Machine perfusion of human donor livers with a focus on the biliary tree

Machine perfusion of human donor livers offers the possibility to protect organs against damage and to select more carefully. The results of normothermic machine perfusion (NMP) based on human blood were presented. Subsequently, an NMP perfusion fluid was developed that circumvented the use of human blood products and was based on bovine hemoglobin (HBOC-201). HBOC-perfused livers showed better function than livers perfused with human blood products. HBOC-201 can also be used as an oxygen carrier at lower temperatures, allowing cold rescuscitation followed by gradual rewarming to NMP. This protocol was tested in seven livers that were initially rejected for transplantation, five of which were successfully transplanted. Subsequently, it was demonstrated that bicarbonate, pH, and glucose in bile are accurate predictors of histological biliary injury during NMP, which is important because biliary injury is correlated to biliary strictures prior to transplantation. Liver and bile duct-derived micro-RNAs in perfusate and bile were also tested. Early release of these micro-RNAs were able to predict late function and damage during NMP. Furthermore, the ability of various preservation fluids to protect against biliary injury was investigated. HTK solution led to higher biliary injury compared to UW solution, which has important clinical implications since HTK solution is used widely and livers with a high risk of biliary complications are increasingly being transplanted. Finally, the so-called ex vivo “precision-cut bile duct slices” model was developed to study human bile ducts, bypassing the use of laboratory animals. This was the first study to show that cells in peribiliary glands – niches of stem cells in the bile duct wall that play a crucial role in the development of biliary strictures - respond with proliferation, migration and differentiation to restore biliary epithelium after biliary damage

Peribiliary glands are key in regeneration of the human biliary epithelium after severe bile duct injury

Peribiliary glands (PBG) are a source of stem/progenitor cells organized in a cellular network encircling large bile ducts. Severe cholangiopathy with loss of luminal biliary epithelium has been proposed to activate PBG, resulting in cell proliferation and differentiation to restore biliary epithelial integrity. However, formal evidence for this concept in human livers is lacking. We, therefore, developed a novel ex vivo model using precision-cut slices of extrahepatic human bile ducts obtained from discarded donor livers, providing an intact anatomical organization of cell structures, to study spatiotemporal differentiation and migration of PBG cells after severe biliary injury. Post-ischemic bile duct slices were incubated in oxygenated culture medium for up to a week. At baseline, severe tissue injury was evident with loss of luminal epithelial lining and mural stroma necrosis. In contrast, PBG remained relatively well preserved and different reactions of PBG were noted, including PBG dilatation, cell proliferation and maturation. Proliferation of PBG cells increased after 24 h of oxygenated incubation, reaching a peak after 72 h. Proliferation of PBG cells was paralleled by a reduction in PBG apoptosis and differentiation from a primitive and pluripotent (Nanog+/Sox9+) to a mature (CFTR+/secretin receptor+) and activated phenotype (increased expression of HIF-1α, Glut-1, and VEGF-A). Migration of proliferating PBG cells in our ex vivo model was unorganized, but resulted in generation of epithelial monolayers at stromal surfaces. CONCLUSION: Human PBG contain biliary progenitor cells and are able to respond to bile duct epithelial loss with proliferation, differentiation, and maturation to restore epithelial integrity. The ex vivo spatiotemporal behaviour of human PBG cells provides evidence for a pivotal role of PBG in biliary regeneration after severe injury. This article is protected by copyright. All rights reserved

Cell-free microRNAs as early predictors of graft viability during ex vivo normothermic machine perfusion of human donor livers

Background Cell-free microRNAs (miRs) have emerged as early and sensitive biomarkers for tissue injury and function. This study aimed to investigate whether the release of hepatocyte-derived microRNAs (HDmiRs) and cholangiocyte-derived miRs (CDmiRs) correlates with hepato-cholangiocellular injury and function during oxygenated, normothermic machine perfusion (NMP) of human liver grafts. Methods Donor livers (n = 12), declined for transplantation, were subjected to oxygenated NMP (6 hours) after a period of static cold storage (median 544 minutes (IQR 421-674)). Perfusate and bile samples were analyzed by qRT-PCR for HDmiR-122 and CDmiR-222. Spearman correlations were performed between miR levels and currently available indicators and classic markers. Results Both HDmiR-122 and CDmiR-222 levels in perfusate at 30 minutes of NMP strongly correlated with hepatocyte injury (peak perfusate AST) and cholangiocyte injury (peak biliary LDH). In bile, only CDmiR-222 correlated with these injury markers. For hepato-cholangiocellular function, both miRs in perfusate correlated with total bilirubin, while HDmiR-122 (in perfusate) and CDmiR-222 (in bile) correlated with bicarbonate secretion. Both the relative ratio of HDmiR-122/CDmiR-222 and AST in perfusate at 30 minutes significantly correlated with cumulative bile production, but only the relative ratio was predictive of histopathological injury after 6 hours NMP. Conclusion Early levels of HDmiR-122 and CDmiR-222, in perfusate and/or bile, are predictive of excretory functions and hepato-cholangiocellular injury after 6 hours NMP. These miRs may represent new biomarkers for graft viability and function during machine perfusion

Cell-free microRNAs as early predictors of graft viability during ex vivo normothermic machine perfusion of human donor livers

Background: Cell-free microRNAs (miRs) have emerged as early and sensitive biomarkers for tissue injury and function. This study aimed to investigate whether the release of hepatocyte-derived microRNAs (HDmiRs) and cholangiocyte-derived miRs (CDmiRs) correlates with hepato-cholangiocellular injury and function during oxygenated, normothermic machine perfusion (NMP) of human liver grafts. Methods: Donor livers (n = 12), declined for transplantation, were subjected to oxygenated NMP (6 hours) after a period of static cold storage (median 544 minutes (IQR 421-674)). Perfusate and bile samples were analyzed by qRT-PCR for HDmiR-122 and CDmiR-222. Spearman correlations were performed between miR levels and currently available indicators and classic markers. Results: Both HDmiR-122 and CDmiR-222 levels in perfusate at 30 minutes of NMP strongly correlated with hepatocyte injury (peak perfusate AST) and cholangiocyte injury (peak biliary LDH). In bile, only CDmiR-222 correlated with these injury markers. For hepato-cholangiocellular function, both miRs in perfusate correlated with total bilirubin, while HDmiR-122 (in perfusate) and CDmiR-222 (in bile) correlated with bicarbonate secretion. Both the relative ratio of HDmiR-122/CDmiR-222 and AST in perfusate at 30 minutes significantly correlated with cumulative bile production, but only the relative ratio was predictive of histopathological injury after 6 hours NMP. Conclusion: Early levels of HDmiR-122 and CDmiR-222, in perfusate and/or bile, are predictive of excretory functions and hepato-cholangiocellular injury after 6 hours NMP. These miRs may represent new biomarkers for graft viability and function during machine perfusion

Opposite acute potassium and sodium shifts during transplantation of hypothermic machine perfused donor livers

Liver transplantation is frequently associated with hyperkalemia, especially after graft reperfusion. Dual hypothermic oxygenated machine perfusion (DHOPE) reduces ischemia/reperfusion injury and improves graft function, compared to conventional static cold storage (SCS). We examined the effect of DHOPE on ex situ and in vivo shifts of potassium and sodium. Potassium and sodium shifts were derived from balance measurements in a preclinical study of livers that underwent DHOPE (n = 6) or SCS alone (n = 9), followed by ex situ normothermic reperfusion. Similar measurements were performed in a clinical study of DHOPE-preserved livers (n = 10) and control livers that were transplanted after SCS only (n = 9). During DHOPE, preclinical and clinical livers released a mean of 17 +/- 2 and 34 +/- 6 mmol potassium and took up 25 +/- 9 and 24 +/- 14 mmol sodium, respectively. After subsequent normothermic reperfusion, DHOPE-preserved livers took up a mean of 19 +/- 3 mmol potassium, while controls released 8 +/- 5 mmol potassium. During liver transplantation, blood potassium levels decreased upon reperfusion of DHOPE-preserved livers while levels increased after reperfusion of SCS-preserved liver, delta potassium levels were -0.77 +/- 0.20 vs. +0.64 +/- 0.37 mmol/L, respectively (P = .002). While hyperkalemia is generally anticipated during transplantation of SCS-preserved livers, reperfusion of hypothermic machine perfused livers can lead to decreased blood potassium or even hypokalemia in the recipient

Hypothermic oxygenated machine perfusion reduces bile duct reperfusion injury after transplantation of donation after circulatory death livers

INTRODUCTION: Dual hypothermic oxygenated machine perfusion (DHOPE) of the liver has been advocated as a method to reduce ischemia-reperfusion injury. This study aimed to determine whether DHOPE reduces IR injury of the bile ducts in DCD liver transplantation. MATERIALS AND METHODS: In a recently performed phase 1-trial, ten DCD livers were preserved with DHOPE after static cold storage (SCS) (www.trialregister.nl NTR4493). Bile duct biopsies were obtained at the end of SCS (before DHOPE; baseline) and after graft reperfusion in the recipient. Histological severity of biliary injury was graded according to an established semi-quantitative grading system. Twenty liver transplantations using DCD livers not preserved with DHOPE served as control. RESULTS: Baseline characteristics and the degree of bile duct injury at baseline (end of SCS) were similar between both groups. In controls, degree of stroma necrosis (P=0.002) and injury of the deep peribiliary glands (P=0.02) increased after reperfusion, compared to baseline. In contrast, in DHOPE preserved livers the degree of bile duct injury did not increase after reperfusion. Moreover, there was less injury of deep peribiliary glands (P=0.04) after reperfusion in the DHOPE group, compared to controls. CONCLUSION: This study suggests that DHOPE reduces ischemia-reperfusion injury of bile ducts after DCD liver transplantation. This article is protected by copyright. All rights reserved

Pretransplant sequential hypo- and normothermic machine perfusion of suboptimal livers donated after circulatory death using a hemoglobin-based oxygen carrier perfusion solution

Ex situ dual hypothermic oxygenated machine perfusion (DHOPE) and normothermic machine perfusion (NMP) of donor livers may have a complementary effect when applied sequentially. While DHOPE resuscitates the mitochondria and increases hepatic adenosine triphosphate (ATP) content, NMP enables hepatobiliary viability assessment prior to transplantation. In contrast to DHOPE, NMP requires a perfusion solution with an oxygen carrier, for which red blood cells (RBC) have been used in most series. RBC, however, have limitations and cannot be used cold. We, therefore, established a protocol of sequential DHOPE, controlled oxygenated rewarming (COR), and NMP using a new hemoglobin-based oxygen carrier (HBOC)-based perfusion fluid (DHOPE-COR-NMP trial, NTR5972). Seven livers from donation after circulatory death (DCD) donors, which were initially declined for transplantation nationwide, underwent DHOPE-COR-NMP. Livers were considered transplantable if perfusate pH and lactate normalized, bile production was >= 10 mL and biliary pH > 7.45 within 150 minutes of NMP. Based on these criteria five livers were transplanted. The primary endpoint, 3-month graft survival, was a 100%. In conclusion, sequential DHOPE-COR-NMP using an HBOC-based perfusion fluid offers a novel method of liver machine perfusion for combined resuscitation and viability testing of suboptimal livers prior to transplantation

A Start on the Development of Near-infrared Fluorescence Imaging of Hilar Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an aggressive cancer arising from the biliary epithelial cells. The hilar type, which occurs at the confluence of the right and left hepatic ducts, is the most common type and carries the worst prognosis. The only potentially curative treatment is surgery, and even then the 5-year survival rate is only 11-40%. Obtaining tumor negative resection margins during surgery increases the survival, though this can often not be achieved. This is due to the invasive growth characteristics of hilar CCA, with perineural- and angio-invasion, and extensive desmoplastic reactions that form around the tumor. A technique that would help surgeons in locating the tumor and obtaining tumor negative margins would be intraoperative near-infrared fluorescence (NIRF) imaging. This is a rapidly developing technique and involves the use of specifically targeted markers attached to a fluorophore, which subsequently make the tumor fluoresce under near-infrared light. This study made a start on the development of NIRF imaging in hilar CCA by searching the literature for potential markers and testing the expression of four of these (EGFR, CEA, Her2 and MUC1) in 29 resected cases of hilar CCA by means of immunohistochemistry. EGFR, CEA, Her2 and MUC1 expression in the tumors were 62% (mostly weak), 0%, 24% (mostly weak), and MUC1 (mostly strong), respectively. EGFR expression was also found in the stroma (17%, mostly weak) and in the liver parenchyma (100%, mostly strong), CEA in none of the surrounding tissue, Her2 in the stroma (7%, weak) and liver parenchyma (25%, weak), and MUC1 in the stroma (3%, weak) and none in the liver parenchyma. MUC1 also stained healthy bile ducts in 92% (mostly weak) of cases. MUC1 appears to be our most promising target for NIRF imaging given the very high and mostly strong expression rate of MUC1 in hilar CCA tumors, with no to low expression in non-cancerous surrounding tissue. Furthermore, MUC1 is an apical surface protein that has been successfully imaged in vivo in the past. If further research supports the potential value of MUC1 as a target, it would benefit from the availability of an already clinically approved anti-MUC1 antibody (pemtumomab).

Production of Physiologically Relevant Quantities of Hemostatic Proteins During Ex Situ Normothermic Machine Perfusion of Human Livers

Ex-situ normothermic machine perfusion (NMP) of donor livers is an emerging innovation in liver transplantation that has made its transition to clinical trials. NMP is applied in an effort to overcome ischemia-reperfusion injury associated with static cold storage, to improve the quality of sub-optimally functioning donor organs and to permit viability testing of potentially transplantable livers. The in-vivo physiological conditions maintained during ex-situ NMP necessitate the need for a perfusion fluid that mimics the composition of whole blood. To date, perfusion fluids used in NMP studies are composed of either plasma-based solutions or plasma-free solutions consisting of colloids such as Gelofusine1 . The latter avoids the use of human plasma which is scarce, costly and logistically challenging. This article is protected by copyright. All rights reserved