AIM: To optimize the perfusates used for hypothermic machine perfusion (HMP).

METHODS: Sprague-Dawley rats were assigned randomly to three groups (n = 12 per group) that received either saline, University of Wisconsin cold-storage solution (UW) or histidine-tryptophan-ketoglutarate solution (HTK) as the perfusate. Each group was divided into two subgroups: static cold storage (SCS) and HMP (n = 6 per subgroup). The liver graft was retrieved according to the method described by Kamada. For the SCS group, the graft was directly placed into cold perfusate (0-4 °C) for 6 h after liver isolation while the portal vein of the graft was connected to the perfusion machine for the HMP group. Then the perfusates were collected at different time points for analysis of aspartate aminotransferase (AST), alanine transaminase (ALT) and lactate dehydrogenase (LDH) levels. Liver tissues were obtained for evaluation of histology, dry/wet weight (D/W) ratio, and malondialdehyde (MDA) and adenosine-triphosphate (ATP) levels. The portal vein pressure and velocity were monitored in real time in all HMP subgroups.

RESULTS: Comparison of HMP and SCS: Regardless of the perfusate, HMP improved the architecture of donor graft in reducing the congestion around sinusoids and central vein and maintaining sinusoid lining in morphology; HMP improved liver function in terms of ALT, AST and LDH, especially during the 3-6 h period (SCS vs HMP using saline: ALT3, 225.00 ± 105.62 vs 49.50 ± 18.50, P = 0.047; LDH3, 1362.17 ± 563.30 vs 325.75 ± 147.43, P = 0.041; UW: LDH6, 2880.14 ± 948.46 vs 2135.00 ± 174.27, P = 0.049; HTK, AST6, 307.50 ± 52.95 vs 185.20 ± 20.46, P = 0.041); HMP decreased MDA level (saline, 2.79 ± 0.30 vs 1.09 ± 0.09, P = 0.008; UW, 3.01 ± 0.77 vs 1.23 ± 0.68, P = 0.005; HTK, 3.30 ± 0.52 vs 1.56 ± 0.22, P = 0.006). Comparison among HMP subgroups: HTK showed less portal vein resistance than UW and saline (vs saline, 3.41 ± 0.49 vs 5.00 ± 0.38, P < 0.001; vs UW, 3.41 ± 0.49 vs 4.52 ± 0.63, P = 0.007); UW reduced edema most efficiently (vs saline, 0.68 ± 0.02 vs 0.79 ± 0.05, P = 0.013), while HTK maintained ATP levels best (vs saline, 622.60 ± 29.11 vs 327.43 ± 44.66, P < 0.001; vs UW, 622.60 ± 29.11 vs 301.80 ± 37.68, P < 0.001).

CONCLUSION: HMP is superior to SCS in maintaining both architecture and function of liver grafts. Further, HTK was found to be the optimal perfusate for HMP.

Static cold storage (SCS) is currently the gold standard method for preservation of the donor graft for liver transplantation (LT), with University of Wisconsin cold-storage solution (UW) and histidine-tryptophan-ketoglutarate solution (HTK) being the most commonly used preservation solutions[1]. The principal aim of SCS is to minimize preservation-related injury by rapidly cooling the graft core temperature to reduce metabolism[2]. Hypothermic machine perfusion (HMP) was first proposed by Belzer in the early 1960s, but the complex nature of this technique, the cumbersome equipment required, and lack of clarity regarding the mechanisms have limited its wide usage in clinical settings. However, in the era of the donor organ shortage, HMP is of increasing interest in transplantation centers because it allows better monitoring of the graft, improves washout of injurious waste products, and allows pharmaceutical intervention, leading to decreased ischemia reperfusion (I/R) injury which could be indirectly measured by malondialdehyde (MDA), and extended preservation, even in cases of resuscitated marginal organs or those obtained from cardiac death donors (DCD)[3,4]. Mounting evidence shows that HMP challenges the limitations of SCS in terms of reduced preservation-related injury and improved LT outcome[5-7]. However, there is no consensus on the basic conditions of HMP, including the optimal perfusate, pressure and flow velocity, double or single vessel perfusion (retrograde or antegrade, pulsatile or not), and with or without oxygen. In this study, we aimed to identify the optimal perfusate by comparison of the influence of different perfusates on liver viability during HMP.

Currently, more than 5500 cases of LT are performed each year in Europe and in the United States[10,11] with 5-year patient survival rates exceeding 70% in most centers. The success of LT has subsequently resulted in a substantial worldwide organ shortage. To reduce the gap between the patients’ need and the availability of donors, the use of “extended criteria donor” (ECD) grafts, which were previously considered unsuitable for transplantation, has regained interest. Consequently, there is a critical need for improved methods of organ preservation. Currently, SCS and HMP are the two major methods used for liver preservation. SCS is widely accepted because of its simplicity and for economic reasons; however, recent ex vivo and in vivo transplantation studies suggest that HMP provides improved preservation compared to SCS, especially for ECD grafts[12,13]. Nevertheless, the optimal conditions for HMP remain controversial due to a lack of consensus regarding the conditions in terms of perfusates, oxygenation, perfusion route (dual vs portal), and reperfusion pressure. Optimizing techniques would help to reduce preservation injury and increase the donor pool.

In this study, we established a stable, simplified HMP system and compared the performance of three perfusate solutions using this system. Two of the perfusates (UW and HTK) are commonly used in the clinical LT setting. Our results showed that HMP is superior to SCS in terms of AST, ALT, LDH release and MDA levels, regardless of the perfusate used, which is in accordance with previous reports[14,15]. Among the three perfusates evaluated, UW and HTK are superior to saline in HMP in terms of D/W ratio, VR and ATP levels. However, UW and HTK are associated with their own individual advantages and disadvantages. For instance, UW reduced edema more efficiently, but was associated with higher VR and ATP consumption than HTK, which may be due to its high viscosity and the hydroxyethyl starch (HES) component. Although UW is the most commonly used perfusate, its high viscosity may result in initial poor perfusion and incomplete distribution of UW between the intravascular space and the liver parenchyma in the donor grafts[16]. Furthermore, HES in UW causes hyperaggregating effects on erythrocytes, which hampers a complete washout from the liver and loss of ATP[17]. Compared with UW, HTK solution contains lower sodium and potassium concentrations, no HES and a much lower viscosity (approximately one-third that of UW), thus facilitating its release into the circulation[18]. Experimentally, studies have recently reported HTK is as good as or even better than UW for short periods of perfusion[19,20]. The low viscosity and absence of HES in HTK partially contribute to the relatively low VR and ATP consumption associated with its use as a perfusate[21].

Historically, a number of perfusates have been developed such as UW, HTK, Celsior, Cardiosol, or Custodiol[22]. Belzer et al[23], who proposed the concept of HMP, developed the first perfusate, UW, which remains the gold standard today. However, the intrinsic shortage of intracellular electrolytes and the HES component imposes marked changes in the ionic environment and produces high viscosity[2], resulting in deterioration of cellular components, with loss of ATP energy stores and alteration in the biochemical function and cellular architecture, which is even more deleterious in ECD[17]. Subsequently, other perfusates such as modified UW alone[24] and later with diluted, heparinized and oxygenated blood[25,26], Polysol[27], HTK or Custodiol-N solution[24] sometimes enriched with Kidney Perfusion Solution-1 and Aqix RS-I[28], were introduced to achieve better preservation. However, these perfusates have not resulted in significant improvements in the preservation of donor organs, with the exception of diluted blood. This perfusate has been shown to be safe and effective as an oxygen carrier in an animal model[29] and the first clinical HMP trial by Oxford University has shown an excellent outcome.

In this study, particular attention has been paid to the energy balance of the liver in its preservation. Recovered levels of ATP appear to be a good indicator of mitochondrial respiratory function. Improvement of mitochondrial condition, reflected here by augmentation of tissue ATP content during HMP, may result in improved tolerance to I/R injury[30]. Moreover, many clinical studies have shown that the preimplantation ATP content correlates with liver failure[31] and post-transplantation graft function[32].

There are some limitations of our study that should be noted. First, the time period for liver preservation (SCS or HMP) was only 6 h, while the benefits of UW associated with the ischemic period may extend to longer than 6 h. The selection of 6 h in our experimental design was mainly based on our clinical experience with 1500 LT cases, in which the optimized cold ischemia time was less than 6-8 h.

In conclusion, innovative and improved perfusates are an important approach to the future development of HMP techniques for LT. We established a stable HMP system for use in optimizing HMP conditions and demonstrated that HMP is superior to SCS in terms of both the architecture and function of the liver. Among the perfusates evaluated in our study, HTK was identified as the optimized machine perfusion solution (MPS). However, some optimized MPS as Polysol and diluted blood might have set a new standard for liver organ preservation solutions.