA hypermethylation and an increased expression of DNA methyltransferase enzymes in human cardiac fibroblasts after chronic exposure to hypoxia at normothermia [44]. Since dyshomeostasis of DNA-methylation is unanimously associated with cellular stress by tissue hypoxia/reperfusion injury, the prevention of hypoxia (e.g. through oxygenated machine perfusion or persufflation) might represent an interesting approach to get hold of these processes. Ongoing experiments looking at individual changes in the methylation status of several genes delineate a notable but not uniform influence of renal preservation on gene promoter methylation (fig. 1). Moreover, preliminary evidence could be produced showing that maintenance of tissue aerobiosis (e.g. by oxygenated machine preservation) might, at particular sites, have effects


Relative promoter methylation

Fig. 1. Change of CpG methylation at 2 individual genes involved in chronic tissue fibrosis (TGFβ1) or the regeneration and reversal of fibrosis (BMP7) in rat kidneys after ischemic preservation in HTK by either cold storage (CS) or oxygenated HMP. The data stem from DNA extracted from renal cortical tissue and are analyzed in duplicate by real-time PCR after cleavage with a methylation-sensitive and/or a methylation-dependent restriction enzyme. Normal tissue is given the status of 1, and the postischemic samples are compared to this (n = 4). * p < 0.05 vs. CS (Tukey-Kramer test).

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relevant to the long-term fate of the graft. BMP7, for instance, contributes to the maintenance of the kidney in promoting the regeneration of tubular cells and in playing a pivotal role in the antagonism/reversal of renal fibrosis [45] induced by TGFβ [46]. A persistent imbalance of TGFβ and BMP7 transcription by altered postischemic promoter methylation hence could potentially affect a graft’s proclivity for chronic kidney dysfunction, even if this has not been detected in short-term experiments. Further research in this field thus seems to be clearly worthwhile.

There is an abundance of reports on the beneficial use of HMP, even in strictly nonoxygenated models that have called for further reflections on the underlying mechanisms in machine perfusion. In pancreas preservation for islet isolation, anoxic machine perfusion has been shown to be beneficial, although it was associated with moderate but significant interstitial edema development. Precisely this edema has been conjectured to facilitate enzymatic digestion and islet isolation, thus producing an enhanced yield after up to 24 h of machine preservation [35]. A possible advantage of machine perfusion storage may relate to progressive equilibration of the tissue with the preservation solution during ongoing perfusion, especially when graft retrieval is performed after cardiocirculatory standstill of the donor. The enhanced equilibration of the preservation fluid would be operative in eliminating areas of incomplete protection that are otherwise encountered upon simple flushout of the organ [47]. Special attention, however, has recently been devoted to the genuine effect of pulsatile perfusion per se. Vascular endothelial cells have been shown to be subject to mechanosensitive gene regulation through mechanoreceptors that are sensitive to laminar shear stress [48]. Several lines of evidence are now suggesting that deprivation of the vascular endothelium from mechanical stimulation during organ ischemia interrupts specific transcriptional activities which converge in an anti-inflammatory and vasculoprotective endothelial phenotype. This regulatory machinery is orchestrated in a pivotal fashion by the transcription factor Krüppel-like factor (KLF)2 triggering the transcription of various genes involved in cellular adhesion, vascular tone or inflammatory response (fig. 2). The loss of physiological concentrations of KLF2 has been observed within hours after cessation of blood flow [49], while adequate stimulation of KLF2 during preservation should maintain vasoprotective programs and improve the viability of machine-perfused kidney grafts [49]. Adequate expression levels of KLF2 could actually be maintained by adding pulsatile stimulation to gaseous oxygen persufflation in rat livers during preservation [50], in line with an improved function upon isolated reperfusion. Likewise, supplementation of the preservation solution with simvastatin, an HMG-CoA reductase inhibitor known to upregulate KLF2-derived transcriptional programs [51], prevented endothelial dysfunction and graft injury in a similar model. Interestingly, the above-mentioned adverse endothelial signal regulations could be reversed even after prolonged periods of static storage. In a recent study on isolated porcine kidneys [52], a short period of pulsatile HMP after 18 h of static storage had shown to significantly increase the molecular expression of KLF2 and eNOS compared to static storage alone. This was associated with a net improvement in the balance between endothelial secretion of endothelin-1 and nitric oxide, resulting in enhanced vascular conductance and function upon early reperfusion.


Machine Perfusion – Pulsatile Stimulation of the Vasculature

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Laminar flow/shear stress

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Fig. 2. Schematic representation of selective mechanoreceptor-induced cellular signaling pathways. ERK = Extracellular-signal-regulated kinase; ET-1 = endothelin-1; PKA = protein kinase A; TM = thrombomodulin; TF = transcription factor; VCAM = vascular cell adhesion molecule.

Most of the tissue injury triggered by the ischemic storage of organ grafts is brought about only upon warm reperfusion after transplantation and not during hypothermic preservation itself. Thus, several groups have made attempts to reestablish cellular homeostasis in the cold-stored graft prior to warm reperfusion in order to prevent the exacerbation of tissue injury during the initial phase of posttransplant reperfusion. These techniques basically comprise artificial oxygenation of the grafts by either aerobic machine perfusion or gaseous oxygenation via the venous vascular system (extensively reviewed by Minor and Paul [53]). From a logistical point of view, this approach has some attractive advantages compared to continuous machine perfusion. It precludes the need for specialized machines being available already during organ retrieval as the graft can be procured and transported according to standard procedures of simple cold storage preservation. Subsequent ‘in-house’ reconditioning of the organ will be possible under controlled stable conditions following its arrival at the implantation center. The hypothesis that a restoration of the physiological mitochondrial redox state during the hypothermia-induced reduction of cell metabolism may elicit more appropriate conditions for warm reperfusion has first been tested in rat livers. One hour of hypothermic oxygen persufflation at the end of 47 h of cold storage, for example, significantly improved the energetic recovery of rat livers and prevented the loss of adenine nucleotides upon isolated reperfusion [54]. The actual impact of hypothermic oxygenation on hepatic cytochrome C oxidation


Hypothermic Reconditioning

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has been nicely shown by Dutkowski et al. [55] using transhepatic spectroscopy. It could thus be demonstrated that the effective improvement of the mitochondrial redox status culminated after approximately 90–120 min of hypothermic perfusion following previous cold storage, and was associated with a net reduction of mitochondrial dysfunction upon reperfusion. Similar observations could be found using gaseous oxygen persufflation of pig livers, showing maximal energetic restoration after 1–2 h of end-ischemic treatment [56]. The extent of protection possibly provided by hypothermic reconditioning has been investigated by Stegemann and Minor [57], who compared the outcomes for rat livers that were reconditioned after 22 h of cold storage with the results obtained for positive controls that had only been preserved for 6 h. It could be shown that the viability of liver grafts that had been preserved for an extended period was restored by transient hypothermic reconditioning using either machine perfusion or gaseous oxygen persufflation, both resulting in postischemic liver recovery comparable to that observed after only 6 h of preservation. The observed effects were closely related to the prevention of initial mitochondrial dysfunction upon reperfusion. Short-term oxygen persufflation has also been shown to restore basal rates of autophagy after ischemic preservation [30, 58]. Autophagy-defective cells were shown to accumulate dysfunctional mitochondria that are responsible for enhanced RIG-I-like receptor signaling and the consecutive secretion of IFN [59]. Interestingly, type I IFN expression was found to be reduced in liver tissue after short-term hypothermic oxygenation prior to transplantation. As type I IFN, generated as part of the innate immune response to TLR activation, may eventually promote adaptive immune responses [60], the effects of hypothermic reconditioning may not be limited to immediate graft function but may also extend to the ulterior reduction of tissue immunogenicity after transplantation. Hypothermic oxygenation after cold storage had further been shown to confer protection to the vascular endothelium by reducing signs of inflammatory activation (expression of von Willebrand factor, ICAM-1 or TLR-4) [56, 61] and improving vascular conductance upon reperfusion [56, 62] as well as endothelial clearance of hyaluronic acid after transplantation [62]. Fewer and more controversial experiences are reported for hypothermic reconditioning in the kidney. Hosgood et al. [63] did not find any protective effects of end-ischemic HMP on functional recovery during isolated reperfusion of porcine kidneys retrieved after cardiac arrest of the donor. In these experiments, however, machine perfusion was performed without active oxygenation of the perfusate and on grafts that had to some extent suffered warm ischemic injury prior to the start of cold preservation. By contrast, in a model of porcine autotransplantation of freshly retrieved kidneys, Gallinat et al. [64] showed significant improvements by 2 h of HMP after 19 h of cold storage. In that model, end-ischemic HMP resulted in a posttransplant kidney function which was at least equivalent to that obtained with continuous machine perfusion during the whole preservation period. Interestingly, in that model, short-term pulsatile machine perfusion was also found to be effective when done without oxygenation [52]. It might be conjectured that oxygenation of the perfusate is only needed for the revitalization of DCD kidneys – but in that case seems to be mandatory.

Rewarming of cold-preserved cells or tissue to physiological temperatures may, on its own, induce cell injury. In an isolated rat liver preparation, Leducq et al. [65] have shown that an abrupt rise in perfusate temperature from 4 to 37 ° C was associated with mitochondrial permeability transition and impairments of the oxidative phosphorylation system. This


‘Rewarming Injury’

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‘rewarming injury’ depends on cellular alterations occurring during extended periods of hypothermia and seems to be mediated by the iron-dependent formation of reactive oxygen species [66]. It has been shown to be related to the duration of preceding cold storage and to the degree of hypothermia. It was not seen after incubation at temperatures above 16 ° C [67]. A conceptual refinement of hypothermic reconditioning, possibly mitigating the rewarming injury after transplantation, has recently been proposed in the gentle warming-up of the ischemic organ during end-ischemic machine perfusion under adapted oxygen delivery [68]. This approach has been tested in a first study in the blood-perfused isolated pig liver [68]. A brief end-ischemic period of gradual rewarming up to 20 ° C by oxygenated machine perfusion with preservation solution prior to normothermic reperfusion actually resulted in a significant reduction of oxygen free radical-induced lipid peroxidation and was associated with a notably better functional recovery in terms of bile production and release of transaminases. The benefits of oxygenated rewarming were evident with respect to cold storage alone as well as in comparison to end-ischemic HMP without rewarming. Primary clinical applications of this procedure have so far been reported for 6 selected cases of rescue allocation liver grafts which had previously been rejected for transplantation in other centers but eventually were all successfully transplanted after reconditioning by controlled rewarming.

Another even more radical approach to preventing rewarming injuries to the graft lies in the complete prevention of extended periods of hypothermia by continuous normothermic machine perfusion (NMP) during transport of the explanted organ. Organ preservation by warm perfusion maintains a physiological environment for the graft and may hence represent an optimal condition for the evaluation of its viability prior to transplantation [69]. In experimental studies, normothermic preservation of liver grafts by machine perfusion with diluted blood has demonstrated impressive benefits as compared to static cold storage, particularly for predamaged or ischemia-sensitive liver grafts. Thus, in a porcine transplant model, Brockmann et al. [70] were able to show life-sustaining function after up to 20 h of NMP in 83% of DCD liver grafts, whereas all the cold-stored grafts failed upon transplantation. Likewise, fatty liver grafts could be successfully preserved by NMP for extended periods of ischemia and concomitantly demonstrated a reversal of the degree of steatosis [71]. However, incidentally, NMP has proven less effective when installed after notable periods of preceding cold storage. In the kidney, 16 h of cold storage and a subsequent 2 h of NMP showed results inferior to those with 18 h of HMP [72], although both methods produced significantly better results than cold storage alone. In the liver, NMP after 4 h of cold storage failed to resuscitate porcine DCD grafts that had been well preserved when NMP had been installed immediately upon retrieval [22]. Recently, sub-NMP at approximately room temperatures has been proposed as a variation to prevent hypothermic-rewarming injury to the graft as this requires less metabolic support during perfusion than NMP. Thus, machine perfusion at 20 ° C led to a marked improvement in hepatic preservation of steatotic rat livers as compared with cold storage and conferred even better protection than machine perfusion at 4 ° C [73]. Similar results had been found in an isolated kidney model where the clearance of creatinine was highest after 7 h of machine preservation at 20 ° C as compared to machine preservation at 4 ° C or simple cold storage [74]. Tolboom et al. [75] compared long-term posttransplantation graft performance in rat livers after machine perfusion at different temperatures. They found equivalent preservation after machine perfusion at 20 or 37 ° C, although serum


Normothermic Machine Perfusion

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levels of bilirubin tended to be higher after subnormothermic than after normothermic preservation. In summary, growing attention is directed towards more dynamic preservation methods instead of simple static storage. Particularly in grafts that are retrieved after cardiac standstill of the donor, the provision of oxygen to enable some kind of regenerative metabolism appears to be desirable, although the optimal temperature for oxygenated preservation/revitalization is still under debate. Hybrid solutions, comprising conventional cold storage for ease of graft procurement and transportation together with more sophisticated in-house reconditioning protocols after arrival at the implantation clinic, might help to minimize graft injury during the critical transition from preservation to reperfusion. Acknowledgement The authors gratefully acknowledge the valuable technical help of P. Efferz in performing the molecular analyses.

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71

Pelvic Organ Prolapse: New Concepts in Pelvic Floor Anatomy.

Emerging concepts in liver graft preservation.

Changing paradigms in organ preservation and resuscitation.

Novel biomarkers in acute coronary syndromes: new molecules, new concepts, but what about new treatment strategies?

Advances and New Concepts in Alcohol-Induced Organelle Stress, Unfolded Protein Responses and Organ Damage.

Current Concepts in Debridement: Science and Strategies.

A new combined therapy for functional organ preservation and survival in lateral oropharyngeal wall cancer.

Organ procurement and preservation: what is new and what is established?

Resistant Malaria : Current Concepts and Therapeutic Strategies.

Proteogenomics: concepts, applications and computational strategies.

Concepts and strategies for human gene therapy.

[Organ and kidney function preservation in renal cell carcinoma].

Current Concepts in Hip Preservation Surgery: Part II--Rehabilitation.

Current Concepts in Hip Preservation Surgery: Part I.

New concepts in immunotherapy.

Vemurafenib resistance signature by proteome analysis offers new strategies and rational therapeutic concepts.

New concepts in fillers.

Plasma cells in immunopathology: concepts and therapeutic strategies.

Strategies for fertility preservation and restoration in the male.

Emerging concepts and strategies in analytical glow discharges.

Comorbidity in child psychopathology: concepts, issues and research strategies.

Organ preservation for heart-lung and lung transplantation.

Organ Preservation for Advanced Larynx Cancer: Issues and Outcomes.

New concepts in cutaneous allergy.