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Machine perfusion in liver transplantation as a tool to prevent non-anastomotic biliary strictures: Rationale, current evidence and future directions

  • Pepijn D. Weeder
    Affiliations
    Section of Hepatobiliary Surgery and Liver Transplantation, Department of Surgery, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands
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  • Rianne van Rijn
    Affiliations
    Section of Hepatobiliary Surgery and Liver Transplantation, Department of Surgery, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands
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  • 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
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Open AccessPublished:March 11, 2015DOI:https://doi.org/10.1016/j.jhep.2015.03.008

      Summary

      The high incidence of non-anastomotic biliary strictures (NAS) after transplantation of livers from extended criteria donors is currently a major barrier to widespread use of these organs. This review provides an update on the most recent advances in the understanding of the etiology of NAS. These new insights give reason to believe that machine perfusion can reduce the incidence of NAS after transplantation by providing more protective effects on the biliary tree during preservation of the donor liver. An overview is presented regarding the different endpoints that have been used for assessment of biliary injury and function before and after transplantation, emphasizing on methods used during machine perfusion. The wide spectrum of different approaches to machine perfusion is discussed, including the many different combinations of techniques, temperatures and perfusates at varying time points. In addition, the current understanding of the effect of machine perfusion in relation to biliary injury is reviewed. Finally, we explore directions for future research such as the application of (pharmacological) strategies during machine perfusion to further improve preservation. We stress the great potential of machine perfusion to possibly expand the donor pool by reducing the incidence of NAS in extended criteria organs.

      Abbreviations:

      NAS (non-anastomotic biliary strictures, ECD, extended criteria donors), DCD (donation after circulatory death), DBD (donation after brain death), PBG (peribiliary glands), PVP (peribiliary vascular plexus), NRP (normothermic regional perfusion), HMP (hypothermic machine perfusion), HOPE (hypothermic oxygenated perfusion), SNMP (subnormothermic machine perfusion), NMP (normothermic machine perfusion), MSCs (mesenchymal stem cells), MRCP (magnetic resonance cholangiopancreatography), ERCP (endoscopic retrograde cholangiopancreatography), PTCD (percutaneous transhepatic cholangiography drainage), COR (controlled oxygenated rewarming)

      Keywords

      Introduction

      Orthotopic liver transplantation is currently the only available life-saving treatment for patients with end-stage liver disease. Unfortunately, the number of organs needed for transplantation greatly surpass the supply, prompting strict selection criteria for transplant candidates and long waiting lists for those patients that reach candidate status. As a result of this shortage about 15% of patients die while on the waiting list [
      • Kim W.R.
      • Therneau T.
      • Benson J.
      • Kremers W.
      • Rosen C.
      • Gores G.
      • et al.
      Deaths on the liver transplant waiting list: an analysis of competing risks.
      ]. Additionally, according to studies of death certificates, about 60,000 patients die of liver disease annually in the United States [
      • Asrani S.K.
      • Larson J.J.
      • Yawn B.
      • Therneau T.M.
      • Kim W.R.
      Underestimation of liver-related mortality in the United States.
      ]; many of whom could (theoretically) have been treated with a liver transplant. When only waitlist mortality is considered, the magnitude of the problem of end-stage liver disease is grossly underestimated. In fact, only about 1.5% of liver disease related mortality concerns waitlisted patients [
      • Kim W.R.
      • Smith J.M.
      • Skeans M.A.
      • Schladt D.P.
      • Schnitzler M.A.
      • Edwards E.B.
      • et al.
      OPTN/SRTR 2012 annual data report: liver.
      ].
      The grave shortage of livers available for transplantation causes the transplant community to continuously push the boundaries to increase the availability of organs. As a part of this effort, criteria for donor liver selection have been progressively expanded, allowing the usage of more suboptimal or compromised grafts. Livers from donors that fall outside of standard criteria, also known as ‘extended criteria donors (ECDs), are increasingly considered for transplantation during the past decades [
      • Domínguez-Gil B.
      • Haase-Kromwijk B.
      • Van Leiden H.
      • Neuberger J.
      • Coene L.
      • Morel P.
      • et al.
      Current situation of donation after circulatory death in European countries.
      ]. For example, in the United Kingdom 42% of transplanted livers now come from donation after circulatory death (DCD) donors, as opposed to donation after brain death (DBD) [

      NHS blood and transplant. Organ donation and transplantation activity report 2012/13. Available at: <http://www.organdonation.nhs.uk/statistics/transplant_activity_report/current_activity_reports/ukt/activity_report_2012_13.pdf> [accessed 04.10.14].

      ]. The most important other criteria that are being widened include donor age, blood type ABO incompatibility, steatosis, and infectious diseases in the donor [
      • Harring T.R.
      • O’Mahony C.A.
      • Goss J.A.
      Extended donors in liver transplantation.
      ,
      • Durand F.
      • Renz J.F.
      • Alkofer B.
      • Burra P.
      • Clavien P.
      • Porte R.J.
      • et al.
      Report of the Paris consensus meeting on expanded criteria donors in liver transplantation.
      ]. The increased acceptance of livers from ECDs in the past two decades has contributed significantly to the expansion of the donor organ pool [
      • Muiesan P.
      • Raffaele G.
      • Jassem W.
      • Melendez H.V.
      • O’Grady J.
      • Bowles M.
      • et al.
      Single-center experience with liver transplantation from controlled non-heartbeating donors: a viable source of grafts.
      ,
      • Deshpande R.
      • Heaton N.
      Can non-heart-beating donors replace cadaveric heart-beating liver donors?.
      ].
      Today, the survival rate of patients that receive a DCD liver graft is approximately similar to DBD recipients [
      • Morrissey P.E.
      • Monaco A.P.
      Donation after circulatory death: current practices, ongoing challenges, and potential improvements.
      ,
      • Dubbeld J.
      • Hoekstra H.
      • Farid W.
      • Ringers J.
      • Porte R.J.
      • Metselaar H.J.
      • et al.
      Similar liver transplantation survival with selected cardiac death donors and brain death donors.
      ,
      • Reich D.H.
      Johnny. Current status of donation after cardiac death liver transplantation.
      ,
      • Mateo R.
      • Cho Y.
      • Singh G.
      • Stapfer M.
      • Donovan J.
      • Kahn J.
      • et al.
      Risk factors for graft survival after liver transplantation from donation after cardiac death donors: an analysis of OPTN/UNOS data.
      ]. However, the transplantation of DCD liver grafts is associated with around 10% lower 1-year graft survival rate, compared to DBD livers and a markedly higher incidence of biliary complications [
      • Foley D.P.
      • Fernandez L.A.
      • Leverson G.
      • Anderson M.A.
      • Mezrich J.
      • Sollinger H.W.
      • et al.
      Biliary complications after liver transplantation from donation after cardiac death donors: an analysis of risk factors and long-term outcomes from a single center.
      ,
      • Abt P.
      • Crawford M.
      • Desai N.
      • Markmann J.
      • Olthoff K.
      • Shaked A.
      Liver transplantation from controlled non-heart-beating donors: an increased incidence of biliary complications.
      ,
      • Gastaca M.
      Biliary complications after orthotopic liver transplantation: a review of incidence and risk factors.
      ,
      • Jay C.L.
      • Lyuksemburg V.
      • Ladner D.P.
      • Wang E.
      • Caicedo J.C.
      • Holl J.L.
      • et al.
      Ischemic cholangiopathy after controlled donation after cardiac death liver transplantation: a meta-analysis.
      ]. The incidence of NAS varies between 4% and 15% after transplantation of DBD livers, but can be as high as 30–50% after transplantation of DCD grafts [
      • Jay C.L.
      • Lyuksemburg V.
      • Ladner D.P.
      • Wang E.
      • Caicedo J.C.
      • Holl J.L.
      • et al.
      Ischemic cholangiopathy after controlled donation after cardiac death liver transplantation: a meta-analysis.
      ,
      • Op den Dries S.
      • Sutton M.
      • Lisman T.
      • Porte R.
      Protection of bile ducts in liver transplantation: looking beyond ischemia.
      ]. The occurrence of NAS in a donor liver critically impacts patients’ long-term survival, rate of re-transplantation, quality of life and costs of care [
      • Sharma S.
      • Gurakar A.
      • Jabbour N.
      Biliary strictures following liver transplantation: past, present and preventive strategies.
      ,
      • Duffy J.P.
      • Kao K.
      • Ko C.
      • Farmer D.
      • McDiarmid S.
      • Hong J.
      • et al.
      Long-term patient outcome and quality of life after liver transplantation: analysis of 20-year survivors.
      ]. Synonyms for NAS that are frequently used in the literature are ischemic–type biliary lesions and ischemic cholangiopathy. In general, all three names refer to the same clinical entity characterized by a combination of narrowing and dilatations (or even intraparenchymal leakage) of the larger intra- and extrahepatic donor bile ducts, either with or without intraluminal sludge and cast formation, and in the presence of a patent hepatic artery (Fig. 1A and B). Severity and distribution of NAS along the biliary tree may vary considerably among patients and clinical symptoms range from no symptoms to recurrent jaundice and/or life-threatening cholangitis with subsequent need for re-transplantation [
      • Buis C.I.
      • Verdonk R.C.
      • Van der Jagt E.J.
      • van der Hilst C.S.
      • Slooff M.J.H.
      • Haagsma E.B.
      • et al.
      Nonanastomotic biliary strictures after liver transplantation, part 1: radiological features and risk factors for early vs. late presentation.
      ].
      Figure thumbnail gr1
      Fig. 1Radiological and histological examples of bile duct injury in a donor liver. (A) Cholangiogram revealing mild non-anastomotic strictures in a donor liver. (B) Cholangiogram showing severe non-anastomotic strictures in a donor liver. (C) Histology of a donor bile duct (hematoxylin & eosin staining) with approximately 50% detachment and loss of the luminal epithelium (lumen indicted by #), but well preserved bile duct wall stroma (asterix) and peribiliary glands (dotted lines). (D) Histology of a donor bile duct (hematoxylin & eosin staining) with a well preserved arteriole (arrow) as part of the PVP. (E and F) Histology of a donor bile duct (hematoxylin & eosin staining) with necrosis of bile duct wall stroma (asterix), arteriolonecrosis, and loss of epithelial cells in the peribiliary glands (dotted lines). The arrow indicates a peribiliary gland with a complete loss of epithelial cells.
      Figure thumbnail fx2

      Risk factors and pathogenesis of NAS

      Traditionally, the degree of biliary injury that occurs during and after transplantation was considered to be the main determining factor for NAS [
      • Buis C.
      • Hoekstra H.
      • Verdonk R.
      • Porte R.
      Causes and consequences of ischemic-type biliary lesions after liver transplantation.
      ]. Warm and cold ischemia, reperfusion injury, bile salt toxicity, and immune-mediated injury have been identified as the most important contributors to this multifactorial process of biliary injury [
      • Op den Dries S.
      • Sutton M.
      • Lisman T.
      • Porte R.
      Protection of bile ducts in liver transplantation: looking beyond ischemia.
      ,
      • Karimian N.
      • Westerkamp A.
      • Porte R.J.
      Biliary complications after orthotopic liver transplantation.
      ]. However, three independent histological studies focusing on the common bile duct of donor livers revealed that in 86%-88% of cases the bile duct epithelium is already significantly injured at the end of cold storage [
      • Hansen T.
      • Hollemann D.
      • Pitton M.
      • Heise M.
      • Hoppe Lotichius M.
      • Schuchmann M.
      • et al.
      Histological examination and evaluation of donor bile ducts received during orthotopic liver transplantation – A morphological clue to ischemic-type biliary lesion?.
      ,
      • Brunner S.M.
      • Junger H.
      • Ruemmele P.
      • Schnitzbauer A.A.
      • Doenecke A.
      • Kirchner G.I.
      • et al.
      Bile duct damage after cold storage of deceased donor livers predicts biliary complications after liver transplantation.
      ,
      • Karimian N.
      • Op den Dries S.
      • Porte R.
      The origin of biliary strictures after liver transplantation: is it the amount of epithelial injury or insufficient regeneration that counts?.
      ]. This observation indicates that the bile ducts of almost every donor liver suffer substantial injury during graft preservation, yet only a minority of them develop NAS after transplantation, suggesting that the lack of regenerative capacity of the donor bile duct may be more important than the initial degree of injury [
      • Karimian N.
      • Op den Dries S.
      • Porte R.
      The origin of biliary strictures after liver transplantation: is it the amount of epithelial injury or insufficient regeneration that counts?.
      ].
      This new concept of the pathogenesis of NAS has incited interest into the mechanisms involved in biliary regeneration. In a study of 128 liver transplants, op den Dries et al. [
      • op den Dries S.
      • Westerkamp A.
      • Karimian N.
      • Gouw A.S.H.
      • Bruinsma B.
      • Markmann J.
      • et al.
      Injury to peribiliary glands and vascular plexus before liver transplantation predicts formation of non-anastomotic biliary strictures.
      ] have demonstrated a strong association between injury of the peribiliary glands (PBG) and the peribiliary vascular plexus (PVP) at the time of transplantation and the later development of NAS in liver transplant recipients. The PBG are glandular structures that are abundantly present in the wall of the extrahepatic and major intrahepatic bile ducts, which are the most common locations of stricture formation [
      • Buis C.I.
      • Verdonk R.C.
      • Van der Jagt E.J.
      • van der Hilst C.S.
      • Slooff M.J.H.
      • Haagsma E.B.
      • et al.
      Nonanastomotic biliary strictures after liver transplantation, part 1: radiological features and risk factors for early vs. late presentation.
      ,
      • Dipaola F.
      • Shivakumar P.
      • Pfister J.
      • Walters S.
      • Sabla G.
      • Bezerra J.
      Identification of intramural epithelial networks linked to peribiliary glands that express progenitor cell markers and proliferate after injury in mice.
      ]. Moreover, the PBG have been shown to be a niche for progenitor cells that can proliferate upon biliary injury [
      • Dipaola F.
      • Shivakumar P.
      • Pfister J.
      • Walters S.
      • Sabla G.
      • Bezerra J.
      Identification of intramural epithelial networks linked to peribiliary glands that express progenitor cell markers and proliferate after injury in mice.
      ,
      • Carpino G.
      • Cardinale V.
      • Onori P.
      • Franchitto A.
      • Berloco P.
      • Rossi M.
      • et al.
      Biliary tree stem/progenitor cells in glands of extrahepatic and intraheptic bile ducts: an anatomical in situ study yielding evidence of maturational lineages.
      ,
      • Cardinale V.
      • Wang Y.
      • Carpino G.
      • Cui C.B.
      • Gatto M.
      • Rossi M.
      • et al.
      Multipotent stem/progenitor cells in human biliary tree give rise to hepatocytes, cholangiocytes, and pancreatic islets.
      ,
      • Semeraro R.
      • Carpino G.
      • Cardinale V.
      • Onori P.
      • Gentile R.
      • Cantafora A.
      • et al.
      Multipotent stem/progenitor cells in the human foetal biliary tree.
      ,
      • Sutton M.
      • op den Dries S.
      • Koster M.
      • Lisman T.
      • Gouw A.S.H.
      • Porte R.
      Regeneration of human extrahepatic biliary epithelium: the peribiliary glands as progenitor cell compartment.
      ]. The fact that; i) extensive epithelial regeneration is necessary after transplantation in the majority of liver grafts; ii) proliferation is observed in the PBG upon biliary injury; and iii) loss of PBG integrity at the time of transplant is associated to the development of NAS post-transplant strongly suggests that the PBG are a key component required for successful recovery from the extensive and multifactorial biliary injury that occurs during transplantation. Possibly, the absence of a healthy epithelial barrier due to the failure of cholangiocyte regeneration increases prolonged bile salt cytotoxicity and inflammation eventually leading to NAS [
      • Op den Dries S.
      • Sutton M.
      • Lisman T.
      • Porte R.
      Protection of bile ducts in liver transplantation: looking beyond ischemia.
      ,
      • Karimian N.
      • Westerkamp A.
      • Porte R.J.
      Biliary complications after orthotopic liver transplantation.
      ,
      • op den Dries S.
      • Westerkamp A.
      • Karimian N.
      • Gouw A.S.H.
      • Bruinsma B.
      • Markmann J.
      • et al.
      Injury to peribiliary glands and vascular plexus before liver transplantation predicts formation of non-anastomotic biliary strictures.
      ,
      • Buis C.I.
      • Geuken E.
      • Visser D.S.
      • Kuipers F.
      • Haagsma E.B.
      • Verkade H.J.
      • et al.
      Altered bile composition after liver transplantation is associated with the development of nonanastomotic biliary strictures.
      ].
      The PVP is essential for the viability of the bile duct as it provides blood with nutrients and oxygen to the bile ducts. Blood flow through the PVP is mainly supplied by the hepatic artery and thrombosis of the hepatic artery early after transplantation generally leads to necrosis of the larger donor bile ducts [
      • Zajko A.B.
      • Campbell W.L.
      • Logsdon G.A.
      • Bron K.M.
      • Tzakis A.
      • Esquivel C.O.
      • et al.
      Cholangiographic findings in hepatic artery occlusion after liver transplantation.
      ,
      • Seehofer D.
      • Eurich D.
      • Veltzke-Schlieker W.
      • Neuhaus P.
      Biliary complications after liver transplantation: Old problems and new challenges.
      ,
      • Gunji H.
      • Cho A.
      • Tohma T.
      • Okazumi S.
      • Makino H.
      • Shuto K.
      • et al.
      The blood supply of the hilar bile duct and its relationship to the communicating arcade located between the right and left hepatic arteries.
      ]. Even in the presence of a patent artery, dysfunction and preservation injury of the PVP is believed to play a role in the etiology of NAS [
      • Hansen T.
      • Hollemann D.
      • Pitton M.
      • Heise M.
      • Hoppe Lotichius M.
      • Schuchmann M.
      • et al.
      Histological examination and evaluation of donor bile ducts received during orthotopic liver transplantation – A morphological clue to ischemic-type biliary lesion?.
      ,
      • Heidenhain C.
      • Pratschke J.
      • Puhl G.
      • Neumann U.
      • Pascher A.
      • Veltzke Schlieker W.
      • et al.
      Incidence of and risk factors for ischemic-type biliary lesions following orthotopic liver transplantation.
      ]. These recent findings suggest a critical role of the PVP and the PBG in biliary regeneration and the pathogenesis of NAS. Histology of bile ducts with various degrees of injury of the luminal biliary epithelium, the PBG, and the PVP are presented in Fig. 1C–F. The regenerative capacity of the bile duct may be negatively influenced by known risk factors for NAS, such as donor age and donor warm ischemia (as occurs in DCD donors), as well as toxic bile salts that may not only affect the luminal epithelium, but also the PBG and PVP. Especially when the luminal biliary epithelium is absent, bile salts can easily enter the bile duct wall stroma and cause injury to these essential structures. Achieving protection or improved recovery of these critical components of the bile duct wall by machine perfusion could therefore contribute to the prevention of NAS. Here, preventing injury altogether would be the ultimate target, because when regeneration is not needed it also cannot fail. If this cannot be accomplished, protection of the PVP and PBG to facilitate successful regeneration through tissue oxygenation and preservation of progenitor cells in the PBG are obvious subsidiary goals. Not surprisingly, optimal preservation of the PVP and PBG has become an important endpoint in machine perfusion studies [
      • Liu Q.
      • Nassar A.
      • Farias K.
      • Buccini L.
      • Baldwin W.
      • Mangino M.
      • et al.
      Sanguineous normothermic machine perfusion improves hemodynamics and biliary epithelial regeneration in DCD porcine livers.
      ,
      • op den Dries S.
      • Sutton M.E.
      • Karimian N.
      • de Boer M.T.
      • Wiersema-Buist J.
      • Gouw A.S.H.
      • et al.
      Hypothermic oxygenated machine perfusion prevents arteriolonecrosis of the peribiliary plexus in pig livers donated after circulatory death.
      ,
      • Muiesan P.F.
      Simon. The bile duct in donation after cardiac death donor liver transplant.
      ]. The proposed pathogenesis of NAS and the various targets for machine perfusion are summarized in Fig. 2.
      Figure thumbnail gr2
      Fig. 2Proposed pathogenesis of NAS after liver transplantation. During warm (in DCD donors) and cold ischemia (in both DCD and DBD livers) bile duct injury occurs almost universally, characterized by injury and loss of the luminal biliary epithelium. This type of injury alone does not necessarily lead to NAS. Critical components of the bile duct wall are the peribiliary glands (PBG) and the PVP. Severe injury to the PBGs and PVP has been associated with the development of NAS
      [
      • op den Dries S.
      • Westerkamp A.
      • Karimian N.
      • Gouw A.S.H.
      • Bruinsma B.
      • Markmann J.
      • et al.
      Injury to peribiliary glands and vascular plexus before liver transplantation predicts formation of non-anastomotic biliary strictures.
      ]
      suggesting that adequate preservation of these structures is essential for a timely regeneration of the biliary epithelium. After transplantation, biliary bile salts may cause additional damage to the remnant luminal epithelium and diffuse into the bile duct wall, causing injury to the PBG and PVP. Secondary influx of immune cells leads to inflammation with subsequent fibrosis and scarring of the bile ducts resulting in NAS. Machine perfusion of donor livers may reduce the incidence of NAS after transplantation by reducing the amount of injury to the various components of the bile duct wall.

      Assessment of the impact of machine perfusion on the biliary tree

      Accurately assessing the effect of machine perfusion on the biliary tree poses a significant challenge. Biliary function and injury are frequently used as surrogate endpoints for the occurrence of NAS. However, in general the overall degree of biliary injury does not correlate very well with the development of NAS [
      • Karimian N.
      • Op den Dries S.
      • Porte R.
      The origin of biliary strictures after liver transplantation: is it the amount of epithelial injury or insufficient regeneration that counts?.
      ]. Unfortunately, NAS do not easily occur after liver transplantation in rats and therefore this definitive endpoint is only obtainable in transplantation studies of large animals or humans, which are more complex and expensive. Nevertheless, a worthy set of endpoints, based on the current understanding of the etiology of NAS, is available for evaluating the biliary tree in the setting of machine perfusion (Table 1).
      Table 1Overview of endpoints suitable for studying biliary preservation by machine perfusion.
      *Level of evidence assessed according to oxford 2011 levels of evidence

      Howick J, Chalmers I, Glasziou P, Greenhalgh T, Heneghan C, Liberati A, et al. The Oxford 2011 levels of evidence; 2011. Available at: <http://www.cebm.net/index.aspx?o=5653> [accessed 12.21.14].

      .
      ERCP, Endoscopic Retrograde Cholangiopancreatography; MRCP, Magnetic Resonance Cholangiopancreatography, PTCD, percutaneous cholangiography drainage; H&E, hematoxillin and eosin, PVP, peribiliary vascular plexus, PBG,s peribiliary gland.
      As discussed previously, multiple studies have indicated an important role of the PVP and PBG in the etiology of NAS [
      • Hansen T.
      • Hollemann D.
      • Pitton M.
      • Heise M.
      • Hoppe Lotichius M.
      • Schuchmann M.
      • et al.
      Histological examination and evaluation of donor bile ducts received during orthotopic liver transplantation – A morphological clue to ischemic-type biliary lesion?.
      ,
      • op den Dries S.
      • Westerkamp A.
      • Karimian N.
      • Gouw A.S.H.
      • Bruinsma B.
      • Markmann J.
      • et al.
      Injury to peribiliary glands and vascular plexus before liver transplantation predicts formation of non-anastomotic biliary strictures.
      ,
      • Heidenhain C.
      • Pratschke J.
      • Puhl G.
      • Neumann U.
      • Pascher A.
      • Veltzke Schlieker W.
      • et al.
      Incidence of and risk factors for ischemic-type biliary lesions following orthotopic liver transplantation.
      ]. Therefore, the effects of machine perfusion on these structures are of particular relevance. They can be investigated using standard light microscopy and a standardized semi-quantitative scoring system [
      • Hansen T.
      • Hollemann D.
      • Pitton M.
      • Heise M.
      • Hoppe Lotichius M.
      • Schuchmann M.
      • et al.
      Histological examination and evaluation of donor bile ducts received during orthotopic liver transplantation – A morphological clue to ischemic-type biliary lesion?.
      ,
      • Brunner S.M.
      • Junger H.
      • Ruemmele P.
      • Schnitzbauer A.A.
      • Doenecke A.
      • Kirchner G.I.
      • et al.
      Bile duct damage after cold storage of deceased donor livers predicts biliary complications after liver transplantation.
      ,
      • op den Dries S.
      • Westerkamp A.
      • Karimian N.
      • Gouw A.S.H.
      • Bruinsma B.
      • Markmann J.
      • et al.
      Injury to peribiliary glands and vascular plexus before liver transplantation predicts formation of non-anastomotic biliary strictures.
      ]. This approach is significantly more objective compared to free-format pathological interpretation and has emerged as an important endpoint [
      • Liu Q.
      • Nassar A.
      • Farias K.
      • Buccini L.
      • Baldwin W.
      • Mangino M.
      • et al.
      Sanguineous normothermic machine perfusion improves hemodynamics and biliary epithelial regeneration in DCD porcine livers.
      ,
      • op den Dries S.
      • Sutton M.E.
      • Karimian N.
      • de Boer M.T.
      • Wiersema-Buist J.
      • Gouw A.S.H.
      • et al.
      Hypothermic oxygenated machine perfusion prevents arteriolonecrosis of the peribiliary plexus in pig livers donated after circulatory death.
      ]. More sophisticated methods such as immunohistochemistry may also be used. For example, Ki67 and CK19 are markers for proliferation in the PBG and the bile duct epithelium respectively [
      • Sutton M.
      • op den Dries S.
      • Koster M.
      • Lisman T.
      • Gouw A.S.H.
      • Porte R.
      Regeneration of human extrahepatic biliary epithelium: the peribiliary glands as progenitor cell compartment.
      ,
      • Liu Q.
      • Nassar A.
      • Farias K.
      • Buccini L.
      • Baldwin W.
      • Mangino M.
      • et al.
      Sanguineous normothermic machine perfusion improves hemodynamics and biliary epithelial regeneration in DCD porcine livers.
      ,
      • Schlegel A.
      • Graf R.
      • Clavien P.
      • Dutkowski P.
      Hypothermic oxygenated perfusion (HOPE) protects from biliary injury in a rodent model of DCD liver transplantation.
      ]. The expression of von Willebrand Factor on the vascular endothelium indicates endothelial cell activation that can lead to platelet aggregation in the PVP [
      • Liu Q.
      • Nassar A.
      • Farias K.
      • Buccini L.
      • Baldwin W.
      • Mangino M.
      • et al.
      Sanguineous normothermic machine perfusion improves hemodynamics and biliary epithelial regeneration in DCD porcine livers.
      ].
      Bile salt toxicity is known to be another important pillar in the mechanism of NAS [
      • Op den Dries S.
      • Sutton M.
      • Lisman T.
      • Porte R.
      Protection of bile ducts in liver transplantation: looking beyond ischemia.
      ]. The epithelial lining forms a specialized barrier to protect the bile duct from the detergent effects of bile. Hepatocytes excrete phospholipids that form mixed micelles, neutralizing the detergent effect of bile salts. When this barrier is impaired by ischemia/reperfusion induced injury or dysfunction, toxic bile salts can cause additional harm to the biliary epithelium, or when this is already absent, to the unprotected sub-mucosal stroma and the deeper located structures such as PBG and PVP [
      • Buis C.I.
      • Geuken E.
      • Visser D.S.
      • Kuipers F.
      • Haagsma E.B.
      • Verkade H.J.
      • et al.
      Altered bile composition after liver transplantation is associated with the development of nonanastomotic biliary strictures.
      ,
      • Geuken E.
      • Visser D.
      • Kuipers F.
      • Blokzijl H.
      • Leuvenink H.G.D.
      • de Jong K.
      • et al.
      Rapid increase of bile salt secretion is associated with bile duct injury after human liver transplantation.
      ]. Consequently, the ratio of bile salts to phospholipids in bile during perfusion is an important marker of the endogenous protective mechanisms against bile salt toxicity. The same is true for the biliary concentration of bicarbonate (HCO3), which prevents the protonation of biliary glycine-conjugated bile salts and uncontrolled cell entry of the corresponding bile acids through cell membranes by raising the pH, a phenomenon known as the “bicarbonate umbrella” [
      • Beuers U.
      • Maroni L.
      • Elferink R.
      The biliary HCO(3)(−) umbrella: experimental evidence revisited.
      ]. Bicarbonate is actively secreted into the bile by the biliary epithelial cells (or cholangiocytes) and, therefore, biliary bicarbonate concentration can also be used as a biochemical marker of cholangiocyte function [
      • Op den Dries S.
      • Sutton M.
      • Lisman T.
      • Porte R.
      Protection of bile ducts in liver transplantation: looking beyond ischemia.
      ]. Moreover, disruption or imbalance of bile salt transportation, which is an ATP dependent process, can contribute to injury by the intracellular accumulation of hydrophobic bile salts [
      • Palmeira C.M.
      • Rolo A.P.
      Mitochondrially-mediated toxicity of bile acids.
      ,
      • Cheng L.
      • Zhao L.
      • Li D.
      • Liu Z.
      • Chen G.
      • Tian F.
      • et al.
      Role of cholangiocyte bile Acid transporters in large bile duct injury after rat liver transplantation.
      ].
      The production of bile involves a cascade of steps that requires good function of all its components, including both hepatocytes and cholangiocytes [
      • Trauner M.B.
      • James L.
      Bile salt transporters: molecular characterization, function, and regulation.
      ]. Therefore, the rate of bile production during machine perfusion, in combination with the characteristics of its composition, is an excellent indicator of both liver and biliary function [
      • Sutton M.E.
      • op den Dries S.
      • Karimian N.
      • Weeder P.D.
      • de Boer M.T.
      • Wiersema-Buist J.
      • et al.
      Criteria for viability assessment of discarded human donor livers during ex-vivo normothermic machine perfusion.
      ]. Glucose is initially present in the canalicular bile at a concentration equal to plasma and is subsequently reabsorbed by the cholangiocytes of the bile ducts. A low glucose concentration (<1.0 mmol/L) in the common bile duct bile, therefore, indicates adequately functioning biliary epithelial cells, and can be used as a target for assessing bile duct integrity during machine perfusion [
      • Tabibian J.H.
      • Masyuk A.I.
      • Masyuk T.V.
      • O’Hara S.P.
      • LaRusso N.F.
      Physiology of cholangiocytes. Comprehensive physiology.
      ,
      • Guzelian P.
      • Boyer J.L.
      Glucose reabsorption from bile. Evidence for a biliohepatic circulation.
      ].
      Markers of cellular injury, such as lactate dehydrogenase and gamma-glutamyltransferase, can be measured in bile and perfusate [
      • Liu Q.
      • Nassar A.
      • Farias K.
      • Buccini L.
      • Baldwin W.
      • Mangino M.
      • et al.
      Sanguineous normothermic machine perfusion improves hemodynamics and biliary epithelial regeneration in DCD porcine livers.
      ,
      • op den Dries S.
      • Karimian N.
      • Sutton M.E.
      • Westerkamp A.C.
      • Nijsten M.W.N.
      • Gouw A.S.H.
      • et al.
      Ex vivo normothermic machine perfusion and viability testing of discarded human donor livers.
      ]. Although they may reflect the degree of biliary injury, it is unknown whether high biliary levels of these injury markers are associated with the later development of NAS. One method that holds promise is the detection of cholangiocyte specific micro RNAs in bile that have been associated with the development of NAS [
      • Verhoeven C.J.
      • Farid W.R.R.
      • de Jonge J.
      • Metselaar H.J.
      • Kazemier G.
      • van der Laan L.J.
      Biomarkers to assess graft quality during conventional and machine preservation in liver transplantation.
      ,
      • Lankisch T.O.
      • Voigtländer T.
      • Manns M.P.
      • Holzmann A.
      • Dangwal S.
      • Thum T.
      MicroRNAs in the bile of patients with biliary strictures after liver transplantation.
      ].
      Obviously, the incidence of NAS during follow up after transplantation of machine-perfused grafts is the most relevant endpoint in determining the effect of machine perfusion on biliary complications. Randomized clinical trials are needed to elucidate the effect of machine perfusion on biliary complications in humans.

      Machine perfusion: a spectrum of techniques

      The term ‘machine perfusion’ is very broad; it covers a spectrum of techniques with mechanical perfusion as their common denominator. Fundamentally, the configuration of machine perfusion depends on three parameters: the timing and duration of its application, and the perfusion temperature. In order to prevent endothelial shear stress, pressure controlled perfusion is considered to be a safer method compared to flow controlled perfusion [
      • Schlegel A.
      • Dutkowski P.
      Role of hypothermic machine perfusion in liver transplantation.
      ].
      Perfusion through the portal vein is only feasible at low temperatures [
      • Guarrera J.V.
      • Henry S.D.
      • Samstein B.
      • Odeh Ramadan R.
      • Kinkhabwala M.
      • Goldstein M.J.
      • et al.
      Hypothermic machine preservation in human liver transplantation: the first clinical series.
      ,
      • Dutkowski Philipp
      • Schlegel Andrea
      • de Oliveira Michelle
      • Müllhaupt Beat
      • Neff Fabienne
      • Clavien Pierre-Alain
      HOPE for human liver grafts obtained from donors after cardiac death.
      ]. But from the perspective of biliary preservation, arterial perfusion and the delivery of oxygen to the bile ducts seems paramount because the biliary system depends mainly on the arterial circulation [
      • Muiesan P.F.
      Simon. The bile duct in donation after cardiac death donor liver transplant.
      ,
      • Noack K.
      • Bronk S.F.
      • Kato A.
      • Gores G.J.
      The greater vulnerability of bile duct cells to reoxygenation injury than to anoxia. Implications for the pathogenesis of biliary strictures after liver transplantation.
      ]. Additionally, oxygenated perfusion of the arterial system has been described to attenuate arteriolonecrosis of the PVP and therefore may contribute to preservation of the vasculature to the bile ducts [
      • op den Dries S.
      • Sutton M.E.
      • Karimian N.
      • de Boer M.T.
      • Wiersema-Buist J.
      • Gouw A.S.H.
      • et al.
      Hypothermic oxygenated machine perfusion prevents arteriolonecrosis of the peribiliary plexus in pig livers donated after circulatory death.
      ]. On the other hand, it should be noted that in contrast to traditional belief, blood is also supplied to the bile ducts by the portal vein [
      • Slieker J.
      • Farid W.R.R.
      • van Eijck C.H.J.
      • Lange J.
      • van Bommel J.
      • Metselaar H.
      • et al.
      Significant contribution of the portal vein to blood flow through the common bile duct.
      ] and insufficient portal perfusion should also be considered a risk factor for the development of NAS [
      • Farid W.R.R.
      • de Jonge J.
      • Slieker J.C.
      • Zondervan M.G.J.
      • Thomeer M.G.J.
      • Metselaar H.J.
      • et al.
      The importance of portal venous blood flow in ischemic-type biliary lesions after liver transplantation.
      ,
      • Farid W.R.R.
      • de Jonge J.
      • Zondervan P.E.
      • Demirkiran A.
      • Metselaar H.J.
      • Tilanus H.W.
      • et al.
      Relationship between the histological appearance of the portal vein and development of ischemic-type biliary lesions after liver transplantation.
      ]. An argument against cannulation and perfusion of the hepatic artery is the potential risk of causing intima injury or vascular dissection. This risk can be minimized by using a cylindrical aortic segment for cannulation, which can be cut off and discarded at the end of perfusion before implantation.
      Machine perfusion can be applied at different time points during the process of procurement, transportation, and transplantation, as summarized in Fig. 3. The earliest possible application of machine perfusion in the chain of events is in situ normothermic regional perfusion (NRP) in the donor, immediately after circulatory arrest. It offers the advantage of restoring cellular levels of ATP that have become depleted during the period of warm ischemia by restoration of normothermic circulation in the donor prior to standard cold storage [
      • Barrou B.
      • Billault C.
      • Nicolas-Robin A.
      The use of extracorporeal membranous oxygenation in donors after cardiac death.
      ]. The technique has been successfully used in clinical DCD organ donation, and its effect on preservation of the bile ducts and the rate of biliary complications seems promising [
      • Butler A.
      • Randle L.
      • Watson C.
      Normothermic regional perfusion for donation after circulatory death without prior heparinization.
      ,
      • Rojas-Peña A.
      • Sall L.E.
      • Gravel M.T.
      • Cooley E.G.
      • Pelletier S.J.
      • Bartlett R.H.
      • et al.
      Donation after circulatory determination of death: the University of Michigan experience with extracorporeal support.
      ,
      • Hessheimer A.J.
      • Billault C.
      • Barrou B.
      • Fondevila C.
      Hypothermic or normothermic abdominal regional perfusion in high-risk donors with extended warm ischemia times: impact on outcomes?.
      ]. Investigators from the Hospital Clinic in Barcelona have reported 42 liver transplant procedures after NRP of DCD donors with a biliary complication rate of 17% [
      • Hessheimer A.J.
      • Billault C.
      • Barrou B.
      • Fondevila C.
      Hypothermic or normothermic abdominal regional perfusion in high-risk donors with extended warm ischemia times: impact on outcomes?.
      ]. Another group, from the University of Michigan, has described 13 liver transplant procedures using this method with an incidence of biliary complications of 14% [
      • Rojas-Peña A.
      • Sall L.E.
      • Gravel M.T.
      • Cooley E.G.
      • Pelletier S.J.
      • Bartlett R.H.
      • et al.
      Donation after circulatory determination of death: the University of Michigan experience with extracorporeal support.
      ]. While these outcomes are very hopeful, both studies lacked a control group and were not randomized. Also, in one report 38% of donor livers were discarded because of technical complications, illustrating the complexity of NRP [
      • Rojas-Peña A.
      • Sall L.E.
      • Gravel M.T.
      • Cooley E.G.
      • Pelletier S.J.
      • Bartlett R.H.
      • et al.
      Donation after circulatory determination of death: the University of Michigan experience with extracorporeal support.
      ]. Randomized controlled trials are necessary to assess the clinical benefits of NRP.
      Figure thumbnail gr3
      Fig. 3Schematic presentation of the various dimensions of machine perfusion. DCD: donation after circulatory death, NRP: normothermic regional perfusion.
      Ex situ machine perfusion of the liver can be performed at the donor site before cold storage (pre-storage perfusion), at the receiving hospital after cold storage prior to implantation (end-ischemic perfusion), or throughout the preservation period (preservation perfusion) (Fig. 3) [
      • de Rougemont O.
      • Lehmann K.
      • Clavien P.
      Preconditioning, organ preservation, and postconditioning to prevent ischemia-reperfusion injury to the liver.
      ,
      • Selzner N.
      • Boehnert M.
      • Selzner M.
      Preconditioning, postconditioning, and remote conditioning in solid organ transplantation: basic mechanisms and translational applications.
      ]. The advantage of pre-storage perfusion is that it has the ability to restore the energy charge of the liver prior to subjecting it to cold ischemia, potentially reducing the synergistic accumulation of injury caused by subsequent warm and cold ischemia. However, accomplishing on-site machine perfusion in donor hospitals is troubled by several logistical challenges.
      These challenges are even more pronounced in preservation perfusion, which requires a machine perfusion device that is portable and self-sufficient for the duration of the preservation period. Theoretically, continuous preservation perfusion is the most optimal choice because it eliminates the need for cold storage. Normothermic preservation perfusion has been successfully used in a clinical trial including liver transplantation of ten patients [

      Ravikumar R, Coussios CC, Holroyd D, Heaton N, Friend PJ, Jassem W. Human Liver transplantation using normothermic machine preservation; 2014; Available at: http://www.abstracts2view.com/ilts/view.php?nu=ILTS14L1_O-1&terms= [accessed 09.11.14].

      ]. Although continuous normothermic preservation perfusion may prove to be the most effective preservation method in the future, it is questionable whether complete elimination of cold preservation is necessary and cost-effective in the current era.
      End-ischemic perfusion offers the advantage that it can be performed in the environment of the transplant center with all the logistic support needed for successful perfusion. Although logistically appealing, irreversible injury may have occurred already to the liver and the biliary tree, which might have been preventable or treatable upon earlier intervention. Finally, through both preservation perfusion and end-ischemic perfusion, the organ can be optimized immediately prior to implantation, which is not the case for pre-storage perfusion. In general, machine perfusion requires specific surgical training and the direct costs are higher compared to simple cold storage. However, the technique could be cost-effective and even lead to lower overall costs due to shorter postoperative length of stay and reduction of the incidence of NAS, but studies of cost-effectiveness are needed to clarify this. Promisingly, renal machine perfusion has been proven to be cost-effective [
      • Groen H.
      • Moers C.
      • Smits J.M.
      • Treckmann J.
      • Monbaliu D.
      • Rahmel A.
      • et al.
      Cost-effectiveness of hypothermic machine preservation versus static cold storage in renal transplantation.
      ,
      • Garfield S.S.
      • Poret A.W.
      • Evans R.W.
      The cost-effectiveness of organ preservation methods in renal transplantation: US projections based on the machine preservation trial.
      ].

      Temperature

      The cellular rate of respiration is greatly dependent on the temperature, following an exponential curve [
      • Robinson W.R.
      • Peters R.H.
      • Zimmermann J.
      The effects of body size and temperature on metabolic rate of organisms.
      ]. Simple static cold storage at 0–4 °C is based on this principle and reduces the metabolic rate of the liver to about 5% of the physiological level. Because of this relationship, the temperature used during machine perfusion dictates the calibration of most other perfusion parameters that should be optimized to meet the dynamics and metabolic needs of the organ at a given temperature.

      Hypothermic machine perfusion

      Under hypothermic conditions (0–10 °C), the use of an acellular perfusion fluid at low pressures is most common. The use of red blood cells or an oxygen carrier is not necessary, because adequate uptake of oxygen into the fluid can be achieved through diffusion at low temperatures [
      • Monbaliu D.B.
      John. Machine perfusion of the liver: past, present and future.
      ]. Hypothermic machine perfusion (HMP) is safe because in case of pump failure, the organ falls back on the ‘default’ conditions of static cold preservation. The low metabolic activity that is associated with hypothermic perfusion conditions also forms a limitation, because it does not allow a functional assessment of the liver. For example, the liver does not produce bile during HMP, which is an important indicator of hepatobiliary function and allows biochemical assessment of biliary markers of biliary epithelial cell function and injury [
      • op den Dries S.
      • Karimian N.
      • Sutton M.E.
      • Westerkamp A.C.
      • Nijsten M.W.N.
      • Gouw A.S.H.
      • et al.
      Ex vivo normothermic machine perfusion and viability testing of discarded human donor livers.
      ,
      • Imber C.J.
      • St. Peter S.D.
      • De Cenarruzabeitia I.L.
      • Lemonde H.
      • Rees M.
      • Butler A.
      • et al.
      Optimisation of bile production during normothermic preservation of porcine livers.
      ].
      A large number of studies using HMP in animal models have been performed; however, few studies have evaluated the effects of HMP on the biliary system [
      • Schlegel A.
      • Dutkowski P.
      Role of hypothermic machine perfusion in liver transplantation.
      ]. Using a non-arterialized model of DCD liver transplantation in rats, Schlegel et al. [
      • Schlegel A.
      • Graf R.
      • Clavien P.
      • Dutkowski P.
      Hypothermic oxygenated perfusion (HOPE) protects from biliary injury in a rodent model of DCD liver transplantation.
      ] compared one hour of end-ischemic hypothermic oxygenated machine perfusion to cold storage alone. After four weeks of follow up, animals from the HMP group presented with superior histological appearance of the intrahepatic bile ducts and significantly lower serum gamma-glutamyltransferase, alkaline phosphatase and bilirubin [
      • Schlegel A.
      • Graf R.
      • Clavien P.
      • Dutkowski P.
      Hypothermic oxygenated perfusion (HOPE) protects from biliary injury in a rodent model of DCD liver transplantation.
      ]. In another experimental study, our group has compared 4 h of hypothermic oxygenated perfusion to cold storage of DCD pig livers, followed by 2 h of normothermic blood reperfusion to simulate transplantation. Although, no differences in biliary epithelial function or injury were observed, oxygenated HMP did lead to superior preservation of the PVP, which may have a positive impact on blood supply to the bile ducts and their regenerative capacity after transplantation [
      • op den Dries S.
      • Sutton M.E.
      • Karimian N.
      • de Boer M.T.
      • Wiersema-Buist J.
      • Gouw A.S.H.
      • et al.
      Hypothermic oxygenated machine perfusion prevents arteriolonecrosis of the peribiliary plexus in pig livers donated after circulatory death.
      ].
      Guarerra and coworkers, who were the first to describe successful clinical transplantation of HMP preserved liver grafts in 2010 [
      • Guarrera J.V.
      • Henry S.D.
      • Samstein B.
      • Odeh Ramadan R.
      • Kinkhabwala M.
      • Goldstein M.J.
      • et al.
      Hypothermic machine preservation in human liver transplantation: the first clinical series.
      ], recently reported the outcome of the first 31 cases performed in their center [
      • Guarrera J.V.
      • Henry S.D.
      • Samstein B.
      • Reznik E.
      • Musat C.
      • Lukose T.I.
      • et al.
      Hypothermic machine preservation facilitates successful transplantation of? Orphan? Extended criteria donor livers.
      ]. Apart from generally improved outcome parameters (shorter length of stay, lower postoperative serum injury markers etc.), the number of biliary strictures in the HMP preserved group was significantly lower compared to matched controls (10% after HMP vs. 33% after SCS). It should be noted that the perfusion system used by Guarerra and coworkers does not provide active oxygenation of the perfusion fluid and the duration of perfusion was not standardized. Dutkowski et al. [
      • Dutkowski Philipp
      • Schlegel Andrea
      • de Oliveira Michelle
      • Müllhaupt Beat
      • Neff Fabienne
      • Clavien Pierre-Alain
      HOPE for human liver grafts obtained from donors after cardiac death.
      ] have applied oxygenated HMP, or hypothermic oxygenated perfusion (HOPE), to eight locally procured DCD livers before transplantation and compared the outcomes with matched DBD controls. The rate of biliary complications was identical between the two groups; however, the numbers were too low to draw any robust conclusion on the incidence of biliary strictures. Despite the fact that livers were perfused through the portal vein only and the study lacks a DCD control group, it suggests a possible protective effect of HOPE against bile duct injury. A clinical trial with hypothermic oxygenated dual perfusion of DCD livers (through both the portal vein and hepatic artery) was recently initiated in our center (Netherlands Trial Registry, NTR4493; www.trialregister.nl). Final assessment of the efficacy of HMP (either oxygenated or non-oxygenated) in reducing biliary injury and subsequent NAS should come from large multicenter randomized clinical trials. Several initiatives to organize such trials have been taken (i.e. ClinicalTrials.gov, ID: NCT01317342).

      Subnormothermic machine perfusion

      Subnormothermic machine perfusion (SNMP, 20–30 °C) is a middle-of-the-road approach between HMP and normothermic machine perfusion (37 °C, NMP) [
      • Berendsen T.
      • Bruinsma B.
      • Lee J.
      • D’Andrea V.
      • Liu Q.
      • Izamis M.
      • et al.
      A simplified subnormothermic machine perfusion system restores ischemically damaged liver grafts in a rat model of orthotopic liver transplantation.
      ]. An advantage of SNMP is its relative simplicity compared to NMP. By inducing a metabolic rate of approximately 25% of physiological levels, it allows for functional assessment of the liver [
      • Bruinsma B.G.
      • Yeh H.
      • Özer S.
      • Martins P.N.
      • Farmer A.
      • Wu W.
      • et al.
      Subnormothermic machine perfusion for ex vivo preservation and recovery of the human liver for transplantation.
      ]. To prevent depletion of nutrients, SMP requires the use of a nutrient enriched perfusate. Surprisingly, an oxygen carrier is not always used; SMP has been performed both with and without red blood cells or an artificial oxygen carrier [
      • Berendsen T.
      • Bruinsma B.
      • Lee J.
      • D’Andrea V.
      • Liu Q.
      • Izamis M.
      • et al.
      A simplified subnormothermic machine perfusion system restores ischemically damaged liver grafts in a rat model of orthotopic liver transplantation.
      ,
      • Gringeri E.
      • Polacco M.
      • D’Amico F.E.
      • Scopelliti M.
      • Bassi D.
      • Bonsignore P.
      • et al.
      A new liver autotransplantation technique using subnormothermic machine perfusion for organ preservation in a porcine model.
      ,
      • Plauth M.
      • Zimmermann B.
      • Raible A.
      • Vieillard Baron D.
      • Bauder Gross D.
      • Hartmann F.
      Use of an artificial oxygen carrier in isolated rat liver perfusion: first demonstration of net glucose uptake at physiological portal glucose concentrations using a hemoglobin-free perfusate.
      ]. The question whether the use of an oxygen carrier at subnormothermic temperatures is essential, therefore, remains a matter of debate and this issue requires further research.
      SNMP has been tested in rats with positive effects on bile production and decreased release of biliary enzymes [
      • Ferrigno A.
      • Rizzo V.
      • Boncompagni E.
      • Bianchi A.
      • Gringeri E.
      • Neri D.
      • et al.
      Machine perfusion at 20 °C reduces preservation damage to livers from non-heart beating donors.
      ,
      • Tolboom H.
      • Izamis M.
      • Sharma N.
      • Milwid J.M.
      • Uygun B.
      • Berthiaume F.
      • et al.
      Subnormothermic machine perfusion at both 20 °C and 30 °C recovers ischemic rat livers for successful transplantation.
      ]. Using a hepatocyte cell culture medium as a perfusate, Bruinsma et al. [
      • Bruinsma B.G.
      • Yeh H.
      • Özer S.
      • Martins P.N.
      • Farmer A.
      • Wu W.
      • et al.
      Subnormothermic machine perfusion for ex vivo preservation and recovery of the human liver for transplantation.
      ] performed SNMP of seven human livers that were declined for transplantation. During 3 h of end-ischemic SNMP, increasing bile flow and biliary bicarbonate excretion were seen. Moreover, the ratio of bile salts to phospholipids in bile decreased over time, indicating improvement of hepatocellular secretory function and a decrease in bile toxicity. Unfortunately, this study had no control group or reperfusion phase; therefore it provides little substantiation for SNMP beyond proof of feasibility [
      • Bruinsma B.G.
      • Yeh H.
      • Özer S.
      • Martins P.N.
      • Farmer A.
      • Wu W.
      • et al.
      Subnormothermic machine perfusion for ex vivo preservation and recovery of the human liver for transplantation.
      ].
      Knaak et al. [
      • Knaak J.M.
      • Spetzler V.N.
      • Goldaracena N.
      • Boehnert M.U.
      • Bazerbachi F.
      • Louis K.S.
      • et al.
      Subnormothermic ex vivo liver perfusion reduces endothelial cell and bile duct injury after DCD pig liver transplantation.
      ] have performed liver transplantation after SNMP of DCD pig livers, comparing it to static cold storage. The three hour perfusion phase in this experiment, using Steen’s solution with washed red blood cells at 33 °C, was preceded by four hours, and followed by three hours of cold ischemia time. The investigators first studied bile output of the liver during eight hours of in situ reperfusion. Subsequently, ten animals (five vs. five) were transplanted and monitored for seven days. Lactate dehydrogenase in the bile was found to be significantly lower immediately after transplantation in the SNMP group. Moreover, cold stored grafts presented with substantial bile duct necrosis after seven days, but those that preserved with SNMP did not. This was also reflected in the serum levels of alkaline phosphatase during the survival period, which were significantly higher in the cold storage group. This study indicates that SNMP has protective effects on the bile ducts in a DCD model of pig liver transplantation.

      Normothermic machine perfusion

      Under normothermic conditions, the liver reaches a physiological metabolic rate and functionality. Sufficient oxygen delivery can only be achieved with the use of an oxygen carrier. This form of machine perfusion has been studied extensively in experimental models by the group of Friend and coworkers [

      Ravikumar R, Coussios CC, Holroyd D, Heaton N, Friend PJ, Jassem W. Human Liver transplantation using normothermic machine preservation; 2014; Available at: http://www.abstracts2view.com/ilts/view.php?nu=ILTS14L1_O-1&terms= [accessed 09.11.14].

      ,
      • Imber C.J.
      • St. Peter S.D.
      • De Cenarruzabeitia I.L.
      • Lemonde H.
      • Rees M.
      • Butler A.
      • et al.
      Optimisation of bile production during normothermic preservation of porcine livers.
      ,
      • St Peter S.D.
      • Imber C.J.
      • Lopez I.
      • Hughes D.
      • Friend P.J.
      Extended preservation of non-heart-beating donor livers with normothermic machine perfusion.
      ,
      • Reddy S.P.
      • Bhattacharjya S.
      • Maniakin N.
      • Greenwood J.
      • Guerreiro D.
      • Hughes D.
      • et al.
      Preservation of porcine non-heart-beating donor livers by sequential cold storage and warm perfusion.
      ] and enables ex situ viability testing of the liver [
      • op den Dries S.
      • Karimian N.
      • Sutton M.E.
      • Westerkamp A.C.
      • Nijsten M.W.N.
      • Gouw A.S.H.
      • et al.
      Ex vivo normothermic machine perfusion and viability testing of discarded human donor livers.
      ]. Some argue that it is superior to cold perfusion because it provides the closest resemblance to the normal physiological situation in vivo [
      • Hessheimer A.J.
      • Fondevila C.
      • García-Valdecasas J.C.
      Extracorporeal machine liver perfusion: are we warming up?.
      ]. However, bringing the organ up to ‘full metabolic speed’ also makes it more vulnerable. Interruption of perfusion or insufficient oxygen delivery will immediately expose the liver to warm ischemia. Remaining mindful of this risk, NMP can be safe and reliable when performed correctly [
      • Schön M.R.
      • Kollmar O.
      • Wolf S.
      • Schrem H.
      • Matthes M.
      • Akkoc N.
      • et al.
      Liver transplantation after organ preservation with normothermic extracorporeal perfusion.
      ,
      • Brockmann J.
      • Reddy S.
      • Coussios C.
      • Pigott D.
      • Guirriero D.
      • Hughes D.
      • et al.
      Normothermic perfusion: a new paradigm for organ preservation.
      ,
      • Fondevila C.
      • Hessheimer A.
      • Maathuis M.
      • Muñoz J.
      • Taurá P.
      • Calatayud D.
      • et al.
      Superior preservation of DCD livers with continuous normothermic perfusion.
      ,
      • Nassar A.
      • Liu Q.
      • Farias K.
      • D’Amico G.
      • Tom C.
      • Grady P.
      • et al.
      Ex vivo normothermic machine perfusion is safe, simple, and reliable: results from a large animal model.
      ].
      NMP appears to be the category of perfusion that provides the most pronounced effects on biliary preservation. In an interesting experiment using pig DCD livers, Liu et al. [
      • Liu Q.
      • Nassar A.
      • Farias K.
      • Buccini L.
      • Baldwin W.
      • Mangino M.
      • et al.
      Sanguineous normothermic machine perfusion improves hemodynamics and biliary epithelial regeneration in DCD porcine livers.
      ] assessed the effect of 10 h of NMP on biliary injury and regeneration during 24 h of ex vivo reperfusion. The study showed lower release of injury markers (gamma-glutamyltransferase and lactate dehydrogenase) in bile and increased biliary bicarbonate secretion in NMP preserved livers, compared to livers that underwent conventional 10 h of static cold storage. Moreover, NMP preserved livers displayed significantly less histological injury of the PBG and PVP, produced more bile, and demonstrated lower thrombogenicity of the microvasculature. Additionally, using Ki-67 staining, these investigators observed proliferation of cholangiocytes during 24 h of reperfusion of NMP preserved grafts, which was not seen in livers preserved by static cold storage. This study provides important evidence that 10 h of NMP yields superior results with regard to biliary injury and regeneration, compared to static cold storage [
      • Liu Q.
      • Nassar A.
      • Farias K.
      • Buccini L.
      • Baldwin W.
      • Mangino M.
      • et al.
      Sanguineous normothermic machine perfusion improves hemodynamics and biliary epithelial regeneration in DCD porcine livers.
      ]. It should however, be noted that this study did not include actual transplantation of machine-perfused livers and, therefore, the occurrence of NAS after transplantation was not assessed.
      Boehnert et al. [
      • Boehnert M.U.
      • Yeung J.C.
      • Bazerbachi F.
      • Knaak J.M.
      • Selzner N.
      • McGilvray I.D.
      • et al.
      Normothermic acellular ex vivo liver perfusion reduces liver and bile duct injury of pig livers retrieved after cardiac death.
      ] have published an experimental study showing extraordinary biliary preservation by NMP in a DCD pig model. Although their experiment offers new perspectives on NMP, such as through the use of CT angiography, this study have been criticized for its design and difficulty to explain the outcomes [
      • op den Dries S.
      • Karimian N.
      • Weeder P.D.
      • Porte R.J.
      Normothermic acellular machine perfusion and bile duct injury in pig livers retrieved after cardiac death.
      ,
      • Martins P.N.A.
      Normothermic machine preservation as an approach to decrease biliary complications of DCD liver grafts.
      ]. Our group was the first to publish on the feasibility of NMP in four discarded human livers using six hours of perfusion in an end-ischemic approach [
      • op den Dries S.
      • Karimian N.
      • Sutton M.E.
      • Westerkamp A.C.
      • Nijsten M.W.N.
      • Gouw A.S.H.
      • et al.
      Ex vivo normothermic machine perfusion and viability testing of discarded human donor livers.
      ]. During the six hours of NMP, bile duct histology remained well preserved and did not deteriorate. After expanding this series to a total of 12 livers, we found that bile production during NMP can be used as a predictor of graft function, enabling viability testing [
      • Sutton M.E.
      • op den Dries S.
      • Karimian N.
      • Weeder P.D.
      • de Boer M.T.
      • Wiersema-Buist J.
      • et al.
      Criteria for viability assessment of discarded human donor livers during ex-vivo normothermic machine perfusion.
      ]. This finding is in line with previous animal studies of NMP, which have recognized bile production as a critical parameter of viability of the perfused liver [
      • Imber C.J.
      • St. Peter S.D.
      • De Cenarruzabeitia I.L.
      • Lemonde H.
      • Rees M.
      • Butler A.
      • et al.
      Optimisation of bile production during normothermic preservation of porcine livers.
      ,
      • Schön M.R.
      • Kollmar O.
      • Wolf S.
      • Schrem H.
      • Matthes M.
      • Akkoc N.
      • et al.
      Liver transplantation after organ preservation with normothermic extracorporeal perfusion.
      ,
      • Xu H.
      • Berendsen T.
      • Kim K.
      • Soto-Gutiérrez A.
      • Bertheium F.
      • Yarmush M.L.
      • et al.
      Excorporeal normothermic machine perfusion resuscitates pig DCD livers with extended warm ischemia.
      ].
      Several (randomized) clinical trials have been initiated recently and hopefully these studies will provide evidence for the beneficial effects of machine perfusion on the incidence of biliary complications after liver transplantation. In one randomized controlled multicenter trial normothermic preservation perfusion will be compared with static cold storage (http://www.controlled-trials.com/ISRCTN39731134/). Another multicenter trial is investigating the possible protective effects of NRP in DCD donors (http://www.controlled-trials.com/ISRCTN89667087/).

      Controlled rewarming

      The abrupt change in temperature that occurs upon reperfusion during transplantation of a hypothermic liver is believed to contribute to reperfusion injury. Many research groups have explored machine perfusion strategies at a constant temperature, still exposing the liver to sudden temperature changes. Minor et al. studied the effect of controlled oxygenated rewarming (COR) of the liver from ice-cold to subnormothermic temperatures during perfusion and compared this method with HMP and SNP alone [
      • Minor T.
      • Efferz P.
      • Fox M.
      • Wohlschlaeger J.
      • Lüer B.
      Controlled oxygenated rewarming of cold stored liver grafts by thermally graduated machine perfusion prior to reperfusion.
      ]. In this experiment, pig livers were harvested and preserved by cold storage for 18 h, followed by 90 min of SNP, HMP or COR. The organs where subsequently re-perfused ex vivo normothermically with autologous blood to simulate transplantation. Energy charge restoration during COR was similar to SMP, but superior to HMP. In addition, COR was associated with significantly better hepatobiliary function (as indicated by bile production) and less injury upon reperfusion. A group from Tokyo has described similar results [
      • Shigeta T.
      • Matsuno N.
      • Obara H.
      • Kanazawa H.
      • Tanaka H.
      • Fukuda A.
      • et al.
      Impact of rewarming preservation by continuous machine perfusion: improved post-transplant recovery in pigs.
      ,
      • Matsuno N.
      • Obara H.
      • Watanabe R.
      • Iwata S.
      • Kono S.
      • Fujiyama M.
      • et al.
      Rewarming preservation by organ perfusion system for donation after cardiac death liver grafts in pigs.
      ]. Integrating controlled rewarming into perfusion protocols could potentially combine the best of cold and warm perfusion techniques and may potentially provide better protection of the bile ducts.

      Perfusate composition

      The composition of the perfusion fluid is guided primarily by the temperature, timing and duration of machine perfusion. Whereas at low temperatures a low-nutrient preservation solution may be used, increasing duration and temperature of perfusion necessitates more additives. Irrespective of these factors, oxygen delivery through the perfusate is a fundamental pillar of the advantages that machine perfusion has to offer to both the liver and the bile ducts [
      • Noack K.
      • Bronk S.F.
      • Kato A.
      • Gores G.J.
      The greater vulnerability of bile duct cells to reoxygenation injury than to anoxia. Implications for the pathogenesis of biliary strictures after liver transplantation.
      ,
      • Lüer B.
      • Koetting M.
      • Efferz P.
      • Minor T.
      Role of oxygen during hypothermic machine perfusion preservation of the liver.
      ,
      • Stegemann J.
      • Minor T.
      Energy charge restoration, mitochondrial protection and reversal of preservation induced liver injury by hypothermic oxygenation prior to reperfusion.
      ,
      • Schlegel A.
      • de Rougemont O.
      • Graf R.
      • Clavien P.
      • Dutkowski P.
      Protective mechanisms of end-ischemic cold machine perfusion in DCD liver grafts.
      ]. When oxygen consumption exceeds the delivery potential of dissolved oxygen in a watery fluid, red blood cells or an artificial oxygen carrier should be added [
      • Liu Q.
      • Nassar A.
      • Farias K.
      • Buccini L.
      • Baldwin W.
      • Mangino M.
      • et al.
      Sanguineous normothermic machine perfusion improves hemodynamics and biliary epithelial regeneration in DCD porcine livers.
      ,
      • op den Dries S.
      • Karimian N.
      • Sutton M.E.
      • Westerkamp A.C.
      • Nijsten M.W.N.
      • Gouw A.S.H.
      • et al.
      Ex vivo normothermic machine perfusion and viability testing of discarded human donor livers.
      ,
      • Bezinover D.
      • Ramamoorthy S.
      • Postula M.
      • Weller G.
      • Mahmoud S.
      • Mani H.
      • et al.
      Effect of cold perfusion and perfluorocarbons on liver graft ischemia in a donation after cardiac death model.
      ]. Because the hepatobiliary transporters that are responsible for bile formation are calcium dependent, it is important to titrate calcium to physiological levels for the optimization of biliary secretory function [
      • Trauner M.B.
      • James L.
      Bile salt transporters: molecular characterization, function, and regulation.
      ,
      • Vairetti M.
      • Ferrigno A.
      • Rizzo V.
      • Boncompagni E.
      • Carraro A.
      • Gringeri E.
      • et al.
      Correlation between the liver temperature employed during machine perfusion and reperfusion damage: role of Ca2+.
      ]. This is especially true when blood products are added to the perfusate, which usually contain calcium-scavenging agents such as citrate. Bile salts excreted by the liver during machine perfusion do no return to the perfusate, which normally happens through the enterohepatic circulation in vivo. Supplementation of bile salts is therefore necessary when applying machine perfusion for extended periods of time to ensure sustained bile production [
      • Imber C.J.
      • St. Peter S.D.
      • De Cenarruzabeitia I.L.
      • Lemonde H.
      • Rees M.
      • Butler A.
      • et al.
      Optimisation of bile production during normothermic preservation of porcine livers.
      ]. Another relevant aspect is the viscosity of the solution. Several studies have indicated that low-viscosity solutions may achieve superior perfusion of the peribiliary vasculature [
      • Moench C.
      • Moench K.
      • Lohse A.W.
      • Thies J.
      • Otto G.
      Prevention of ischemic-type biliary lesions by arterial back-table pressure perfusion.
      ,
      • Olschewski P.
      • Gaß P.
      • Ariyakhagorn V.
      • Jasse K.
      • Hunold G.
      • Menzel M.
      • et al.
      The influence of storage temperature during machine perfusion on preservation quality of marginal donor livers.
      ,
      • Feng L.
      • Zhao N.
      • Yao X.
      • Sun X.
      • Du L.
      • Diao X.
      • et al.
      Histidine-tryptophan-ketoglutarate solution vs. University of Wisconsin solution for liver transplantation: a systematic review.
      ,
      • Moench C.
      • Heimann A.
      • Foltys D.
      • Schneider B.
      • Minouchehr S.
      • Schwandt E.
      • et al.
      Flow and pressure during liver preservation under ex situ and in situ perfusion with University of Wisconsin solution and histidine-tryptophan-ketoglutarate solution.
      ]. However, the effect of low-viscosity cold flush solutions on biliary complications is controversial [
      • Stewart Z.A.
      • Cameron A.M.
      • Singer A.L.
      • Montgomery R.A.
      • Segev D.L.
      Histidine-Tryptophan-Ketoglutarate (HTK) is associated with reduced graft survival in deceased donor livers, especially those donated after cardiac death.
      ]. Other constituents of machine perfusion solutions may be oncotic agents (e.g. albumin), parenteral nutrition, antibiotics, insulin, heparin, and vasoactive drugs [
      • Liu Q.
      • Nassar A.
      • Farias K.
      • Buccini L.
      • Baldwin W.
      • Mangino M.
      • et al.
      Sanguineous normothermic machine perfusion improves hemodynamics and biliary epithelial regeneration in DCD porcine livers.
      ,
      • op den Dries S.
      • Karimian N.
      • Sutton M.E.
      • Westerkamp A.C.
      • Nijsten M.W.N.
      • Gouw A.S.H.
      • et al.
      Ex vivo normothermic machine perfusion and viability testing of discarded human donor livers.
      ,
      • Butler A.
      • Rees M.
      • Wight D.G.D.
      • Casey N.
      • Alexander G.
      • White D.J.G.
      • et al.
      Successful extracorporeal porcine liver perfusion for 72 h.
      ]. Machine perfusion also opens alleyways towards ex situ treatment of the liver to enhance the preservation even further. As these techniques are still speculative, they will be discussed as ‘future directions’.
      Many combinations of perfusion temperatures, perfusates, time points and durations have been explored. For a more detailed overview of particular configurations, we suggest further reading of specific reviews [
      • Schlegel A.
      • Dutkowski P.
      Role of hypothermic machine perfusion in liver transplantation.
      ,
      • Vogel T.
      • Brockmann J.G.
      • Coussios C.
      • Friend P.J.
      The role of normothermic extracorporeal perfusion in minimizing ischemia reperfusion injury.
      ,
      • García-Valdecasas J.C.
      • Fondevila C.
      In-vivo normothermic recirculation: an update.
      ,
      • Rubbini M.
      Perfusion machines for liver transplantation: technology and multifunctionality.
      ,
      • Graham J.A.
      • Guarrera J.V.
      “Resuscitation” of marginal liver allografts for transplantation with machine perfusion technology.
      ]. Which combination of these parameters provides optimal protection for the biliary tree currently remains unclear.

      Future directions

      Machine perfusion provides a platform that may facilitate the treatment of donor livers, uncovering specific targets that previously remained out of reach for the transplant surgeon. One possibility that quickly comes to mind is the addition of pharmacological agents to the perfusion fluid. Such interventions could be specifically targeted at factors that have been shown to play a role in the pathogenesis of NAS, such as bile composition, immunological factors, bicarbonate secretion, or even factors that may stimulate biliary epithelial regeneration.
      With the aim of reducing the Kupffer cell-mediated insult to the liver after transplantation, one group has administered gadolinium chloride (GdCl3) to rats in an autologous rat transplant model. This intervention reduced the amount of apoptosis observed in the bile duct after warm ischemia/reperfusion injury [
      • Wang B.
      • Zhang Q.
      • Zhu B.
      • Cui Z.
      • Zhou J.
      Protective effect of gadolinium chloride on early warm ischemia/reperfusion injury in rat bile duct during liver transplantation.
      ]. Another prospect could be the addition of hydrophilic bile salts or other compounds to stimulate a less cytotoxic bile composition [
      • Imber C.J.
      • St. Peter S.D.
      • De Cenarruzabeitia I.L.
      • Lemonde H.
      • Rees M.
      • Butler A.
      • et al.
      Optimisation of bile production during normothermic preservation of porcine livers.
      ].
      Finally, it has been proposed that the infusion of MSCs into the perfusion circuit could help restore a donor organ [
      • 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?.
      ]. Although the exact mechanisms are unknown, MSCs are believed to have anti-inflammatory and pro-regenerative effects mostly through the excretion of paracrine factors without requiring colonization [
      • English K.
      • Mahon B.
      Allogeneic mesenchymal stem cells: agents of immune modulation.
      ,
      • Lanzoni G.
      • Oikawa T.
      • Wang Y.
      • Cui C.
      • Carpino G.
      • Cardinale V.
      • et al.
      Concise review: clinical programs of stem cell therapies for liver and pancreas.
      ]. The clinical safety of MSC infusion in liver transplant recipients has been suggested, but the efficacy is still strongly debated [
      • Popp F.
      • Fillenberg B.
      • Eggenhofer E.
      • Renner P.
      • Dillmann J.
      • Benseler V.
      • et al.
      Safety and feasibility of third-party multipotent adult progenitor cells for immunomodulation therapy after liver transplantation – A phase I study (MISOT-I).
      ,
      • Franquesa M.
      • Hoogduijn M.
      • Reinders M.
      • Eggenhofer E.
      • Engela A.
      • Mensah F.
      • et al.
      Mesenchymal Stem Cells in Solid Organ Transplantation (MiSOT) Fourth Meeting: lessons learned from first clinical trials.
      ]. Machine perfusion provides an exceptional opportunity for the ex situ application of MSCs.

      Conclusion

      Liver machine perfusion comprises a spectrum of promising techniques of liver graft preservation that are currently making the transition into clinical practice. It is anticipated that the use of machine perfusion can lead to a significant increase of the availability of transplant livers as well as a reduction in postoperative complications such as early graft dysfunction and biliary complications. Existing data from animal models and preliminary data from (discarded) human donor livers provide promising evidence that machine perfusion has relevant protective effects on the bile ducts. In animal studies, liver machine perfusion did not only reduce the amount of injury to the bile ducts, but it also helped to restore the endogenous regenerative capacity of the biliary epithelium and bile duct wall. Whether this results in a decreased incidence of biliary complications, such as NAS, after clinical transplantation needs to be awaited. Randomized clinical trials are being initiated and will be necessary to demonstrate the efficacy of machine perfusion in clinical transplantation. Finally, machine perfusion provides a platform for the exploration of putative ex situ treatments, using interventions that are aimed at specific targets in the liver and the biliary system.

      Conflict of interest

      The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

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