INTRODUCTION
Ischemic injury encountered during organ preservation or reperfusion still prevails as a major impediment in clinical liver transplantation. The increasing use of extended criteria donor organs that are more prone to ischemia–reperfusion injury has driven efforts to optimize organ transport and preservation.
Ex vivo organ perfusion has come up as a tool to reduce preservation injury or to enhance the resilience of the graft to reperfusion injury upon transplantation.1,2 Normothermic machine perfusion (NMP) provides a near natural environment to the liver graft that allows for physiological metabolism to be pursued during the ex vivo period. Prolonged storage periods can be obtained without compromising the outcome after transplantation.3 Thus, upfront NMP, providing organ transport and perfusion under near-physiological conditions, has been associated with a 50% lower level of graft injury in a randomized controlled multicenter trial.4
Moreover, NMP might provide the opportunity to evaluate liver graft function before transplantation and thereby help the surgeon to decide upon acceptance or discard of an individual graft. Thus, assessment of biomarkers, perfusate enzymes, or coagulation factors during NMP may help to predict allograft function after liver transplantation.5,6
However, perfusion devices are difficult to transport, and they require extensive additional surveillance compared with simple cold storage (CS) on ice.
Therefore, recent developments tend to combine favorable transport logistics of simple CS and the advantage of organ conditioning by NMP before reperfusion using a postponed application of ex vivo graft perfusion.7-9
But then again, the procedures heavily impose on local center infrastructure, including the availability of equipment, skilled staff personnel, and most probably an additional specific on-duty service for the execution of the MP.
Technical preparation of the graft for connecting it on an MP device exceeds the routine back table preparation performed for preparing the liver for transplantation. The above-mentioned facilities and expertise may not be available or desired in every implant clinic.
The concentration of MP at a limited number of regional perfusion centers (hubs) that bundle expertise and economize the utilization of perfusionist personnel on call may be an attractive opportunity to maximize the efficiency of ex vivo graft perfusion while simultaneously minimizing the material and personal expense.
Donor organs would be retrieved and transported to the perfusion center by conventional methods and without interfering with the established procurement protocols.
After a given time of MP at the regional hub center, the organs will be transported to a local implantation clinic, associated with and in proximity of the respective hub, where the organ could be transplanted without further manipulations.
During the transfer from the hub to the patient, organs have to incur a second short period of CS of approximately some hours, which may or may not have a notable effect on the graft’s recovery upon reperfusion.
Therefore, the present study was undertaken to evaluate this putative effect of a secondary short period of static preservation after graft reconditioning by NMP.
This could be done by comparing liver graft recovery upon reperfusion after cold preservation and subsequent MP either immediately before reperfusion (simulating local MP in the implantation clinic) or followed by another 3 h of CS (simulating terminal transfer to the implantation clinic after MP at a regional hub center).
Livers that were ordinarily preserved only by CS served as controls.
MATERIALS AND METHODS
The study is reported in accordance with the recommendations in the ARRIVE guidelines (PLoS Bio 8(6), e1000412,2010). Experiments were performed on isolated livers retrieved from male Wistar rats with a weight between 250 and 300 g. Animals were treated according to the rules and after approval of the local authorities. The federal law regulating the protection of animals and the principles of laboratory animal care (National Institutes of Health publication vol 25, No 28, revised 1996) were followed. Animals were killed in deep anesthesia by intracardiac injection of KCl.
Livers were then exposed by midline abdominal incision. The infrarenal vena cava was incised for exsanguination of the animal and 20 min after cardiac arrest, the portal vein was cannulated and the liver was retrieved from the carcass.
All livers were rinsed via the portal vein with 60 mL of histidine–tryptophan–ketoglutarate (HTK) solution at 4 °C, cold stored in HTK, and randomly assigned to the experimental groups.
After 18 h of CS, livers were put on a MP set up for oxygenated, recirculating MP with 150 mL of Aqix RS I solution as previously described.10 The perfusion was effectuated at constant pressure, and the flow rate was adjusted by a servo-controlled roller pump connected to a pressure transducer attached to the portal inflow line. The temperature was slowly elevated from 8 °C to 35 °C within the first 60 min to provide maximal protection to the mitochondria11 and perfusion continued at 35 °C at a pressure of 6 mm Hg via the portal vein. Oxygenation of the perfusate was achieved by means of an interposed membrane oxygenator fed with 100% oxygen.
After 2 h of MP, the livers were disconnected from the device, flushed with 20 mL of HTK solution, and either directly used for the experiment (terminal MP [TMP]) or preserved again by static CS at 4 °C for another 3 h (preterminal machine perfusion [PTMP]). A schematic representation of the experimental groups is given in Figure 1.
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FIGURE 1.: Schematic representation of the experimental groups. After 20 min of WI, livers were retrieved and flushed with HTK solution before randomization into one of the following groups: (1) static CS for 18 h, (2) CS for 18 h + 2 h of TMP on the machine, (3) CS for 18 h + 2 h of TMP on the machine + 3 h of secondary cold storage, that simulates a short transit from a regional pump center to a nearby transplantation clinic (PTMP). At the end of respective preservation protocol, grafts were flushed with 20 mL of saline solution and connected to the reperfusion device for evaluation. CS, cold storage; HTK, histidine-tryptophan-ketoglutarate; MP, machine perfusion; PTMP, preterminal machine perfusion; TMP, terminal machine perfusion; WI, warm ischemia.
Livers that were only preserved by CS in HTK for 18 h served as controls. The livers were thoroughly rinsed with 20 mL of saline solution before connection to the reperfusion setup to wash out residual preservation solution along with all enzymes accumulated during static preservation.
Viability Testing After Preservation
Organ recovery after preservation was evaluated upon warm reperfusion according to previously described techniques10,12 with Williams E solution (Sigma Aldrich, Germany), supplemented with 3 g/100 mL of bovine serum albumin in a recirculating system for 60 min at 37 °C.
In brief, Williams E solution, supplemented with 3 g/100 mL of bovine serum albumin, was oxygenated with a 95% O2 and 5% CO2 gas mixture and pumped through the portal vein at a constant flow of 3 mL/g min in a recirculating manner while the liver was placed floating in a bath of perfusion solution at 37 °C.
Before reperfusion, all livers were exposed to room temperature on a Petri dish for 20 min before reperfusion to simulate the ischemic period during surgical implantation in vivo. Perfusate samples were taken after 15, 30, 45, and 60 min of warm reperfusion.
Detection of Injury Markers
Enzyme activities of alanine aminotransferase (ALT) and glutamate dehydrogenase (GLDH) in the perfusate were assessed in a routine manner at the laboratory center of the University Hospital.
Liver Function
Bile was drained from a 27-gauge polyethylene tubing, inserted into the common bile duct, and collected during the whole reperfusion period. Hepatic bile production was then calculated as microliter per gram per hour.
Energetic Status
Tissue specimens for assessment of high-energy phosphates were taken with precooled steel tongs, immersed in liquid nitrogen, and stored at –80 °C for later analysis. Wet weight of the frozen tissue samples was measured before they were lyophilized in a vacuum freezer (–60 °C; <0.025 mbar) for at least 7 d to evaporate tissue water. Freeze-dried specimens were weighed again and proteins were extracted with perchloric acid as described previously.13 Aliquots of the neutralized supernatant were used for determination of ATP by means of a commercial test kit (Abcam, Cambridge, United Kingdom) according to the manufacturer’s instructions. The results were corrected for the respective dry weight to wet weight ratio of the tissue samples and expressed as micromole per gram dry weight.
Histology
Liver tissue was collected after the experiments, cut into small blocks (3-mm thickness), and fixed by immersion in 4% buffered formalin. The blocks were embedded in paraffin, and 2- to 4-mm tissue slides were prepared using a microtome (SM 2000R, Leica Instruments, Nußloch, Germany). Hematoxylin and eosin staining was conducted adherent to in-house standards and used to assess morphological integrity of the parenchyma. Sections were analyzed at 200-fold magnification (Nikon Eclipse E800, Nikon, Tokyo, Japan), and the extent of the necrotic injury was semiquantitatively graded in a 4-stage system ranging from 0 (no necrosis) to 3 (severe necrosis with disintegration of hepatic cords) as described elsewhere14 by 2 independent examiners.
Statistics
All values were expressed as means ± standard deviation of 6 animals per group. Differences among the groups were first tested by analysis of variances and post hoc testing using the Student-Newman-Keuls test unless otherwise indicated. Statistical significance was set at a P value of <0.05.
RESULTS
Liver Behavior During MP
No apparent differences were disclosed concerning the behavior of the livers during PTMP or TMP. Hepatic vascular resistance amounted to 864 ± 365 versus 798 ± 152 Pa s/min, enzyme leakage of ALT averaged 12.9 ± 6.6 versus 12.3 ± 4.5 U/L, and concentrations of lactic acid in the perfusate amounted to 1.9 ± 0.4 versus 1.5 ± 1.0 mmol/L (PTMP versus TMP; P > 0.05, respectively).
Liver Recovery During Reperfusion
Mean portal vascular resistance upon constant flow reperfusion showed no significant differences among the groups but a tendency toward higher values in the control group as compared with the livers that were subjected to intermediate MP or TMP (1333 ± 178 versus 1022 ± 41 versus 933 ± 83 Pa s/mL, control versus PTMP versus TMP).
Hepatocellular integrity upon reperfusion was approximated by the release of cytosolic enzyme ALT as well as the leakage of the mitochondrial enzyme GLDH.
As depicted in Figure 2, there was a significant reduction of ALT leakage after TMP with respect to the untreated controls. However, the addition of a brief period of CS after MP in the PTMP group did not lessen the benefit obtained in the former group.
FIGURE 2.: Perfusate activities of ALT upon terminal reperfusion of livers after 18 h of CS, 18 h of CS and 2 h of TMP immediately before reperfusion, or 18 h of CS, 2 h of PTMP, and another 3 h of CS. *P < 0.05 versus CS; by comparison of the AUC with the Student-Newman-Keuls test. ALT, alanine aminotransferase; AUC, areas under the curve; CS, cold storage; PTMP, preterminal machine perfusion; TMP, terminal machine perfusion.
Similar results were also evidenced with regard to the hepatic leakage of GLDH (Figure 3). Of note, although a steady increase of GLDH in the perfusate was notable in the control group, the slope of the curve remained flat in both treatment groups.
FIGURE 3.: Perfusate activities of GLDH upon terminal reperfusion of livers after 18 h of CS, 18 h of CS and 2 h of TMP immediately before reperfusion, or 18 h of CS, 2 h of PTMP, and another 3 h of CS. *P < 0.05 vs CS; by comparison of the AUC with the Student-Newman-Keuls test. AUC, areas under the curve; CS, cold storage; GLDH, glutamate dehydrogenase; PTMP, preterminal machine perfusion; TMP, terminal machine perfusion.
Functional recovery of the livers was quite in line with the data on enzyme leakage (Table 1).
TABLE 1. -
Functional recovery of livers upon reperfusion after 18 h of CS (controls), 18 h of CS and 2 h of TMP immediately before reperfusion, or 18 h of CS, 2 h of PTMP, and another 3 h of CS
|
Control |
PTMP |
TMP |
| Bile production (µL/g/h): |
7.3 ± 5.1 |
16.1 ± 2.8*
|
16.8 ± 3.8*
|
| Lactic acid (µmol/L) |
3.2 ± 0.7 |
1.7 ± 1.3*
|
1.3 ± 0.8*
|
| ATP (µmol/g dw) |
0.42 ± 0.28 |
1.07 ± 0.53 |
0.91 ± 0.37 |
| Dry/wet weight ratio (%) |
22.8 ± 2.2 |
23.0 ± 1.6 |
23.6 ± 2.2 |
Values given as mean ± standard deviation of n = 6 experiments per group.
*P < 0.05 ANOVA and SNK test.
ANOVA, analysis of variance; ATP, XXX; CS, cold storage; dw, dry weight; PTMP, preterminal machine perfusion; SNK, Student-Newman-Keuls; TMP, terminal machine perfusion.
MP after 18 h of CS significantly increased hepatic bile production in comparison with the control livers, and this was quite independent of the intercalation of a brief period of CS between PTMP and actual reperfusion of the livers.
Likewise, clearance of lactic acid was significantly improved by PTMP as well as TMP, whereas energetic recovery (hepatocellular content of ATP) did show substantially higher values after either of the 2 reconditioning protocols.
Histology
Morphological analysis did not disclose relevant differences between the 3 groups in our experimental setting. Representative histological sections are depicted in Figure 4. Structural alterations were rather limited and sinusoidal architecture was well preserved in all of the groups. The quantitative injury score in the CS group was 1.2 ± 0.1 versus 1.0 ± 0.2 after PTMP and 1.0 ± 0.3 after TMP.
FIGURE 4.: Liver tissue morphology at the end of the experiments (H&E staining, 200-fold original magnification: (A) cold stored livers; (B) PTMP; (C) TMP. H&E, hematoxylin and eosin; PTMP, preterminal machine perfusion; TMP, terminal machine perfusion.
DISCUSSION
This is not the first study to demonstrate that a brief period of TMP of donor organs immediately before transplantation alleviates reperfusion injury upon implantation.
Our results concerning the benefit of TMP versus simple CS are well in line with previous experimental reports10,15 and qualitatively corroborated by first clinical investigations.7,16
From there, we might conclude that our model quite accurately reflects the clinical situation and could be considered a valid approach for a screening comparison of the PTMP concept and the terminal reconditioning method.
Thus, looking at the entirety of our present results, PTMP with ensuing short period of conventional static CS actually proved to be quite similar effective than putting the graft on the machine immediately before reperfusion.
A similar observation has previously been made in a clinical case of renal MP, although not in the context of a systematic investigation. Hosgood and Nicholson17 report the case of an extended criteria donor kidney that was put on the machine while the recipient was prepared for transplantation. Because the recipient became unfit for transplantation during preparation, the kidney graft was disconnected from the machine reflushed and put on ice for another 5 h before being transplanted into another recipient with immediate graft function.
It has previously been shown that 1 to 2 h of MP after preceding 18 h of liver CS abrogated any pathological alterations associated with the extended ischemic period and rendered the livers functionally comparable with control grafts.13
It is hence conjectured that the short period of 3 h CS, known to be excellently tolerated by a health liver graft,18 does not represent a harmful challenge for a reconditioned graft either.
The virtual absence of untoward effects attributable to the additional brief cold ischemic period would allow to change the organization of MP from being occasionally done at the individual implantation center to a more specialized and dedicated logistic routine at regional pump centers, which may serve as a hub for graft perfusion, evaluation, and ex situ therapy.
This option will become even more important because novel therapeutic procedures have already been pioneered in preclinical models that may soon be translated into clinical application. Such attempts of graft bioengineering include the delivery of silencing RNA using nanoparticles as a vehicle during MP19 aiming at transiently modifying proinflammatory reactivity of the graft.
A permanent silencing of endothelial major histocompatibility complex expression could be achieved by lentoviral vector transmission of short hairpin RNA20 with the final effect to render the graft immunologically invisible.
Cellular therapy during MP comprises the application of mesenchymal stroma cells or mesenchymal cell-derived extracellular vesicles during MP to confer additional protection against reperfusion injury.21,22
All these interventions share a relevant degree of complexity and specialized expertise that will be necessary for successful routine usage and the 24/7 availability of such expertise seems unlikely as well as logistically unadvisable at each individual transplantation center.
Economization with regard to specialized personnel as well as the provision of local facilities would however be possible by concentration of the MP process to a small numbers of regional hub centers.
To this end, the present study gives first indication that the secondary cold preservation period, which will necessarily incur during transport of the organ from the regional pump center to the final transplantation clinic, would not be associated with a notable reduction of graft viability.
Limitations of our study are related to the drawbacks of any in vitro experiment. Thus, data on long-term experiences are lacking, and some aspects important for ulterior liver graft survival, like posttransplant ischemic cholangiopathy, could not yet be addressed. However, a first line of evidence has been produced, suggesting that PTMP might become a promising strategy justifying further research on this topic.
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