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Given the current severe shortage of available livers for transplantation, there is an urgent need to maximize the utilization of donor organs. One of the strategies to increase the number of available livers for transplantation is to improve organ utilization through the use of elderly, overweight, or organs donated after circulatory death. However, the utilization of these "marginal" organs was associated with an increased risk of early allograft dysfunction, primary nonfunction, ischemic biliary complications, or even re-transplantation. Ischemia-reperfusion injury is a key mechanism in the pathogenesis of these complications., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background:  For 50 years, static cold storage (SCS) has been the gold standard for solid organ preservation in transplantation. Although logistically convenient, this preservation method presents important constraints in terms of duration and cold ischemia-induced lesions. We aimed to develop a machine perfusion (MP) protocol for recovery of vascularized composite allografts (VCA) after static cold preservation and determine its effects in a rat limb transplantation model., Methods:  Partial hindlimbs were procured from Lewis rats and subjected to SCS in Histidine-Tryptophan-Ketoglutarate solution for 0, 12, 18, 24, and 48 hours. They were then either transplanted (Txp), subjected to subnormothermic machine perfusion (SNMP) for 3 hours with a modified Steen solution, or to SNMP + Txp. Perfusion parameters were assessed for blood gas and electrolytes measurement, and flow rate and arterial pressures were monitored continuously. Histology was assessed at the end of perfusion. For select SCS durations, graft survival and clinical outcomes after transplantation were compared between groups at 21 days., Results:  Transplantation of limbs preserved for 0, 12, 18, and 24-hour SCS resulted in similar survival rates at postoperative day 21. Grafts cold-stored for 48 hours presented delayed graft failure ( p  = 0.0032). SNMP of limbs after 12-hour SCS recovered the vascular resistance, potassium, and lactate levels to values similar to limbs that were not subjected to SCS. However, 18-hour SCS grafts developed significant edema during SNMP recovery. Transplantation of grafts that had undergone a mixed preservation method (12-hour SCS + SNMP + Txp) resulted in better clinical outcomes based on skin clinical scores at day 21 post-transplantation when compared to the SCS + Txp group ( p  = 0.01613)., Conclusion:  To date, VCA MP is still limited to animal models and no protocols are yet developed for graft recovery. Our study suggests that ex vivo SNMP could help increase the preservation duration and limit cold ischemia-induced injury in VCA transplantation., Competing Interests: The authors declare competing interests. Drs. A.G.L., C.L.C., K.U., S.N.T., R.J.d.V., and C.A.P. have provisional patent applications relevant to this study. K.U. and S.N.T. have financial interests in Sylvatica Biotech Inc., a company focused on developing organ preservation technology. All competing interests are managed by Mass General Brigham in accordance with their conflict-of-interest policies., (Thieme. All rights reserved.)

Access to liver transplantation is limited by a significant organ shortage. The recent introduction of machine perfusion technology allows surgeons to monitor and assess ex situ liver function prior to transplantation. However, many donated organs are of inadequate quality for transplant, though opportunities exist to rehabilitate organ function with adjunct therapeutics during normothermic machine perfusion. In this preclinical study, we targeted the apoptosis pathway as a potential method of improving hepatocellular function. Treatment of discarded human livers during normothermic perfusion with an irreversible pan-caspase inhibitor, emricasan, resulted in significant mitigation of innate immune and pro-inflammatory responses at both the transcriptional and protein level. This was evidenced by significantly decreased circulating levels of the pro-inflammatory cytokines, interleukin-6, interleukin-8, and interferon-gamma, compared to control livers. Compared to emricasan-treated livers, untreated livers demonstrated transcriptional changes notable for enrichment in pathways involved in innate immunity, leukocyte migration, and cytokine-mediated signaling. Targeting of unregulated apoptosis may represent a viable therapeutic intervention for immunomodulation during machine perfusion., Competing Interests: SR, HY, and KU are inventors on pending patents relevant to this study and have a provisional patent application relevant to this study. KU has a financial interest in Sylvatica, a company focused on developing organ preservation technology. KU’s interests are managed by the Massachusetts General Hospital and Mass General Brigham in accordance with their conflict of interest policies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Raigani, Santiago, Ohman, Heaney, Baptista, Coe, de Vries, Rosales, Shih, Markmann, Gruppuso, Uygun, Sanders and Yeh.)

The current gold standard of Static Cold Storage (SCS), which is static cold storage on ice (about + 4 °C) in a specialized media such as the University of Wisconsin solution (UW), limits storage to few hours for vascular and metabolically active tissues such as the liver and the heart. The liver is arguably the pinnacle of metabolism in human body and therefore metabolic pathway analysis immediately becomes very relevant. In this article, a Nash Equilibrium (NE) approach, which is a first principles approach, is used to model and simulate the static cold storage and warm ischemia of a proposed model of liver cells. Simulations of energy depletion in the liver in static cold storage measured by ATP content and energy charge are presented along with comparisons to experimental data. In addition, conversion of Nash Equilibrium iterations to time are described along with an uncertainty analysis for the parameters in the model. Results in this work show that the Nash Equilibrium approach provides a good match to experimental data for energy depletion and that the uncertainty in model parameters is very small with percent variances less than 0.1%., (© 2022. The Author(s).)

Compared to donation after brain death (DBD), livers procured for transplantation from donation after circulatory death (DCD) donors experience more ischemia-reperfusion injury and higher rates of ischemic cholangiopathy due to the period of warm ischemic time (WIT) following withdrawal of life support. As a result, utilization of DCD livers for liver transplant (LT) has generally been limited to short WITs and younger aged donor grafts, causing many recovered DCD organs to be discarded without consideration for transplant. This study assesses how DCD liver utilization and outcomes have changed over time, using OPTN data from adult, first-time, deceased donor, whole-organ LTs between January 1995 and December 2019. Results show that increased clinical experience with DCD LT has translated into increased use of livers from DCD donors, shorter ischemic times, shorter lengths of hospitalization after transplant, and lower rates of retransplantation. The data also reveal that over the past decade, the rate of increase in DCD LTs conducted in the United States has outpaced that of DBD. Together, these trends signal an opportunity for the field of liver transplantation to mitigate the organ shortage by capitalizing on DCD liver allografts that are currently not being utilized., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Over 700 donor livers are discarded annually in the United States due to high risk of poor graft function. The objective of this study was to determine the impact of using normothermic machine perfusion to identify transplantable livers among those currently discarded., Study Design: A series of 21 discarded human livers underwent viability assessment during normothermic machine perfusion. Cross-sectional analysis of the Scientific Registry of Transplant Recipients database and cost analysis was performed to extrapolate the case series to national experience., Results: 21 discarded human livers were included in the perfusion cohort. 11 of 20 (55%) eligible grafts met viability criteria for transplantation. Grafts in the perfusion cohort had a similar donor risk index compared with discarded grafts (n = 1402) outside of New England in 2017 and 2018 (median [IQR]: 2.0 [1.5, 2.4] vs. 2.0 [1.7, 2.3], P = .40). 705 (IQR 677-741) livers were discarded annually in the United States since 2005, translating to the potential for 398 additional transplants nationally. The median cost to identify a transplantable graft with machine perfusion was $28,099 USD., Conclusions: Normothermic machine perfusion of discarded livers could identify a significant number of transplantable grafts, significantly improving access to liver transplantation., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Cellular rejection of liver transplant allografts remains a concern despite immunosuppressant use. Existing transplant biomarkers are often not sensitive enough to detect acute or chronic rejection at an early enough stage to allow successful clinical intervention. We herein developed a cell-based sensor that can potentially be used for monitoring local events following liver transplantation. Utilizing a machine perfusion system as a platform to engraft the cells into a donor liver, we effectively established the biocompatibility of the biosensor cells and confirmed efficient delivery of cells distributed throughout the organ. This work proves an innovative concept of integrating synthetic reporter cells ex vivo into organs as a transplant-within-a-transplant during functional organ preservation with a vision to use cell biosensors as a broad way to monitor and treat tissue transplants., Competing Interests: A patent on engineered cell technology and uses in organ transplantation entitled “Personalized and time release of biomolecules“ has been filed. The authors declare no other competing financial interests. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Donation after circulatory death (DCD) liver grafts are known to be predisposed to primary nonfunction and ischemic cholangiopathy. Many DCD grafts are discarded because of older donor age or long warm ischemia times. Thus, it is critical to improve the quality of DCD liver grafts. Here, we have tested whether an enriched oxygen carrier added to the preservation solution can prolong graft survival and reduce biliary damage., Methods: We assessed the adenosine triphosphate (ATP) content decay of mouse liver grafts after cold ischemia, warm ischemia, and combined warm+cold ischemia. In addition, we used a rat model of liver transplantation to compare survival of DCD grafts preserved in high-oxygen solution (preoxygenated perfluorocarbon [PFC] + University of Wisconsin [UW] solution) versus lower oxygen solution (preoxygenated UW solution)., Results: Adenosine triphosphate levels under UW preservation fall to less than 10% after 30 minutes of warm ischemia. Preoxygenated UW solution with PFC reached a significantly higher PaO2. After 45 minutes of warm ischemia in oxygenated UW + PFC solution, grafts showed 63% higher levels of ATP (P = 0.011). In addition, this was associated with better preservation of morphology when compared to grafts stored in standard UW solution. Animals that received DCD grafts preserved in higher oxygenation solution showed improved survival: 4 out of 6 animals survived long-term whereas all control group animals died within 24 hours., Conclusions: The additional oxygen provided by PFC during static cold preservation of DCD livers can better sustain ATP levels, and thereby reduce the severity of ischemic tissue damage. PFC-based preservation solution extends the tolerance to warm ischemia, and may reduce the rate of ischemic cholangiopathy.

Background: The ongoing shortage of donor livers for transplantation and the increased use of marginal livers necessitate the development of accurate pretransplant tests of viability. Considering the importance energy status during transplantation, we aimed to correlate peritransplant energy cofactors to posttransplant outcome and subsequently model this in an ex vivo setting., Methods: Sequential biopsies were taken from 19 donor livers postpreservation, as well as 30 minutes after portal venous reperfusion and hepatic arterial reperfusion and analyzed by liquid chromatography-mass spectrometry for energetic cofactors (adenosine triphosphate [ATP]/adenosine diphosphate [ADP]/adenosine monophosphate [AMP], nicotinamide adenine dinucleotide /NAD, nicotinamide adenine dinucleotide phosphate / nicotinamide adenine dinucleotide phosphate , flavin adenine dinucleotide , glutathione disulfide/glutathione). Energy status was correlated to posttransplant outcome. In addition, 4 discarded human donation after circulatory death livers were subjected to ex vivo reperfusion, modeling reperfusion injury and were similarly analyzed for energetic cofactors., Results: A rapid shift toward higher energy adenine nucleotides was observed following clinical reperfusion, with a 2.45-, 3.17- and 2.12-fold increase in ATP:ADP, ATP:AMP and energy charge after portal venous reperfusion, respectively. Seven of the 19 grafts developed early allograft dysfunction. Correlation with peritransplant cofactors revealed a significant difference in EC between early allograft dysfunction and normal functioning grafts (0.09 vs 0.31, P < 0.05). In the simulated reperfusion model, a similar trend in adenine nucleotide changes was observed., Conclusions: A preserved energy status appears critical in the peritransplant period. Levels of adenine nucleotides change rapidly after reperfusion and ratios of ATP/ADP/AMP after reperfusion are significantly correlated to graft function. Using these markers as a viability test in combination with ex vivo reperfusion may provide a useful predictor of outcome that incorporates donor, preservation, and reperfusion factors.

Purpose of Review: Herein, we review the field of subzero organ preservation with a focus on recent developments in hepatic supercooling., Recent Findings: Organ preservation is making a rapid shift from the decade old standard of storage on ice toward techniques that improve organ availability as well as preservation time. Long-term organ preservation would have tremendous benefits to the organ transplantation field, including better organ allocation, donor-recipient matching, as well as reduced preservation injury, and subsequent improvement of donor organ use. The formation of ice has proven an important limiting factor and novel techniques attempt to control or prevent freezing using cryoprotective agents, and highly controlled cooling regimens. Various techniques have been employed over the previous decades, including true organ freezing, vitrification, and subzero nonfreezing or supercooling. For most techniques, successful transplantation following long-term subzero preservation has remained elusive. Supercooling, however, recently delivered the first promising results, yielding survival after up to 4 days of supercooled preservation at -6°C., Summary: As the field of organ preservation undergoes significant development, the field of subzero preservation also receives renewed interest. Although many obstacles remain to be overcome to make subzero preservation feasible, novel techniques are beginning to show their potential in achieving long-term preservation.

Ex vivo machine perfusion (MP) is a promising way to better preserve livers prior to transplantation. Currently, no methodology has a verified benefit over simple cold storage. Before becoming clinically feasible, MP requires validation in models that reliably predict human performance. Such a model has been found in porcine liver, whose physiological, anatomical, and immunological characteristics closely resemble the human liver. Since the 1930s, researchers have explored MP as preservation, but only recently have clinical trials been performed. Making this technology clinically available holds the promise of expanding the donor pool through more effective preservation of extended criteria donor (ECD) livers. MP promises to decrease delayed graft function, primary nonfunction, and biliary strictures, which are all common failure modes of transplanted ECD livers. Although hypothermic machine perfusion (HMP) has become the standard for kidney ex vivo preservation, the precise settings and clinical role for liver MP have not yet been established. In research, there are 2 schools of thought: normothermic machine perfusion, closely mimicking physiologic conditions, and HMP, to maximize preservation. Here, we review the literature for porcine ex vivo MP, with an aim to summarize perfusion settings and outcomes pertinent to the clinical establishment of MP. Liver Transplantation 23 679-695 2017 AASLD., (© 2017 by the American Association for the Study of Liver Diseases.)

In contrast to conventional static cold preservation (0-4 °C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 °C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 °C and 37 °C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.

There is currently a severe shortage of liver grafts available for transplantation. Novel organ preservation techniques are needed to expand the pool of donor livers. Machine perfusion of donor liver grafts is an alternative to traditional cold storage of livers and holds much promise as a modality to expand the donor organ pool. We have recently described the potential benefit of subnormothermic machine perfusion of human livers. Machine perfused livers showed improving function and restoration of tissue ATP levels. Additionally, machine perfusion of liver grafts at subnormothermic temperatures allows for objective assessment of the functionality and suitability of a liver for transplantation. In these ways a great many livers that were previously discarded due to their suboptimal quality can be rescued via the restorative effects of machine perfusion and utilized for transplantation. Here we describe this technique of subnormothermic machine perfusion in detail. Human liver grafts allocated for research are perfused via the hepatic artery and portal vein with an acellular oxygenated perfusate at 21 °C.

The current standard for liver preservation involves cooling of the organ on ice (0-4 °C). Although it is successful for shorter durations, this method of preservation does not allow long-term storage of the liver. The gradual loss of hepatic viability during preservation puts pressure on organ sharing and allocation, may limit the use of suboptimal grafts and necessitates rushed transplantation to achieve desirable post-transplantation outcomes. In an attempt to improve and prolong liver viability during storage, alternative preservation methods are under investigation. For instance, ex vivo machine perfusion systems aim to sustain and even improve viability by supporting hepatic function at warm temperatures, rather than simply slowing down deterioration by cooling. Here we describe a novel subzero preservation technique that combines ex vivo machine perfusion with cryoprotectants to facilitate long-term supercooled preservation. The technique improves the preservation of rat livers to prolong storage times as much as threefold, which is validated by successful long-term recipient survival after orthotopic transplantation. This protocol describes how to load rat livers with cryoprotectants to prevent both intracellular and extracellular ice formation and to protect against hypothermic injury. Cryoprotectants are loaded ex vivo using subnormothermic machine perfusion (SNMP), after which livers can be cooled to -6 °C without freezing and kept viable for up to 96 h. Cooling to a supercooled state is controlled, followed by 3 h of SNMP recovery and orthotopic liver transplantation.

The realization of long-term human organ preservation will have groundbreaking effects on the current practice of transplantation. Herein we present a new technique based on subzero nonfreezing preservation and extracorporeal machine perfusion that allows transplantation of rat livers preserved for up to four days, thereby tripling the viable preservation duration.

Background & Aims: The peribiliary glands of large bile ducts have been identified as a niche of progenitor cells that contribute to regeneration of biliary epithelium after injury. We aimed to determine whether injury to the peribiliary glands of donor livers is a risk factor for development of non-anastomotic biliary strictures (NAS) after liver transplantation., Methods: In 128 liver transplant procedures, biopsies were taken from the donor bile duct and injury was assessed using an established histological grading system. Histological severity of injury was subsequently compared in liver grafts that later developed biliary structures vs. uncomplicated liver grafts., Results: Luminal biliary epithelial loss >50% was observed in 91.8% of the grafts before transplantation, yet NAS occurred in only 16.4%. Periluminal peribiliary glands were more severely injured than deep peribiliary glands located near the fibromuscular layer (>50% loss in 56.9% vs. 17.5%, respectively; p<0.001). Injury of deep peribiliary glands was more prevalent and more severe in livers that later developed NAS, compared to grafts without NAS (>50% loss in 50.0% vs. 9.8%, respectively; p=0.004). In parallel, injury of the peribiliary vascular plexus was more severe in livers that developed NAS, compared to grafts without NAS (>50% vascular changes in 57.1% vs. 20.3%; p=0.006)., Conclusion: Injury of peribiliary glands and vascular plexus before transplantation is strongly associated with the occurrence of biliary strictures after transplantation. This suggests that insufficient regeneration due to loss of peribiliary glands or impaired blood supply may explain the development of biliary strictures., (Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The shortage of donor livers has led to an increased use of organs from expanded criteria donors. Included are livers with steatosis, a metabolic abnormality that increases the likelihood of graft complications post-transplantation. After a brief introduction on the etiology, pathophysiology, categories and experimental models of hepatic steatosis, we herein review the methods to rescue steatotic donor livers before transplantation applied in clinical and experimental studies. The methods span the spectrum of encouraging donor weight loss, employing drug therapy, heat shock preconditioning, ischemia preconditioning and selective anesthesia on donors, and the treatment on isolated grafts during preservation. These methods work at different stages of transplantation process, although share similar molecular mechanisms including lipid metabolism stimulation through enzymes or nuclear receptor e.g., peroxisomal proliferator-activated receptor, or anti-inflammation through suppressing cytokines e.g., tumor necrosis factor-α, or antioxidant therapies to alleviate oxidative stress. This similarity of molecular mechanisms implies possible future attempts to reinforce each approach by repeating the same treatment approach at several stages of procurement and preservation, as well as utilizing these alternative approaches in tandem.

Background: The 110,000 patients currently on the transplant waiting list reflect the critical shortage of viable donor organs. However, a large pool of unused organs, from donors after cardiac death (DCD) that are disqualified because of extensive ischemic injury, may prove transplantable after machine perfusion treatment, fundamentally impacting the availability of treatment for end-stage organ failure. Machine perfusion is an ex-vivo organ preservation and treatment procedure that has the capacity to quantitatively evaluate and resuscitate cadaveric organs for transplantation., Methods: To diagnose whether an organ was fresh or ischemic, an initial assessment of liver quality was conducted via dynamic discriminant analysis. Subsequently, to determine whether the organs were sufficiently viable for successful implantation, fitness indices for transplantation were calculated based on squared prediction errors (SPE) for fresh and ischemic livers., Results: With just three perfusate metabolites, glucose, urea and lactate, the developed MPLSDA model distinguished livers as fresh or ischemic with 90% specificity. The SPE analyses revealed that fresh livers with SPE(F) < 10.03 and WI livers with SPE(WI) < 3.92 yield successful transplantation with 95% specificity., Conclusions: The statistical methods used here can discriminate between fresh and ischemic livers based on simple metabolic indicators measured during perfusion. The result is a predictive fitness index for transplantation of rat livers procured after cardiac death. The translational implications of this study are that any donor organ procured from controlled, but most especially from uncontrolled cardiac death donors, will be objectively assessed and its recovery monitored over time, minimizing the critical loss of otherwise viable organs.

Background: Utilizing livers from donors after cardiac death could significantly expand the donor pool. We have previously shown that normothermic (37°C) extracorporeal liver perfusion significantly improves transplantation outcomes of ischemic rat livers. Here we investigate whether recovery of ischemic livers is possible using sub-normothermic machine perfusion at 20°C and 30°C., Methods: Livers from male Lewis rats were divided into five groups after 1 h of warm ischemia (WI): (1) WI only, (2) 5 h of static cold storage (SCS), or 5 h of MP at (3) 20°C, (4) 30°C, and (5) 37°C. Long-term graft performance was evaluated for 28 d post-transplantation. Acute graft performance was evaluated during a 2 h normothermic sanguineous reperfusion ex vivo. Fresh livers with 5 h of SCS were positive transplant controls while fresh livers were positive reperfusion controls., Results: Following machine perfusion (MP) (Groups 3, 4, and 5), ischemically damaged livers could be orthotopically transplanted into syngeneic recipients with 100% survival (N ≥ 4) after 4 wk. On the other hand, animals from WI only, or WI + SCS groups all died within 24 h of transplantation. Fresh livers preserved using SCS had the highest alanine aminotransferase (ALT), aspartate aminotransferase (AST), and the lowest bile production during reperfusion, while at 28 d post-transplantation, livers preserved at 20°C and 30°C had the highest total bilirubin values., Conclusions: MP at both 20°C and 30°C eliminated temperature control in perfusion systems and recovered ischemically damaged rat livers. Postoperatively, low transaminases suggest a beneficial effect of sub-normothermic perfusion, while rising total bilirubin levels suggest inadequate prevention of ischemia- or hypothermia-induced biliary damage., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Orthotopic liver transplantation is the only available treatment for severe liver failure, but it is currently limited by organ shortage. One technical challenge that has thus far limited the development of a tissue-engineered liver graft is oxygen and nutrient transport. Here we demonstrate a novel approach to generate transplantable liver grafts using decellularized liver matrix. The decellularization process preserves the structural and functional characteristics of the native microvascular network, allowing efficient recellularization of the liver matrix with adult hepatocytes and subsequent perfusion for in vitro culture. The recellularized graft supports liver-specific function including albumin secretion, urea synthesis and cytochrome P450 expression at comparable levels to normal liver in vitro. The recellularized liver grafts can be transplanted into rats, supporting hepatocyte survival and function with minimal ischemic damage. These results provide a proof of principle for the generation of a transplantable liver graft as a potential treatment for liver disease.

Liver transplantation is currently the only established treatment of end-stage liver disease, but it is limited by a severe shortage of viable donor livers. Donors after cardiac death (DCD) are an untapped source that could significantly increase the pool of available livers. Preservation of these DCD livers by conventional static cold storage (SCS) is associated with an unacceptable risk of primary nonfunction and delayed graft failure. Normothermic extracorporeal liver perfusion (NELP) has been suggested as an improvement over SCS. Livers recovered from male Lewis rats were subjected to 1 hr of warm ischemia and preserved with 5 hr of SCS or NELP, and transplanted into syngeneic recipients. As additional controls, non-ischemic livers preserved with 6 hr of SCS or NELP and unpreserved ischemic livers were transplanted. After NELP, ischemically damaged livers could be orthotopically transplanted into syngeneic recipients with 92% survival (n=13) after 4 weeks, which was comparable with control animals that received healthy livers preserved by SCS (n=9) or NELP (n=11) for 6 hr. On the other hand, animals from ischemia/SCS control group all died within 12 hr postoperatively (n=6). Similarly, animals that received ischemic livers without preservation all died within 24 hr after transplantation (n=6). These results suggest that NELP has the potential to reclaim warm ischemic livers that would not be transplantable otherwise. The rat model in this study is a useful platform to further optimize NELP as a method of recovery and preservation of DCD livers.

Liver transplantation is hampered by a severe shortage of donor organs. Normothermic machine perfusion (NMP) of donor livers allows dynamic preservation in addition to viability assessment before transplantation. Little is known about the injury and repair mechanisms induced during NMP. To investigate these mechanisms, we examined gene and protein expression changes in a cohort of discarded human livers, stratified by hepatocellular function, during NMP. Six human livers acquired through donation after circulatory death (DCD) underwent 12 h of NMP. Of the six livers, three met predefined criteria for adequate hepatocellular function. We applied transcriptomic profiling and protein analysis to evaluate temporal changes in gene expression during NMP between functional and nonfunctional livers. Principal component analysis segregated the two groups and distinguished the various perfusion time points. Transcriptomic analysis of biopsies from functional livers indicated robust activation of innate immunity after 3 h of NMP followed by enrichment of prorepair and prosurvival mechanisms. Nonfunctional livers demonstrated delayed and persistent enrichment of markers of innate immunity. Functional livers demonstrated effective induction of autophagy, a cellular repair and homeostasis pathway, in contrast to nonfunctional livers. In conclusion, NMP of discarded DCD human livers results in innate immune-mediated injury, while also activating autophagy, a presumed mechanism for support of cellular repair. More pronounced activation of autophagy was seen in livers that demonstrated adequate hepatocellular function. [ABSTRACT FROM AUTHOR]

Ischemia reperfusion injury (IRI) is a critical problem in liver transplantation that can lead to life-threatening complications and substantially limit the utilization of livers for transplantation. However, because there are no early diagnostics available, fulminant injury may only become evident post-transplant. Mitochondria play a central role in IRI and are an ideal diagnostic target. During ischemia, changes in the mitochondrial redox state form the first link in the chain of events that lead to IRI. In this study we used resonance Raman spectroscopy to provide a rapid, non-invasive, and label-free diagnostic for quantification of the hepatic mitochondrial redox status. We show this diagnostic can be used to significantly distinguish transplantable versus non-transplantable ischemically injured rat livers during oxygenated machine perfusion and demonstrate spatial differences in the response of mitochondrial redox to ischemia reperfusion. This novel diagnostic may be used in the future to predict the viability of human livers for transplantation and as a tool to better understand the mechanisms of hepatic IRI. [ABSTRACT FROM AUTHOR]

There is a significant organ shortage in the field of liver transplantation, partly due to a high discard rate of steatotic livers from donors. These organs are known to function poorly if transplanted but make up a significant portion of the available pool of donated livers. This study demonstrates the ability to improve the function of steatotic rat livers using a combination of ex situ machine perfusion and a "defatting" drug cocktail. After 6 hours of perfusion, defatted livers demonstrated lower perfusate lactate levels and improved bile quality as demonstrated by higher bile bicarbonate and lower bile lactate. Furthermore, defatting was associated with decreased gene expression of pro-inflammatory cytokines and increased expression of enzymes involved in mitochondrial fatty acid oxidation. Rehabilitation of marginal or discarded steatotic livers using machine perfusion and tailored drug therapy can significantly increase the supply of donor livers for transplantation. [ABSTRACT FROM AUTHOR]

Due to the growth of cell-based therapeutic alternatives addressing the shortage of livers for transplant, there is necessity for a reliable source of human hepatocytes. In addition, pharmaceutical research often requires human hepatocytes to assess new drug therapies during development or to achieve FDA approval. Whole human livers producing large quantities of cells from the same donor are ideal, enhancing reproducibility for all purposes, while also allowing for capturing variances in drug-metabolism across different demographics for pharmaceutical testing and development but are limited in availability and quality for research purposes. The present study investigates the effect of donor and liver procurement factors of 16 human livers on cell viability and yield, showing that typical exclusion criteria for transplant still produce viable hepatocytes with significant yields. Although limited in number of data points, which should be taken into consideration, the conclusions of this study could be utilized as indications, allowing for expansion of liver selection criteria for hepatocyte isolation and provide the necessary quality hepatocytes in large quantities for the growing pharmaceutical, biomedical, and therapeutic research fields. [ABSTRACT FROM AUTHOR]

Machine perfusion-based organ preservation techniques are prudently transitioning into clinical practice. Although experimental data is compelling, the outcomes in the highly variable clinical donation-transplantation setting are unpredictable. Here, we offer an intermediate tool for pre-clinical assessment of human donor livers. We present a model for ex situ reperfusion of discarded human livers and report on its application in three human livers that have undergone subnormothermic (21°C) machine perfusion as an experimental preservation method. During reperfusion, the livers macroscopically reperfused in the first 15 minutes, and remained visually well-perfused for 3 hours of ex situ reperfusion. Bile production and oxygen consumption were observed throughout ex situ reperfusion. ATP levels increased 4.25-fold during SNMP. Between the end of SNMP and the end of reperfusion ATP levels dropped 45%. ALT levels in blood increased rapidly in the first 30 minutes and ALT release continued to taper off towards the end of perfusion. Release of CRP, TNF-, IL-1, and IL-12, IFN- was sustained during reperfusion. These findings support the use of this model for the evaluation of novel human liver preservation techniques. [ABSTRACT FROM AUTHOR]

Background: Liver transplantation plays a pivotal role in the treatment of patients with end-stage liver disease. Despite excellent outcomes, the field is strained by a severe shortage of viable liver grafts. To meet high demands, attempts are made to increase the use of suboptimal livers by both pretransplant recovery and assessment of donor livers. Here we aim to assess hepatic injury in the measurement of routine markers in the post-ischemic flush effluent of discarded human liver with a wide warm ischemic range. Methods: Six human livers discarded for transplantation with variable warm and cold ischemia times were flushed at the end of preservation. The liver grafts were flushed with NaCl or Lactated Ringer’s, 2 L through the portal vein and 1 L through the hepatic artery. The vena caval effluent was sampled and analyzed for biochemical markers of injury; lactate dehydrogenase (LDH), alanine transaminase (ALT), and alkaline phosphatase (ALP). Liver tissue biopsies were analyzed for ATP content and histologically (H&E) examined. Results: The duration of warm ischemia in the six livers correlated significantly to the concentration of LDH, ALT, and ALP in the effluent from the portal vein flush. No correlation was found with cold ischemia time. Tissue ATP content at the end of preservation correlated very strongly with the concentration of ALP in the arterial effluent (P<0.0007, R2 = 0.96). Conclusion: Biochemical injury markers released during the cold preservation period were reflective of the duration of warm ischemic injury sustained prior to release of the markers, as well as the hepatic energy status. As such, assessment of the flush effluent at the end of cold preservation may be a useful tool in evaluating suboptimal livers prior to transplantation, particularly in situations with undeterminable ischemic durations. [ABSTRACT FROM AUTHOR]

There continues to be significant debate regarding the most effective mode of ex situ machine perfusion of livers for transplantation. Subnormothermic (SNMP) and normothermic machine perfusion (NMP) are two methods with different benefits. We examined the metabolomic profiles of discarded steatotic human livers during three hours of subnormothermic or normothermic machine perfusion. Steatotic livers regenerate higher stores of ATP during SNMP than NMP. However, there is a significant depletion of available glutathione during SNMP, likely due to an inability to overcome the high energy threshold needed to synthesize glutathione. This highlights the increased oxidative stress apparent in steatotic livers. Rescue of discarded steatotic livers with machine perfusion may require the optimization of redox status through repletion or supplementation of reducing agents. [ABSTRACT FROM AUTHOR]

The only treatment available for end stage liver diseases is orthotopic liver transplantation. Although there is a big donor scarcity, many donor livers are discarded as they do not qualify for transplantation. Alternatively, decellularization of discarded livers can potentially render them transplantable upon recellularization and functional testing. The success of this approach will heavily depend on the quality of decellularized scaffolds which might show variability due to factors including age. Here we assessed the age-dependent differences in liver extracellular matrix (ECM) using rat and human livers. We show that the liver matrix has higher collagen and glycosaminoglycan content and a lower growth factor content with age. Importantly, these changes lead to deterioration in primary hepatocyte function potentially due to ECM stiffening and integrin-dependent signal transduction. Overall, we show that ECM changes with age and these changes significantly affect cell function thus donor age should be considered as an important factor for bioengineering liver substitutes. [ABSTRACT FROM AUTHOR]

Background High-quality human hepatocytes form the basis of drug safety and efficacy tests, cell-based therapies, and bridge-to-transplantation devices. Presently the only supply of cells derives from an inadequate pool of suboptimal disqualified donor livers. Here we evaluated whether machine perfusion could ameliorate ischemic injury that many of these livers experience prior to hepatocyte isolation. Methods Non-heparinized female Lewis rat livers were exposed to an hour of warm ischemia (34 °C) and then perfused for 3 h. Five different perfusion conditions that utilized the cell isolation apparatus were investigated, namely: (1) modified Williams Medium E and (2) Lifor, both with active oxygenation (95%O 2 /5%CO 2 ), as well as (3) Lifor passively oxygenated with ambient air (21%O 2 /0.04%CO 2 ), all at ambient temperatures (20 ± 2 °C). At hypothermic temperatures (5 ± 1 °C) and under passive oxygenation were (4) University of Wisconsin solution (UW) and (5) Vasosol. Negative and positive control groups comprised livers that had ischemia (WI) and livers that did not (Fresh) prior to cell isolation, respectively. Results Fresh livers yielded 32 ± 9 million cells/g liver while an hour of ischemia reduced the cell yield to 1.6 ± 0.6 million cells/g liver. Oxygenated Williams Medium E and Lifor recovered yields of 39 ± 11 and 31 ± 2.3 million cells/g liver, respectively. The passively oxygenated groups produced 15 ± 7 (Lifor), 13 ± 7 (Vasosol), and 10 ± 6 (UW) million cells/g liver. Oxygenated Williams Medium E was most effective at sustaining pH values, avoiding the accumulation of lactate, minimizing edematous weight gain and producing bile during perfusion. Conclusions Machine perfusion results in a dramatic increase in cell yields from livers that have had up to an hour of warm ischemia, but perfusate choice significantly impacts the extent of recovery. Oxygenated Williams Medium E at room temperature is superior to Lifor, UW and Vasosol, largely facilitated by its high oxygen content and low viscosity. [ABSTRACT FROM AUTHOR]