Static cold storage (SCS) has been the standard of care for the preservation of liver grafts before transplantation for the past 4 decades. Simply, SCS involves flushing the organ with a preservation solution and keeping it in an ice bath at 4°C.1 SCS is low cost, effective, and practical, and although it has helped establish transplantation as the only life-saving modality of treatment for end-stage organ failure, preservation remains far from perfect. Indeed, cold ischemia triggers a cascade of cytotoxic pathways, leading to sinusoidal endothelial cell swelling, membrane damage, and injury to other components of the hepatic microvasculature.2,3 Clinically, it may be associated with early graft dysfunction and limits the time between organ retrieval and implantation.4,5
Machine perfusion (MP) techniques are an alternative to SCS, in which a perfusate solution (typically dilute oxygenated blood) is actively circulated through the organ to maintain cellular metabolism and prevent the effects of cold ischemia.6 MP featured prominently in the early days of liver transplantation, including the first successful human transplants by Starzl, in which a hypothermic, hyperbaric setup with low-flow perfusion by diluted blood was applied.7,8 It subsequently fell by the wayside, however, partly due to its relative complexity, but more due to the introduction of simple, effective cold storage solutions, most notably Belzer’s University of Wisconsin (UW) solution.9
There has been a recent resurgence of interest in MP, beginning with preclinical trials of several systems in the early 2000s to its current use in the clinical setting.10,11 A recently published randomized controlled trial (RCT) by Nasralla et al12 demonstrated efficacy over SCS using a normothermic MP (NMP) system, by showing lower levels of graft injury despite a lower rate of organ discard and longer preservation times. Similar trials are underway using an alternative hypothermic setup.13 The aim of these initial clinical investigations was to demonstrate both safety and protective efficacy with preservation of liver grafts, with the hope and expectation that the increasing use of higher risk, marginal liver grafts including donation after circulatory death (DCD), would be afforded with additional protection to reduce patient risk. However, this only begins to scratch the surface of potential uses for this remarkable technology.
This article reviews the rationale and relevant preclinical studies that support the use of ex situ liver perfusion for extended applications adjacent to and beyond the realm of liver transplantation (Figure 1). This includes the use of MP to assess graft function and treat grafts before implantation, for indications such as the eradication of hepatitis C and “defatting” of steatotic livers. Other potential areas of opportunity include use as extracorporeal liver support devices, either with marginal grafts or genetically engineered xenografts, cancer research and therapeutics, and preclinical studies of hepatic toxicology and injury. Many avenues exist for the development and refinement of this technology for use in both clinical and translational research.
OVERVIEW OF MACHINE PERFUSION TECHNOLOGY
The main difference between MP devices is the temperature at which the organ is perfused.6 With hypothermic MP (HMP), the graft is kept as cold as 4°C, similar to SCS.14 The suggested benefit over SCS is improvement of microcirculation by mobilization and dilution of metabolic waste. HMP does not provide supplemental oxygen, whereas its oxygenated alternative is referred to as hypothermic oxygenated perfusion (HOPE).15 HOPE typically perfuses at a slightly higher temperature (10–12°C), though both use preservation solutions, such as UW solution or Vasosol (a modification of UW solution containing additional vasodilatory and antioxidant agents), as the perfusate.16 The oxygen supplied in HOPE is only that dissolved in the perfusate (ie, there is no additional oxygen-carrying molecule). A disadvantage of the hypothermic approach is that the low metabolism at these temperatures makes assessment of functional viability more challenging.
Subnormothermic MP (SMP) maintains the graft at a warm, but still subphysiologic temperature, typically between 25°C and 34°C.17 Similar to HOPE, the graft is perfused with an oxygenated preservation solution. It essentially attempts to find the middle ground between HOPE and NMP. At this temperature, the graft may still benefit from lower metabolic demand, while retaining enough metabolic function to allow for viability testing. Preclinical studies have demonstrated improved mitochondrial function and replenishment of ATP stores with this approach.18 A variant of this technique is controlled oxygenated rewarming, in which the perfusion temperature is gradually increased from HMP to SMP levels, has demonstrated improved efficacy over SMP alone in preclinical studies.19 The authors hypothesized that the gentler rewarming mitigated the effects of reperfusion injury.
NMP, by contrast, aims to target an environment as close to physiologic as possible. The graft is kept between 35–38°C and the perfusate contains erythrocytes, either from stored blood products (for human grafts) or diluted whole blood, in the case of animal models. NMP seeks to minimize cold ischemic time and maintains a liver that is fully metabolically active, which allows for accurate functional viability assessments, such as via glucose metabolism and bile production.
Each of these approaches has been used clinically, with subsequent transplantation of the perfused livers. NMP is the only approach to have been evaluated in an RCT, as mentioned previously.12 Nonrandomized clinical series have been reported for HMP, HOPE, and SMP (in the context of controlled oxygenated rewarming), which demonstrated safety and several improved outcomes compared with SCS controls, including lower peak transaminases, fewer biliary complications, and increased graft survival.20-24 Of note, a nonrandomized clinical trial comparing 50 DCD livers transplanted after HOPE to 50 matched controls from brain dead donors demonstrated similar graft survival at 5 years.25 Recruitment for larger RCTs involving HOPE (NCT03484455, NCT02584283, NCT03929523) has already been initiated. Commercial adaptations of these devices include the OrganOx metra and Organ Care SystemTM Liver (by TransMedics), which use NMP, the LifePort Liver Transporter (by Organ Recovery Systems), which uses now use HOPE, and the Liver Assist, which is adaptable to different temperatures. Neither clinical nor preclinical studies have been conducted that compare different MP strategies against one another.
EVALUATION OF LIVER GRAFTS EX SITU
An important strategy to maximize the utility of NMP in liver donation involves assessment and treatment of marginal liver grafts. Marginal grafts refer to organs that are at increased risk of graft failure and primary nonfunction or with an increased potential to transmit diseases based on donor factors.26 These include grafts obtained from DCD donors, elderly donors, steatotic livers, donors with prolonged stay in the intensive care unit (especially if ionotropic support is required), and donors with positive viral serology, malignancy, or active bacterial infections. In the previously mentioned RCT, Nasralla et al12 demonstrated that the use of NMP decreased the rate of organ discard by 50% as compared to SCS, without compromising graft quality and function.
With SCS, assessment of the graft itself is limited to biopsy, which is considered in the context of donor characteristics and laboratory tests. Conversely, NMP (and SMP) allows for additional evaluation, which can be as simple as monitoring physiologic parameters and basic laboratory investigations (pH, lactate, transaminases, etc) to more complicated scoring systems. The validation of viability markers on NMP will assist in making the decision to transplant the graft. As well, these markers could be used to justify deferment of transplantation to daylight hours and in prolonged transport from regional hospitals to transplantation centers.
Many potential biomarkers have been proposed for evaluation of graft function on NMP, including bile production, transaminase levels, ATP content, and lipid metabolism.27 Analysis of bile composition has been suggested as a means to predict ischemic-type biliary lesions in particular. Preclinical studies have associated increased bile pH and bicarbonate concentration with decreased biliary epithelial injury.28,29
Mergental et al30 published a clinical series, in which previously discarded grafts were successfully transplanted after undergoing assessment on NMP. They judged graft viability based on lactate concentration in the perfusate, bile production, vascular flow, and overall appearance (eg, macroscopic evidence of fibrosis, cirrhosis, steatosis, etc). The same group has already initiated a nonrandomized, prospective clinical trial (NCT02740608).31 In this trial, livers are considered viable if they meet 2 or more of the following criteria within the first 4 hours of perfusion—lactate in the perfusate ≤2.5 mmol/L, any evidence of bile production, metabolism of glucose, hepatic artery flow ≥150 mL/min and portal vein flow ≥500 mL/min, pH ≥7.30, or homogeneous perfusion (ie, all segments of the liver appear to be perfused equally). Because this trial makes use of NMP, it is unclear if and how these criteria could be applied to an alternative setup, such as hypothermic-oxygenated MP (HOPE).13
As this area of research progresses, both experimentally and clinically, it may be necessary to develop 2 sets of criteria: one that is used to determine donor suitability before procurement and a second used to judge graft viability, once it has been applied to MP, before implantation. The former would likely be a modification of existing donor criteria, informed by preclinical and clinical experience with MP. Of the many potential candidates for the latter, the optimal solution should ultimately be one that combines predictive ability with clinical feasibility. An ideal scoring system should integrate donor and organ factors simultaneously.
MACHINE PERFUSION FOR OPTIMIZATION OF LIVER GRAFTS
Using MP as a platform for optimization of liver grafts has not yet progressed to clinical applications. Several approaches have been explored in preclinical models, though there is still much research to be done. Among the most promising techniques include “defatting” of steatotic livers, gene therapy, and eradication of hepatitis C virus.
Treatment of steatotic grafts or “defatting” is a promising application in liver transplantation, given the impact of steatosis on graft viability. A study of data from European transplant centers found high-grade hepatic steatosis to be the reason for transplant cancelation in 63.6% of cases in 2016.32 A preclinical study by Nagrath et al33 details the development of a “defatting cocktail,” which they tested during NMP of steatotic rat livers. Their solution contained a combination of amino acids, visfatin (an adipocyte hormone that promotes glucose uptake and cell proliferation, among other functions), forskolin (a plant derivative used to raise levels of cAMP), and several nuclear receptor ligands, acting on peroxisome proliferator-activated enzyme alpha, peroxisome proliferator-activated enzyme delta, pregnane X receptor, and carboxylic acid reductase. The Nagrath group were able to show a 50% reduction in intracellular lipid content after 3 hours of NMP. Boteon et al34 validated the same combination in human grafts that had been discarded for steatosis. They found a reduction in tissue triglycerides (TG) and macrovesicular steatosis by nearly 40% within 6 hours compared with controls. This was also associated with enhanced metabolic function, lower vascular resistance, lower levels of transaminases in the perfusate, and higher bile production. As hepatic steatosis is primarily associated with TG accumulation, the challenge is to increase TG breakdown via lipolysis, while preserving structural and functional lipids important for normal liver function.35 Another consideration is the removal of byproducts of desteatification from the closed system, though this could conceivably be achieved by the addition of a dialysis circuit or a novel, targeted filtration system. Interestingly, Jamieson et al36 showed a 50% reduction in the size of lipid deposits after 48 hours of NMP alone (ie, with no lipolytic additives) in a porcine model of mild hepatic steatosis. In this case, however, the length of perfusion required is clearly prohibitive, and it is unclear how representative the porcine livers are of discarded steatotic grafts.
MP may also be a suitable platform for the eradication of viral infections from donor livers, such as hepatitis C virus (HCV). This has been shown in porcine livers undergoing NMP by Goldaracena et al,37 who used Miravirsen to inhibit viral replication via sequestration of miRNA-122, on which HCV replication is dependent. A comparatively simple approach for inactivation of HCV by circulating perfusate around a lamp producing germicidal UV light was shown to be successful in ex situ lung perfusion.38 However, this approach may not be effective in livers, given that HCV replication primarily occurs intracellularly in hepatocytes.39
Beyond the simple addition of pharmaceutical compounds to the MP circuit, of which there is any number of reasonable candidates (antiapoptotic agents, antioxidants, vasoactive drugs, etc), more advanced therapies could be used to modify the graft on a genetic level. Gene therapy, with the aim of either improving graft quality or suppressing immunogenicity, has been used to modify several types of machine-perfused organs. Proof of concept has been recently demonstrated in large-animal ex situ heart models, with the successful integration of the reporter gene luciferase.40 Machuca et al,41 working with a porcine ex situ lung model, were able to show successful delivery of human interleukin-10, with decreased inflammation and improved gas exchange posttransplantation. A study by de Roos et al42 demonstrated the successful transfection of ex situ rat livers using the 2 reporter genes (Escherichia coli lacZ and firefly luciferase). However, we failed to find any gene therapy studies using MP of livers from large animals. The advent of CRISPR/Cas9 may allow for more sophisticated and reliable gene-editing techniques.43 Depending on the intended duration of effect, techniques, such as RNA interference, could be used to downregulate whole pathways. Suggested targets are mainly aimed at preventing apoptosis caused by ischemia-reperfusion injury include p53, fas ligand (FAS), caspase-3, and complement component C3.44 Proof of concept was recently reported by Gillooly et al45 in isolated rat livers, in which they used lipid-based nanoparticles to achieve hepatocyte uptake of small interfering RNAs designed to target FAS receptor expression. Cellular therapy is an additional avenue that merits further investigation. Verstegen et al46 have recently reported the successful delivery of human mesenchymal stem cells at therapeutic doses to porcine liver grafts undergoing HOPE, including the retention of paracrine signaling function.
EXTRACORPOREAL LIVER SUPPORT USING MACHINE-PERFUSED LIVERS
The first potential application of MP, which extends beyond its direct use in liver transplantation, involves an integrative approach using livers perfused ex situ to provide metabolic support for patients in liver failure. This would involve using a healthy liver, most likely a xenograft, on an MP circuit and connecting it with the patient’s circulation, either directly or separated by a filter (Figure 2). The ex situ liver would compensate for the patient’s failing liver, allowing it time to recover while providing normal liver functions, such as glucose regulation, drug metabolism, and synthesis of albumin and clotting factors. This could potentially be applied to several groups of patients, including those awaiting a liver transplant, and patients presenting with acute or acute-on-chronic liver failure who are not immediate transplant candidates. It is even conceivable that it could be used for routine treatment of chronic liver failure patients, akin to renal dialysis, with the goal of prolonging or improving quality of life. However, this would require significant investment in supporting infrastructure. The recent study by Eshmuminov et al,47 demonstrating viability of a liver graft after 7 days of ex situ perfusion, supports the feasibility of this idea, as a single liver could be used to support multiple patients within this time period.
The idea behind application of MP for liver support stems from research on bioartificial livers (BALs). Clinical reports of BALs appear in the literature as early as 1987, where one was used at a Japanese center to treat hepatic failure in a patient with inoperable cholangiocarcinoma.48 BALs have been used in many individual cases as a bridge therapy to transplant, but a benefit in mortality has not been convincingly shown. A recent systematic review and metaanalysis by He et al49 identified 2 RCTs, 16 nonrandomized clinical trials, and 12 preclinical large-animal studies. The pooled analysis from the RCTs included 195 patients (97 treated with BAL) and showed a trend toward improved mortality that was not statistically significant. The preclinical studies all reported benefit in terms of mortality and metabolic function.
The main limitation with BALs has been the functional mass of hepatocytes. It has been estimated that 10–40 billion functional hepatocytes are required to provide adequate metabolic support.50 Hepatocytes used in BALs have come from 4 main sources: primary human hepatocytes, hepatoblastoma cell lines, human-induced hepatocytes, and porcine hepatocytes.51 Each source comes with its own challenges. Human hepatocytes are difficult to maintain in culture. They are also limited in availability and often of inconsistent quality. Interestingly, MP has been proposed as a platform to enhance the yield of isolated hepatocytes.52 Cancer and animal cell lines are typically less metabolically active than normal human hepatocytes, and with animal hepatocytes, there is risk of zoonoses, such as porcine endogenous retrovirus. Induced hepatocytes (ie, from pluripotent stem cells) can function well but are expensive, time-consuming, and require special expertise to produce.53
Novel techniques have been used to enhance hepatocyte function, such as the use of bio-scaffolds and 3D tissue culture.54 The general premise behind these innovations is that enhanced hepatocyte survival and function can be achieved by better mimicking the in vivo microenvironment. Specifically, extracellular biomatrices derived from porcine liver, cryogel matrix, hepatocyte spheroids, and coculture with sinusoidal endothelial and stellate cells have been successfully used to improve the functional capacity of BALs.55-58 Some groups have even made use of decellularized human livers from discarded grafts.59 Ex situ may offer a solution to this problem, though it comes with its own challenges of organ availability and procurement.
There are several preclinical studies that directly support the application of MP to extracorporeal liver support. A study by Nakamura et al,60 published in 1999, connected a healthy porcine liver on an NMP circuit directly to the circulation of a second animal, in which ischemic hepatopathy had been induced by 2 hours of hepatic artery and portal vein clamping. Their study included both positive and negative controls. The former consisted of an animal without liver injury attached to the NMP circuit, while the latter was made up of animals with ischemic hepatopathy, to which no treatment was given. They found evidence that the ex situ liver assumed some of the physiological functions in the injured animal, including the production of ketone bodies, clearance of bilirubin, and regulation of glucose (all of the control animals died of hypoglycemia before 24 h). Carraro et al61 reported a similar study in 2007, with a more severe hepatic injury induced by complete hepatic artery ligation in a porcine model. They were able to successfully maintain perfusion for 6 hours, though the animal eventually succumbed to lactic acidosis. Unfortunately, they did not provide any controls for comparison. Naruse et al62 developed a xenogeneic model using a canine recipient that relied on separate leukocyte- and immunoglobulin-adsorbent filtration columns. They were able to perfuse the animal for 3 hours without acute rejection and demonstrated reduction in ammonia levels. The sole clinical report was described by Levy et al,63 in which they used MP of a transgenic porcine liver to treat 2 patients with acute liver failure (ALF) awaiting transplantation. They were able to demonstrate safety and feasibility over a total perfusion time of 16.5 hours, including the lack of zoonotic transmission. No similar studies have been reported in the literature since, presumably due to the complexity of the models and the number of technical and logistical hurdles to be overcome before they could be applied clinically.
A simpler extracorporeal liver support device, dubbed L.E.O.NARDO, which provides oxygenation directly via the portal vein, has been described by Nardo et al.64 They were able to demonstrate benefit in a porcine model of ALF induced by subtotal hepatectomy, with the treatment group showing lower serum transaminases, improvement of coagulopathy, and increased 7-day survival (75% versus 0%). They subsequently published a clinical case report using this device in a patient who underwent an extended hepatectomy for colorectal metastases.65 Although it appears quite resource-intensive (requiring application in the operating room for 6 consecutive days), the patient reportedly had an uneventful postoperative course and was discharged home on postoperative day 10. Additionally, it is unclear whether this treatment would be effective outside of ischemic liver injury.
A potential concern with the use of MP for extracorporeal support is the immunologic effect of the ex situ–perfused liver. The most likely source would be large-animal xenografts. Discarded human grafts could potentially be used, which would be less immunogenic, but would also be limited by their sporadic availability and potentially decreased function (for the same reason they were discarded). Immunosorbent filters (ie, filters capable of absorbing immunoglobulins) used in BALs have been shown to mitigate immunogenic hepatocyte injury when treating across species.66
More promising is the extensive research that has been done in the genetic engineering of animals with the express purpose of xenotransplanting various organs, from large solid organs, like liver and kidney, to heart and lungs to smaller volume tissues, like corneas and pancreatic islets.67 There has been remarkable success in xenotransplantation between animals. Of note, a model of orthotopic heart transplantation from a genetically engineered pig into a baboon has been reported, which survived for up to 195 days.68 Swine are often the animal of choice for such models, given their similarity to humans in terms of size and metabolism. Typically, porcine models of xenotransplantation rely on the knockout of α1,3-galactosyltransferase with or without the addition of human CD46.69 α1,3-Galactosyltransferase is the enzyme responsible for the synthesis of α1,3Gal epitopes on the cell surface.70 These are present in most mammals, with the exception of apes (including humans) and old-world monkeys. These epitopes were identified in early research in xenotransplantation as playing a major role in hyperacute rejection in pig-to-human transplants.71 CD46 is a membrane-bound protein that plays an inhibitory role in the complement system.72 Though CD46 is the most widely studied in this capacity, other human complement inhibitors, such as CD55 and CD59, have also been investigated for their utility in models of xenotransplantation.
Porcine livers from animals genetically engineered to be less immunogenic could serve as the basis for extracorporeal liver support via MP. There has been success in preclinical studies with the transplantation of such organs between animals, and because liver support would be of relatively short duration, immunogenicity would potentially be less of a concern. Immunosuppressive agents could still be given during therapy to mitigate any residual or idiosyncratic response. The use of CRISPR/Cas9 gene-editing technology could greatly benefit the further development of suitable transgenic animals for liver support.73 The gene-editing technology has already successfully been used to inactivate porcine endogenous retrovirus in porcine models.74
THE ROLE OF MACHINE PERFUSION IN CANCER RESEARCH AND THERAPEUTICS
MP of livers and other organs has the potential for benefits in the field of oncology, both with regard to research and therapeutics. Both systemic and regional therapies could be tested for efficacy on ex situ–perfused livers, healthy and diseased. Organs could come from discarded grafts or even from resected specimens from cancer patients. Alternatively, researchers could potentially test therapies on animal livers that had been xenografted with human tumors.75 MP is unlikely to be a feasible platform to guide personalized cancer therapeutics (ie, using patient-derived xenografts), but this is a conceivable possibility.
One study was identified in the literature that made use of an NMP circuit along these lines. Cyzmek et al76 used an ex situ liver model to establish dose–response relationships with electrochemical ablative techniques. The effects of other regional therapies, such as radiofrequency ablation or transarterial chemoembolization could likewise be studied in this way. Systemic chemotherapy could also be tested for efficacy within the circuit. Ex situ liver perfusion limits animal suffering compared with in vivo models while maintaining representative physiology.
MP could potentially play a role in the treatment of patients, such as with high-dose chemotherapy with or without the addition of radiotherapy, with the goal being to maximize the therapeutic window for exposure to the liver while minimizing systemic exposure.77 This would be similar to in vivo isolated hepatic perfusion, which has been studied clinically. With in vivo isolated hepatic perfusion, a closed circuit is made between an extracorporeal perfusion pump, with inflow via the hepatic arterial system and outflow via the inferior vena cava (IVC).78 Vascular clamps are applied to occlude other inflow and outflow tracts, and a second circuit is required to shunt blood from the infrarenal IVC back to the heart. Applying this idea to ex situ perfusion, the patient’s liver could be resected, attached to the circuit, treated, and then reimplanted. The advantage of ex situ over in vivo hepatic perfusion would be the avoidance of systemic leakage, as well as the potential for more targeted techniques. Currently, however, the literature does not support the use of in vivo isolated hepatic perfusion for the treatment of malignancies. A phase I clinical trial using high-dose melphalan to treat colorectal metastases showed only a 29% response rate, with only 1 complete response among 24 patients.79 The same treatment has been reported for hepatic metastases of uveal melanoma, with better, but still variable, response rates.80 Effectiveness of the chemotherapeutic agent would have to be firmly established before investigating this application of ex situ MP clinically.
A second application of MP in relation to oncologic therapies would be to use this technology for otherwise unresectable tumors. With the exception of hepatocellular carcinoma, which is regularly treated with liver transplantation, palliative treatments are often the only options for surgically unresectable hepatic tumors.81 Similar to the high-dose chemotherapy treatment described above, it would require hepatectomy, resection of the tumor, while the organ undergoes MP, followed by autotransplantation. Gringeri et al82 described a preclinical model using MP at 20°C, in which they demonstrate the feasibility of this technique. A partial hepatectomy was performed ex situ during MP, which lasted 2 hours. The liver remnant was reimplanted, and the animals were recovered. They reported peak transaminases and lactate levels at 3 hours postreperfusion and negligible histologic damage upon examination of the reimplanted segment. A clinical series by Forni and Meriggi details 4 cases of ex vivo tumor resection, 1 of whom underwent ex situ hypothermic perfusion at 4°C.83 The evidence for ex vivo liver resection, in general, is limited to case reports and series. It is mainly used in cases requiring complex vascular or biliary reconstruction, such as tumors adjacent to the IVC or involving the porta hepatis.84-86 Success is variable, though tumor-free survival has been reported as far out as 17 months.87 The application of MP to this technique could mitigate ischemia and reperfusion injury of the remnant liver, potentially reducing the high morbidity and mortality in the early postoperative period.
TOXICOLOGY STUDIES USING MACHINE-PERFUSED LIVERS
A variety of models exist that are routinely used for the preclinical evaluation of hepatoxicity.88 These include in vitro techniques from basic hepatocyte culture, to coculture with nonparenchymal cells, to liver slices to 3D cell culture and BALs, in addition to in vivo studies.89 Isolated perfusion of rat livers was developed as a model for hepatotoxicity in the 1950s.90 However, the increased setup and finesse required may not be justified given the differences in rat metabolism, including resistance to acetaminophen toxicity.91,92 It is generally assumed that studies in high-order species are more representative of humans, with rodent studies being less relevant than porcine or canine studies, which are less relevant than studies in nonhuman primates. Clinical correlation with animal models can often be disappointingly low, with concordance of approximately 40% for rodent models alone.93,94 Though the addition of a second animal model can raise concordance rates to over 70%, this still results in a number of unpredicted effects. A review of FDA reports found hepatotoxicity to be the primary reason for the withdrawal of a drug from the market in 32% of cases from 1975 to 2007.95 This was second only to cardiovascular side-effects.
MP could offer an alternative platform for hepatotoxicity testing, serving as a bridge between preclinical and clinical studies. It would make use of porcine grafts, whose hepatic metabolism is generally more comparable with humans than that of other animal models, or potentially deceased donor grafts, subject to availability.96 Studies investigating drug metabolism as a marker of graft viability have shown ex situ–perfused grafts to be metabolically active. Linares-Cervantes et al97 recently demonstrated metabolism of rocuronium in an ex situ liver on a NMP circuit.
There is the possibility of further refinement using genetically engineered animals, such as a model expressing human cytochrome p450 genes, the enzymes responsible for metabolism of up to 55% of drugs.98 Compared with an in vivo model, this approach reduces animal suffering and eliminates the need for care and husbandry, while maintaining representative physiology. A longer duration of perfusion, such as the 7 days demonstrated by Eshmuminov et al,47 may prove better suited for this purpose. Though, it would be limited to the use of intravenous formulations. Beyond hepatotoxicity, MP could potentially be developed into models of specific diseases, such as ALF.
OTHER PROPOSED USES
The main potential for MP of the liver outside of transplantation involves research and clinical applications of novel therapeutic agents. However, some have suggested a role in surgical education. Liu et al99 recently published a report of a laparoscopic training system using an ex situ–perfused porcine liver. This is an innovative use of a novel technology, though it is not clear that this use would offer much advantage over live animals. The animal may not have to be maintained under anesthesia for a prolonged period, but it would still need to undergo a hepatectomy. This setup would also offer lower fidelity than laparoscopy in a live pig and would still be single use. Perhaps if multiple parts of the animal were needed simultaneously for teaching purposes or if organs were obtained from an abattoir at the time of commercial sacrifice, this could be a viable option.
The past 2 decades have seen the rise of remarkable technology for the preservation of whole organs outside of the body. MP of the liver has entered the clinical sphere as a tool to improve organ preservation between removal and implantation, in an effort to reduce complications of ischemia and make efficient use of the limited organ supply. Recently completed and ongoing RCTs make it likely that MP will remain a part of liver transplantation for the foreseeable future. The assessment and modification of externally perfused grafts are at the frontlines of research in this field. Beyond transplantation, this technology has the potential to be applied toward other problems in healthcare, including the use of extracorporeal liver support, oncologic research and therapeutics, and toxicology testing.
Rena L. Pawlick for her input during the editing process.
1. Terrace JD, Oniscu GC. Oniscu GC, Forsythe JLR, Pomfret EA. Abdominal multiorgan retrieval. Springer Surgery Atlas Series In: Transplantation Surgery. 2019, Berlin, Heidelberg: Springer, 3–32
2. Huet PM, Nagaoka MR, Desbiens G, et al. Sinusoidal endothelial cell and hepatocyte death following cold ischemia-warm reperfusion of the rat liver. Hepatology. 2004; 39:1110–1119
3. Schön MR, Kollmar O, Akkoc N, et al. Cold ischemia affects sinusoidal endothelial cells while warm ischemia affects hepatocytes in liver transplantation. Transplant Proc. 1998; 30:2318–2320
4. Sibulesky L, Li M, Hansen RN, et al. Impact of cold ischemia time on outcomes of liver transplantation: a single center experience. Ann Transplant. 2016; 21:145–151
5. Stahl JE, Kreke JE, Malek FA, et al. Consequences of cold-ischemia time on primary nonfunction and patient and graft survival in liver transplantation: a meta-analysis. PLoS One. 2008; 3:e2468
6. Bral M, Gala-Lopez B, Bigam DL, et al. Ex situ liver perfusion: organ preservation into the future. Transplant Rev (Orlando). 2018; 32:132–141
7. Brettschneider L, Daloze PM, Huguet C, et al. Successful orthotopic transplantation of liver homografts after eight to twenty-five hours preservation. Surg Forum. 1967; 18:376–378
8. Starzl TE, Groth CG, Brettschneider L, et al. Extended survival in 3 cases of orthotopic homotransplantation of the human liver. Surgery. 1968; 63:549–563
9. Southard JH, Belzer FO. The University of Wisconsin organ preservation solution: components, comparisons, and modifications Transplantation Reviews. 1993; 7:176–190
10. Butler AJ, Rees MA, Wight DG, et al. Successful extracorporeal porcine liver perfusion for 72 hr. Transplantation. 2002; 73:1212–1218
11. Schön MR, Kollmar O, Wolf S, et al. Liver transplantation after organ preservation with normothermic extracorporeal perfusion. Ann Surg. 2001; 233:114–123
12. Nasralla D, Coussios CC, Mergental H, et al.; Consortium for Organ Preservation in Europe. A randomized trial of normothermic preservation in liver transplantation. Nature. 2018; 557:50–56
13. Czigany Z, Schöning W, Ulmer TF, et al. Hypothermic oxygenated machine perfusion (HOPE) for orthotopic liver transplantation of human liver allografts from extended criteria donors (ECD) in donation after brain death (DBD): a prospective multicentre randomised controlled trial (HOPE ECD-DBD). BMJ Open. 2017; 7:e017558
14. Schlegel A, Kron P, Dutkowski P. Hypothermic machine perfusion in liver transplantation. Curr Opin Organ Transplant. 2016; 21:308–314
15. Polyak MM, Grosche A. Comparison of vasosol and University of Wisconsin solutions on early kidney function after 24 hours of cold ischemia in a canine autotransplantation model. J Surg Res. 2008; 150:255–260
16. Schlegel A, Kron P, Dutkowski P. Hypothermic oxygenated liver perfusion: basic mechanisms and clinical application. Curr Transplant Rep. 2015; 2:52–62
17. Bruinsma BG, Yeh H, Ozer S, et al. Subnormothermic machine perfusion for ex vivo preservation and recovery of the human liver for transplantation. Am J Transplant. 2014; 14:1400–1409
18. Fontes P, Lopez R, van der Plaats A, et al. Liver preservation with machine perfusion and a newly developed cell-free oxygen carrier solution under subnormothermic conditions. Am J Transplant. 2015; 15:381–394
19. Minor T, Efferz P, Fox M, et al. Controlled oxygenated rewarming of cold stored liver grafts by thermally graduated machine perfusion prior to reperfusion. Am J Transplant. 2013; 13:1450–1460
20. Guarrera JV, Henry SD, Samstein B, et al. Hypothermic machine preservation in human liver transplantation: the first clinical series. Am J Transplant. 2010; 10:372–381
21. Guarrera JV, Henry SD, Samstein B, et al. Hypothermic machine preservation facilitates successful transplantation of “orphan” extended criteria donor livers. Am J Transplant. 2015; 15:161–169
22. Dutkowski P, Schlegel A, de Oliveira M, et al. HOPE for human liver grafts obtained from donors after cardiac death. J Hepatol. 2014; 60:765–772
23. Dutkowski P, Polak WG, Muiesan P, et al. First comparison of hypothermic oxygenated perfusion versus static cold storage of human donation after cardiac death liver transplants: an international-matched case analysis. Ann Surg. 2015; 262:764–70. discussion 770
24. Hoyer DP, Mathé Z, Gallinat A, et al. Controlled oxygenated rewarming of cold stored livers prior to transplantation: first clinical application of a new concept. Transplantation. 2016; 100:147–152
25. Schlegel A, Muiesan P. Reply to: “The UK DCD Risk Score: still no consensus on futility in DCD liver transplantation.” J Hepatol. 2019; 70:1036–1038
26. Attia M, Silva MA, Mirza DF. The marginal liver donor—an update. Transpl Int. 2008; 21:713–724
27. Verhoeven CJ, Farid WR, de Jonge J, et al. Biomarkers to assess graft quality during conventional and machine preservation in liver transplantation. J Hepatol. 2014; 61:672–684
28. Op den Dries S, Karimian N, Westerkamp AC, et al. Normothermic machine perfusion reduces bile duct injury and improves biliary epithelial function in rat donor livers. Liver Transpl. 2016; 22:994–1005
29. Westerkamp AC, Mahboub P, Meyer SL, et al. End-ischemic machine perfusion reduces bile duct injury in donation after circulatory death rat donor livers independent of the machine perfusion temperature. Liver Transpl. 2015; 21:1300–1311
30. Mergental H, Perera MT, Laing RW, et al. Transplantation of declined liver allografts following normothermic ex-situ evaluation. Am J Transplant. 2016; 16:3235–3245
31. Laing RW, Mergental H, Yap C, et al. Viability testing and transplantation of marginal livers (VITTAL) using normothermic machine perfusion: study protocol for an open-label, non-randomised, prospective, single-arm trial. BMJ Open. 2017; 7:e017733
32. Moosburner S, Gassner JMGV, Nösser M, et al. Prevalence of steatosis hepatis in the eurotransplant region: impact on graft acceptance rates. HPB Surg. 2018; 2018:6094936
33. Nagrath D, Xu H, Tanimura Y, et al. Metabolic preconditioning of donor organs: defatting fatty livers by normothermic perfusion ex vivo. Metab Eng. 2009; 11:274–283
34. Boteon YL, Attard J, Boteon APCS, et al. Manipulation of lipid metabolism during normothermic machine perfusion: effect of defatting therapies on donor liver functional recovery. Liver Transpl. 2019; 25:1007–1022
35. Perla FM, Prelati M, Lavorato M, et al. The role of lipid and lipoprotein metabolism in non-alcoholic fatty liver disease Children (Basel). 2017; 4:46
36. Jamieson RW, Zilvetti M, Roy D, et al. Hepatic steatosis and normothermic perfusion-preliminary experiments in a porcine model. Transplantation. 2011; 92:289–295
37. Goldaracena N, Spetzler VN, Echeverri J, et al. Inducing hepatitis C virus resistance after pig liver transplantation—a proof of concept of liver graft modification using warm ex vivo perfusion. Am J Transplant. 2017; 17:970–978
38. Galasso M, Feld JJ, Watanabe Y, et al. Inactivating hepatitis C virus in donor lungs using light therapies during normothermic ex vivo lung perfusion. Nat Commun. 2019; 10:481
39. Dustin LB, Bartolini B, Capobianchi MR, et al. Hepatitis C virus: life cycle in cells, infection and host response, and analysis of molecular markers influencing the outcome of infection and response to therapy. Clin Microbiol Infect. 2016; 22:826–832
40. Bishawi M, Roan JN, Milano CA, et al. A normothermic ex vivo organ perfusion delivery method for cardiac transplantation gene therapy. Sci Rep. 2019; 9:8029
41. Machuca TN, Cypel M, Bonato R, et al. Safety and efficacy of ex vivo donor lung adenoviral IL-10 gene therapy in a large animal lung transplant survival model. Hum Gene Ther. 2017; 28:757–765
42. de Roos WK, Fallaux FJ, Marinelli AW, et al. Isolated-organ perfusion for local gene delivery: efficient adenovirus-mediated gene transfer into the liver. Gene Ther. 1997; 4:55–62
43. Pickar-Oliver A, Gersbach CA. The next generation of CRISPR-Cas technologies and applications. Nat Rev Mol Cell Biol. 2019; 20:490–507
44. Glebova K, Reznik ON, Reznik AO, et al. siRNA technology in kidney transplantation: current status and future potential. Biodrugs. 2014; 28:345–361
45. Gillooly AR, Perry J, Martins PN. First report of siRNA uptake (for RNA interference) during ex vivo hypothermic and normothermic liver machine perfusion. Transplantation. 2019; 103:e56–e57
46. Verstegen MMA, Mezzanotte L, Ridwan RY, et al. First report on ex vivo delivery of paracrine active human mesenchymal stromal cells to liver grafts during machine perfusion. Transplantation. 2020; 104:e5–e7
47. Eshmuminov D, Becker D, Borrego LB, et al. An integrated perfusion machine preserves injured human livers for 1 week Nat Biotechnol. 2020; 38:189–198
48. Matsumura KN, Guevara GR, Huston H, et al. Hybrid bioartificial liver in hepatic failure: preliminary clinical report. Surgery. 1987; 101:99–103
49. He YT, Qi YN, Zhang BQ, et al. Bioartificial liver support systems for acute liver failure: a systematic review and meta-analysis of the clinical and preclinical literature. World J Gastroenterol. 2019; 25:3634–3648
50. Bianconi E, Piovesan A, Facchin F, et al. An estimation of the number of cells in the human body. Ann Hum Biol. 2013; 40:463–471
51. Carpentier B, Gautier A, Legallais C. Artificial and bioartificial liver devices: present and future. Gut. 2009; 58:1690–1702
52. Izamis ML, Perk S, Calhoun C, et al. Machine perfusion enhances hepatocyte isolation yields from ischemic livers. Cryobiology. 2015; 71:244–255
53. Sakiyama R, Blau BJ, Miki T. Clinical translation of bioartificial liver support systems with human pluripotent stem cell-derived hepatic cells. World J Gastroenterol. 2017; 23:1974–1979
54. Soloviev V, Hassan AN, Akatov V, et al. A novel bioartificial liver containing small tissue fragments: efficiency in the treatment of acute hepatic failure induced by carbon tetrachloride in rats. Int J Artif Organs. 2003; 26:735–742
55. Ambrosino G, Varotto S, Basso S, et al. ALEX (artificial liver for extracorporeal xenoassistance): a new bioreactor containing a porcine autologous biomatrix as hepatocyte support. Preliminary results in an ex vivo experimental model. Int J Artif Organs. 2002; 25:960–965
56. Damania A, Hassan M, Shirakigawa N, et al. Alleviating liver failure conditions using an integrated hybrid cryogel based cellular bioreactor as a bioartificial liver support. Sci Rep. 2017; 7:40323
57. Lorenti A, Barbich M, de Santibáñes M, et al. Ammonium detoxification performed by porcine hepatocyte spheroids in a bioartificial liver for pediatric use: preliminary report. Artif Organs. 2003; 27:665–670
58. Wei G, Wang J, Lv Q, et al. Three-dimensional coculture of primary hepatocytes and stellate cells in silk scaffold improves hepatic morphology and functionality in vitro. J Biomed Mater Res A. 2018; 106:2171–2180
59. Mazza G, Rombouts K, Rennie Hall A, et al. Decellularized human liver as a natural 3D-scaffold for liver bioengineering and transplantation. Sci Rep. 2015; 5:13079
60. Nakamura N, Kamiyama Y, Takai S, et al. Ex vivo liver perfusion with arterial blood from a pig with ischemic liver failure. Artif Organs. 1999; 23:153–160
61. Carraro A, Gringeri E, Calabrese F, et al. A new experimental model of isolated perfused pig liver to support acute hepatic failure. Transplant Proc. 2007; 39:2028–2030
62. Naruse K, Sakai Y, Natori T, et al. Xenogeneic direct hemoperfusion using whole swine liver for liver failure in dogs. J Surg Res. 2003; 111:229–235
63. Levy MF, Crippin J, Sutton S, et al. Liver allotransplantation after extracorporeal hepatic support with transgenic (hCD55/hCD59) porcine livers: clinical results and lack of pig-to-human transmission of the porcine endogenous retrovirus. Transplantation. 2000; 69:272–280
64. Nardo B, Puviani L, Prezzi D, et al. Protective effect of portal vein arterialization in acute liver failure induced by hepatectomy in normal and fatty liver rat. Transplant Proc. 2006; 38:3249–3250
65. Nardo B, Bertelli R, Cavallari G, et al. Analysis of 80 dual-kidney transplantations: a multicenter experience. Transplant Proc. 2011; 43:1559–1565
66. Shindoh J, Naruse K, Sakai Y, et al. Efficacy of immunoadsorbent devices for maintaining hepatic function in ex vivo direct xenogenic hemoperfusion. Int J Artif Organs. 2004; 27:294–302
67. Wang Y, Lei T, Wei L, et al. Xenotransplantation in China: present status. Xenotransplantation. 2019; 26:e12490
68. Längin M, Mayr T, Reichart B, et al. Consistent success in life-supporting porcine cardiac xenotransplantation. Nature. 2018; 564:430–433
69. Phelps CJ, Koike C, Vaught TD, et al. Production of alpha 1,3-galactosyltransferase-deficient pigs. Science. 2003; 299:411–414
70. Galili U. The α-Gal epitope (Galα1-3Galβ1-4GlcNAc-R) in xenotransplantation. Biochimie. 2001; 83:557–563
71. Begum NA, Murakami Y, Mikata S, et al. Molecular remodelling of human CD46 for xenotransplantation: designing a potent complement regulator without measles virus receptor activity. Immunology. 2000; 100:131–139
72. Fischer K, Kraner-Scheiber S, Petersen B, et al. Efficient production of multi-modified pigs for xenotransplantation by ‘combineering’, gene stacking and gene editing. Sci Rep. 2016; 6:29081
73. Cowan PJ, Hawthorne WJ, Nottle MB. Xenogeneic transplantation and tolerance in the era of CRISPR-Cas9. Curr Opin Organ Transplant. 2019; 24:5–11
74. Niu D, Wei HJ, Lin L, et al. Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science. 2017; 357:1303–1307
75. Watson AL, Carlson DF, Largaespada DA, et al. Engineered swine models of cancer. Front Genet. 2016; 7:78
76. Czymek R, Dinter D, Löffler S, et al. Electrochemical treatment: an investigation of dose-response relationships using an isolated liver perfusion model. Saudi J Gastroenterol. 2011; 17:335–342
77. Pace M, Gattai R, Matteini M, et al. Toxicity and morbility after isolated lower limb perfusion in 242 chemo-hyperthermal treatments for cutaneous melanoma: the experience of the Tuscan Reference Centre. J Exp Clin Cancer Res. 2008; 27:67
78. Reddy SK, Kesmodel SB, Alexander HR Jr. Isolated hepatic perfusion for patients with liver metastases. Ther Adv Med Oncol. 2014; 6:180–194
79. Vahrmeijer AL, van Dierendonck JH, Keizer HJ, et al. Increased local cytostatic drug exposure by isolated hepatic perfusion: a phase I clinical and pharmacologic evaluation of treatment with high dose melphalan in patients with colorectal cancer confined to the liver. Br J Cancer. 2000; 82:1539–1546
80. Ben-Shabat I, Hansson C, Sternby Eilard M, et al. Isolated hepatic perfusion as a treatment for liver metastases of uveal melanoma J Vis Exp. 2015; 95:52490
81. Byam J, Renz J, Millis JM. Liver transplantation for hepatocellular carcinoma. Hepatobiliary Surg Nutr. 2013; 2:22–30
82. Gringeri E, Bassi D, D’Amico FE, et al. Neoadjuvant therapy protocol and liver transplantation in combination with pancreatoduodenectomy for the treatment of hilar cholangiocarcinoma occurring in a case of primary sclerosing cholangitis: case report with a more than 8-year disease-free survival. Transplant Proc. 2011; 43:1187–1189
83. Forni E, Meriggi F. Bench surgery and liver autotransplantation. Personal experience and technical considerations. G Chir. 1995; 16:407–413
84. Chui AKK, Rao ARN, Wong J, et al. Ex situ ex vivo liver resection, partial liver autotransplantation for advanced hilar cholangiocarcinoma: a case report Transplant Proc. 2003; 35:402–403
85. Soejima Y, Takeishi K, Ikegami T, et al. All-in-one ex vivo self-reconstruction technique using an autologous inferior vena cava for a right lobe liver graft with multiple and complex venous orifices. Liver Transpl. 2010; 16:909–913
86. Vicente E, Quijano Y, Ielpo B, et al. Ex situ hepatectomy and liver autotransplantation for cholangiocarcinoma. Ann Surg Oncol. 2017; 24:3990
87. Chui AKK, Island ER, Rao ARN, et al. The longest survivor and first potential cure of an advanced cholangiocarcinoma by ex vivo resection and autotransplantation: a case report and review of the literature. Am Surg. 2003; 69:441–444
88. Zhang D, Luo G, Ding X, et al. Preclinical experimental models of drug metabolism and disposition in drug discovery and development Acta Pharmaceutica Sinica B. 2012; 2:549–561
89. Godoy P, Hewitt NJ, Albrecht U, et al. Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol. 2013; 87:1315–1530
90. Miller LL, Bly CG, Watson ML, et al. The dominant role of the liver in plasma protein synthesis; a direct study of the isolated perfused rat liver with the aid of lysine-epsilon-C14. J Exp Med. 1951; 94:431–453
91. Bessems M, ‘t Hart NA, Tolba R, et al. The isolated perfused rat liver: standardization of a time-honoured model. Lab Anim. 2006; 40:236–246
92. McGill MR, Williams CD, Xie Y, et al. Acetaminophen-induced liver injury in rats and mice: comparison of protein adducts, mitochondrial dysfunction, and oxidative stress in the mechanism of toxicity. Toxicol Appl Pharmacol. 2012; 264:387–394
93. Greaves P, Williams A, Eve M. First dose of potential new medicines to humans: how animals help. Nat Rev Drug Discov. 2004; 3:226–236
94. Olson H, Betton G, Robinson D, et al. Concordance of the toxicity of pharmaceuticals in humans and in animals. Regul Toxicol Pharmacol. 2000; 32:56–67
95. Stevens JL, Baker TK. The future of drug safety testing: expanding the view and narrowing the focus. Drug Discov Today. 2009; 14:162–167
96. Linares-Cervantes I, Echeverri J, Cleland S, et al. Predictor parameters of liver viability during porcine normothermic ex situ liver perfusion in a model of liver transplantation with marginal grafts. Am J Transplant. 2019; 19:2991–3005
97. Helke KL, Nelson KN, Sargeant AM, et al. Pigs in toxicology: breed differences in metabolism and background findings. Toxicol Pathol. 2016; 44:575–590
98. Schelstraete W, Clerck L, Govaert E, et al. Characterization of porcine hepatic and intestinal drug metabolizing CYP450: comparison with human orthologues from A quantitative, activity and selectivity perspective. Sci Rep. 2019; 9:9233
99. Liu W, Zheng X, Wu R, et al. Novel laparoscopic training system with continuously perfused ex-vivo porcine liver for hepatobiliary surgery. Surg Endosc. 2018; 32:743–750