Advertisement

Normothermic liver machine perfusion as a dynamic platform for regenerative purposes: What does the future have in store for us?

  • Bianca Lascaris
    Affiliations
    Section of Hepatobiliary Surgery and Liver Transplantation, Department of Surgery, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands
    Search for articles by this author
  • Vincent E. de Meijer
    Affiliations
    Section of Hepatobiliary Surgery and Liver Transplantation, Department of Surgery, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands
    Search for articles by this author
  • Robert J. Porte
    Correspondence
    Corresponding author. Address: Section of Hepatobiliary Surgery and Liver Transplantation, Department of Surgery, University Medical Center Groningen, P.O. Box 30.001, 9700 RB Groningen, The Netherlands. Tel.: +31 503612896; fax: +31 503611745.
    Affiliations
    Section of Hepatobiliary Surgery and Liver Transplantation, Department of Surgery, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands
    Search for articles by this author
Open AccessPublished:May 06, 2022DOI:https://doi.org/10.1016/j.jhep.2022.04.033

      Summary

      Liver transplantation has become an immense success; nevertheless, far more recipients are registered on waiting lists than there are available donor livers for transplantation. High-risk, extended criteria donor livers are increasingly used to reduce the discrepancy between organ demand and supply. Especially for high-risk livers, dynamic preservation using machine perfusion can decrease post-transplantation complications and may increase donor liver utilisation by improving graft quality and enabling viability testing before transplantation. To further increase the availability of donor livers suitable for transplantation, new strategies are required that make it possible to use organs that are initially too damaged to be transplanted. With the current progress in experimental liver transplantation research, (long-term) normothermic machine perfusion may be used in the future as a dynamic platform for regenerative medicine approaches, enabling repair and regeneration of injured donor livers. Currently explored therapeutics such as defatting cocktails, RNA interference, senolytics, and stem cell therapy may assist in the repair and/or regeneration of injured livers before transplantation. This review will provide a forecast of the future utility of normothermic machine perfusion in decreasing the imbalance between donor liver demand and supply by enabling the repair and regeneration of damaged donor livers.

      Keywords

      Linked Article

      Introduction

      Liver transplantation offers the only definite cure for end-stage liver disease but primarily relies on the supply of suitable donor livers. Because of the ongoing imbalance between demand and supply, up to 20% of patients die while on the transplant waiting list.
      • Kwong A.J.
      • Kim W.R.
      • Lake J.R.
      • Smith J.M.
      • Schladt D.P.
      • Skeans M.A.
      • et al.
      OPTN/SRTR 2019 annual data report: liver.
      The discrepancy between organ availability and demand has forced an increasing use of “extended criteria donor” (ECD) livers for transplantation, including fatty livers, livers from donors of older age, or livers donated after circulatory death (DCD). However, a significant number of ECD livers are currently discarded.
      • Orman E.S.
      • Barritt ASt
      • Wheeler S.B.
      • Hayashi P.H.
      Declining liver utilization for transplantation in the United States and the impact of donation after cardiac death.
      ,
      • Busuttil R.W.
      • Tanaka K.
      The utility of marginal donors in liver transplantation.
      Meanwhile, fatty liver disease has emerged as the most common chronic liver disease, affecting up to a quarter of the global population. It is expected that fatty liver-induced cirrhosis will become the leading indication for liver transplantation worldwide.
      • Wong R.J.
      • Aguilar M.
      • Cheung R.
      • Perumpail R.B.
      • Harrison S.A.
      • Younossi Z.M.
      • et al.
      Nonalcoholic steatohepatitis is the second leading etiology of liver disease among adults awaiting liver transplantation in the United States.
      ,
      • Rajaram R.B.
      • Jayaraman T.
      • Yoong B.K.
      • Koh P.S.
      • Loh P.S.
      • Koong J.K.
      • et al.
      Non-alcoholic fatty liver disease and obesity among adult donors are major challenges to living donor liver transplantation: a single-centre experience.
      Besides, due to the success of transplant oncology, indications for liver transplantation are expanding.
      • Sapisochin G.
      • Hibi T.
      • Toso C.
      • Man K.
      • Berenguer M.
      • Heimbach J.
      • et al.
      Transplant oncology in primary and metastatic liver tumors: principles, evidence, and opportunities.
      ,
      • Ceresa C.D.L.
      • Nasralla D.
      • Pollok J.M.
      • Friend P.J.
      Machine perfusion of the liver: applications in transplantation and beyond.
      A practical solution to this conundrum is ex situ dynamic preservation of livers before transplantation to aid in the careful selection of ECD livers, thereby optimising utilisation rates. Also, ex situ dynamic preservation has shown that many livers that were initially discarded can be safely used, leading to the extension of standard criteria for donor livers. Compared to traditional static cold preservation, ex situ machine perfusion improves the quality of donor livers, and allows for longer preservation times and viability testing.
      • Ceresa C.D.L.
      • Nasralla D.
      • Pollok J.M.
      • Friend P.J.
      Machine perfusion of the liver: applications in transplantation and beyond.
      • de Meijer V.E.
      • Fujiyoshi M.
      • Porte R.J.
      Ex situ machine perfusion strategies in liver transplantation.
      • van Rijn R.
      • Schurink I.J.
      • de Vries Y.
      • van den Berg A.P.
      • Cortes Cerisuelo M.
      • Darwish Murad S.
      • et al.
      Hypothermic machine perfusion in liver transplantation - a randomized trial.
      • Nasralla D.
      • Coussios C.C.
      • Mergental H.
      • Akhtar M.Z.
      • Butler A.J.
      • Ceresa C.D.L.
      • et al.
      A randomized trial of normothermic preservation in liver transplantation.
      • Czigany Z.
      • Pratschke J.
      • Fronek J.
      • Guba M.
      • Schoning W.
      • Raptis D.A.
      • et al.
      Hypothermic oxygenated machine perfusion (HOPE) reduces early allograft injury and improves post-transplant outcomes in extended criteria donation (ECD) liver transplantation from donation after brain death (DBD): results from a multicenter randomized controlled trial (HOPE ECD-DBD).
      By using normothermic (37°C) machine perfusion (NMP), high-risk ECD livers have been successfully transplanted with excellent short-term outcomes.
      • Ceresa C.D.L.
      • Nasralla D.
      • Pollok J.M.
      • Friend P.J.
      Machine perfusion of the liver: applications in transplantation and beyond.
      ,
      • Nasralla D.
      • Coussios C.C.
      • Mergental H.
      • Akhtar M.Z.
      • Butler A.J.
      • Ceresa C.D.L.
      • et al.
      A randomized trial of normothermic preservation in liver transplantation.
      ,
      • van Leeuwen O.B.
      • de Vries Y.
      • Fujiyoshi M.
      • Nijsten M.W.N.
      • Ubbink R.
      • Pelgrim G.J.
      • et al.
      Transplantation of high-risk donor livers after ex situ resuscitation and assessment using combined hypo- and normothermic machine perfusion: a prospective clinical trial.
      ,
      • Markmann J.F.
      • Abouljoud M.S.
      • Ghobrial R.M.
      • Bhati C.S.
      • Pelletier S.J.
      • Lu A.D.
      • et al.
      Impact of portable normothermic blood-based machine perfusion on outcomes of liver transplant: the OCS liver PROTECT randomized clinical trial.
      Several clinical trials have already been conducted, and many more are ongoing, primarily to investigate the feasibility of NMP, patient and graft survival, postoperative and graft complications, and to perform graft viability assessments. A detailed overview of different (ongoing) clinical trials is presented elsewhere.
      • Bonaccorsi-Riani E.
      • Bruggenwirth I.M.A.
      • Buchwald J.E.
      • Iesari S.
      • Martins P.N.
      Machine perfusion: cold versus warm, versus neither. Update on clinical trials.
      ,
      • de Goeij F.H.C.
      • Schlegel A.
      • Muiesan P.
      • Guarrera J.V.
      • Dutkowski P.
      Hypothermic oxygenated machine perfusion protects from cholangiopathy in donation after circulatory death liver transplantation.
      However, to further increase the availability of donor livers suitable for transplantation, new strategies are required that make it possible to use organs that are initially too damaged to be transplanted. With the current progress in NMP research, whereby experimental human donor livers have been preserved for up to 7 days on a custom-made NMP device,
      • Eshmuminov D.
      • Becker D.
      • Bautista Borrego L.
      • Hefti M.
      • Schuler M.J.
      • Hagedorn C.
      • et al.
      An integrated perfusion machine preserves injured human livers for 1 week.
      NMP may become a platform for regenerative medicine approaches, enabling repair and reconditioning of injured donor livers, immunomodulation, and other treatments in the future.
      This review provides a forecast of the potential future utility of long-term (≥24 h) NMP in decreasing the discrepancy between donor liver demand and supply (Fig. 1, Fig. 2). The use of NMP outside liver transplantation, for instance as a platform to test the safety and effectiveness of new therapeutics in metabolically active livers before the start of clinical trials, is outside the scope of this review.
      Figure thumbnail gr1
      Fig. 1NMP as a platform for repair and regenerative medicine purposes.
      To obtain a better balance between the supply and demand for donor livers, NMP can be used as a platform for repair and regenerative medicine to improve the quality of extended criteria donors and make them suitable for transplantation. (A) Donor liver taken out. The liver can be damaged during life, procurement, and/or preservation. (B) The liver is placed on machine perfusion after procurement. Machine perfusion can be used for several purposes, depending on the (expected) degree of injury to the donor liver and/or logistical circumstances. (C) D-HOPE: livers from DCD donors can be placed on hypothermic perfusion to decrease post-transplantation complications (e.g., due to IRI). This is already implemented in clinical care in some clinics. (D) NMP: the viability of high risk, ECD livers of questionable quality can be tested with NMP: i) When the liver fulfils the viability criteria, the liver proceeds to transplantation (current practice). ii) When the viability criteria are not met, the liver will be discarded (current practice). The liver could remain on NMP for treatment to repair or regenerate it in the future. iii) When viability criteria are not met, the donor liver remains on NMP for therapeutic interventions. Different treatments (e.g., defatting cocktails, RNAi, senolytics) can be used during NMP for donor livers that are too damaged for transplantation. Because during NMP the liver is perfused in isolation, the therapeutics will only affect the liver and no other parts of the human, reducing potential side effects and decreasing the amount of drug needed, and thus the cost. When the liver subsequently meets the viability criteria after the interventions, the repaired donor liver proceeds to transplantation. When criteria are not met, the liver will be discarded. iv) When criteria are not met, the liver remains on NMP for the regeneration interventions. Regeneration can take place with decellularised scaffolds and/or organoids. Also, NMP can be used for the regeneration of partial livers. Livers from deceased donors can be split into 2 parts, or partial livers from living donors can be placed on NMP for regeneration to a full size, passively or with treatment (not shown). (E) Livers that were initially discarded are now transplanted after the use of (long-term) NMP. DCD, donation after circulatory death; D-HOPE, dual hypothermic oxygenated machine perfusion; ECD, extended criteria donor; NMP, normothermic machine perfusion; RNAi, RNA interference.
      Figure thumbnail gr2
      Fig. 2Regenerative medicine possibilities for the liver during NMP.
      We envision that any donor liver that is too injured for transplantation could be repaired or regenerated during NMP using several regenerative medicine techniques, such as defatting cocktails, senolytics, RNA interference, stem cell, and progenitor cell therapy, and decellularising drugs to obtain functional liver scaffolds for recellularisation, for example with organoids. These therapeutics can be added to the perfusate solution or injected directly into the liver or vessels. The continuous blood/perfusate flow creates an optimal environment for the therapeutics to work and provide oxygen and nutrients for the liver. NMP, normothermic machine perfusion.

      Definitions for liver repair and regeneration

      Although organ repair and regeneration are commonly used terms in the field of machine perfusion, there is currently no consensus about their exact definition. In a review, Resch et al. did not find uniform terminology and concluded that only the term organ preservation should be used for now because organ reconditioning, repair, and regeneration have not yet been established clinically.
      • Resch T.
      • Cardini B.
      • Oberhuber R.
      • Weissenbacher A.
      • Dumfarth J.
      • Krapf C.
      • et al.
      Transplanting marginal organs in the era of modern machine perfusion and advanced organ monitoring.
      High-risk donor livers are increasingly being used to reduce the discrepancy between organ supply and demand.
      According to the Cambridge dictionary, repair means: “to put something that is damaged, broken, or not working correctly, back into good condition or make it work again”. This would mean that repairing damaged livers is to correct the damage that has occurred during the donor's life, the procurement, or the preservation period. For regeneration, the Cambridge dictionary definition in biology is: “to grow again, or to make something grow again, for example, new tissue or a new part”. This would imply that it is necessary to renew and/or replace the old/damaged tissue to regenerate damaged livers. This review will adhere to the aforementioned terminology for repair and regeneration.

      NMP as a platform for repair and regeneration

      Promising therapeutics to treat ECD livers are defatting strategies, RNA interference (RNAi), and senolytics. These therapies mainly focus on repairing the damage that has occurred to the donor liver during life (e.g. steatosis, old age) or during procurement and/or reperfusion (e.g. DCD). When donor livers are too damaged, and repair during NMP is not possible or desirable, assisted regeneration of a part of the liver (e.g. the bile ducts) or the whole liver could be an alternative. The use of regeneration with autologous cells from the recipient can also minimise the complications of rejection and the use of immunosuppressants.
      • Zomer H.D.
      • Vidane A.S.
      • Goncalves N.N.
      • Ambrosio C.E.
      Mesenchymal and induced pluripotent stem cells: general insights and clinical perspectives.
      ,
      • Elisseeff J.
      • Badylak S.F.
      • Boeke J.D.
      Immune and genome engineering as the future of transplantable tissue.
      In deceased donor liver transplantation, it is currently not possible to use autologous cells because of the limited time between the procurement of the liver allograft and subsequent transplantation. However, with the advent of long-term NMP, this might become a possibility. Whole liver regeneration of large animals or humans has not yet been accomplished, but rapid developments in whole organ decellularisation, generation of scaffolds, and creation of human organoids, in combination with long-term NMP, may create a range of possibilities.
      Also, after a reduction (e.g. after partial hepatectomy or liver split) in mass, the liver has the ability to regenerate almost all of its original mass within a week.
      • Nadalin S.
      • Testa G.
      • Malago M.
      • Beste M.
      • Frilling A.
      • Schroeder T.
      • et al.
      Volumetric and functional recovery of the liver after right hepatectomy for living donation.
      ,
      • Michalopoulos G.K.
      • DeFrances M.C.
      Liver regeneration.
      Mueller et al. have shown that long-term perfusion of partial human livers is possible, which could allow for the use of isolated perfused hemi-livers in the future.
      • Mueller M.
      • Hefti M.
      • Eshmuminov D.
      • Schuler M.J.
      • Silva R.
      • Petrowsky H.
      • et al.
      Long-term normothermic machine preservation of partial livers: first experience with 21 human hemi-livers.

      Conditions to support liver repair and regeneration

      In clinical settings, NMP was initially used for a duration of 3-6 h, but (experimental) perfusion of 24 h and longer is not uncommon anymore.
      • Eshmuminov D.
      • Becker D.
      • Bautista Borrego L.
      • Hefti M.
      • Schuler M.J.
      • Hagedorn C.
      • et al.
      An integrated perfusion machine preserves injured human livers for 1 week.
      ,
      • Butler A.J.
      • Rees M.A.
      • Wight D.G.
      • Casey N.D.
      • Alexander G.
      • White D.J.
      • et al.
      Successful extracorporeal porcine liver perfusion for 72 hr.
      ,
      • Liu Q.
      • Nassar A.
      • Buccini L.
      • Grady P.
      • Soliman B.
      • Hassan A.
      • et al.
      Ex situ 86-hour liver perfusion: pushing the boundary of organ preservation.
      However, to support liver repair and mainly regeneration, long-term NMP (>24 h) will be necessary. To keep a liver metabolically active for more than 24 h, the machine has to mimic the human body to maintain a physiologic environment for the liver (Fig. 3). However, several developments are required to make long-term NMP a success. These include long-term oxygenators that last for at least 7 days, pumps that minimise haemolysis, an artificial kidney to remove waste products, an artificial pancreas and nutrients to meet the demands of the liver, a container where the liver stays undamaged and sterile, and a perfusate containing an oxygen carrier, but also medication to prevent or limit activation of coagulation and the immune system.
      • Lascaris B.
      • Thorne A.M.
      • Lisman T.
      • Nijsten M.W.N.
      • Porte R.J.
      • de Meijer V.E.
      Long-term normothermic machine preservation of human livers: what is needed to succeed?.
      As Eshmuminov et al. showed in their research on 7-day ex situ liver NMP, 5 major obstacles needed to be addressed by them: i) control of glucose metabolism, ii) prevention of haemolysis, iii) removal of waste products, iv) control of perfusate oxygenation, and v) simulation of diaphragm movement to prevent pressure necrosis.
      • Eshmuminov D.
      • Becker D.
      • Bautista Borrego L.
      • Hefti M.
      • Schuler M.J.
      • Hagedorn C.
      • et al.
      An integrated perfusion machine preserves injured human livers for 1 week.
      Also, sterility was a problem that needed to be addressed.
      • Eshmuminov D.
      • Mueller M.
      • Brugger S.D.
      • Bautista Borrego L.
      • Becker D.
      • Hefti M.
      • et al.
      Sources and prevention of graft infection during long-term ex situ liver perfusion.
      Nevertheless, these investigators succeeded in long-term NMP of porcine and human (discarded) livers for up to 7 days, which shows the potential for repair and regeneration of discarded donor livers in combination with NMP.
      Figure thumbnail gr3
      Fig. 3Graphic presentation of a system for long-term liver NMP.
      Graphical presentation of an example of a machine that will make long-term (>24 h) normothermic perfusion possible. The machine has to mimic the human body by integrating multiple core physiological functions. The machine will need pumps to mimic the heart to provide controllable blood flow and pressure and an oxygenator for oxygenation and CO2 removal to replace the lungs. Hormones and nutrients, usually provided by the intestines and pancreas, will be supplemented into the perfusate. It will also need an artificial kidney to remove waste products and a leukocyte filter, mimicking the spleen, for “washing” the blood. The container (skin and abdominal cavity) will provide a sterile environment with an integrated diaphragm movement to help avoid pressure necrosis. Finally, the machine must be automated and monitored from a distance with sensors providing information about the pressure, flow, temperature, and perfusion solution’s composition to maintain a physiological environment. NMP, normothermic machine perfusion.
      NMP may also have limitations, such as inducing ischaemia-reperfusion injury (IRI) while the liver perfuses on the machine.
      • Boteon Y.L.
      • Afford S.C.
      Machine perfusion of the liver: which is the best technique to mitigate ischaemia-reperfusion injury?.
      Therefore it may be helpful to combine NMP with other machine perfusion techniques. Several groups, including the Birmingham group and our group, have applied hypothermic machine perfusion before NMP.
      • van Leeuwen O.B.
      • de Vries Y.
      • Fujiyoshi M.
      • Nijsten M.W.N.
      • Ubbink R.
      • Pelgrim G.J.
      • et al.
      Transplantation of high-risk donor livers after ex situ resuscitation and assessment using combined hypo- and normothermic machine perfusion: a prospective clinical trial.
      ,
      • Boteon Y.L.
      • Laing R.W.
      • Schlegel A.
      • Wallace L.
      • Smith A.
      • Attard J.
      • et al.
      Combined hypothermic and normothermic machine perfusion improves functional recovery of extended criteria donor livers.
      This combined approach may lead to improved mitochondrial function during hypothermic machine perfusion,
      • Schlegel A.
      • Muller X.
      • Mueller M.
      • Stepanova A.
      • Kron P.
      • de Rougemont O.
      • et al.
      Hypothermic oxygenated perfusion protects from mitochondrial injury before liver transplantation.
      resulting in reduced oxidative and inflammatory damage during subsequent NMP.
      • Boteon Y.L.
      • Laing R.W.
      • Schlegel A.
      • Wallace L.
      • Smith A.
      • Attard J.
      • et al.
      Combined hypothermic and normothermic machine perfusion improves functional recovery of extended criteria donor livers.
      Especially for long-term NMP, hypothermic machine perfusion prior to NMP might be of particular value.

      Ex situ treatment strategies

      Defatting strategies

      Hepatic steatosis can be classified as mild (<30%), moderate (30-60%), or severe (>60%), and it is often
      To allow for safe transplantation of high-risk donor livers, NMP enables ex situ viability assessment.
      subcategorised as macro- and microvesicular steatosis.
      • McCormack L.
      • Dutkowski P.
      • El-Badry A.M.
      • Clavien P.A.
      Liver transplantation using fatty livers: always feasible?.
      Severely steatotic livers are generally not used for transplantation because of the increased risk for IRI and primary non-function.
      • McCormack L.
      • Dutkowski P.
      • El-Badry A.M.
      • Clavien P.A.
      Liver transplantation using fatty livers: always feasible?.
      Several defatting strategies have been studied which might decrease the amount of hepatic steatosis. Some promising results have already been achieved in isolated hepatocytes and precision-cut liver slices, as well as in rat and discarded human livers in combination with NMP.
      An often studied defatting cocktail is the combination of forskolin, scoparone, nuclear receptor ligands GW7647 and GW501516, hypericin, and visfatin, which all play a role in fat metabolism, uptake, or accumulation.
      • Xu M.
      • Zhou F.
      • Ahmed O.
      • Upadhya G.A.
      • Jia J.
      • Lee C.
      • et al.
      A novel multidrug combination mitigates rat liver steatosis through activating AMPK pathway during normothermic machine perfusion.
      This cocktail led to a 4-fold faster decrease in macrovesicular steatosis in isolated rat hepatocytes and a triglyceride content reduction of 65% in rat livers after only 3 h NMP, compared to a 30% reduction with a control perfusate.
      • Nativ N.I.
      • Yarmush G.
      • Chen A.
      • Dong D.
      • Henry S.D.
      • Guarrera J.V.
      • et al.
      Rat hepatocyte culture model of macrosteatosis: effect of macrosteatosis induction and reversal on viability and liver-specific function.
      ,
      • Nagrath D.
      • Xu H.
      • Tanimura Y.
      • Zuo R.
      • Berthiaume F.
      • Avila M.
      • et al.
      Metabolic preconditioning of donor organs: defatting fatty livers by normothermic perfusion ex vivo.
      Histology showed a significant decrease in lipid vesicles in hepatocytes, especially in the periportal area (zone 1), and a restored hepatocellular cytoplasmic volume.
      • Nagrath D.
      • Xu H.
      • Tanimura Y.
      • Zuo R.
      • Berthiaume F.
      • Avila M.
      • et al.
      Metabolic preconditioning of donor organs: defatting fatty livers by normothermic perfusion ex vivo.
      The addition of L-carnitine to this cocktail led to even better results, by increasing fatty acid transportation to the mitochondria.
      • Xu M.
      • Zhou F.
      • Ahmed O.
      • Upadhya G.A.
      • Jia J.
      • Lee C.
      • et al.
      A novel multidrug combination mitigates rat liver steatosis through activating AMPK pathway during normothermic machine perfusion.
      ,
      • Yarmush G.
      • Santos L.
      • Yarmush J.
      • Koundinyan S.
      • Saleem M.
      • Nativ N.I.
      • et al.
      CFD assessment of the effect of convective mass transport on the intracellular clearance of intracellular triglycerides in macrosteatotic hepatocytes.
      Reductions in tissue triglyceride levels and macrovesicular steatosis were seen in discarded human steatotic livers after 6 h NMP. The addition of L-carnitine also decreased IRI, expression of oxidative injury markers, activation of immune cells, and inflammatory cytokines in the perfusate.
      • Boteon Y.L.
      • Attard J.
      • Boteon A.
      • Wallace L.
      • Reynolds G.
      • Hubscher S.
      • et al.
      Manipulation of lipid metabolism during normothermic machine perfusion: effect of defatting therapies on donor liver functional recovery.
      The defatting combination of L-carnitine and exendin-4 led to an increased triglyceride concentration in the perfusate in human steatotic livers after 8 h NMP, but information on changes in tissue fat content was not reported.
      • Banan B.
      • Watson R.
      • Xu M.
      • Lin Y.
      • Chapman W.
      Development of a normothermic extracorporeal liver perfusion system toward improving viability and function of human extended criteria donor livers.
      However, these promising results with NMP might also reflect the effect of a continuous flow passing the hepatocytes. Yarmush et al. showed that flow is an important catalyst for the effectiveness of a defatting cocktail. By adding flow conditions to a defatting cocktail in isolated hepatocytes, the defatting time decreased from 48 h to 4-6 h (70% defatting), probably because of the increase in L-carnitine uptake by the cells.
      • Yarmush G.
      • Santos L.
      • Yarmush J.
      • Koundinyan S.
      • Saleem M.
      • Nativ N.I.
      • et al.
      CFD assessment of the effect of convective mass transport on the intracellular clearance of intracellular triglycerides in macrosteatotic hepatocytes.
      Another possibility to increase the speed of defatting passively might be the use of mild hyperthermia during machine perfusion.
      • Thorne A.M.
      • Ubbink R.
      • Bruggenwirth I.M.A.
      • Nijsten M.W.
      • Porte R.J.
      • de Meijer V.E.
      Hyperthermia-induced changes in liver physiology and metabolism: a rationale for hyperthermic machine perfusion.
      Unfortunately, some of these compounds are not approved for clinical use yet and might actually be harmful despite these encouraging results. The first mentioned defatting cocktail has been reported to increase lactic dehydrogenase activity, and the GW-compounds are associated with hepatic carcinogenesis and mitochondrial dysfunction.
      • Xu M.
      • Zhou F.
      • Ahmed O.
      • Upadhya G.A.
      • Jia J.
      • Lee C.
      • et al.
      A novel multidrug combination mitigates rat liver steatosis through activating AMPK pathway during normothermic machine perfusion.
      ,
      • Taba Taba Vakili S.
      • Kailar R.
      • Rahman K.
      • Nezami B.G.
      • Mwangi S.M.
      • Anania F.A.
      • et al.
      Glial cell line-derived neurotrophic factor-induced mice liver defatting: a novel strategy to enable transplantation of steatotic livers.
      For this reason, other agents have also been tested, with success. The replacement of the GW-compounds with 2 polyphenols, epigallocatechin-3-gallate, and resveratrol, led to less hepatotoxicity in rat livers, and glial cell line-derived neurotrophic factor led to no alteration in lactic dehydrogenase activity in steatotic mouse livers after 4 h NMP, while both were still effective.
      • Xu M.
      • Zhou F.
      • Ahmed O.
      • Upadhya G.A.
      • Jia J.
      • Lee C.
      • et al.
      A novel multidrug combination mitigates rat liver steatosis through activating AMPK pathway during normothermic machine perfusion.
      ,
      • Taba Taba Vakili S.
      • Kailar R.
      • Rahman K.
      • Nezami B.G.
      • Mwangi S.M.
      • Anania F.A.
      • et al.
      Glial cell line-derived neurotrophic factor-induced mice liver defatting: a novel strategy to enable transplantation of steatotic livers.
      In summary, in experimental studies, defatting cocktails can decrease steatosis in livers in just a few hours. However, with long-term NMP, it might become possible to defat livers without extra medication because a “spontaneous” decrease in hepatic triglyceride content and steatosis is also seen without defatting cocktails.
      • Nagrath D.
      • Xu H.
      • Tanimura Y.
      • Zuo R.
      • Berthiaume F.
      • Avila M.
      • et al.
      Metabolic preconditioning of donor organs: defatting fatty livers by normothermic perfusion ex vivo.
      ,
      • Jamieson R.W.
      • Zilvetti M.
      • Roy D.
      • Hughes D.
      • Morovat A.
      • Coussios C.C.
      • et al.
      Hepatic steatosis and normothermic perfusion-preliminary experiments in a porcine model.
      To make this work, we propose that the fat that is transported to the perfusion solution (e.g., as very-low-density lipoprotein) should be removed from the perfusate with a fat filter. Otherwise, the perfusate will become saturated with fat, a defatting limit will be reached, and potentially, fat emboli may occlude the oxygenators leading to impaired gas exchange.

      RNA interference

      RNAi is a natural process of post-transcriptional gene regulation that inhibits the translation of mRNA into proteins by adding a complementary strand. By explicitly targeting genes related to the causes of donor liver damage or post-transplantation complications, such as IRI or graft rejection, these genes can be transiently silenced to prevent such problems from arising.
      • Bruggenwirth I.M.A.
      • Martins P.N.
      RNA interference therapeutics in organ transplantation: the dawn of a new era.
      Several molecules, including microRNA, small interfering RNA (siRNA), and short-hairpin RNA, can be used for therapeutic RNAi.
      • Bruggenwirth I.M.A.
      • Martins P.N.
      RNA interference therapeutics in organ transplantation: the dawn of a new era.
      IRI is associated with liver apoptosis, mediated by death receptors such as Fas and tumour necrosis factor α, and mitochondrial dysfunction induced by cellular stress. Also, apoptotic genes, such as caspase-8 and caspase-3, and those involved in the nuclear factor-κB pathways can induce IRI.
      • Contreras J.L.
      • Vilatoba M.
      • Eckstein C.
      • Bilbao G.
      • Anthony Thompson J.
      • Eckhoff D.E.
      Caspase-8 and caspase-3 small interfering RNA decreases ischemia/reperfusion injury to the liver in mice.
      • Li X.
      • Zhang J.F.
      • Lu M.Q.
      • Yang Y.
      • Xu C.
      • Li H.
      • et al.
      Alleviation of ischemia-reperfusion injury in rat liver transplantation by induction of small interference RNA targeting Fas.
      • Thijssen M.F.
      • Bruggenwirth I.M.A.
      • Gillooly A.
      • Khvorova A.
      • Kowalik T.F.
      • Martins P.N.
      Gene silencing with siRNA (RNA interference): a new therapeutic option during ex vivo machine liver perfusion preservation.
      Silencing these genes in rodent liver and transplantation models of IRI has resulted in less Fas, caspase-8, and caspase-3 protein expression and activity and better preservation of the liver architecture, indicating diminishing IRI.
      • Contreras J.L.
      • Vilatoba M.
      • Eckstein C.
      • Bilbao G.
      • Anthony Thompson J.
      • Eckhoff D.E.
      Caspase-8 and caspase-3 small interfering RNA decreases ischemia/reperfusion injury to the liver in mice.
      ,
      • Li X.
      • Zhang J.F.
      • Lu M.Q.
      • Yang Y.
      • Xu C.
      • Li H.
      • et al.
      Alleviation of ischemia-reperfusion injury in rat liver transplantation by induction of small interference RNA targeting Fas.
      Many more experiments have been successfully performed on different targets to diminish IRI and graft rejection, as reviewed by Brüggenwirth et al.
      • Bruggenwirth I.M.A.
      • Martins P.N.
      RNA interference therapeutics in organ transplantation: the dawn of a new era.
      The downside of these agents is that they have to be administrated to the donor a few hours to days before organ procurement.
      • Bruggenwirth I.M.A.
      • Martins P.N.
      RNA interference therapeutics in organ transplantation: the dawn of a new era.
      This is where NMP may become an attractive solution. During NMP, the medication to target these genes can be administrated ex situ to the isolated liver before transplantation. This has already been tested in a few experimental studies.
      • Gillooly A.R.
      • Perry J.
      • Martins P.N.
      First report of siRNA uptake (for RNA interference) during ex vivo hypothermic and normothermic liver machine perfusion.
      • Moore C.
      • Thijssen M.
      • Wang X.
      • Mandrekar P.
      • Xiaofei E.
      • Abdi R.
      • et al.
      Gene silencing with p53 si-RNA downregulates inflammatory markers in the liver: potential utilization during normothermic machine preservation.
      • Cui J.
      • Qin L.
      • Zhang J.
      • Abrahimi P.
      • Li H.
      • Li G.
      • et al.
      Ex vivo pretreatment of human vessels with siRNA nanoparticles provides protein silencing in endothelial cells.
      • Goldaracena N.
      • Spetzler V.N.
      • Echeverri J.
      • Kaths J.M.
      • Cherepanov V.
      • Persson R.
      • et al.
      Inducing hepatitis C virus resistance after pig liver transplantation-A proof of concept of liver graft modification using warm ex vivo perfusion.
      siRNA against the Fas receptor, coated with invivofectamine lipid nanoparticles, were added directly to the perfusion solution and were taken up by hepatocytes in a rat liver after 4 h NMP.
      • Gillooly A.R.
      • Perry J.
      • Martins P.N.
      First report of siRNA uptake (for RNA interference) during ex vivo hypothermic and normothermic liver machine perfusion.
      Ablation of endothelial cell class II major histocompatibility complex molecules, which can reduce protein expression in the allograft and protect the graft against rejection, has been achieved by administering siRNA-releasing poly(amine-co-ester) nanoparticles during NMP – this led to effective and reliable particle uptake and major histocompatibility complex class II molecule silencing in vascular endothelial cells in mouse livers, without causing toxic effects.
      • Cui J.
      • Qin L.
      • Zhang J.
      • Abrahimi P.
      • Li H.
      • Li G.
      • et al.
      Ex vivo pretreatment of human vessels with siRNA nanoparticles provides protein silencing in endothelial cells.
      NMP also provides an optimal platform for regenerative medicine purposes because of oxygen and nutrient delivery.
      RNAi can also be used for antiviral treatment of the donor liver, such as for hepatitis C. The hepatitis C virus is dependent on the presence of microRNA-122, which can be silenced by miravirsen, a locked-nucleic acid oligonucleotide that can inhibit hepatitis C virus replication. After 4 h of NMP, miravirsen led to microRNA-122 sequestration and target gene derepression in porcine livers, preventing hepatitis C infection after liver transplantation.
      • Goldaracena N.
      • Spetzler V.N.
      • Echeverri J.
      • Kaths J.M.
      • Cherepanov V.
      • Persson R.
      • et al.
      Inducing hepatitis C virus resistance after pig liver transplantation-A proof of concept of liver graft modification using warm ex vivo perfusion.
      The success of these experiments can be enhanced using ex situ NMP, which allows for the delivery of therapeutics into the isolated liver. This will minimise systemic toxicity and reduce costs, as only the dose required for a single organ will be needed. In this way, RNAi can directly act inside the liver, thereby potentially curing, protecting, or repairing damaged livers before transplantation. While the current literature on the application of RNAi during NMP is limited to experimental studies, RNAi in other fields is already being explored in clinical trials.
      • Adams D.
      • Gonzalez-Duarte A.
      • O'Riordan W.D.
      • Yang C.C.
      • Ueda M.
      • Kristen A.V.
      • et al.
      Patisiran, an RNAi therapeutic, for hereditary transthyretin amyloidosis.
      The application of RNAi in liver transplantation is still in its infancy, but successes in other areas make RNAi a serious candidate to become a therapeutic agent for use during NMP in the future.

      Senolytics

      Senescent cells (SCs) are cells in an irreversible state of cell-cycle arrest that triggers the production of a range of inflammatory cytokines and chemokines, matrix metalloproteinases, and growth factors referred to as the senescence-associated secretory phenotype.
      • van Willigenburg H.
      • de Keizer P.L.J.
      • de Bruin R.W.F.
      Cellular senescence as a therapeutic target to improve renal transplantation outcome.
      ,
      • Kirkland J.L.
      • Tchkonia T.
      Senolytic drugs: from discovery to translation.
      Cellular senescence results from a stress response, such as inflammatory or metabolic signals, DNA damage, reactive oxygen species, and mitochondrial dysfunction.
      • van Willigenburg H.
      • de Keizer P.L.J.
      • de Bruin R.W.F.
      Cellular senescence as a therapeutic target to improve renal transplantation outcome.
      For this reason, SCs play an important role in age-related and chronic diseases, as well as in solid organ transplantation due to the systemic response to IRI.
      • van Willigenburg H.
      • de Keizer P.L.J.
      • de Bruin R.W.F.
      Cellular senescence as a therapeutic target to improve renal transplantation outcome.
      ,
      • Prata L.
      • Ovsyannikova I.G.
      • Tchkonia T.
      • Kirkland J.L.
      Senescent cell clearance by the immune system: emerging therapeutic opportunities.
      Thus, targeting SCs to limit the senescence-associated secretory phenotype during and shortly after liver transplantation is garnering interest.
      Senolytics are a group of drugs that selectively target SCs to remove their protection from apoptosis and hence induce apoptosis.
      • Prata L.
      • Ovsyannikova I.G.
      • Tchkonia T.
      • Kirkland J.L.
      Senescent cell clearance by the immune system: emerging therapeutic opportunities.
      Because SCs can form from all kinds of cells, different senolytics can target specific tissue types. Several different senolytics are currently being investigated, and some are already being tested in clinical trials, such as dasatinib and quercetin, navitoclax, and heat-shock protein 90 inhibitors.
      • van Willigenburg H.
      • de Keizer P.L.J.
      • de Bruin R.W.F.
      Cellular senescence as a therapeutic target to improve renal transplantation outcome.
      Senescence plays a major role in liver dysfunction. Accumulation of SCs promotes hepatic fat accumulation and steatosis, which were significantly reduced after dasatinib administration in mouse livers and human adipose tissue.
      • Ogrodnik M.
      • Miwa S.
      • Tchkonia T.
      • Tiniakos D.
      • Wilson C.L.
      • Lahat A.
      • et al.
      Cellular senescence drives age-dependent hepatic steatosis.
      ,
      • Xu M.
      • Pirtskhalava T.
      • Farr J.N.
      • Weigand B.M.
      • Palmer A.K.
      • Weivoda M.M.
      • et al.
      Senolytics improve physical function and increase lifespan in old age.
      The same treatment also cleared SCs and diminished cell-free mitochondrial DNA release in old mice, thereby dampening the senescence-associated secretory phenotype and prolonging the survival of old cardiac allografts.
      • Iske J.
      • Seyda M.
      • Heinbokel T.
      • Maenosono R.
      • Minami K.
      • Nian Y.
      • et al.
      Senolytics prevent mt-DNA-induced inflammation and promote the survival of aged organs following transplantation.
      Furthermore, the treatment with the senescence-inhibiting drug ABT-737 improved regeneration by disrupting p21 expression, a senescence-associated gene.
      • Ritschka B.
      • Knauer-Meyer T.
      • Goncalves D.S.
      • Mas A.
      • Plassat J.L.
      • Durik M.
      • et al.
      The senotherapeutic drug ABT-737 disrupts aberrant p21 expression to restore liver regeneration in adult mice.
      Besides hepatocytes, cholangiocytes are also influenced by SCs. Senolytics, such as B-cell lymphoma–extra-large inhibitors (A-1331852 and navitoclax) that induce apoptosis in senescent murine biliary epithelial cells or AP20187 and fisetin that reduce senescence markers associated with cholangiopathy could be used to treat cholangiopathy.
      • Sasaki M.
      • Sato Y.
      • Nakanuma Y.
      Increased p16(INK4a)-expressing senescent bile ductular cells are associated with inadequate response to ursodeoxycholic acid in primary biliary cholangitis.
      ,
      • Alsuraih M.
      • O'Hara S.P.
      • Woodrum J.E.
      • Pirius N.E.
      • LaRusso N.F.
      Genetic or pharmacological reduction of cholangiocyte senescence improves inflammation and fibrosis in the Mdr2 (-/-) mouse.
      Senolytics can potentially be administrated to the donor or the recipient. However, during NMP, senolytics will directly target the isolated liver, thereby enabling optimal dosing to achieve adequate treatment effects and reduce the risk of side effects. To date, no experimental or clinical research is available on the combination of senolytics with NMP; future work will need to demonstrate its efficacy to diminish or prevent post-transplantation complications, such as post-transplant cholangiopathy.

      Stem cell/progenitor cell therapy

      Stem cells are cells with the ability to divide for an indefinite period and can differentiate into different kinds of cells under specific conditions and signals.
      • Zomer H.D.
      • Vidane A.S.
      • Goncalves N.N.
      • Ambrosio C.E.
      Mesenchymal and induced pluripotent stem cells: general insights and clinical perspectives.
      Mesenchymal stem cells (MSCs) are present in various adult organs and often function to support organs. MSCs can be isolated from various tissues and organs, such as bone marrow and adipose tissue, and may be used to treat diseases. MSCs have immunosuppressive functions, participate in the anti-inflammatory response, and secrete cytokines that inhibit the macrophage-mediated inflammatory response and reduce hepatic IRI because of their reparative immunomodulatory effects.
      • Yang L.
      • Cao H.
      • Sun D.
      • Hou B.
      • Lin L.
      • Shen Z.Y.
      • et al.
      Bone marrow mesenchymal stem cells combine with normothermic machine perfusion to improve rat donor liver quality-the important role of hepatic microcirculation in donation after circulatory death.
      • Haga H.
      • Yan I.K.
      • Borrelli D.A.
      • Matsuda A.
      • Parasramka M.
      • Shukla N.
      • et al.
      Extracellular vesicles from bone marrow-derived mesenchymal stem cells protect against murine hepatic ischemia/reperfusion injury.
      • Wang Y.
      • Zhang A.
      • Ye Z.
      • Xie H.
      • Zheng S.
      Bone marrow-derived mesenchymal stem cells inhibit acute rejection of rat liver allografts in association with regulatory T-cell expansion.
      MSCs are most often used in experimental settings to reduce IRI and to overcome rejection via their immunomodulatory action.
      • Van Raemdonck D.
      • Neyrinck A.
      • Rega F.
      • Devos T.
      • Pirenne J.
      Machine perfusion in organ transplantation: a tool for ex-vivo graft conditioning with mesenchymal stem cells?.
      In a preclinical setting with rats, injection of MSCs after reperfusion preserved hepatocyte integrity and suppressed inflammatory responses, oxidative stress, and apoptosis, and inhibited acute allograft rejection after liver transplantation.
      • Wang Y.
      • Zhang A.
      • Ye Z.
      • Xie H.
      • Zheng S.
      Bone marrow-derived mesenchymal stem cells inhibit acute rejection of rat liver allografts in association with regulatory T-cell expansion.
      ,
      • Sun C.K.
      • Chang C.L.
      • Lin Y.C.
      • Kao Y.H.
      • Chang L.T.
      • Yen C.H.
      • et al.
      Systemic administration of autologous adipose-derived mesenchymal stem cells alleviates hepatic ischemia-reperfusion injury in rats.
      • Kanazawa H.
      • Fujimoto Y.
      • Teratani T.
      • Iwasaki J.
      • Kasahara N.
      • Negishi K.
      • et al.
      Bone marrow-derived mesenchymal stem cells ameliorate hepatic ischemia reperfusion injury in a rat model.
      • Wu B.
      • Song H.L.
      • Yang Y.
      • Yin M.L.
      • Zhang B.Y.
      • Cao Y.
      • et al.
      Improvement of liver transplantation outcome by heme oxygenase-1-transduced bone marrow mesenchymal stem cells in rats.
      Some studies showed promising results with MSCs in combination with machine perfusion. Human bone marrow MSCs (BMMSCs) have been injected into the hepatic artery or portal vein of a porcine liver kept on hypothermic machine perfusion for 30 min, where a wide range and patchy distribution was shown, with preserved paracrine activity. After 4 h of NMP, to mimic the reperfusion phase of liver transplantation, an increase in the cytokines IL-6 and IL-8 was observed, showing regenerative and immunomodulatory effects.
      • Verstegen M.M.A.
      • Mezzanotte L.
      • Ridwan R.Y.
      • Wang K.
      • de Haan J.
      • Schurink I.J.
      • et al.
      First report on ex vivo delivery of paracrine active human mesenchymal stromal cells to liver grafts during machine perfusion.
      Injection of BMMSCs during NMP of DCD rat livers led to improved liver function with reduced histological damage, serum and liver pro-inflammatory cytokine levels, oxidative stress injury, mitochondrial damage and biliary epithelial cell injury, and prolonged survival time.
      • Yang L.
      • Cao H.
      • Sun D.
      • Hou B.
      • Lin L.
      • Shen Z.Y.
      • et al.
      Bone marrow mesenchymal stem cells combine with normothermic machine perfusion to improve rat donor liver quality-the important role of hepatic microcirculation in donation after circulatory death.
      ,
      • Yang L.
      • Cao H.
      • Sun D.
      • Lin L.
      • Zheng W.P.
      • Shen Z.Y.
      • et al.
      Normothermic machine perfusion combined with bone marrow mesenchymal stem cells improves the oxidative stress response and mitochondrial function in rat donation after circulatory death livers.
      • Sun D.
      • Yang L.
      • Zheng W.
      • Cao H.
      • Wu L.
      • Song H.
      Protective effects of bone marrow mesenchymal stem cells (BMMSCS) combined with normothermic machine perfusion on liver grafts donated after circulatory death via reducing the ferroptosis of hepatocytes.
      • Hou B.
      • Song H.
      • Cao H.
      • Yang L.
      • Sun D.
      • Shen Z.
      [Effects of bone marrow mesenchymal stem cells combined with normothermic mechanical perfusion on biliary epithelial cells donated after cardiac death in rats].
      Regenerative therapies delivered during NMP may rescue donor livers that currently cannot be transplanted.
      These improvements were enhanced by administering heme oxygenase-1-modified BMMSCs during NMP in rats, which improved post-transplant survival
      • Cao H.
      • Yang L.
      • Hou B.
      • Sun D.
      • Lin L.
      • Song H.L.
      • et al.
      Heme oxygenase-1-modified bone marrow mesenchymal stem cells combined with normothermic machine perfusion to protect donation after circulatory death liver grafts.
      and promoted activation of peribiliary glands to facilitate repair of injured bile duct epithelium in recipient rats.
      • Tian X.
      • Cao H.
      • Wu L.
      • Zheng W.
      • Yuan M.
      • Li X.
      • et al.
      Heme oxygenase-1-modified bone marrow mesenchymal stem cells combined with normothermic machine perfusion repairs bile duct injury in a rat model of DCD liver transplantation via activation of peribiliary glands through the wnt pathway.
      These promising results were also seen in discarded human livers. Multi-potent adult progenitor cells were infused directly into the right lobe via the right hepatic artery or the portal vein during NMP. The introduction of the cells directly into the target organ was successful, without any noticeable adverse effect on the perfusion itself.
      • Laing R.W.
      • Stubblefield S.
      • Wallace L.
      • Roobrouck V.D.
      • Bhogal R.H.
      • Schlegel A.
      • et al.
      The delivery of multipotent adult progenitor cells to extended criteria human donor livers using normothermic machine perfusion.
      Extracellular vesicles (EVs) released by stem cells have also been used in a preclinical setting to mitigate IRI.
      • Haga H.
      • Yan I.K.
      • Borrelli D.A.
      • Matsuda A.
      • Parasramka M.
      • Shukla N.
      • et al.
      Extracellular vesicles from bone marrow-derived mesenchymal stem cells protect against murine hepatic ischemia/reperfusion injury.
      ,
      • Rigo F.
      • De Stefano N.
      • Navarro-Tableros V.
      • David E.
      • Rizza G.
      • Catalano G.
      • et al.
      Extracellular vesicles from human liver stem cells reduce injury in an ex vivo normothermic hypoxic rat liver perfusion model.
      ,
      • Anger F.
      • Camara M.
      • Ellinger E.
      • Germer C.T.
      • Schlegel N.
      • Otto C.
      • et al.
      Human mesenchymal stromal cell-derived extracellular vesicles improve liver regeneration after ischemia reperfusion injury in mice.
      EVs play an important role in cell-to-cell communication and contain several materials, such as proteins, lipids, and RNA, that can transfer to other cells.
      • Raposo G.
      • Stoorvogel W.
      Extracellular vesicles: exosomes, microvesicles, and friends.
      Human liver stem cell-derived EVs were added during NMP of rat livers. After 4 h of NMP, EV uptake by hepatocytes was shown, and the rat liver allograft showed less histological damage and injury markers.
      • Rigo F.
      • De Stefano N.
      • Navarro-Tableros V.
      • David E.
      • Rizza G.
      • Catalano G.
      • et al.
      Extracellular vesicles from human liver stem cells reduce injury in an ex vivo normothermic hypoxic rat liver perfusion model.

      Regeneration techniques

      Even though a liver itself has regenerative properties, for assisted regeneration, a good basic structure that can provide biomechanical support for tissue to regenerate is preferred. A scaffold, which is a template on which (stem) cells can be attached to proliferate, differentiate, migrate, repair, and regenerate new tissue, is frequently used for this purpose.
      • Elisseeff J.
      • Badylak S.F.
      • Boeke J.D.
      Immune and genome engineering as the future of transplantable tissue.
      ,
      • Cesaretti M.
      • Zarzavajian Le Bian A.
      • Moccia S.
      • Iannelli A.
      • Schiavo L.
      • Diaspro A.
      From deceased to bioengineered graft: new frontiers in liver transplantation.
      ,
      • Willemse J.
      • Lieshout R.
      • van der Laan L.J.W.
      • Verstegen M.M.A.
      From organoids to organs: bioengineering liver grafts from hepatic stem cells and matrix.
      Because scaffolds have to mimic the properties of the extracellular matrix (ECM), regarding both structure and cellular behaviour/interactions, scaffolds made by decellularisation of natural tissues or organs have many advantages, such as maintaining natural geometric morphology, vasculature structure, and active ECM proteins that are beneficial for recellularisation.
      • Cesaretti M.
      • Zarzavajian Le Bian A.
      • Moccia S.
      • Iannelli A.
      • Schiavo L.
      • Diaspro A.
      From deceased to bioengineered graft: new frontiers in liver transplantation.
      • Willemse J.
      • Lieshout R.
      • van der Laan L.J.W.
      • Verstegen M.M.A.
      From organoids to organs: bioengineering liver grafts from hepatic stem cells and matrix.
      • Kajbafzadeh A.M.
      • Javan-Farazmand N.
      • Monajemzadeh M.
      • Baghayee A.
      Determining the optimal decellularization and sterilization protocol for preparing a tissue scaffold of a human-sized liver tissue.
      • Uygun B.E.
      • Soto-Gutierrez A.
      • Yagi H.
      • Izamis M.L.
      • Guzzardi M.A.
      • Shulman C.
      • et al.
      Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.
      • Gupta S.K.
      • Mishra N.C.
      • Dhasmana A.
      Decellularization methods for scaffold fabrication.
      • Coronado R.E.
      • Somaraki-Cormier M.
      • Natesan S.
      • Christy R.J.
      • Ong J.L.
      • Halff G.A.
      Decellularization and solubilization of porcine liver for use as a substrate for porcine hepatocyte culture: method optimization and comparison.
      • Hynes R.O.
      The extracellular matrix: not just pretty fibrils.
      The downside is the need for (discarded) organ donors. Although porcine livers might also be used for decellularisation and recellularisation with human cells in the future.
      • Lang R.
      • Stern M.M.
      • Smith L.
      • Liu Y.
      • Bharadwaj S.
      • Liu G.
      • et al.
      Three-dimensional culture of hepatocytes on porcine liver tissue-derived extracellular matrix.
      ,
      • Barakat O.
      • Abbasi S.
      • Rodriguez G.
      • Rios J.
      • Wood R.P.
      • Ozaki C.
      • et al.
      Use of decellularized porcine liver for engineering humanized liver organ.
      Different protocols can achieve decellularisation, including chemical and enzymatic reagents and physical methods.
      • Cesaretti M.
      • Zarzavajian Le Bian A.
      • Moccia S.
      • Iannelli A.
      • Schiavo L.
      • Diaspro A.
      From deceased to bioengineered graft: new frontiers in liver transplantation.
      ,
      • Gupta S.K.
      • Mishra N.C.
      • Dhasmana A.
      Decellularization methods for scaffold fabrication.
      ,
      • Mazza G.
      • Rombouts K.
      • Rennie Hall A.
      • Urbani L.
      • Vinh Luong T.
      • Al-Akkad W.
      • et al.
      Decellularized human liver as a natural 3D-scaffold for liver bioengineering and transplantation.
      ,
      • Willemse J.
      • Verstegen M.M.A.
      • Vermeulen A.
      • Schurink I.J.
      • Roest H.P.
      • van der Laan L.J.W.
      • et al.
      Fast, robust and effective decellularization of whole human livers using mild detergents and pressure controlled perfusion.
      For whole organs, the perfusion method is preferred and is possible because the basic structure of the vascular system stays intact. This method shows better preservation of the ECM and has the advantage of supplying the liver with oxygen and nutrients.
      • Kajbafzadeh A.M.
      • Javan-Farazmand N.
      • Monajemzadeh M.
      • Baghayee A.
      Determining the optimal decellularization and sterilization protocol for preparing a tissue scaffold of a human-sized liver tissue.
      ,
      • Gupta S.K.
      • Mishra N.C.
      • Dhasmana A.
      Decellularization methods for scaffold fabrication.
      ,
      • Mirmalek-Sani S.H.
      • Sullivan D.C.
      • Zimmerman C.
      • Shupe T.D.
      • Petersen B.E.
      Immunogenicity of decellularized porcine liver for bioengineered hepatic tissue.
      Besides whole livers, bile ducts can also be decellularised to serve as a scaffold.
      • Willemse J.
      • Roos F.J.M.
      • Voogt I.J.
      • Schurink I.J.
      • Bijvelds M.
      • de Jonge H.R.
      • et al.
      Scaffolds obtained from decellularized human extrahepatic bile ducts support organoids to establish functional biliary tissue in a dish.
      While liver decellularisation is a delicate procedure, promising experimental results have already been reported with animal and discarded human livers. The basic architecture of the liver allograft, consisting of the ECM with the biliary drainage network and the vascular structure, is preserved and immunological compounds are removed.
      • Uygun B.E.
      • Soto-Gutierrez A.
      • Yagi H.
      • Izamis M.L.
      • Guzzardi M.A.
      • Shulman C.
      • et al.
      Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.
      ,
      • Coronado R.E.
      • Somaraki-Cormier M.
      • Natesan S.
      • Christy R.J.
      • Ong J.L.
      • Halff G.A.
      Decellularization and solubilization of porcine liver for use as a substrate for porcine hepatocyte culture: method optimization and comparison.
      ,
      • Mazza G.
      • Rombouts K.
      • Rennie Hall A.
      • Urbani L.
      • Vinh Luong T.
      • Al-Akkad W.
      • et al.
      Decellularized human liver as a natural 3D-scaffold for liver bioengineering and transplantation.
      • Willemse J.
      • Verstegen M.M.A.
      • Vermeulen A.
      • Schurink I.J.
      • Roest H.P.
      • van der Laan L.J.W.
      • et al.
      Fast, robust and effective decellularization of whole human livers using mild detergents and pressure controlled perfusion.
      • Mirmalek-Sani S.H.
      • Sullivan D.C.
      • Zimmerman C.
      • Shupe T.D.
      • Petersen B.E.
      Immunogenicity of decellularized porcine liver for bioengineered hepatic tissue.
      ,
      • Verstegen M.M.A.
      • Willemse J.
      • van den Hoek S.
      • Kremers G.J.
      • Luider T.M.
      • van Huizen N.A.
      • et al.
      Decellularization of whole human liver grafts using controlled perfusion for transplantable organ bioscaffolds.
      • Sabetkish S.
      • Kajbafzadeh A.M.
      • Sabetkish N.
      • Khorramirouz R.
      • Akbarzadeh A.
      • Seyedian S.L.
      • et al.
      Whole-organ tissue engineering: decellularization and recellularization of three-dimensional matrix liver scaffolds.
      • Robertson M.J.
      • Soibam B.
      • O'Leary J.G.
      • Sampaio L.C.
      • Taylor D.A.
      Recellularization of rat liver: an in vitro model for assessing human drug metabolism and liver biology.
      • Zhou P.
      • Huang Y.
      • Guo Y.
      • Wang L.
      • Ling C.
      • Guo Q.
      • et al.
      Decellularization and recellularization of rat livers with hepatocytes and endothelial progenitor cells.
      • Yagi H.
      • Fukumitsu K.
      • Fukuda K.
      • Kitago M.
      • Shinoda M.
      • Obara H.
      • et al.
      Human-scale whole-organ bioengineering for liver transplantation: a regenerative medicine approach.
      • Pla-Palacin I.
      • Sainz-Arnal P.
      • Morini S.
      • Almeida M.
      • Baptista P.M.
      Liver bioengineering using decellularized whole-liver scaffolds.
      After decellularisation, recellularisation has to take place. Rebuilding the scaffold can be achieved by several cells. Induced pluripotent stem cells (iPSCs) are most often used, but hepatic and endothelial progenitor cells, and embryonic, foetal and mesenchymal stem cells have also been used, as have foetal and adult/mature hepatocytes.
      • Uygun B.E.
      • Soto-Gutierrez A.
      • Yagi H.
      • Izamis M.L.
      • Guzzardi M.A.
      • Shulman C.
      • et al.
      Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.
      ,
      • Robertson M.J.
      • Soibam B.
      • O'Leary J.G.
      • Sampaio L.C.
      • Taylor D.A.
      Recellularization of rat liver: an in vitro model for assessing human drug metabolism and liver biology.
      ,
      • Zhou P.
      • Huang Y.
      • Guo Y.
      • Wang L.
      • Ling C.
      • Guo Q.
      • et al.
      Decellularization and recellularization of rat livers with hepatocytes and endothelial progenitor cells.
      ,
      • Asadi M.
      • Khalili M.
      • Lotfi H.
      • Vaghefi Moghaddam S.
      • Zarghami N.
      • Andre H.
      • et al.
      Liver bioengineering: recent trends/advances in decellularization and cell sheet technologies towards translation into the clinic.
      • Ko I.K.
      • Peng L.
      • Peloso A.
      • Smith C.J.
      • Dhal A.
      • Deegan D.B.
      • et al.
      Bioengineered transplantable porcine livers with re-endothelialized vasculature.
      • Zhang W.
      • Li W.
      • Liu B.
      • Wang P.
      • Li W.
      • Zhang H.
      Efficient generation of functional hepatocyte-like cells from human fetal hepatic progenitor cells in vitro.
      Because the liver is composed of many different cell types, it is a challenging task to rebuild a whole functioning liver. Approximately 30 billion hepatocytes are required for a full-size liver, which is not easily achieved.
      • Willemse J.
      • Lieshout R.
      • van der Laan L.J.W.
      • Verstegen M.M.A.
      From organoids to organs: bioengineering liver grafts from hepatic stem cells and matrix.
      ,
      • Uygun B.E.
      • Soto-Gutierrez A.
      • Yagi H.
      • Izamis M.L.
      • Guzzardi M.A.
      • Shulman C.
      • et al.
      Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.
      ,
      • Uygun B.E.
      • Izamis M.L.
      • Jaramillo M.
      • Chen Y.
      • Price G.
      • Ozer S.
      • et al.
      Discarded livers find a new life: engineered liver grafts using hepatocytes recovered from marginal livers.
      The sheer number of cells required is not the only problem, which cell types to use, the method, sequence/timing of seeding, and the culture media also have to be studied before whole livers can be regenerated.
      • Elisseeff J.
      • Badylak S.F.
      • Boeke J.D.
      Immune and genome engineering as the future of transplantable tissue.
      ,
      • Asadi M.
      • Khalili M.
      • Lotfi H.
      • Vaghefi Moghaddam S.
      • Zarghami N.
      • Andre H.
      • et al.
      Liver bioengineering: recent trends/advances in decellularization and cell sheet technologies towards translation into the clinic.
      ,
      • Jaramillo M.
      • Yeh H.
      • Yarmush M.L.
      • Uygun B.E.
      Decellularized human liver extracellular matrix (hDLM)-mediated hepatic differentiation of human induced pluripotent stem cells (hIPSCs).
      Major challenges and barriers still need to be overcome before NMP can be used for this purpose.
      Different recellularisation methods have been used, but the continuous perfusion method has shown many advantages and is now commonly used.
      • Asadi M.
      • Khalili M.
      • Lotfi H.
      • Vaghefi Moghaddam S.
      • Zarghami N.
      • Andre H.
      • et al.
      Liver bioengineering: recent trends/advances in decellularization and cell sheet technologies towards translation into the clinic.
      During machine perfusion, cells are either injected into the perfusion fluid or directly into the portal vein in a single or multi-step approach, with the multi-step approach leading to a higher number of cells attaching to the liver.
      • Uygun B.E.
      • Soto-Gutierrez A.
      • Yagi H.
      • Izamis M.L.
      • Guzzardi M.A.
      • Shulman C.
      • et al.
      Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.
      ,
      • Robertson M.J.
      • Soibam B.
      • O'Leary J.G.
      • Sampaio L.C.
      • Taylor D.A.
      Recellularization of rat liver: an in vitro model for assessing human drug metabolism and liver biology.
      ,
      • Zhou P.
      • Huang Y.
      • Guo Y.
      • Wang L.
      • Ling C.
      • Guo Q.
      • et al.
      Decellularization and recellularization of rat livers with hepatocytes and endothelial progenitor cells.
      ,
      • Asadi M.
      • Khalili M.
      • Lotfi H.
      • Vaghefi Moghaddam S.
      • Zarghami N.
      • Andre H.
      • et al.
      Liver bioengineering: recent trends/advances in decellularization and cell sheet technologies towards translation into the clinic.
      ,
      • Soto-Gutierrez A.
      • Zhang L.
      • Medberry C.
      • Fukumitsu K.
      • Faulk D.
      • Jiang H.
      • et al.
      A whole-organ regenerative medicine approach for liver replacement.
      The machine perfusion method also provides a better distribution of cells. One study showed that the primary rat hepatocytes remained around the vessels for the first 4 h, but after 1-2 days, they were distributed to the whole decellularised liver scaffold.
      • Uygun B.E.
      • Soto-Gutierrez A.
      • Yagi H.
      • Izamis M.L.
      • Guzzardi M.A.
      • Shulman C.
      • et al.
      Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.
      Also, the direction in which the seeding occurs can have an influence on the distribution of cells, which is better with antegrade flow (through the portal vein) than with retrograde flow (through the vena cava and hepatic veins).
      • Baptista P.M.
      • Siddiqui M.M.
      • Lozier G.
      • Rodriguez S.R.
      • Atala A.
      • Soker S.
      The use of whole organ decellularization for the generation of a vascularized liver organoid.
      Machine perfusion already plays an important part in this method of experimental liver regeneration. For decellularisation, either hypothermic machine perfusion alone or combined with NMP can be used, but for the regeneration process, NMP is more appropriate.
      • Uygun B.E.
      • Soto-Gutierrez A.
      • Yagi H.
      • Izamis M.L.
      • Guzzardi M.A.
      • Shulman C.
      • et al.
      Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.
      ,
      • Willemse J.
      • Verstegen M.M.A.
      • Vermeulen A.
      • Schurink I.J.
      • Roest H.P.
      • van der Laan L.J.W.
      • et al.
      Fast, robust and effective decellularization of whole human livers using mild detergents and pressure controlled perfusion.
      ,
      • Verstegen M.M.A.
      • Willemse J.
      • van den Hoek S.
      • Kremers G.J.
      • Luider T.M.
      • van Huizen N.A.
      • et al.
      Decellularization of whole human liver grafts using controlled perfusion for transplantable organ bioscaffolds.
      ,
      • Zhou P.
      • Huang Y.
      • Guo Y.
      • Wang L.
      • Ling C.
      • Guo Q.
      • et al.
      Decellularization and recellularization of rat livers with hepatocytes and endothelial progenitor cells.
      This method of regeneration can not only be used for the generation of whole livers but can also be used as a therapeutic option for the administration of liver cubes in hepatic diseases or after hepatectomy.
      • Mazza G.
      • Rombouts K.
      • Rennie Hall A.
      • Urbani L.
      • Vinh Luong T.
      • Al-Akkad W.
      • et al.
      Decellularized human liver as a natural 3D-scaffold for liver bioengineering and transplantation.
      ,
      • Shimoda H.
      • Yagi H.
      • Higashi H.
      • Tajima K.
      • Kuroda K.
      • Abe Y.
      • et al.
      Decellularized liver scaffolds promote liver regeneration after partial hepatectomy.
      However, despite major progress in this field, no large animal or human livers have been regenerated and transplanted yet. This is mainly because of the diversity and very high number of different cells that need to be generated in line with good manufacturing practices and the difficulties of re-endothelialisation of the vascular system to prevent coagulation activation.
      • Asadi M.
      • Khalili M.
      • Lotfi H.
      • Vaghefi Moghaddam S.
      • Zarghami N.
      • Andre H.
      • et al.
      Liver bioengineering: recent trends/advances in decellularization and cell sheet technologies towards translation into the clinic.
      The development of organoids has been increasing over the last decade. An organoid has recently been defined by consensus as a “three-dimensional structure derived from (pluripotent) stem cells, progenitor, and/or differentiated cells that self-organize through cell-cell and cell-matrix interactions to recapitulate aspects of the native tissue architecture and function in vitro”.
      • Marsee A.
      • Roos F.J.M.
      • Verstegen M.M.A.
      • Consortium H.P.B.O.
      • Gehart H.
      • de Koning E.
      • et al.
      Building consensus on definition and nomenclature of hepatic, pancreatic, and biliary organoids.
      Liver organoids can be developed from the same cells used to recellularise scaffolds. The cells can differentiate into different cells depending on the culture medium and conditions used.
      • Marsee A.
      • Roos F.J.M.
      • Verstegen M.M.A.
      • Consortium H.P.B.O.
      • Gehart H.
      • de Koning E.
      • et al.
      Building consensus on definition and nomenclature of hepatic, pancreatic, and biliary organoids.
      • Takebe T.
      • Sekine K.
      • Kimura M.
      • Yoshizawa E.
      • Ayano S.
      • Koido M.
      • et al.
      Massive and reproducible production of liver buds entirely from human pluripotent stem cells.
      • Sampaziotis F.
      • Justin A.W.
      • Tysoe O.C.
      • Sawiak S.
      • Godfrey E.M.
      • Upponi S.S.
      • et al.
      Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids.
      • Wu F.
      • Wu D.
      • Ren Y.
      • Huang Y.
      • Feng B.
      • Zhao N.
      • et al.
      Generation of hepatobiliary organoids from human induced pluripotent stem cells.
      Also, autologous cells can be obtained from patients to decrease graft rejection and immunological problems after transplantation.
      • Willemse J.
      • Roos F.J.M.
      • Voogt I.J.
      • Schurink I.J.
      • Bijvelds M.
      • de Jonge H.R.
      • et al.
      Scaffolds obtained from decellularized human extrahepatic bile ducts support organoids to establish functional biliary tissue in a dish.
      However, primary hepatocytes remain scarce, which is why iPSCs are still commonly used.
      • Takebe T.
      • Sekine K.
      • Kimura M.
      • Yoshizawa E.
      • Ayano S.
      • Koido M.
      • et al.
      Massive and reproducible production of liver buds entirely from human pluripotent stem cells.
      The advantage of a 3D structure is that organoids resemble the actual tissue better, including the architecture and ECM, which is important for cell-cell interactions.
      • Willemse J.
      • Lieshout R.
      • van der Laan L.J.W.
      • Verstegen M.M.A.
      From organoids to organs: bioengineering liver grafts from hepatic stem cells and matrix.
      ,
      • Gupta S.K.
      • Mishra N.C.
      • Dhasmana A.
      Decellularization methods for scaffold fabrication.
      ,
      • Hynes R.O.
      The extracellular matrix: not just pretty fibrils.
      However, a problem with large 3D organoids is the vascularisation of these structures and not receiving enough oxygen and nutrients, resulting in necrosis. This problem might be solved by NMP, where the perfusion machine acts as a platform for the implantation and distribution of organoids in injured liver allografts. In an experimental setting, Sampaziotis et al. have used NMP to deliver cholangiocyte organoids to donor livers and demonstrated repair of injured bile ducts. Cholangiocyte organoids, made from primary human cholangiocytes, exhibit plasticity and can keep their in vivo signatures when placed around the “original” biliary tree. The organoids were injected into a terminal branch of the intrahepatic ducts. After only 100 h of NMP, the organoids were still in place and had been regenerating so that after NMP, the intrahepatic ducts consisted of native and transplanted cells, without signs of cholangiopathy and with higher pH and higher bile volume than control livers.
      • Sampaziotis F.
      • Muraro D.
      • Tysoe O.C.
      • Sawiak S.
      • Beach T.E.
      • Godfrey E.M.
      • et al.
      Cholangiocyte organoids can repair bile ducts after transplantation in the human liver.
      This success represents an early example of the increasing possibilities of long-term NMP in the development of organoids and the regeneration of damaged livers.

      Challenges and barriers

      The aforementioned future ideas for possible treatments and therapies to treat damaged livers with NMP can only become a reality if more research is done and some major challenges and barriers are overcome.
      A major barrier is having clinically certified long-term NMP devices available to apply these treatments. Although no such machine is currently available, both clinically certified short-term machines as well as custom-made devices are used for perfusion for up to 7 days for experimental research.
      • Eshmuminov D.
      • Becker D.
      • Bautista Borrego L.
      • Hefti M.
      • Schuler M.J.
      • Hagedorn C.
      • et al.
      An integrated perfusion machine preserves injured human livers for 1 week.
      ,
      • Liu Q.
      • Nassar A.
      • Buccini L.
      • Grady P.
      • Soliman B.
      • Hassan A.
      • et al.
      Ex situ 86-hour liver perfusion: pushing the boundary of organ preservation.
      ,
      • Vogel T.
      • Brockmann J.G.
      • Pigott D.
      • Neil D.A.H.
      • Muthusamy A.S.R.
      • Coussios C.C.
      • et al.
      Successful transplantation of porcine liver grafts following 48-hour normothermic preservation.
      In addition to most of the treatments described, devices that enable long-term NMP are not yet clinically approved, which could potentially take several years.
      Another challenge is the translation of these treatments with (long-term) NMP into clinical practice. Even if an NMP machine becomes fully automated, it will require trained personnel who will need to be able to adjust the machine at any time of day as needed. The NMP device must also be readily accessible to certified personnel at all times while maintaining sterility.
      For long-term NMP, viability assessment criteria need to be established to evaluate treatments and decide whether the liver is suitable for transplantation or not. Currently, (non-validated) hepatocellular and/or cholangiocellular viability markers are used.
      • Mergental H.
      • Laing R.W.
      • Hodson J.
      • Boteon Y.L.
      • Attard J.A.
      • Walace L.L.
      • et al.
      Introduction of the concept of diagnostic sensitivity and specificity of normothermic perfusion protocols to assess high-risk donor livers.
      ,
      • Panconesi R.
      • Flores Carvalho M.
      • Mueller M.
      • Meierhofer D.
      • Dutkowski P.
      • Muiesan P.
      • et al.
      Viability assessment in liver transplantation-what is the impact of dynamic organ preservation?.
      However, when using NMP for repair and regeneration, new biomarkers will have to be identified to act as viability criteria in order to evaluate the effect of the treatments in addition to the current criteria (Box 1).
      Overview of potential biomarkers for assessment of viability during NMP.
      ALT, alanine aminotransferase; AST, aspartate aminotransferase; FMN, flavin mononucleotide; LDH, lactate dehydrogenase; miRNA, microRNA; NMP, normothermic machine perfusion; RNAi, RNA interference; TNF-α, tumour necrosis factor-α.
      • Bruggenwirth I.M.A.
      • de Meijer V.E.
      • Porte R.J.
      • Martins P.N.
      Viability criteria assessment during liver machine perfusion.
      • Bhogal R.H.
      • Mirza D.F.
      • Afford S.C.
      • Mergental H.
      Biomarkers of liver injury during transplantation in an era of machine perfusion.
      • Kesseli S.J.
      • Gloria J.N.
      • Abraham N.
      • Halpern S.E.
      • Cywinska G.N.
      • Zhang M.
      • et al.
      Point-of-Care assessment of DCD livers during normothermic machine perfusion in a nonhuman primate model.
      • Karangwa S.A.
      • Burlage L.C.
      • Adelmeijer J.
      • Karimian N.
      • Westerkamp A.C.
      • Matton A.P.
      • et al.
      Activation of fibrinolysis, but not coagulation, during end-ischemic ex situ normothermic machine perfusion of human donor livers.
      • Thorgersen E.B.
      • Barratt-Due A.
      • Haugaa H.
      • Harboe M.
      • Pischke S.E.
      • Nilsson P.H.
      • et al.
      The role of complement in liver injury, regeneration, and transplantation.
      Not unimportantly, costs associated with the implementation of long-term NMP should be evaluated, especially in light of exponentially increasing overall healthcare costs. In the end, evidence of the clinical and economic advantages of this new technology will be needed to drive its implementation.
      • Boteon Y.L.
      • Hessheimer A.J.
      • Bruggenwirth I.M.A.
      • Boteon A.
      • Padilla M.
      • de Meijer V.E.
      • et al.
      The economic impact of machine perfusion technology in liver transplantation.
      Despite an increase in experimental research on repair and regeneration strategies of damaged donor livers in combination with NMP, many challenges and barriers need to be addressed first. Table 1 shows an agenda outlining the topics that need to be explored before the treatments described in this review can become a reality.
      Table 1Proposed research agenda to enable repair and regeneration of damaged livers with NMP in the future.
      SubjectWhat remains to be done?
      Long-term (≥24 h) NMP machineAssessment of safety, feasibility and efficacy, and obtaining clinical approval for long-term NMP devices
      Trained personnelDevelopment of clinical training programs
      Ex situ treatment strategiesAssessment of safety, feasibility and efficacy, and obtaining clinical approval
      Viability criteriaValidation of previously established viability criteria Development of new viability criteria
      CostsEconomic cost-utility analysis of new treatments and technology
      NMP, normothermic machine perfusion.

      Conclusion

      To reduce the discrepancy between organ demand and supply in liver transplantation, NMP may increasingly be used in the future as a dynamic platform to optimise organ utilisation by enabling the repair and regeneration of damaged donor livers. Machine preservation has already been shown to be of great value in diminishing post-transplantation complications and increasing the donor pool via increased use of high-risk ECD livers (following pre-transplant viability testing). In the last few years, experimental research on the repair and regeneration of organs has increased exponentially. Several therapeutics, such as defatting cocktails, RNAi, senolytics, and stem cell therapy, might repair damaged livers, whereas stem cell therapy, scaffolds, and organoids may assist in the regeneration of injured livers before transplantation.
      Nevertheless, some major challenges and barriers need to be overcome before NMP can be used as a platform for the repair and regeneration of damaged donor livers. Currently, only a minority of liver transplant centres worldwide have employed NMP in their clinical practice. If future high-level evidence supports the widespread introduction of these techniques, overcoming logistical constraints, including optimal utilisation of these relatively expensive devices and the availability of specially trained perfusion personnel, will be essential. In addition, most of the aforementioned therapies are based on experimental research and require extensive clinical testing and approval before being used in clinical practice. This review may guide the future research agenda on NMP as a dynamic platform for the repair and regeneration of damaged donor livers, potentially leading to better utilisation of the current donor pool, ultimately reducing transplant waiting lists and improving patient outcomes.

      Abbreviations

      BMMSCs, bone marrow mesenchymal stem cells; DCD, donation after circulatory death; ECD, extended criteria donor; ECM, extracellular matrix; EVs, extracellular vesicles; iPSCs, induced pluripotent stem cells; IRI, ischaemia-reperfusion injury; MSCs, mesenchymal stem cells; NMP, normothermic machine perfusion; RNAi, RNA interference; SCs, senescent cells; siRNA, small-interfering RNA.

      Financial support

      V.E.d.M reports a VENI research grant by the Dutch Research Council (NWO; grant #09150161810030 ), a Research grant from the Dutch Ministry of Economic Affairs (Health∼Holland Public Private Partnership grant #PPP-2019-024 ), and a Research grant from the Dutch Society for Gastroenterology (NVGE #01-2021 ).

      Authors’ contributions

      Concept and design: B.L., V.E.d.M and R.J.P. Writing of article & preparing figures and tables: B.L. Editing and revision & approval of the final version: B.L., V.E.d.M and R.J.P.

      Conflict of interest

      The authors declare no conflicts of interest that pertain to this work.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

      The following are the supplementary data to this article:

      References

        • Kwong A.J.
        • Kim W.R.
        • Lake J.R.
        • Smith J.M.
        • Schladt D.P.
        • Skeans M.A.
        • et al.
        OPTN/SRTR 2019 annual data report: liver.
        Am J Transpl. 2021; 21: 208-315
        • Orman E.S.
        • Barritt ASt
        • Wheeler S.B.
        • Hayashi P.H.
        Declining liver utilization for transplantation in the United States and the impact of donation after cardiac death.
        Liver Transpl. 2013; 19: 59-68
        • Busuttil R.W.
        • Tanaka K.
        The utility of marginal donors in liver transplantation.
        Liver Transpl. 2003; 9: 651-663
        • Wong R.J.
        • Aguilar M.
        • Cheung R.
        • Perumpail R.B.
        • Harrison S.A.
        • Younossi Z.M.
        • et al.
        Nonalcoholic steatohepatitis is the second leading etiology of liver disease among adults awaiting liver transplantation in the United States.
        Gastroenterology. 2015; 148: 547-555
        • Rajaram R.B.
        • Jayaraman T.
        • Yoong B.K.
        • Koh P.S.
        • Loh P.S.
        • Koong J.K.
        • et al.
        Non-alcoholic fatty liver disease and obesity among adult donors are major challenges to living donor liver transplantation: a single-centre experience.
        Asian J Surg. 2021;
        • Sapisochin G.
        • Hibi T.
        • Toso C.
        • Man K.
        • Berenguer M.
        • Heimbach J.
        • et al.
        Transplant oncology in primary and metastatic liver tumors: principles, evidence, and opportunities.
        Ann Surg. 2021; 273: 483-493
        • Ceresa C.D.L.
        • Nasralla D.
        • Pollok J.M.
        • Friend P.J.
        Machine perfusion of the liver: applications in transplantation and beyond.
        Nat Rev Gastroenterol Hepatol. 2022;
        • de Meijer V.E.
        • Fujiyoshi M.
        • Porte R.J.
        Ex situ machine perfusion strategies in liver transplantation.
        J Hepatol. 2019; 70: 203-205
        • van Rijn R.
        • Schurink I.J.
        • de Vries Y.
        • van den Berg A.P.
        • Cortes Cerisuelo M.
        • Darwish Murad S.
        • et al.
        Hypothermic machine perfusion in liver transplantation - a randomized trial.
        N Engl J Med. 2021; 384: 1391-1401
        • Nasralla D.
        • Coussios C.C.
        • Mergental H.
        • Akhtar M.Z.
        • Butler A.J.
        • Ceresa C.D.L.
        • et al.
        A randomized trial of normothermic preservation in liver transplantation.
        Nature. 2018; 557: 50-56
        • Czigany Z.
        • Pratschke J.
        • Fronek J.
        • Guba M.
        • Schoning W.
        • Raptis D.A.
        • et al.
        Hypothermic oxygenated machine perfusion (HOPE) reduces early allograft injury and improves post-transplant outcomes in extended criteria donation (ECD) liver transplantation from donation after brain death (DBD): results from a multicenter randomized controlled trial (HOPE ECD-DBD).
        Ann Surg. 2021;
        • van Leeuwen O.B.
        • de Vries Y.
        • Fujiyoshi M.
        • Nijsten M.W.N.
        • Ubbink R.
        • Pelgrim G.J.
        • et al.
        Transplantation of high-risk donor livers after ex situ resuscitation and assessment using combined hypo- and normothermic machine perfusion: a prospective clinical trial.
        Ann Surg. 2019; 270: 906-914
        • Markmann J.F.
        • Abouljoud M.S.
        • Ghobrial R.M.
        • Bhati C.S.
        • Pelletier S.J.
        • Lu A.D.
        • et al.
        Impact of portable normothermic blood-based machine perfusion on outcomes of liver transplant: the OCS liver PROTECT randomized clinical trial.
        JAMA Surg. 2022;
        • Bonaccorsi-Riani E.
        • Bruggenwirth I.M.A.
        • Buchwald J.E.
        • Iesari S.
        • Martins P.N.
        Machine perfusion: cold versus warm, versus neither. Update on clinical trials.
        Semin Liver Dis. 2020; 40: 264-281
        • de Goeij F.H.C.
        • Schlegel A.
        • Muiesan P.
        • Guarrera J.V.
        • Dutkowski P.
        Hypothermic oxygenated machine perfusion protects from cholangiopathy in donation after circulatory death liver transplantation.
        Hepatology. 2021; 74: 3525-3528
        • Eshmuminov D.
        • Becker D.
        • Bautista Borrego L.
        • Hefti M.
        • Schuler M.J.
        • Hagedorn C.
        • et al.
        An integrated perfusion machine preserves injured human livers for 1 week.
        Nat Biotechnol. 2020; 38: 189-198
        • Resch T.
        • Cardini B.
        • Oberhuber R.
        • Weissenbacher A.
        • Dumfarth J.
        • Krapf C.
        • et al.
        Transplanting marginal organs in the era of modern machine perfusion and advanced organ monitoring.
        Front Immunol. 2020; 11: 631
      1. “repair”. Cambridge Dictionary, 2021 ([cited Retrieved 21 July 2021]; Available from:)
      2. “regeneration”. Cambridge Dictionary, 2021 ([cited Retrieved 21 July 2021]; Available from:)
        • Zomer H.D.
        • Vidane A.S.
        • Goncalves N.N.
        • Ambrosio C.E.
        Mesenchymal and induced pluripotent stem cells: general insights and clinical perspectives.
        Stem Cells Cloning. 2015; 8: 125-134
        • Elisseeff J.
        • Badylak S.F.
        • Boeke J.D.
        Immune and genome engineering as the future of transplantable tissue.
        N Engl J Med. 2021; 385: 2451-2462
        • Nadalin S.
        • Testa G.
        • Malago M.
        • Beste M.
        • Frilling A.
        • Schroeder T.
        • et al.
        Volumetric and functional recovery of the liver after right hepatectomy for living donation.
        Liver Transpl. 2004; 10: 1024-1029
        • Michalopoulos G.K.
        • DeFrances M.C.
        Liver regeneration.
        Science. 1997; 276: 60-66
        • Mueller M.
        • Hefti M.
        • Eshmuminov D.
        • Schuler M.J.
        • Silva R.
        • Petrowsky H.
        • et al.
        Long-term normothermic machine preservation of partial livers: first experience with 21 human hemi-livers.
        Ann Surg. 2021;
        • Butler A.J.
        • Rees M.A.
        • Wight D.G.
        • Casey N.D.
        • Alexander G.
        • White D.J.
        • et al.
        Successful extracorporeal porcine liver perfusion for 72 hr.
        Transplantation. 2002; 73: 1212-1218
        • Liu Q.
        • Nassar A.
        • Buccini L.
        • Grady P.
        • Soliman B.
        • Hassan A.
        • et al.
        Ex situ 86-hour liver perfusion: pushing the boundary of organ preservation.
        Liver Transpl. 2018; 24: 557-561
        • Lascaris B.
        • Thorne A.M.
        • Lisman T.
        • Nijsten M.W.N.
        • Porte R.J.
        • de Meijer V.E.
        Long-term normothermic machine preservation of human livers: what is needed to succeed?.
        Am J Physiol Gastrointest Liver Physiol. 2021;
        • Eshmuminov D.
        • Mueller M.
        • Brugger S.D.
        • Bautista Borrego L.
        • Becker D.
        • Hefti M.
        • et al.
        Sources and prevention of graft infection during long-term ex situ liver perfusion.
        Transpl Infect Dis. 2021; e13623
        • Boteon Y.L.
        • Afford S.C.
        Machine perfusion of the liver: which is the best technique to mitigate ischaemia-reperfusion injury?.
        World J Transpl. 2019; 9: 14-20
        • Boteon Y.L.
        • Laing R.W.
        • Schlegel A.
        • Wallace L.
        • Smith A.
        • Attard J.
        • et al.
        Combined hypothermic and normothermic machine perfusion improves functional recovery of extended criteria donor livers.
        Liver Transpl. 2018; 24: 1699-1715
        • Schlegel A.
        • Muller X.
        • Mueller M.
        • Stepanova A.
        • Kron P.
        • de Rougemont O.
        • et al.
        Hypothermic oxygenated perfusion protects from mitochondrial injury before liver transplantation.
        EBioMedicine. 2020; 60103014
        • McCormack L.
        • Dutkowski P.
        • El-Badry A.M.
        • Clavien P.A.
        Liver transplantation using fatty livers: always feasible?.
        J Hepatol. 2011; 54: 1055-1062
        • Xu M.
        • Zhou F.
        • Ahmed O.
        • Upadhya G.A.
        • Jia J.
        • Lee C.
        • et al.
        A novel multidrug combination mitigates rat liver steatosis through activating AMPK pathway during normothermic machine perfusion.
        Transplantation. 2021;
        • Nativ N.I.
        • Yarmush G.
        • Chen A.
        • Dong D.
        • Henry S.D.
        • Guarrera J.V.
        • et al.
        Rat hepatocyte culture model of macrosteatosis: effect of macrosteatosis induction and reversal on viability and liver-specific function.
        J Hepatol. 2013; 59: 1307-1314
        • Nagrath D.
        • Xu H.
        • Tanimura Y.
        • Zuo R.
        • Berthiaume F.
        • Avila M.
        • et al.
        Metabolic preconditioning of donor organs: defatting fatty livers by normothermic perfusion ex vivo.
        Metab Eng. 2009; 11: 274-283
        • Yarmush G.
        • Santos L.
        • Yarmush J.
        • Koundinyan S.
        • Saleem M.
        • Nativ N.I.
        • et al.
        CFD assessment of the effect of convective mass transport on the intracellular clearance of intracellular triglycerides in macrosteatotic hepatocytes.
        Biomech Model Mechanobiol. 2017; 16: 1095-1102
        • Boteon Y.L.
        • Attard J.
        • Boteon A.
        • Wallace L.
        • Reynolds G.
        • Hubscher S.
        • 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
        • Banan B.
        • Watson R.
        • Xu M.
        • Lin Y.
        • Chapman W.
        Development of a normothermic extracorporeal liver perfusion system toward improving viability and function of human extended criteria donor livers.
        Liver Transpl. 2016; 22: 979-993
        • Thorne A.M.
        • Ubbink R.
        • Bruggenwirth I.M.A.
        • Nijsten M.W.
        • Porte R.J.
        • de Meijer V.E.
        Hyperthermia-induced changes in liver physiology and metabolism: a rationale for hyperthermic machine perfusion.
        Am J Physiol Gastrointest Liver Physiol. 2020; 319: G43-G50
        • Taba Taba Vakili S.
        • Kailar R.
        • Rahman K.
        • Nezami B.G.
        • Mwangi S.M.
        • Anania F.A.
        • et al.
        Glial cell line-derived neurotrophic factor-induced mice liver defatting: a novel strategy to enable transplantation of steatotic livers.
        Liver Transpl. 2016; 22: 459-467
        • Jamieson R.W.
        • Zilvetti M.
        • Roy D.
        • Hughes D.
        • Morovat A.
        • Coussios C.C.
        • et al.
        Hepatic steatosis and normothermic perfusion-preliminary experiments in a porcine model.
        Transplantation. 2011; 92: 289-295
        • Bruggenwirth I.M.A.
        • Martins P.N.
        RNA interference therapeutics in organ transplantation: the dawn of a new era.
        Am J Transpl. 2020; 20: 931-941
        • Contreras J.L.
        • Vilatoba M.
        • Eckstein C.
        • Bilbao G.
        • Anthony Thompson J.
        • Eckhoff D.E.
        Caspase-8 and caspase-3 small interfering RNA decreases ischemia/reperfusion injury to the liver in mice.
        Surgery. 2004; 136: 390-400
        • Li X.
        • Zhang J.F.
        • Lu M.Q.
        • Yang Y.
        • Xu C.
        • Li H.
        • et al.
        Alleviation of ischemia-reperfusion injury in rat liver transplantation by induction of small interference RNA targeting Fas.
        Langenbecks Arch Surg. 2007; 392: 345-351
        • Thijssen M.F.
        • Bruggenwirth I.M.A.
        • Gillooly A.
        • Khvorova A.
        • Kowalik T.F.
        • Martins P.N.
        Gene silencing with siRNA (RNA interference): a new therapeutic option during ex vivo machine liver perfusion preservation.
        Liver Transpl. 2019; 25: 140-151
        • Gillooly A.R.
        • Perry J.
        • Martins P.N.
        First report of siRNA uptake (for RNA interference) during ex vivo hypothermic and normothermic liver machine perfusion.
        Transplantation. 2019; 103: e56-e57
        • Moore C.
        • Thijssen M.
        • Wang X.
        • Mandrekar P.
        • Xiaofei E.
        • Abdi R.
        • et al.
        Gene silencing with p53 si-RNA downregulates inflammatory markers in the liver: potential utilization during normothermic machine preservation.
        Am J Transpl. 2017; 18
        • Cui J.
        • Qin L.
        • Zhang J.
        • Abrahimi P.
        • Li H.
        • Li G.
        • et al.
        Ex vivo pretreatment of human vessels with siRNA nanoparticles provides protein silencing in endothelial cells.
        Nat Commun. 2017; 8: 191
        • Goldaracena N.
        • Spetzler V.N.
        • Echeverri J.
        • Kaths J.M.
        • Cherepanov V.
        • Persson R.
        • 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 Transpl. 2017; 17: 970-978
        • Adams D.
        • Gonzalez-Duarte A.
        • O'Riordan W.D.
        • Yang C.C.
        • Ueda M.
        • Kristen A.V.
        • et al.
        Patisiran, an RNAi therapeutic, for hereditary transthyretin amyloidosis.
        N Engl J Med. 2018; 379: 11-21
        • van Willigenburg H.
        • de Keizer P.L.J.
        • de Bruin R.W.F.
        Cellular senescence as a therapeutic target to improve renal transplantation outcome.
        Pharmacol Res. 2018; 130: 322-330
        • Kirkland J.L.
        • Tchkonia T.
        Senolytic drugs: from discovery to translation.
        J Intern Med. 2020; 288: 518-536
        • Prata L.
        • Ovsyannikova I.G.
        • Tchkonia T.
        • Kirkland J.L.
        Senescent cell clearance by the immune system: emerging therapeutic opportunities.
        Semin Immunol. 2018; 40101275
        • Ogrodnik M.
        • Miwa S.
        • Tchkonia T.
        • Tiniakos D.
        • Wilson C.L.
        • Lahat A.
        • et al.
        Cellular senescence drives age-dependent hepatic steatosis.
        Nat Commun. 2017; 815691
        • Xu M.
        • Pirtskhalava T.
        • Farr J.N.
        • Weigand B.M.
        • Palmer A.K.
        • Weivoda M.M.
        • et al.
        Senolytics improve physical function and increase lifespan in old age.
        Nat Med. 2018; 24: 1246-1256
        • Iske J.
        • Seyda M.
        • Heinbokel T.
        • Maenosono R.
        • Minami K.
        • Nian Y.
        • et al.
        Senolytics prevent mt-DNA-induced inflammation and promote the survival of aged organs following transplantation.
        Nat Commun. 2020; 11: 4289
        • Ritschka B.
        • Knauer-Meyer T.
        • Goncalves D.S.
        • Mas A.
        • Plassat J.L.
        • Durik M.
        • et al.
        The senotherapeutic drug ABT-737 disrupts aberrant p21 expression to restore liver regeneration in adult mice.
        Genes Dev. 2020; 34: 489-494
        • Sasaki M.
        • Sato Y.
        • Nakanuma Y.
        Increased p16(INK4a)-expressing senescent bile ductular cells are associated with inadequate response to ursodeoxycholic acid in primary biliary cholangitis.
        J Autoimmun. 2020; 107102377
        • Alsuraih M.
        • O'Hara S.P.
        • Woodrum J.E.
        • Pirius N.E.
        • LaRusso N.F.
        Genetic or pharmacological reduction of cholangiocyte senescence improves inflammation and fibrosis in the Mdr2 (-/-) mouse.
        JHEP Rep. 2021; 3100250
        • Yang L.
        • Cao H.
        • Sun D.
        • Hou B.
        • Lin L.
        • Shen Z.Y.
        • et al.
        Bone marrow mesenchymal stem cells combine with normothermic machine perfusion to improve rat donor liver quality-the important role of hepatic microcirculation in donation after circulatory death.
        Cell Tissue Res. 2020; 381: 239-254
        • Haga H.
        • Yan I.K.
        • Borrelli D.A.
        • Matsuda A.
        • Parasramka M.
        • Shukla N.
        • et al.
        Extracellular vesicles from bone marrow-derived mesenchymal stem cells protect against murine hepatic ischemia/reperfusion injury.
        Liver Transpl. 2017; 23: 791-803
        • Wang Y.
        • Zhang A.
        • Ye Z.
        • Xie H.
        • Zheng S.
        Bone marrow-derived mesenchymal stem cells inhibit acute rejection of rat liver allografts in association with regulatory T-cell expansion.
        Transpl Proc. 2009; 41: 4352-4356
        • Van Raemdonck D.
        • Neyrinck A.
        • Rega F.
        • Devos T.
        • Pirenne J.
        Machine perfusion in organ transplantation: a tool for ex-vivo graft conditioning with mesenchymal stem cells?.
        Curr Opin Organ Transpl. 2013; 18: 24-33
        • Sun C.K.
        • Chang C.L.
        • Lin Y.C.
        • Kao Y.H.
        • Chang L.T.
        • Yen C.H.
        • et al.
        Systemic administration of autologous adipose-derived mesenchymal stem cells alleviates hepatic ischemia-reperfusion injury in rats.
        Crit Care Med. 2012; 40: 1279-1290
        • Kanazawa H.
        • Fujimoto Y.
        • Teratani T.
        • Iwasaki J.
        • Kasahara N.
        • Negishi K.
        • et al.
        Bone marrow-derived mesenchymal stem cells ameliorate hepatic ischemia reperfusion injury in a rat model.
        PLoS One. 2011; 6e19195
        • Wu B.
        • Song H.L.
        • Yang Y.
        • Yin M.L.
        • Zhang B.Y.
        • Cao Y.
        • et al.
        Improvement of liver transplantation outcome by heme oxygenase-1-transduced bone marrow mesenchymal stem cells in rats.
        Stem Cells Int. 2016; 20169235073
        • Verstegen M.M.A.
        • Mezzanotte L.
        • Ridwan R.Y.
        • Wang K.
        • de Haan J.
        • Schurink I.J.
        • 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
        • Yang L.
        • Cao H.
        • Sun D.
        • Lin L.
        • Zheng W.P.
        • Shen Z.Y.
        • et al.
        Normothermic machine perfusion combined with bone marrow mesenchymal stem cells improves the oxidative stress response and mitochondrial function in rat donation after circulatory death livers.
        Stem Cells Dev. 2020; 29: 835-852
        • Sun D.
        • Yang L.
        • Zheng W.
        • Cao H.
        • Wu L.
        • Song H.
        Protective effects of bone marrow mesenchymal stem cells (BMMSCS) combined with normothermic machine perfusion on liver grafts donated after circulatory death via reducing the ferroptosis of hepatocytes.
        Med Sci Monit. 2021; 27e930258
        • Hou B.
        • Song H.
        • Cao H.
        • Yang L.
        • Sun D.
        • Shen Z.
        [Effects of bone marrow mesenchymal stem cells combined with normothermic mechanical perfusion on biliary epithelial cells donated after cardiac death in rats].
        Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2019; 31: 1137-1142
        • Cao H.
        • Yang L.
        • Hou B.
        • Sun D.
        • Lin L.
        • Song H.L.
        • et al.
        Heme oxygenase-1-modified bone marrow mesenchymal stem cells combined with normothermic machine perfusion to protect donation after circulatory death liver grafts.
        Stem Cell Res Ther. 2020; 11: 218
        • Tian X.
        • Cao H.
        • Wu L.
        • Zheng W.
        • Yuan M.
        • Li X.
        • et al.
        Heme oxygenase-1-modified bone marrow mesenchymal stem cells combined with normothermic machine perfusion repairs bile duct injury in a rat model of DCD liver transplantation via activation of peribiliary glands through the wnt pathway.
        Stem Cells Int. 2021; 20219935370
        • Laing R.W.
        • Stubblefield S.
        • Wallace L.
        • Roobrouck V.D.
        • Bhogal R.H.
        • Schlegel A.
        • et al.
        The delivery of multipotent adult progenitor cells to extended criteria human donor livers using normothermic machine perfusion.
        Front Immunol. 2020; 11: 1226
        • Rigo F.
        • De Stefano N.
        • Navarro-Tableros V.
        • David E.
        • Rizza G.
        • Catalano G.
        • et al.
        Extracellular vesicles from human liver stem cells reduce injury in an ex vivo normothermic hypoxic rat liver perfusion model.
        Transplantation. 2018; 102: e205-e210
        • Anger F.
        • Camara M.
        • Ellinger E.
        • Germer C.T.
        • Schlegel N.
        • Otto C.
        • et al.
        Human mesenchymal stromal cell-derived extracellular vesicles improve liver regeneration after ischemia reperfusion injury in mice.
        Stem Cells Dev. 2019; 28: 1451-1462
        • Raposo G.
        • Stoorvogel W.
        Extracellular vesicles: exosomes, microvesicles, and friends.
        J Cell Biol. 2013; 200: 373-383
        • Cesaretti M.
        • Zarzavajian Le Bian A.
        • Moccia S.
        • Iannelli A.
        • Schiavo L.
        • Diaspro A.
        From deceased to bioengineered graft: new frontiers in liver transplantation.
        Transpl Rev (Orlando). 2019; 33: 72-76
        • Willemse J.
        • Lieshout R.
        • van der Laan L.J.W.
        • Verstegen M.M.A.
        From organoids to organs: bioengineering liver grafts from hepatic stem cells and matrix.
        Best Pract Res Clin Gastroenterol. 2017; 31: 151-159
        • Kajbafzadeh A.M.
        • Javan-Farazmand N.
        • Monajemzadeh M.
        • Baghayee A.
        Determining the optimal decellularization and sterilization protocol for preparing a tissue scaffold of a human-sized liver tissue.
        Tissue Eng C Methods. 2013; 19: 642-651
        • Uygun B.E.
        • Soto-Gutierrez A.
        • Yagi H.
        • Izamis M.L.
        • Guzzardi M.A.
        • Shulman C.
        • et al.
        Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.
        Nat Med. 2010; 16: 814-820
        • Gupta S.K.
        • Mishra N.C.
        • Dhasmana A.
        Decellularization methods for scaffold fabrication.
        Methods Mol Biol. 2018; 1577: 1-10
        • Coronado R.E.
        • Somaraki-Cormier M.
        • Natesan S.
        • Christy R.J.
        • Ong J.L.
        • Halff G.A.
        Decellularization and solubilization of porcine liver for use as a substrate for porcine hepatocyte culture: method optimization and comparison.
        Cell Transpl. 2017; 26: 1840-1854
        • Hynes R.O.
        The extracellular matrix: not just pretty fibrils.
        Science. 2009; 326: 1216-1219
        • Lang R.
        • Stern M.M.
        • Smith L.
        • Liu Y.
        • Bharadwaj S.
        • Liu G.
        • et al.
        Three-dimensional culture of hepatocytes on porcine liver tissue-derived extracellular matrix.
        Biomaterials. 2011; 32: 7042-7052
        • Barakat O.
        • Abbasi S.
        • Rodriguez G.
        • Rios J.
        • Wood R.P.
        • Ozaki C.
        • et al.
        Use of decellularized porcine liver for engineering humanized liver organ.
        J Surg Res. 2012; 173: e11-e25
        • Mazza G.
        • Rombouts K.
        • Rennie Hall A.
        • Urbani L.
        • Vinh Luong T.
        • Al-Akkad W.
        • et al.
        Decellularized human liver as a natural 3D-scaffold for liver bioengineering and transplantation.
        Sci Rep. 2015; 513079
        • Willemse J.
        • Verstegen M.M.A.
        • Vermeulen A.
        • Schurink I.J.
        • Roest H.P.
        • van der Laan L.J.W.
        • et al.
        Fast, robust and effective decellularization of whole human livers using mild detergents and pressure controlled perfusion.
        Mater Sci Eng C Mater Biol Appl. 2020; 108110200
        • Mirmalek-Sani S.H.
        • Sullivan D.C.
        • Zimmerman C.
        • Shupe T.D.
        • Petersen B.E.
        Immunogenicity of decellularized porcine liver for bioengineered hepatic tissue.
        Am J Pathol. 2013; 183: 558-565
        • Willemse J.
        • Roos F.J.M.
        • Voogt I.J.
        • Schurink I.J.
        • Bijvelds M.
        • de Jonge H.R.
        • et al.
        Scaffolds obtained from decellularized human extrahepatic bile ducts support organoids to establish functional biliary tissue in a dish.
        Biotechnol Bioeng. 2021; 118: 836-851
        • Verstegen M.M.A.
        • Willemse J.
        • van den Hoek S.
        • Kremers G.J.
        • Luider T.M.
        • van Huizen N.A.
        • et al.
        Decellularization of whole human liver grafts using controlled perfusion for transplantable organ bioscaffolds.
        Stem Cells Dev. 2017; 26: 1304-1315
        • Sabetkish S.
        • Kajbafzadeh A.M.
        • Sabetkish N.
        • Khorramirouz R.
        • Akbarzadeh A.
        • Seyedian S.L.
        • et al.
        Whole-organ tissue engineering: decellularization and recellularization of three-dimensional matrix liver scaffolds.
        J Biomed Mater Res A. 2015; 103: 1498-1508
        • Robertson M.J.
        • Soibam B.
        • O'Leary J.G.
        • Sampaio L.C.
        • Taylor D.A.
        Recellularization of rat liver: an in vitro model for assessing human drug metabolism and liver biology.
        PLoS One. 2018; 13e0191892
        • Zhou P.
        • Huang Y.
        • Guo Y.
        • Wang L.
        • Ling C.
        • Guo Q.
        • et al.
        Decellularization and recellularization of rat livers with hepatocytes and endothelial progenitor cells.
        Artif Organs. 2016; 40: E25-E38
        • Yagi H.
        • Fukumitsu K.
        • Fukuda K.
        • Kitago M.
        • Shinoda M.
        • Obara H.
        • et al.
        Human-scale whole-organ bioengineering for liver transplantation: a regenerative medicine approach.
        Cell Transpl. 2013; 22: 231-242
        • Pla-Palacin I.
        • Sainz-Arnal P.
        • Morini S.
        • Almeida M.
        • Baptista P.M.
        Liver bioengineering using decellularized whole-liver scaffolds.
        Methods Mol Biol. 2018; 1577: 293-305
        • Asadi M.
        • Khalili M.
        • Lotfi H.
        • Vaghefi Moghaddam S.
        • Zarghami N.
        • Andre H.
        • et al.
        Liver bioengineering: recent trends/advances in decellularization and cell sheet technologies towards translation into the clinic.
        Life Sci. 2021; 276119373
        • Ko I.K.
        • Peng L.
        • Peloso A.
        • Smith C.J.
        • Dhal A.
        • Deegan D.B.
        • et al.
        Bioengineered transplantable porcine livers with re-endothelialized vasculature.
        Biomaterials. 2015; 40: 72-79
        • Zhang W.
        • Li W.
        • Liu B.
        • Wang P.
        • Li W.
        • Zhang H.
        Efficient generation of functional hepatocyte-like cells from human fetal hepatic progenitor cells in vitro.
        J Cell Physiol. 2012; 227: 2051-2058
        • Uygun B.E.
        • Izamis M.L.
        • Jaramillo M.
        • Chen Y.
        • Price G.
        • Ozer S.
        • et al.
        Discarded livers find a new life: engineered liver grafts using hepatocytes recovered from marginal livers.
        Artif Organs. 2017; 41: 579-585
        • Jaramillo M.
        • Yeh H.
        • Yarmush M.L.
        • Uygun B.E.
        Decellularized human liver extracellular matrix (hDLM)-mediated hepatic differentiation of human induced pluripotent stem cells (hIPSCs).
        J Tissue Eng Regen Med. 2018; 12: e1962-e1973
        • Soto-Gutierrez A.
        • Zhang L.
        • Medberry C.
        • Fukumitsu K.
        • Faulk D.
        • Jiang H.
        • et al.
        A whole-organ regenerative medicine approach for liver replacement.
        Tissue Eng Part C Methods. 2011; 17: 677-686
        • Baptista P.M.
        • Siddiqui M.M.
        • Lozier G.
        • Rodriguez S.R.
        • Atala A.
        • Soker S.
        The use of whole organ decellularization for the generation of a vascularized liver organoid.
        Hepatology. 2011; 53: 604-617
        • Shimoda H.
        • Yagi H.
        • Higashi H.
        • Tajima K.
        • Kuroda K.
        • Abe Y.
        • et al.
        Decellularized liver scaffolds promote liver regeneration after partial hepatectomy.
        Sci Rep. 2019; 912543
        • Marsee A.
        • Roos F.J.M.
        • Verstegen M.M.A.
        • Consortium H.P.B.O.
        • Gehart H.
        • de Koning E.
        • et al.
        Building consensus on definition and nomenclature of hepatic, pancreatic, and biliary organoids.
        Cell Stem Cell. 2021; 28: 816-832
        • Takebe T.
        • Sekine K.
        • Kimura M.
        • Yoshizawa E.
        • Ayano S.
        • Koido M.
        • et al.
        Massive and reproducible production of liver buds entirely from human pluripotent stem cells.
        Cell Rep. 2017; 21: 2661-2670
        • Sampaziotis F.
        • Justin A.W.
        • Tysoe O.C.
        • Sawiak S.
        • Godfrey E.M.
        • Upponi S.S.
        • et al.
        Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids.
        Nat Med. 2017; 23: 954-963
        • Wu F.
        • Wu D.
        • Ren Y.
        • Huang Y.
        • Feng B.
        • Zhao N.
        • et al.
        Generation of hepatobiliary organoids from human induced pluripotent stem cells.
        J Hepatol. 2019; 70: 1145-1158
        • Sampaziotis F.
        • Muraro D.
        • Tysoe O.C.
        • Sawiak S.
        • Beach T.E.
        • Godfrey E.M.
        • et al.
        Cholangiocyte organoids can repair bile ducts after transplantation in the human liver.
        Science. 2021; 371: 839-846
        • Vogel T.
        • Brockmann J.G.
        • Pigott D.
        • Neil D.A.H.
        • Muthusamy A.S.R.
        • Coussios C.C.
        • et al.
        Successful transplantation of porcine liver grafts following 48-hour normothermic preservation.
        PLoS One. 2017; 12e0188494
        • Mergental H.
        • Laing R.W.
        • Hodson J.
        • Boteon Y.L.
        • Attard J.A.
        • Walace L.L.
        • et al.
        Introduction of the concept of diagnostic sensitivity and specificity of normothermic perfusion protocols to assess high-risk donor livers.
        Liver Transpl. 2021;
        • Panconesi R.
        • Flores Carvalho M.
        • Mueller M.
        • Meierhofer D.
        • Dutkowski P.
        • Muiesan P.
        • et al.
        Viability assessment in liver transplantation-what is the impact of dynamic organ preservation?.
        Biomedicines. 2021; 9
        • Boteon Y.L.
        • Hessheimer A.J.
        • Bruggenwirth I.M.A.
        • Boteon A.
        • Padilla M.
        • de Meijer V.E.
        • et al.
        The economic impact of machine perfusion technology in liver transplantation.
        Artif Organs. 2021;
        • Bruggenwirth I.M.A.
        • de Meijer V.E.
        • Porte R.J.
        • Martins P.N.
        Viability criteria assessment during liver machine perfusion.
        Nat Biotechnol. 2020; 38: 1260-1262
        • Bhogal R.H.
        • Mirza D.F.
        • Afford S.C.
        • Mergental H.
        Biomarkers of liver injury during transplantation in an era of machine perfusion.
        Int J Mol Sci. 2020; : 21
        • Kesseli S.J.
        • Gloria J.N.
        • Abraham N.
        • Halpern S.E.
        • Cywinska G.N.
        • Zhang M.
        • et al.
        Point-of-Care assessment of DCD livers during normothermic machine perfusion in a nonhuman primate model.
        Hepatol Commun. 2021; 5: 1527-1542
        • Karangwa S.A.
        • Burlage L.C.
        • Adelmeijer J.
        • Karimian N.
        • Westerkamp A.C.
        • Matton A.P.
        • et al.
        Activation of fibrinolysis, but not coagulation, during end-ischemic ex situ normothermic machine perfusion of human donor livers.
        Transplantation. 2017; 101: e42-e48
        • Thorgersen E.B.
        • Barratt-Due A.
        • Haugaa H.
        • Harboe M.
        • Pischke S.E.
        • Nilsson P.H.
        • et al.
        The role of complement in liver injury, regeneration, and transplantation.
        Hepatology. 2019; 70: 725-736