Current and Future Applications of Machine Perfusion and Other Dynamic Preservation Strategies in Liver Transplantation (2024)

1. Introduction

Liver transplantation is one of the most successful forms of solid organ transplantation and continues to be the only curative treatment for end-stage liver disease. Developed as a standard medical practice in the mid-1980s, liver transplantation has been remarkably effective for the treatment of end-stage liver disease, bestowing a quality of life that nearly mirrors normalcy upon individuals who would otherwise face imminent mortality [1]. Consequently, the need for liver transplantation has increased globally.

Approaches to minimize the disparity between the supply and demand for liver grafts have included the utilization of what was once considered non-utilizable as "marginal" or "extended criteria" liver grafts, along with livers obtained from donation after cardiac death (DCD) [2]. However, these grafts are underutilized because they are associated with increased risks of post-transplant primary graft failure. Moreover, these grafts are more susceptible to ischemia–reperfusion injuries and are associated with an increased risk of biliary system complications [3].

Static cold storage (SCS) has been the gold standard for graft preservation [4]. However, the technique for the procurement of organs from DCD donors typically involves discontinuing all life-support measures. The donors experience hemodynamic instability and hypoxia during this period until circulatory arrest is confirmed. Following this event, a mandatory 5-minute period of waiting is required prior to organ procurement, which further compromises the quality of the graft [5]. The interval from the withdrawal of life-sustaining therapy to the introduction of a cold preservation solution is called warm ischemia [6]. Although SCS reduces metabolic activity, anaerobic processes still persist, leading to the depletion of adenosine triphosphate and the build-up of reactive oxygen species [6]. SCS offers significant benefits, such as ease of use and affordability, and is suitable for the procurement of low-risk organs. However, SCS has four main limitations: it does not reverse ongoing organ damage, it may cause additional harm to the organ during storage, organ viability cannot be evaluated during storage, and the storage duration is restricted [6]. These issues become more critical in cases of livers obtained from high-risk donors, which comprise an increasing proportion of liver donors. Severe ischemia–reperfusion-related injuries pose significant challenges in fulfilling the need for critical transplantation [7]. Machine perfusion (MP) is being adopted with greater frequency to address the constraints of SCS. This technique expands the pool of usable higher-risk donor organs and can reduce the cold ischemia time, which is particularly beneficial for obtaining marginal organs that are more prone to damage caused by ischemia and reperfusion.

Two primary MP methods are currently in use: hypothermic MP (HMP), which slows cellular metabolism using cold temperatures while flushing out metabolites and toxins; and normothermic MP (NMP), which maintains cellular functions at normal body temperatures. NMP allows the assessment of organ viability under near-physiological conditions. Innovative variations of MP, such as normothermic regional perfusion (NRP), ischemia-free liver transplant, sub-normothermic MP, and long-term NMP, have also been introduced recently.

This review article examined MP as an innovative advancement in the field of liver transplantation, reviewing the possible solutions to the limited availability of organs and improving the viability of donated livers.

2. Hypothermic Machine Perfusion (HMP)

HMP refers to various mechanical perfusion techniques that are based on the use of cold perfusion. Hypothermic oxygenated perfusion (HOPE) involves delivering the perfusate through the portal vein; while dual HOPE (D-HOPE) delivers perfusate through both the portal vein and the hepatic artery [8].

The first prospective trial on ex-situ HMP was published in 2010, which reported a significant reduction in patients’ post-operative liver transaminase and serum bilirubin levels [9]. Another milestone in HOPE was achieved after a study showed that liver grafts obtained from DCD have comparable postoperative outcomes to matched liver grafts from brain death (DBD) [10]. A further study including a larger cohort of the same patient groups substantiated these findings [11].

HOPE and SCS have been compared in randomized controlled trials (RCTs). The trials demonstrated that the use of HOPE-treated DCD allografts reduced non-anastomotic biliary strictures, the incidence of post-reperfusion syndrome, early allograft injury, graft failure [12,13], and liver-related complications [12]. Additionally, HOPE was found to improve long-term graft survival and reduce late-onset morbidity [14].

These previous studies demonstrated that HOPE enhances graft survival and lowers the rate of complications (Table 1). The protective effects of this method may facilitate increased use of marginal organs, thereby addressing the growing need for donor organs.

Table 1 Overview of published randomized controlled trials on the use of hypothermic machine perfusion in liver transplantation.

3. Normothermic Machine Perfusion (NMP)

NMP restores cell metabolism and maintains the physiological state of the liver by keeping the graft at a normal temperature and supplying sufficient oxygen and nutrients throughout the entire liver preservation process [19]. The main components of NMP devices are a blood reservoir, heat exchanger, oxygenator, and one or two pumps for the portal venous and hepatic artery [19,20].

The creation of a perfusion chamber in 1935 for the preservation of organs at room temperature marked the beginning of NMP's history [21]. Ravikumar et al. investigated liver transplantation utilizing NMP-preserved livers in a phase I trial in 2016, showing that this approach is safe and feasible [22]. Subsequently, a phase III multicenter RCT wherein NMP and SCS were compared indicated that NMP is associated with a lower incidence of graft injury, organ loss, and longer mean preservation time [23]. In 2019, Ceresa et al. conducted a study of 31 liver transplants to assess NMP after cold storage. The results indicated a 94% graft survival and 13% incidence of early allograft dysfunction within 30 days. The graft and patient survival rates at 12 months were 84% and 90%, respectively [24]. In a study by Ghinolfi et al., 20 liver transplant recipients who received livers from donors aged 70 years or older were divided into NMP and cold storage (CS) groups for evaluation. At 6 months, both groups showed similar survival rates and biopsy results. No major histological advantages of NMP were noted [25]. Bral et al. compared NMP with SCS for liver transplantation and found no difference in short- or mid-term graft survival; however, the NMP group had a longer hospital stay than the SCS group [26].

The primary justification for NMP is its ability to measure objective graft function parameters and identify marginal organs that would otherwise be eliminated as transplantable. NMP is used to assess the graft survival rate by assessing liver and bile duct function parameters, liver injury parameters, and hemodynamic analysis [27]. The survival rate test offers quantifiable predict of liver function following liver transplantation [28].

The viability of high-risk donor livers that underwent NMP prior to transplantation was investigated by Mergental et al. The results showed that despite initial preservation by static refrigeration, the transplanted livers functioned well immediately post-transplant, with normal liver function test results recorded over an average follow-up period of 7 months. This pilot study demonstrated that after viability assessment using NMP, livers previously deemed unsuitable for transplantation could be successfully transplanted to low-risk recipients without compromising safety [29]. Mergental et al. further conducted a single-center phase II trial evaluating high-risk livers previously deemed “non-transplantable” using NMP and found the 1-year patient and graft survival rates to be 100% and 86%, respectively; the 5-year patient and graft survival rates were 82% and 72%, respectively, based on long-term follow-up of the NMP group [30]. Furthermore, Quintini et al. found that of 21 livers that were declined for transplant, 15 (71.5%) were deemed transplantable after assessment using NMP. There were no instances of primary non-function post-transplant; however, seven livers showed early dysfunction that recovered quickly [31]. Although there are no definitive criteria for pre-transplant assessment of liver function using NMP, the metrics commonly used included perfusate lactate clearance, pH stability without the need for bicarbonate supplements, stable hemodynamics in the hepatic artery and portal vein; liver transaminase levels, and serum glucose levels [32]. By employing these techniques for pre-transplant viability assessment, the hazards of using marginal donor livers can be minimized, the probability of organ rejection and primary non-function after transplant can be decreased, and more organs can be made available for patients in need.

A primary limitation of NMP is that organs often undergo SCS before NMP is performed at the transplant center, owing to logistical issues. Notably, a portable NMP device was employed to avoid using SCS in a recently published RCT. The use of the portable NMP device also allowed the use of livers from DCD [33]. Another drawback of NMP is its high cost compared to HMP/HOPE or SCS; however, it should be noted that its cost-effectiveness was demonstrated by fewer post-transplant complications and increases in the donor pool. A summary of published studies on NMP LT is presented in Table 2.

Table 2 Overview of published studies on the use of NMP in liver transplantation.

4. Normothermic Regional Perfusion (NRP)

NRP is used in potentially transplantable organs as a way of in situ perfusion after circulatory arrest [37,38]. It has been used increasingly to reduce biliary complications and graft failure post-transplantation. Unlike other MP technologies (HOPE and NMP) that can be applied in DBD and DCD liver allograft management, NRP can only be used in DCD liver allograft management.

After circulatory arrest and a hands-off period that can range from 5 to 20 minutes depending on local regulations, extracorporeal membrane oxygenation is initiated with cannulation of the femoral artery or aorta and with venous return from the femoral vein or inferior vena cava [39]. It is also important to distinguish between abdominal normothermic regional perfusion (A-NRP) and thoracoabdominal normothermic regional perfusion (TA-NRP). A-NRP specifically targets the abdominal organs by providing in situ perfusion through the infrarenal aorta and IVC, or the femoral artery and vein, without initiating cardiac activity by applying supraceliac aortic cross-clamp/occlusion balloon. In contrast, TA-NRP performs in situ perfusion of both thoracic and abdominal organs via the aortic arch and right atrium and includes the restoration of cardiac activity, with the aortic arch vessels clamped to prevent brain perfusion [40]. Initial studies show promising outcomes for DCD livers obtained through TA-NRP [41]. However, ethical concerns about restarting cardiac activity during the process have led to a temporary pause of TA-NRP in some organ procurement organizations, pending further ethical review [42]. Conversely, A-NRP faces fewer ethical concerns because it does not reinitiate cardiac activity, and perfusion is confined regionally to only the abdominal organ.

Though an RCT on NRP has not been published yet, retrospective data have demonstrated that NRP transplantation produces better results than SCS transplantation. Overall, preliminary data showed that NRP can enhance the results of DCD organ transplantation through the mitigation of early allograft dysfunction, enhancement of graft survival, reduction of biliary problems, and mitigation of retransplantation risk (Table 3). Owing to positive clinical outcomes, France and other European nations now require NRP for all DCD donations [39,43].

Table 3 Overview of recent studies on normothermic regional perfusion.

5. Combination of Machine Perfusion Techniques

The combination of HOPE with controlled oxygenated rewarming (COR) and NMP could leverage their benefits. This is because HOPE reduces ischemia–reperfusion injury, COR eases the transition from cold to warm states, minimizing organ damage, and NMP at 37°C allows for functional assessment while mitigating ischemic injury. De Vries et al. introduced a protocol in which D-HOPE, COR, and NMP were combined with a new hemoglobin-based oxygen carrier. Based on specific parameters (perfusate pH, lactate levels, bile production, and biliary pH), 5 of 7 livers that were previously declined were transplanted, and all showed a 100% graft survival rate at 3 months [54]. Van Leeuwen et al. studied 16 DCD livers subjected to a sequence of D-HOPE-COR-NMPs after SCS during transportation; consequently, 11 were deemed suitable for transplantation [55]. In a prospective observational cohort study conducted by the same group, the authors noted that ex situ machine perfusion employing sequential D-HOPE-NMP for the resuscitation and viability assessment of high-risk donor livers achieved excellent transplant outcomes [56].

Many centers in Italy have implemented in situ abdominal NRP followed by ex situ MP of DCD organs. Despite the significant hurdle presented by prolonged warm ischemia time, the Italian centers reported good outcomes of DCD live transplantation using this approach [57,58].

Patrono et al. compared abdominal NMP plus D-HOPE in controlled DCD livers versus DBD livers and found similar early outcomes between groups. Biliary complication rates were 15% for DCD and 22% for DBD, with comparable ischemic cholangiopathy incidences (DCD, 5%; DBD, 2%). One-year patient survival was 100% for DCD and 95% for DBD, while graft survival rates were 90% and 95%, respectively. Furthermore, the combination of abdominal NRP and D-HOPE in DCD liver with a prolonged warm ischemia time resulted in outcomes comparable to those of DBD [59].

6. Novel Approaches

6.1 Ischemia-Free Liver Transplant

The complex nature of ischemia–reperfusion injury makes it challenging to advance new scientific concepts in its management. Various MP techniques, such as HMP, HOPE, and NMP, have been introduced to enhance organ preservation in clinical settings, with studies confirming their safety and effectiveness. However, these methods still involve a period of ischemia during the procurement and implantation of the allograft because they are implemented following a cold storage period. Ischemia-free liver transplantation is an innovative technique that maintains continuous blood supply under continuous in-situ NMP during the procurement and transplantation process, thus reducing post-reperfusion syndrome upon allograft revascularization [60]. In 2018, He et al. described an MP method that initiates blood flow in the donor liver before circulatory arrest. This technique involves the cannulation of the common bile duct, infrahepatic inferior vena cava, portal vein, and hepatic artery to establish an in-situ NMP circuit, while harvesting the liver. During transplantation, the liver was connected while still being perfused by the machine, ensuring continuous oxygenated blood flow and preventing ischemia and reperfusion injury [61]. An RCT showed that compared to traditional methods, this technique reduces complications from ischemia-reperfusion injury in liver transplant recipients. This approach could advance transplantation practices, improve outcomes, increase organ utilization, and offer insights into how organ injury affects alloimmunity [62].

6.2 Long-Term Normothermic Machine Perfusion (NMP)

Current MP technology allows for short-term ex-situ preservation and viability assessment of the liver before transplantation. The field of long-term normothermic perfusion is growing and offers significant prospects for evaluating, recovering, and improving organ function. Clavien et al. described a case wherein a human liver graft was successfully transplanted after being kept for 3 days by utilizing ex situ NMP [63]. After initial experiments with partial swine livers, Mueller et al. applied the protocol to 21 partial human livers and achieved a perfusion duration goal of 1 week. The liver sections demonstrated stable perfusion and normal function, while maintaining their structural integrity for up to 1 week [64]. Lau et al. focused on creating a long-term ex-situ perfusion model and achieved a median viability of 125 hours and a median survival of 165 hours during ex-situ perfusion. This study demonstrated the feasibility of long-term ex-situ liver perfusion and perfusing livers using a standardized approach [65].

6.3 Extracorporeal Liver Perfusion (ECLP)

Beyond its application in liver transplantation, MP potentially can assist liver metabolism for patients with liver failure. A circuit that circulates the patient's blood through an entire liver—which could be human or animal—is used in ECLP [66,67,68]. An MP circuit with a healthy liver connected to the patient's circulation would make this feasible. In situations where spontaneous recovery might occur, for example in those with acetaminophen toxicity, treatment utilizing daily ECLP via an isolated genetically modified pig liver could offer a vital support. This approach would eliminate the need for extensive immunosuppressive treatments or a liver transplant. Preclinical investigations shown ECLP using pig livers can potentially preserve injured human livers for 1 week [69]. Although the numbers were small, using ECLP with pig livers in ALF patients showed a survival benefit [70,71,72]. Research indicates this method is safe and practical for bridging patients to liver transplantation [73].

7. Current Challenges

The growing interest in MP owing to its beneficial effects on organ quality and recipient outcomes has not yet been translated into the clinical practice of liver transplantation. The factors determining the wider adoption of specific technologies in clinical practice remain unclear. For example, the benefits of MP for low-risk liver grafts, which already yield favorable short and long-term outcomes with SCS, are not well-studied [74]. It is unsure at what threshold of risk should MP be involved. Even though there are many RCTs already published or ongoing, more information about allograft function, liver use rate, recipients' outcomes, and cost-effectiveness should be evaluated for clinical applications and impact. Furthermore, it should be mentioned that most currently available evidence is restricted to 1-year follow-up, which seems to be one of the primary drawbacks in the current literature [75]. Consensus and RCTs are critical for validating aspects of MP, such as infusion pressure, solution components, perfusion duration, and biological indicators for organ quality assessment.

Criticism has been raised that the aspartate transaminase/alanine aminotransferase level is inconsistent with transplant outcomes, especially in DCD liver grafts [76]. Hospital and intensive care unit stay durations may differ significantly between centers owing to varying discharge policies and facility availability, potentially weakening their reliability as endpoints [75,77]. Therefore, clinically relevant endpoints and future trial guidelines need to be established [78].

To fully comprehend how MP influences the utilization of donor livers and identify areas for improvement, it is vital to understand the reasons behind liver discard [79]. Establishing clear criteria for donor, liver, and recipient risk factors are necessary for routine applications of MP. Creating simpler device registration policies and innovative financial models is essential to facilitate the adoption of MP globally. Moreover, the significant variations in the costs and affordability of new technologies will undoubtedly impact their broader use [79,80].

8. Future Applications

Enhancing liver preservation, organ usage, functional assessment, and outcomes are areas where MP has shown promise. Future perspectives in MP include ex vivo liver repair and potential personalized organ preservation [81]. Recondition steatotic grafts is a promising application of MP in liver transplantation, given the impact of steatosis on graft viability [82]. Nagrath et al. conducted a preclinical study using a “defatting co*cktail” on steatotic rat livers during NMP, which significantly lowered intracellular lipid levels by 50% after 3 hours [83]. MP could also serve as an effective method for removing viral infections from donated livers, including hepatitis C virus infections [84]. Furthermore, with long-term MP, previously wasted livers could be treated with stem cells, organoids, senolytics, or compounds that target the mitochondria and downstream signaling to modulate repair mechanisms and regeneration [85,86].

9. Conclusion

In this review, we summarize findings from clinical trials on MP techniques, explore emerging MP approaches, address obstacles to standardizing MP protocols, and highlight the critical role of clinical trials in validating various aspects of the perfusion process. Furthermore, we discuss the potential of cutting-edge MP techniques in preserving and enhancing graft quality.

Abbreviations

Author Contributions

Yue Qiu MD: writing – original draft. Yinqian Kang MD: review and editing. Hao Liu MD: review and editing. Ibtesam Hilmi, MBCHB, FRCA: Conceptualization, review, and editing. All authors have read and approved the published version of the manuscript.

Competing Interests

The authors have declared that no competing interests exist.