Cholangiocyte Function Research Articles

A reproducible extended ex-vivo normothermic machine liver perfusion protocol utilising improved nutrition and targeted vascular flows

BackgroundNormothermic machine perfusion of donor livers has become standard practice in the field of transplantation, allowing the assessment of organs and safe extension of preservation times. Alongside its clinical uses, there has been expanding interest in extended normothermic machine perfusion (eNMP) of livers as a potential vehicle for medical research. Reproducible extended normothermic machine perfusion has remained elusive due to its increased complexity and monitoring requirements. We set out to develop a reproducible protocol for the extended normothermic machine perfusion of whole human livers.MethodsHuman livers declined for transplantation were perfused using a blood-based perfusate at 36 °C using the Liver Assist device (XVIVO, Sweden), with continuous veno-venous haemofiltration in-parallel. We developed the protocol in a stepwise fashion.ResultsPerfusion techniques utilised included: targeted physiological vascular flows, phosphate replacement (to prevent hypophosphataemia), N-acetylcysteine (to prevent methaemoglobin accumulation), and the utilisation of sodium lactate as both a nutritional source and real-time measure of hepatocyte function. All five human livers perfused with the developed protocol showed preserved function with a median perfusion time of 168 h (range 120–184 h), with preserved viability throughout.ConclusionsLivers can be reproducibly perfused in excess of 120 (range 121–184) hours with evidence of preserved hepatocyte and cholangiocyte function.

Single-Cell Transcriptomic Profiling of Cholangiocyte Organoids Derived from Bile Ducts of Primary Sclerosing Cholangitis Patients

Background and AimsPrimary sclerosing cholangitis (PSC) is a chronic inflammatory liver disorder without effective medical treatment which is characterized by inflammation and fibrotic structures around the bile ducts. Biliary epithelial cells (cholangiocytes) are the target and potential disease drivers in PSC, yet little is known if cholangiocytes from PSC patients differ from non-PSC controls. To characterize cholangiocytes at early rather than end-stage disease, cholangiocyte organoids (COs) were derived from diseased bile ducts of PSC patients and compared to organoids generated from disease controls.MethodsCholangiocytes were obtained during endoscopic retrograde cholangiopancreatography (ERCP) brushing of diseased bile duct areas and expanded as organoids using previously established culture methods. Stable CO lines were analyzed for cell type identity, basic cholangiocyte function, and transcriptomic signature.ResultsWe demonstrate that cholangiocytes, derived from the damaged area within the bile ducts of PSC patients, can be expanded in culture without displaying functional or genetic disease-related features. We further show that COs from patients who later were diagnosed with dysplasia exhibit higher expression of the cancer-associated genes PGC, FXYD2, MIR4435-2HG, and HES1.ConclusionsOur results demonstrate that PSC organoids are largely similar to control organoids after culture and highlight the significance of COs as a tool for regenerative medicine approaches as well as their potential for discovering new potential biomarkers for diagnosing cholangiocarcinoma.

Three-dimensional human bile duct formation from chemically induced human liver progenitor cells.

Background: The intrahepatic bile ducts (BDs) play an important role in the modification and transport of bile, and the integration between the BD and hepatocytes is the basis of the liver function. However, the lack of a source of cholangiocytes limits in vitro research. The aim of the present study was to establish three-dimensional BDs combined with human mature hepatocytes (hMHs) in vitro using chemically induced human liver progenitor cells (hCLiPs) derived from hMHs. Methods: In this study, we formed functional BDs from hCLiPs using hepatocyte growth factor and extracellular matrix. BDs expressed the typical biliary markers CK-7, GGT1, CFTR and EpCAM and were able to transport the bile-like substance rhodamine 123 into the lumen. The established three-dimensional BDs were cocultured with hMHs. These cells were able to bind to the BDs, and the bile acid analog CLF was transported from the culture medium through the hMHs and accumulated in the lumen of the BDs. The BDs generated from the hCLiPs showed a BD function and a physiological system (e.g., the transport of bile within the liver) when they were connected to the hMHs. Conclusion: We present a novel in vitro three-dimensional BD combined with hMHs for study, drug screening and the therapeutic modulation of the cholangiocyte function.

Proteomic Modulation in TGF-β-Treated Cholangiocytes Induced by Curcumin Nanoparticles.

Curcumin is a natural polyphenol that exhibits a variety of beneficial effects on health, including anti-inflammatory, antioxidant, and hepato-protective properties. Due to its poor water solubility and membrane permeability, in the present study, we prepared and characterized a water-stable, freely dispersible nanoformulation of curcumin. Although the potential of curcumin nanoformulations in the hepatic field has been studied, there are no investigations on their effect in fibrotic pathological conditions involving cholangiocytes. Exploiting an in vitro model of transforming growth factor-β (TGF-β)-stimulated cholangiocytes, we applied the Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS)-based quantitative proteomic approaches to study the proteome modulation induced by curcumin nanoformulation. Our results confirmed the well-documented anti-inflammatory properties of this nutraceutic, highlighting the induction of programmed cell death as a mechanism to counteract the cellular damages induced by TGF-β. Moreover, curcumin nanoformulation positively influenced the expression of several proteins involved in TGF-β-mediated fibrosis. Given the crucial importance of deregulated cholangiocyte functions during cholangiopathies, our results provide the basis for a better understanding of the mechanisms associated with this pathology and could represent a rationale for the development of more targeted therapies.

Finite volume simulation of calcium distribution in a cholangiocyte cell

Cholangiocytes are the cells of the liver having a major role in the conditioning of bile used in digestion. Other functions of cholangiocytes are in apoptosis and bicarbonate secretion. The Calcium in the intracellular environment of various cells including cholangiocytes regulates a large number of functions. This regulating mechanism in cholangiocytes has been poorly understood to date. In order to analyze the calcium regulation in cholangiocyte cells, a mathematical model for a one-dimensional steady-state case is constructed in this study. This involves a non-linear reaction-diffusion equation with appropriate boundary conditions. The influx from IP$_{3}$ receptor, ryanodine receptor (RYR), and plasma membrane as well as the efflux of calcium from SERCA pump and plasma membrane have been employed in the model. The finite volume method and Newton-Raphson method have been used to solve the problem. Numerical findings have been used to examine the effects of parameters like diffusion coefficient, rate of SERCA pump efflux, buffer, and influx from plasma membrane on calcium concentration in cholangiocyte cells. The information generated from the model can be useful for understanding the mechanism of cholestatic disorders which can be further useful in the diagnosis and treatment of these disorders.

Bile Acids and Biliary Fibrosis.

Biliary fibrosis is the driving pathological process in cholangiopathies such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). Cholangiopathies are also associated with cholestasis, which is the retention of biliary components, including bile acids, in the liver and blood. Cholestasis may worsen with biliary fibrosis. Furthermore, bile acid levels, composition and homeostasis are dysregulated in PBC and PSC. In fact, mounting data from animal models and human cholangiopathies suggest that bile acids play a crucial role in the pathogenesis and progression of biliary fibrosis. The identification of bile acid receptors has advanced our understanding of various signaling pathways involved in regulating cholangiocyte functions and the potential impact on biliary fibrosis. We will also briefly review recent findings linking these receptors with epigenetic regulatory mechanisms. Further detailed understanding of bile acid signaling in the pathogenesis of biliary fibrosis will uncover additional therapeutic avenues for cholangiopathies.

Ultrasound-Targeted Microbubble Cavitation During Machine Perfusion Reduces Microvascular Thrombi and Graft Injury in a Rat Liver Model of Donation After Circulatory Death

Ischemic cholangiopathy is a process of bile duct injury that might result from peribiliary vascular plexus (PBP) thrombosis and remains a dreaded complication in liver transplantation from donors after circulatory death (DCD). The aim of this study was to propose a mechanical method of clot destruction to clear microvascular thrombi in DCD livers before transplantation. Sonothrombolysis (STL) is a process by which inertial cavitation of circulating microbubbles entering an ultrasound field create a high-energy shockwave at a microbubble-thrombus interface, causing mechanical clot destruction. The effectiveness of STL in DCD liver treatment remains unclear. We carried out STL treatment during normothermic, oxygenated, ex vivo machine perfusion (NMP), introducing microbubbles into the perfusate with the liver enveloped in an ultrasound field. The STL livers showed reduction in hepatic arterial and PBP thrombus and decreases in hepatic arterial and portal venous flow resistance, reduced parenchymal injury as measured by aspartate transaminase release and oxygen consumption, and improved cholangiocyte function. Light and electron microscopy showed reduction of hepatic arterial and PBP thrombus in STL livers compared with controls and preserved hepatocyte structure, sinusoid endothelial morphology, and biliary epithelial microvilli. In this model, STL improved flow and functional measures in DCD livers undergoing NMP. These data suggest a novel therapeutic approach to treat PBP injury in DCD livers, which may ultimately increase the pool of grafts available to patients awaiting liver transplantation.

Predicting Early Allograft Function After Normothermic Machine Perfusion.

Normothermic ex situ liver perfusion is increasingly used to assess donor livers, but there remains a paucity of evidence regarding criteria upon which to base a viability assessment or criteria predicting early allograft function. Perfusate variables from livers undergoing normothermic ex situ liver perfusion were analyzed to see which best predicted the Model for Early Allograft Function score. One hundred fifty-four of 203 perfused livers were transplanted following our previously defined criteria. These comprised 84/123 donation after circulatory death livers and 70/80 donation after brain death livers. Multivariable analysis suggested that 2-h alanine transaminase, 2-h lactate, 11 to 29 mmol supplementary bicarbonate in the first 4 h, and peak bile pH were associated with early allograft function as defined by the Model for Early Allograft Function score. Nonanastomotic biliary strictures occurred in 11% of transplants, predominantly affected first- and second-order ducts, despite selection based on bile glucose and pH. This work confirms the importance of perfusate alanine transaminase and lactate at 2-h, as well as the amount of supplementary bicarbonate required to keep the perfusate pH > 7.2, in the assessment of livers undergoing perfusion. It cautions against the use of lactate as a sole indicator of viability and also suggests a role for cholangiocyte function markers in predicting early allograft function.

Molecular determinants of peri-apical targeting of inositol 1,4,5-trisphosphate receptor type 3 in cholangiocytes.

Fluid and bicarbonate secretion is a principal function of cholangiocytes, and impaired secretion results in cholestasis. Cholangiocyte secretion depends on peri‐apical expression of the type 3 inositol trisphosphate receptor (ITPR3), and loss of this intracellular Ca2+ release channel is a final common event in most cholangiopathies. Here we investigated the mechanism by which ITPR3 localizes to the apical region to regulate secretion. Isolated bile duct units, primary mouse cholangiocytes, and polarized Madin‐Darby canine kidney (MDCK) cells were examined using a combination of biochemical and fluorescence microscopy techniques to investigate the mechanism of ITPR3 targeting to the apical region. Apical localization of ITPR3 depended on the presence of intact lipid rafts as well as interactions with both caveolin 1 (CAV1) and myosin heavy chain 9 (MYH9). Chemical disruption of lipid rafts or knockdown of CAV1 or MYH9 redistributed ITPR3 away from the apical region. MYH9 interacted with the five c‐terminal amino acids of the ITPR3 peptide. Disruption of lipid rafts impaired Ca2+ signaling, and absence of CAV1 impaired both Ca2+ signaling and fluid secretion. Conclusion: A cooperative mechanism involving MYH9, CAV1, and apical lipid rafts localize ITPR3 to the apical region to regulate Ca2+ signaling and secretion in cholangiocytes.

Human Cholangiocytes Form a Polarized and Functional Bile Duct on Hollow Fiber Membranes.

Liver diseases affect hundreds of millions of people worldwide; most often the hepatocytes or cholangiocytes are damaged. Diseases of the biliary tract cause severe patient burden, and cholangiocytes, the cells lining the biliary tract, are sensitive to numerous drugs. Therefore, investigations into proper cholangiocyte functions are of utmost importance, which is restricted, in vitro, by the lack of primary human cholangiocytes allowing such screening. To investigate biliary function, including transepithelial transport, cholangiocytes must be cultured as three-dimensional (3D) ductular structures. We previously established murine intrahepatic cholangiocyte organoid-derived cholangiocyte-like cells (CLCs) and cultured them onto polyethersulfone hollow fiber membranes (HFMs) to generate 3D duct structures that resemble native bile ducts at the structural and functional level. Here, we established an efficient, stepwise method for directed differentiation of human intrahepatic cholangiocyte organoids (ICOs) into CLCs. Human ICO-derived CLCs showed key characteristics of cholangiocytes, such as the expression of structural and functional markers, formation of primary cilia, and P-glycoprotein-mediated transport in a polarized fashion. The organoid cultures exhibit farnesoid X receptor (FXR)-dependent functions that are vital to liver bile acid homeostasis in vivo. Furthermore, human ICO-derived CLCs cultured on HFMs in a differentiation medium form tubular architecture with some tight, confluent, and polarized monolayers that better mimic native bile duct characteristics than differentiated cultures in standard 2D or Matrigel-based 3D culture plates. Together, our optimized differentiation protocol to obtain CLC organoids, when applied on HFMs to form bioengineered bile ducts, will facilitate studying cholangiopathies and allow developing therapeutic strategies.

Normothermic Machine Perfusion—Improving the Supply of Transplantable Livers for High-Risk Recipients

The effectiveness of liver transplantation to cure numerous diseases, alleviate suffering, and improve patient survival has led to an ever increasing demand. Improvements in preoperative management, surgical technique, and postoperative care have allowed increasingly complicated and high-risk patients to be safely transplanted. As a result, many patients are safely transplanted in the modern era that would have been considered untransplantable in times gone by. Despite this, more gains are possible as the science behind transplantation is increasingly understood. Normothermic machine perfusion of liver grafts builds on these gains further by increasing the safe use of grafts with suboptimal features, through objective assessment of both hepatocyte and cholangiocyte function. This technology can minimize cold ischemia, but prolong total preservation time, with particular benefits for suboptimal grafts and surgically challenging recipients. In addition to more physiological and favorable preservation conditions for grafts with risk factors for poor outcome, the extended preservation time benefits operative logistics by allowing a careful explant and complicated vascular reconstruction when presented with challenging surgical scenarios. This technology represents a significant advancement in graft preservation techniques and the transplant community must continue to incorporate this technology to ensure the benefits of liver transplant are maximized.

Primary Sclerosing Cholangitis and Inflammatory Bowel Disease: A Review.

Primary sclerosing cholangitis is a disease affecting around 0.006–0.016% of the population. Of these, around 75% have concomitant inflammatory bowel disease (IBD) according to the most recent epidemiological studies. Several theories have been proposed regarding the pathogenesis of primary sclerosing cholangitis (PSC). These include changes in the function of cholangiocytes, effects of the gut microbiome, association with specific human leukocyte antigen haplotypes and dysregulation of the immune system. However, these do not explain the observed association with IBD. Moreover, there are considerable differences in the frequency and outcomes between patients with PSC and ulcerative colitis compared with PSC and Crohn’s disease. The aim of this review is to appraise the most recent studies that have contributed to the epidemiology, advances in the pathophysiology, and characterization of important clinical aspects of the association of PSC and IBD.

Piezo1-Pannexin1 complex couples force detection to ATP secretion in cholangiocytes.

Cholangiocytes actively contribute to the final composition of secreted bile. These cells are exposed to abnormal mechanical stimuli during obstructive cholestasis, which has a deep impact on their function. However, the effects of mechanical insults on cholangiocyte function are not understood. Combining gene silencing and pharmacological assays with live calcium imaging, we probed molecular candidates essential for coupling mechanical force to ATP secretion in mouse cholangiocytes. We show that Piezo1 and Pannexin1 are necessary for eliciting the downstream effects of mechanical stress. By mediating a rise in intracellular Ca2+, Piezo1 acts as a mechanosensor responsible for translating cell swelling into activation of Panx1, which triggers ATP release and subsequent signal amplification through P2X4R. Co-immunoprecipitation and pull-down assays indicated physical interaction between Piezo1 and Panx1, which leads to stable plasma membrane complexes. Piezo1-Panx1-P2X4R ATP release pathway could be reconstituted in HEK Piezo1 KO cells. Thus, our data suggest that Piezo1 and Panx1 can form a functional signaling complex that controls force-induced ATP secretion in cholangiocytes. These findings may foster the development of novel therapeutic strategies for biliary diseases.

Viability Assessment in Liver Transplantation-What Is the Impact of Dynamic Organ Preservation?

Based on the continuous increase of donor risk, with a majority of organs classified as marginal, quality assessment and prediction of liver function is of utmost importance. This is also caused by the notoriously lack of effective replacement of a failing liver by a device or intensive care treatment. While various parameters of liver function and injury are well-known from clinical practice, the majority of specific tests require prolonged diagnostic time and are more difficult to assess ex situ. In addition, viability assessment of procured organs needs time, because the development of the full picture of cellular injury and the initiation of repair processes depends on metabolic active tissue and reoxygenation with full blood over several hours or days. Measuring injury during cold storage preservation is therefore unlikely to predict the viability after transplantation. In contrast, dynamic organ preservation strategies offer a great opportunity to assess organs before implantation through analysis of recirculating perfusates, bile and perfused liver tissue. Accordingly, several parameters targeting hepatocyte or cholangiocyte function or metabolism have been recently suggested as potential viability tests before organ transplantation. We summarize here a current status of respective machine perfusion tests, and report their clinical relevance.

Bile Biochemistry Following Liver Reperfusion in the Recipient and Its Association With Cholangiopathy.

Cholangiocytes secrete bicarbonate and absorb glucose, producing bile with alkaline pH and low glucose content. These functions of cholangiocytes have been suggested as a marker of bile duct viability during normothermic ex situ liver perfusion, and they are now monitored routinely after reperfusion in our center. In this study, we reviewed the composition of bile immediately after reperfusion in liver transplant recipients to determine normal posttransplant parameters and the predictive value of bile biochemistry for the later development of cholangiopathy. After reperfusion of the liver graft, a cannula was placed in the bile duct to collect bile over a median 44‐minute time period. The bile produced was analyzed using a point‐of‐care blood gas analyzer (Cobas b221, Roche Diagnostics, Indianapolis, IN). A total of 100 liver transplants (35 from donation after circulatory death and 65 from donation after brain death) were studied. Median bile pH was 7.82 (interquartile range [IQR], 7.67‐7.98); median bile glucose was 2.1 (1.4‐3.7) mmol/L; median blood‐bile‐blood pH difference was 0.50 (0.37‐0.62); and median blood‐bile glucose difference was 7.1 (5.6‐9.1) mmol/L. There were 12 recipients who developed cholangiopathy over a median follow‐up of 15 months (IQR, 11‐20 months). Bile sodium (142 versus 147 mmol/L; P = 0.02) and blood‐bile glucose concentration differences (5.2 versus 7.6 mmol/L; P = 0.001) were significantly lower and were associated with ischemic cholangiopathy. In conclusion, bile biochemistry may provide useful insights into cholangiocyte function and, hence, bile duct viability. Our results suggest bile glucose is the most sensitive predictor of cholangiopathy.

Extended hypothermic oxygenated machine perfusion enables ex situ preservation of porcine livers for up to 24 hours.

Background & AimsEnd-ischemic hypothermic oxygenated machine perfusion (HOPE) of the donor liver for 1–2 h mitigates ischemia-reperfusion injury during subsequent liver transplantation. Extended preservation time may be preferred to facilitate difficult recipient hepatectomy or to optimize logistics. We therefore investigated whether end-ischemic dual HOPE (DHOPE) could extend preservation time for up to 24 h using a porcine liver reperfusion model.MethodsFollowing 30 min warm ischemia, porcine livers were subjected to 2 h static cold storage (SCS), followed by 2 h, 6 h, or 24 h DHOPE (n = 6 per group). Subsequent normothermic reperfusion was performed for 4 h using autologous blood. Two livers preserved by 24 h SCS served as additional controls. A proof of principle confirmation was carried out in 2 discarded human livers subjected to extended DHOPE. Hepatocellular and cholangiocyte injury and function were assessed. Oxidative stress levels and histology were compared between groups.ResultsPerfusion flows remained stable during DHOPE, regardless of duration. After normothermic reperfusion, livers perfused for 24 h by DHOPE had similar lactate clearance, blood pH, glucose, and alanine aminotransferase levels, and biliary pH, bicarbonate, and LDH levels, as livers perfused for 2 h and 6 h. Levels of malondialdehyde and high-mobility group box 1 in serum and liver parenchyma were similar for all groups. Histological analysis of bile ducts and liver parenchyma revealed no differences between the groups. Extended DHOPE in discarded human livers preserved hepatocellular and cholangiocyte function and histology after reperfusion. In contrast, livers preserved by 24 h SCS were non-functioning.ConclusionExtended end-ischemic DHOPE enabled successful preservation of porcine and discarded human donor livers for up to 24 h. Extended DHOPE enables safe extension of preservation time, which may facilitate allocation and transplantation from a logistical perspective, and further expand the donor pool.Lay summaryIt has been suggested that preserving liver grafts with a technique called (dual) hypothermic oxygenated machine perfusion ([D]HOPE) leads to better outcomes after transplantation than if livers are stored on ice, especially if an organ is of lesser quality. In this study, we showed that DHOPE could be used to preserve liver grafts for up to 24 h. This extended procedure could be used globally to facilitate transplantation and expand the donor pool.

Pluripotent stem cell-derived cholangiocytes and cholangiocyte organoids.

The development of protocols for pluripotent stem cell (PSC) differentiation into cholangiocytes and cholangiocyte organoids in three-dimensional structures represent a huge advance in both research and medical fields because of the limited access to primary human cholangiocytes and the potential bias induced by animal models used to study cholangiopathies in vivo. PSC-derived cholangiocyte organoids consisting of either cysts with luminal space or branching tubular structures are composed of cells with apico-basal polarity that can fulfill cholangiocyte functions like the transport of bile salts. Several protocols of PSC differentiation have already been published but we added to the detailed protocol we describe here some notes or advice to facilitate its handling by new users. We also propose detailed protocols to carry out some of the characterization analyses using immunofluorescence to study the expression of specific markers and a functionality test to visualize bile acid transport using cholyl-lysyl-fluorescein (CLF).

Predictor parameters of liver viability during porcine normothermic ex situ liver perfusion in a model of liver transplantation with marginal grafts.

Normothermic ex situ liver perfusion (NEsLP) offers the opportunity to assess biomarkers of graft function and injury. We investigated NEsLP parameters (biomarkers and markers) for the assessment of liver viability in a porcine transplantation model. Grafts from heart-beating donors (HBD), and from donors with 30 minutes (donation after cardiac death [DCD]30'), 70 minutes (DCD70'), and 120 minutes (DCD120') of warm ischemia were studied. The HBD, DCD30', and DCD70'-groups had 100% survival. In contrast, 70% developed primary nonfunction (PNF) and died in the DCD120'-group. Hepatocellular function during NEsLP showed low lactate (≤1.1mmol/L) in all the groups except the DCD120'-group (>2mmol/L) at 4hours of perfusion (P=.04). The fold-urea increase was significantly lower in the DCD120'-group (≤0.4) compared to the other groups (≥0.65) (P=.01). As for cholangiocyte function, bile/perfusate glucose ratio was significantly lower (<0.6) in all the groups except the DCD120'-group (≥0.9) after 3hours of perfusion (<0.01). Bile/perfusate Na+ ratio was significantly higher (≥1.2) after 3hours of perfusion in all the groups except for the DCD120'-group (≤1) (P<.01). Three hours after transplantation, the DCD120'-group had a significantly higher international normalized ratio (>5) compared to the rest of the groups (≤1.9) (P=.02). Rocuronium levels were higher at all the time-points in the animals that developed PNF during NEsLP and after transplantation. This study demonstrates that biomarkers and markers of hepatocellular and cholangiocyte function during NEsLP correlate with the degree of ischemic injury and posttransplant function.

Use of Biliary Organoids in Cholestasis Research.

Cholangiocytes play a crucial role in the pathophysiology of cholestasis. However, research on human cholangiocytes has been restricted by challenges in long-term propagation and large-scale expansion of primary biliary epithelium. The advent of organoid technology has overcome this limitation allowing long-term culture of a variety of epithelia from multiple organs. Here, we describe two methods for growing human cholangiocytes in organoid format. The first applies to the generation of intrahepatic bile ducts using human induced pluripotent stem cells using a protocol of differentiation that recapitulates physiological bile duct development. The second method allows the propagation of primary biliary epithelium from the extrahepatic ducts or gallbladder. Both protocols result in large numbers of cholangiocyte organoids expressing biliary markers and maintaining key cholangiocyte functions.

Bioengineered bile ducts recapitulate key cholangiocyte functions

Investigation of diseases of the bile duct system and identification of potential therapeutic targets are hampered by the lack of tractable in vitro systems to model cholangiocyte biology. Here, we show a step-wise method for the differentiation of murine Lgr5+ liver stem cells (organoids) into cholangiocyte-like cells (CLCs) using a combination of growth factors and extracellular matrix components. Organoid-derived CLCs display key properties of primary cholangiocytes, such as expressing cholangiocyte markers, forming primary cilia, transporting small molecules and responding to farnesoid X receptor agonist. Integration of organoid-derived cholangiocytes with collagen-coated polyethersulfone hollow fiber membranes yielded bioengineered bile ducts that morphologically resembled native bile ducts and possessed polarized bile acid transport activity. As such, we present a novel in vitro model for studying and therapeutically modulating cholangiocyte function.