During organ procurement and SCS, cessation of oxygen delivery arrests mitochondrial oxidative phosphorylation. Hepatocytes shift to anaerobic glycolysis, rapidly depleting intracellular glycogen and adenosine triphosphate (ATP). As ATP declines, the Na⁺/K⁺-ATPase membrane pump fails, leading to intracellular sodium accumulation, cellular edema, and cytosolic calcium overload [6]. A key biochemical event during the ischemic phase is the supraphysiological accumulation of succinate within the mitochondria, a metabolic intermediate that remains relatively inert under cold conditions but constitutes a critical liability upon reperfusion [7].

Reperfusion at normothermia triggers rapid oxidation of accumulated succinate via mitochondrial complex II (succinate dehydrogenase). Due to incomplete recovery of electron transport chain components, reverse electron transfer to mitochondrial complex 1 happens, which generates a burst of reactive oxygen species (ROS), predominantly superoxide, at the flavin mononucleotide (FMN) site [5]. This oxidative cascade produces three principal consequences: (i) mitochondrial dysfunction through opening of the permeability transition pore (MPTP), leading to uncoupling of oxidative phosphorylation and necrosis [8]; (ii) sterile inflammation via release of damage-associated molecular patterns (DAMPs-HMGB1 and DNA), Kupffer cell activation, and pro-inflammatory cytokine production (TNF-±, IL-1β, IL-6) [9,10]; and (iii) microvascular dysfunction through endothelial swelling and “no-reflow,” particularly pronounced in DCD grafts [2].

The biliary epithelium is uniquely susceptible to IRI due to its exclusive dependence on the peribiliary vascular plexus supplied by the hepatic artery. In DCD grafts, the combined effects of warm ischemia and microvascular thrombosis frequently result in biliary epithelial necrosis. Clinically, this manifests as non-anastomotic biliary strictures (NAS) or ischemic cholangiopathy-both of which are major contributors to graft failure and the need for re-transplantation [9,11].

Hypothermic oxygenated perfusion (HOPE) is most commonly applied in the end-ischemic phase at the recipient center. It involves perfusion of the liver with a cold (4-10°C), oxygenated, acellular solution via the portal vein alone (HOPE) or in combination with the hepatic artery (dual-HOPE, D-HOPE) [2,12].

HOPE promotes controlled mitochondrial recovery under hypothermic, oxygenated conditions by gradually reactivating the electron transport chain, allowing slow metabolism of accumulated succinate and reducing the burst of reactive oxygen species during reperfusion. Controlled oxygen delivery restores intracellular ATP stores before implantation, while continuous perfusion clears damage-associated molecular patterns (DAMPs), cytokines, and metabolic waste, thereby reducing graft immunogenicity and ischemia-reperfusion injury [5,13].

The Zurich multicenter randomized controlled trial conducted by Schlegel et al. evaluated the impact of HOPE in extended-criteria donation-after-brain-death (DBD) grafts. A total of 170 transplants were randomized to either SCS or HOPE. Although the primary endpoint-Clavien-Dindo grade ≥ III complications within one year-did not differ significantly between groups (54.1% vs. 51.8%; p = 0.76), post hoc analyses revealed a significant reduction in severe liver-related complications in the HOPE group (risk ratio 0.26; p = 0.027). Notably, graft failure due to liver-related causes occurred in 0% of HOPE-treated grafts compared with 7% in the SCS group (p = 0.004) [12]. Post hoc analyses carry an inherent risk of type I error and should be interpreted cautiously.

The benefits of HOPE are most pronounced in DCD grafts. In the D-HOPE DCD trial, a multicenter randomized study published in the New England Journal of Medicine, 156 DCD grafts were randomized to SCS or D-HOPE [2]. The study met its primary endpoint, demonstrating a significant reduction in non-anastomotic biliary strictures (6% vs. 18%; hazard ratio 0.36; p = 0.03). Secondary outcomes included reductions in EAD and post-reperfusion syndrome, with improved graft function and hemodynamic stability. These findings have established D-HOPE as a standard preservation strategy for DCD livers in many European centers.

A 2023 meta-analysis of 11 studies (including 5 RCTs), which demonstrated that HOPE significantly reduced biliary complications (RR 0.74), NAS (RR 0.34), EAD (RR 0.54), and acute rejection (RR 0.54) compared with SCS [14,15]. A Cochrane review similarly concluded that both HOPE and NMP outperform SCS, with stronger evidence supporting HOPE for improving graft survival and reducing ischemic cholangiopathy in DCD grafts [15].

There are some limitations with the above trials due to considerable difference in study population in donor age, warm ischemic time, center experience and lack of long-term follow up. Additionally, the lack of standardized oxygenation protocols (HOPE vs D-HOPE) introduces technical heterogeneity.

NMP maintains the liver at 37°C using an oxygenated, nutrient-enriched perfusate-typically red-blood-cell based-to sustain physiological metabolism and allow viability assessment. It can be applied as a transport strategy (upfront NMP) or as an end-ischemic platform [16].

e Consortium for Organ Preservation in Europe (COPE) trial, the first large randomized controlled trial evaluating normothermic machine perfusion (NMP), demonstrated a 50% reduction in graft injury and donor liver discard rates, while extending preservation time by 54%. These results indicate improved graft utilization of marginal grafts and surgical logistics enabling a shift toward semi-elective transplantation [1].

In the United States, the OCS Liver PROTECT trial evaluated a portable NMP system, showed significantly reduced EAD (18% vs 31%; p = 0.01) and biliary complications at 6 and 12 months (2.6% vs 9.9%), along with increased DCD utilization (51% vs 26%) [17,18].

The VITTAL trial extended the application of NMP to livers deemed “untransplantable”. Using strict viability criteria, 71% of these discarded grafts were successfully transplanted, achieving 100% 90-day patient survival, though this non-randomized single-arm design limits causal inference [19].

Evidence for NMP remains heterogeneous, with differences between major trials in perfusion systems, donor populations, transport strategies, and comparator arms. Lack of standardized viability criteria, high operational costs, and logistical complexity limit widespread adoption. Additionally, the optimal timing of NMP and its comparative benefits versus HOPE in specific donor subgroups remain uncertain and are being investigated in ongoing trials [15].

NRP represents a distinct in situ preservation strategy specifically applied in donors after circulatory death (DCD), which involves the initiation of Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) to re-establish oxygenated blood flow within the donor’s abdominal compartment while excluding cerebral circulation.

NRP resuscitates the liver from the deleterious effects of warm ischemia by restoring physiological perfusion conditions. This resuscitative phase is typically maintained for approximately two hours, during which metabolic recovery occurs and organ function can be assessed using systemic biochemical parameters. Following this period, the liver is flushed with cold preservation solution and retrieved using standard surgical techniques.

NRP has demonstrated remarkable efficacy in preserving the integrity of the peribiliary vascular plexus, thereby significantly reducing the incidence of ischemic cholangiopathy. Large retrospective registry analyses from both the United Kingdom and the United States have shown that outcomes following NRP in DCD donors-including graft survival and biliary complication rates-are comparable to those achieved with donation after brain death (DBD) grafts [20]. Avoiding selection bias and large prospective RCTs would support the evidences of NRP.

Sequential perfusion protocols integrate complementary benefits of hypothermic and normothermic modalities. The DHOPE-COR-NMP sequence, developed for high-risk ECD-DCD grafts, begins with end-ischemic dual hypothermic oxygenated perfusion (DHOPE) for mitochondrial recovery with ATP repletion, followed by controlled oxygenated rewarming (COR) from 10°C to 37°C, and concludes with full NMP for viability assessment [21]. This approach leverages mitochondrial protection (HOPE phase), avoids abrupt temperature changes (COR phase) and functional assessment (NMP phase) sequentially. However, this approach is resource intensive and time consuming. There is no defined rewarming rate or COR duration.

While both HOPE and NMP significantly mitigate ischemia-reperfusion injury, they do not completely eliminate ischemic periods during organ procurement and implantation. Ischemia-Free Liver Transplantation (IFLT), pioneered by He Xiaoshun and colleagues, represents a paradigm shift aimed at abolishing ischemia entirely [22].

In IFLT, the liver is cannulated and perfused in situ within the donor using normothermic machine perfusion. The organ remains continuously perfused throughout procurement, transport, and implantation, with vascular anastomoses performed while the graft remains connected to the perfusion system. This uninterrupted perfusion effectively eliminates both cold and warm ischemic phases.

Randomized studies showed, improved one-year patient survival (95.5% vs 86.1%), reduced EAD. Long term follow-up studies showed reduced recurrence of hepatocellular carcinoma. IRI promotes tumor recurrence through CXCL10/CXCR3 signaling pathway and IFLT exhibit downregulation of this pathways, providing the biological plausibility for the observed clinical advantages [23,24,25].

IFLT remains a single-center technique from China, requiring substantial infrastructure and surgical expertise. International multicenter validation is essential before broader adoption can be recommended. Nevertheless, long-term follow-up data from 5- to 7-year cohorts are emerging and support its potential as a transformative strategy for selected grafts [23,24].

There are some limitations with the above trials due to considerable difference in study population in donor age, warm ischemic time, center experience and lack of long-term follow up. Additionally, the lack of standardized oxygenation protocols (HOPE vs D-HOPE) introduces technical heterogeneity.

Under normothermic conditions, the liver is metabolically active, enabling direct measurement of functional performance. Established viability criteria, including those used in the VITTAL trial, incorporate multiple physiological and biochemical parameters. Lactate clearance is the most important marker, with perfusate lactate levels ≤2.5 mmol/L within 4 hours strongly predicting graft viability and failure to achieve this threshold associated with primary non-function. Bile chemistry provides additional information, as a biliary pH > 7.4 reflects active bicarbonate secretion and functional biliary epithelium. Hemodynamic stability, indicated by hepatic artery flow >150 mL/min and portal vein flow >500 mL/min, confirms preserved vascular integrity, while dynamic glucose metabolism, demonstrated by declining perfusate glucose levels after 2 hours or maintenance below 10 mmol/L, reflects ongoing metabolic activity and graft viability [19,26,27].

Cholangiocellular viability is critical for preventing late biliary complications. Functional cholangiocytes actively secrete bicarbonate and reabsorb glucose. Accordingly, viability is indicated by a biliary pH > 7.45, elevated bile bicarbonate concentrations (≥18 mmol/L), and bile glucose levels significantly lower than perfusate glucose (<3 mmol/L). These parameters have been incorporated into standardized viability protocols developed by major transplant centers, including the Birmingham, Cambridge, and Groningen criteria [27].

Emerging evidence underscores the importance of endothelial integrity in graft viability. The endothelial glycocalyx plays a critical role in maintaining microvascular function. Elevated levels of syndecan-1-a structural component of the glycocalyx-in the perfusate are indicative of endothelial injury.

High syndecan-1 concentrations (e.g., >4,796.13 ng/mL after six hours of NMP) are strongly associated with early allograft dysfunction, particularly in DCD grafts, and serve as a predictive biomarker of microvascular compromise [28]. A validated consensus protocol through multi-centric study would help for better decision making.

During hypothermic perfusion, metabolic activity is markedly suppressed, precluding the use of functional markers such as bile production. Instead, viability assessment relies on biomarkers of mitochondrial injury. Flavin Mononucleotide (FMN) has emerged as a robust and clinically validated biomarker. During ischemia, FMN dissociates from mitochondrial complex I and is released into the perfusate upon reperfusion [29,30].

Real-time quantification of FMN using fluorescence spectroscopy enables rapid assessment within 30 minutes. Elevated FMN levels are strongly predictive of graft loss, early allograft dysfunction, and biliary complications, with reported area under the curve (AUC) values of 0.93. This allows for reliable “go/no-go” decisions prior to recipient anesthesia [29,30].

Transcriptomic profiling of perfusate and tissue biopsies during NMP has identified gene expression signatures-including CD274 (PD-L1), LEAP2, and IFIT1-achieving predictive accuracy of AUC 0.99 in preliminary data [31]. While these results are compelling, they are derived from small exploratory studies and are not yet applicable in routine clinical practice. Prospective validation with standardized methodology is essential before these tools influence transplant decision-making.

Hepatic steatosis exceeding 30% remains a major cause of organ discard due to its association with IRI, EAD, and PNF. NMP enables functional assessment of steatotic grafts by providing real-time metabolic data, allowing transplantation of organs that demonstrate adequate lactate clearance and hemodynamic stability despite donor histology. A recent multicenter cohort study from Mayo Clinic (2022-2025) demonstrated that NMP was associated with reduced post-reperfusion syndrome and improved patient and graft survival in recipients of livers with moderate macrosteatosis (30-60%), providing contemporary real-world evidence for the utility of NMP in this challenging population [32].

Beyond viability assessment, NMP offers a platform for pharmacological “defatting”-the ex vivo reduction of hepatocellular lipid content prior to implantation. Preclinical studies incorporating L-carnitine, fenofibrate, and forskolin into the perfusate have demonstrated experimental reductions in macrovesicular steatosis of up to 40% within six hours [33]. More recently, the DeFat Study-a multicentre RCT protocol-has been registered to prospectively evaluate pharmacological defatting with forskolin (NKH477) and L-carnitine combined with lipoprotein apheresis during NMP in discarded steatotic human livers, with transplantation as the primary endpoint. This constitutes the most rigorous ongoing effort to establish defatting as a clinical strategy, and results from this trial are required before defatting can be recommended beyond experimental settings [34]. Proof-of-concept cases have also demonstrated successful transplantation of livers with severe steatosis (>70%) using upfront NMP, further supporting the therapeutic potential of this approach [35].

DCD grafts are particularly susceptible to biliary complications due to warm ischemic injury. HOPE, through controlled mitochondrial recovery without oxidative stress, is ideally suited to these grafts, as demonstrated by the D-HOPE trial [2]. NMP is valuable for functional assessment of DCD grafts with uncertain viability, particularly those with prolonged warm ischemia or borderline hemodynamic parameters [36]. NRP, when available, provides in situ resuscitation prior to procurement and has been associated with outcomes approaching DBD grafts in large registry analyses [20]. The choice between these modalities-or their sequential combination-should be guided by institutional expertise, donor characteristics, and the findings of ongoing comparative trials.

The HOPE-NMP Trial is a landmark multicenter RCT designed to address the “cold versus warm perfusion” debate in liver transplantation. In this study, extended criteria donor (ECD) grafts from donation after brain death (DBD) donors are randomized into three groups: end-ischemic hypothermic oxygenated perfusion (HOPE), end-ischemic normothermic machine perfusion (NMP), and a smaller control arm using SCS.

The primary endpoint is the 90-day Comprehensive Complication Index (CCI), a validated composite measure capturing overall postoperative morbidity. This design enables a holistic evaluation of recipient outcomes rather than focusing on isolated complications.

Importantly, this trial directly compares hypothermic and normothermic strategies within a single randomized framework, allowing definitive assessment of whether controlled mitochondrial resuscitation (HOPE) or maintenance of physiological metabolism (NMP) provides superior outcomes in ECD grafts. The findings are expected to significantly influence clinical practice and standardize preservation strategies [43].

The DCD-Net Trial investigates the optimal ex situ management of donation after circulatory death (DCD) livers following initial in situ resuscitation with normothermic regional perfusion (NRP).

In this trial, DCD grafts successfully resuscitated with NRP are randomized 1:1 to either ex vivo hypothermic machine perfusion (HMP) or normothermic machine perfusion (NMP). This design isolates the incremental benefit of the subsequent perfusion modality after initial recovery.

Primary endpoints include graft survival and the incidence of ischemic type biliary lesions (ITBL), both critical determinants of long-term outcomes. By comparing hypothermic versus normothermic support post-NRP, the trial aims to determine whether metabolic suppression or sustained physiological activity offers superior protection against biliary injury and graft dysfunction.

In this trial, DCD grafts successfully resuscitated with NRP are randomized 1:1 to either ex vivo hypothermic machine perfusion (HMP) or normothermic machine perfusion (NMP). This design isolates the incremental benefit of the subsequent perfusion modality after initial recovery.

With the increasing adoption of NRP, results from this trial are expected to have immediate clinical relevance in refining multi-stage perfusion strategies and improving outcomes in high-risk DCD grafts [44].

The Netherlands Sequential Trial is a large, pragmatic, multicenter observational study evaluating real-world, risk-adapted sequential perfusion strategies. Unlike traditional RCTs, this study uses protocol-driven allocation based on donor characteristics, particularly age and graft risk.

Extremely marginal ECD-DCD grafts are assigned to different pathways, including NRP followed by dual hypothermic oxygenated perfusion (DHOPE), standard retrieval followed by DHOPE for intermediate-risk donors (50-60 years), and advanced sequential strategies incorporating controlled oxygenated rewarming followed by normothermic machine perfusion (COR-NMP) for older, high-risk donors (>60 years).

The primary outcomes are organ utilization rates and cost-effectiveness, addressing both clinical and health system considerations. This structured, personalized approach reflects a shift toward tailored organ preservation strategies and is expected to guide optimal allocation of perfusion resources [42].

Collectively, these trials highlight a paradigm shift in liver transplantation. The focus has moved beyond establishing superiority over SCS toward defining the optimal modality, sequence, and timing of perfusion. This evolution underscores the emergence of precision-based organ preservation, integrating donor characteristics, injury profiles, and logistical factors to guide individualized strategies. As these studies mature, they are expected to establish a new generation of evidence-based guidelines for machine perfusion in liver transplantation.