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Tissue engineering of the biliary tract and modelling of cholestatic disorders

  • Teresa Brevini
    Affiliations
    Wellcome Trust-Medical Research Council Stem Cell Institute, Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
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  • Olivia C. Tysoe
    Affiliations
    Wellcome Trust-Medical Research Council Stem Cell Institute, Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK

    Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge, UK
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  • Fotios Sampaziotis
    Correspondence
    Corresponding author. Address: Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, University of Cambridge, Puddicombe Way, Cambridge CB2 0AW, United Kingdom. Tel.: 44.1223.747489, fax: 44.1223.763.350.
    Affiliations
    Wellcome Trust-Medical Research Council Stem Cell Institute, Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK

    Department of Hepatology, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK

    Department of Medicine, University of Cambridge, Cambridge, UK
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      Summary

      Our insight into the pathogenesis of cholestatic liver disease remains limited, partly owing to challenges in capturing the multitude of factors that contribute to disease pathogenesis in vitro. Tissue engineering could address this challenge by combining cells, materials and fabrication strategies into dynamic modelling platforms, recapitulating the multifaceted aetiology of cholangiopathies. Herein, we review the advantages and limitations of platforms for bioengineering the biliary tree, looking at how these can be applied to model biliary disorders, as well as exploring future directions for the field.

      Keywords

      Introduction

      Cholestatic liver disorders remain a significant challenge for hepatology. They affect both children and young adults,
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      • Spirli C.
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      • Mariotti V.
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      • et al.
      Pathobiology of inherited biliary diseases: a roadmap to understand acquired liver diseases.
      carry significant morbidity and mortality
      • Karlsen T.H.
      • Folseraas T.
      • Thorburn D.
      • Vesterhus M.
      Primary sclerosing cholangitis – a comprehensive review.
      and account for some of the highest cancer burdens of all liver diseases.
      • Razumilava N.
      • Gores G.J.
      Cholangiocarcinoma.
      ,
      • Fung B.M.
      • Lindor K.D.
      • Tabibian J.H.
      Cancer risk in primary sclerosing cholangitis: Epidemiology, prevention, and surveillance strategies.
      However, therapeutic modalities remain mostly limited to liver transplantation,
      • Gallo A.
      • Esquivel C.O.
      Current options for management of biliary atresia.
      • Masyuk T.V.
      • Ritman E.L.
      • LaRusso N.F.
      Quantitative assessment of the rat intrahepatic biliary system by three-dimensional reconstruction.
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      • Lo S.
      • Colquhoun S.D.
      Hepaticojejunostomy using short-limb roux-en-Y reconstruction.
      which can be complicated by disease recurrence or transplantation-related bile duct disorders, such as ischaemic cholangiopathy.
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      • Fernandez L.A.
      • Leverson G.
      • Anderson M.
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      • Sollinger H.W.
      • et al.
      Biliary complications after liver transplantation from donation after cardiac death donors.
      ,
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      Biliary complications following liver transplantation.
      Although there is a pressing clinical need for new therapeutic approaches, their development has been restricted by our limited insight into disease pathogenesis and the lack of optimal platforms for disease modelling and drug screening.
      Herein, we review recent advances in modelling cholestatic liver diseases, focusing on tissue engineering approaches. We provide a brief overview of cholestasis and cholestatic liver diseases, define the properties of an optimal model system and explore how these compare to existing modelling platforms. Finally, we discuss how tissue engineering can address some of the limitations of existing models, review current state-of-the-art bioengineered platforms, and explore future directions for the field.

      Biliary anatomy and physiology

      Bile and bile acids

      The main function of the biliary tree is the modification, storage and transport of bile from the liver to the intestine. Bile is a digestive fluid responsible for fat emulsification; it is composed of water, bile acids/bile salts, bilirubin, lipids, organic ions and xenobiotic compounds.
      • Esteller A.
      Physiology of bile secretion.
      Bile acids play a key role in the physiological function of bile. They are produced by hepatocytes, modified by the intestinal microbiota, reabsorbed by the bile ducts and the terminal ileum and circulated back to the liver through the portal vein (cholehepatic and enterohepatic circulation).
      • Hofmann A.F.
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      Bile acids: chemistry, pathochemistry, biology, pathobiology, and therapeutics.
      In addition to fat emulsification, they represent the primary route of cholesterol catabolism.
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      Studies of intestinal digestion and absorption in the human.
      Importantly, bile acids also have immunomodulatory
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      • Yu D.
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      • Huang W.
      The G-protein-coupled bile acid receptor, Gpbar1(TGR5), negatively regulates hepatic inflammatory response through antagonizing nuclear factor kappa light-chain enhancer of activated B cells (NF-jB)in mice.
      ,
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      Bile acid metabolites control TH17 and Treg cell differentiation.
      and endocrine functions,
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      Pleiotropic roles of bile acids in metabolism.
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      • Fonseca V.A.
      Bile acids and metabolic regulation.
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      • Watanabe M.
      • Auwerx J.
      Endocrine functions of bile acids.
      regulating various metabolic processes such as glucose and lipid metabolism and energy homeostasis through their receptors, such as farnesoid X receptor (FXR) and Takeda G protein receptor 5 (TGR5).
      • Trauner M.
      • Fuchs C.D.
      • Halilbasic E.
      • Paumgartner G.
      New therapeutic concepts in bile acid transport and signaling for management of cholestasis.
      Furthermore, bile acids act beyond the liver and intestine in peripheral tissues, such as the muscle, pancreas and adipose tissue.
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      • Wahlström A.
      • Marschall H.U.
      Role of bile acids in metabolic control.
      In the biliary tree, bile acids are taken up by the apical sodium-dependent bile acid transporter (ASBT) and represent the key signal regulating bile duct secretion, proliferation, survival and response to changes in the concentration of bile in health and disease. Furthermore, through the cholehepatic and enterohepatic circulation, bile acids regulate the crosstalk between the liver, the biliary system and the intestine. Recapitulating this intricate interplay remains a major challenge to in vitro modelling.

      Anatomy and physiology of the biliary tree

      Bile is initially secreted by hepatocytes in the bile canaliculi, which are 0.5–1.0 μm canals formed in the intercellular spaces between the apical surfaces of adjacent hepatocytes.
      • Boyer J.L.
      Bile formation and secretion.
      Bile flows from the canaliculi into spaces formed at the interface of hepatocytes with small cuboidal biliary epithelial cells (cholangiocytes), known as the canals of Hering. The canals of Hering drain into bile ducts with progressively increasing diameter, merging into the common hepatic duct, which leads into the common bile duct and the duodenum through the sphincter of Oddi. Between meals, the sphincter remains closed and bile flows through the cystic duct in the gallbladder where it is concentrated and stored, while during meals, the gallbladder contracts under the effect of cholecystokinin, emptying bile into the duodenum.
      The epithelium of the bile ducts plays a key role in this process by determining the composition of bile and protecting the liver parenchyma and surrounding tissue from its cytotoxic effects. Indeed, cholangiocytes secrete up to 40% of bile volume,
      • Boyer J.L.
      Canalicular bile secretion.
      corresponding predominantly to water and electrolytes, and indirectly control canalicular bile flow by absorbing bile acids and recycling them to hepatocytes via the peribiliary plexus (cholehepatic shunt).
      • Tabibian J.H.
      • Masyuk A.I.
      • Masyuk T.V.
      • O'Hara S.P.
      • LaRusso N.F.
      Physiology of cholangiocytes.
      Furthermore, they provide a barrier against bile cytotoxicity through tight junction formation and modify its composition via a series of secretory and absorptive functions.
      • Boyer J.L.
      Bile formation and secretion.
      ,
      • Tabibian J.H.
      • Masyuk A.I.
      • Masyuk T.V.
      • O'Hara S.P.
      • LaRusso N.F.
      Physiology of cholangiocytes.
      Importantly, cholangiocyte morphology and function varies along the biliary tree (Fig. 1).
      • Yuyan H.
      • Glaser S.S.
      • Meng F.
      • Francis H.
      • Marzioni M.
      • McDaniel K.
      • et al.
      Recent advances in the morphological and functional heterogeneity of the biliary epithelium.
      Small intrahepatic ductules (diameter <15 μm) are characterised by small cuboidal cholangiocytes, while larger intrahepatic ducts (diameter >15 μm), extrahepatic ducts and the gallbladder are populated by large columnar cholangiocytes.
      • Strazzabosco M.
      • Fabris L.
      Functional anatomy of normal bile ducts.
      ,
      • Maroni L.
      • Haibo B.
      • Ray D.
      • Zhou T.
      • Wan Y.
      • Meng F.
      • et al.
      Functional and structural features of cholangiocytes in health and disease.
      Small cholangiocytes are highly proliferative and can give rise to large cholangiocytes in response to acute injury of large bile ducts.
      • Maroni L.
      • Haibo B.
      • Ray D.
      • Zhou T.
      • Wan Y.
      • Meng F.
      • et al.
      Functional and structural features of cholangiocytes in health and disease.
      • Sato K.
      • Marzioni M.
      • Meng F.
      • Francis H.
      • Glaser S.
      • Alpini G.
      Ductular reaction in liver diseases: pathological mechanisms and translational Significances.
      • Mancinelli R.
      • Franchitto A.
      • Gaudio E.
      • Onori P.
      • Glaser S.
      • Francis H.
      After damage of large bile ducts by gamma-aminobutyric acid, small ducts replenish the biliary tree by amplification of calcium-dependent signaling and de novo acquisition of large cholangiocyte phenotypes.
      They modify bile fluidity predominantly through the Ca2+-mediated Cl channel transmembrane protein 16A (TMEM16A) in response to apical nucleotide signalling
      • Rodrigues M.A.
      • Gomes D.A.
      • Nathanson M.H.
      Calcium signaling in cholangiocytes: methods, mechanisms, and effects.
      ,
      • Dutta A.K.
      • Al-karim Khimji
      • Kresge C.
      • Bugde A.
      • Dougherty M.
      • Esser V.
      • et al.
      Identification and functional characterization of TMEM16A, a Ca2+-activated Cl− channel activated by extracellular nucleotides, in biliary epithelium.
      and basolateral cholinergic stimulation,
      • Banales J.M.
      • Prieto J.
      • Medina J.F.
      Cholangiocyte anion exchange and biliary bicarbonate excretion.
      but lack many of the channels and receptors associated with bile modification that are expressed in large cholangiocytes, such as cystic fibrosis transmembrane conductance regulator (CFTR), anion exchanger 2 (AE2) and secretin receptor.
      • Maroni L.
      • Haibo B.
      • Ray D.
      • Zhou T.
      • Wan Y.
      • Meng F.
      • et al.
      Functional and structural features of cholangiocytes in health and disease.
      Conversely, large cholangiocytes demonstrate more secretory and reabsorptive functions. They modify the alkalinity and fluidity of bile through both Ca2+-mediated and cAMP-mediated secretory pathways,
      • Yuyan H.
      • Glaser S.S.
      • Meng F.
      • Francis H.
      • Marzioni M.
      • McDaniel K.
      • et al.
      Recent advances in the morphological and functional heterogeneity of the biliary epithelium.
      ,
      • Maroni L.
      • Haibo B.
      • Ray D.
      • Zhou T.
      • Wan Y.
      • Meng F.
      • et al.
      Functional and structural features of cholangiocytes in health and disease.
      ,
      • Banales J.M.
      • Huebert R.C.
      • Karlsen T.
      • Strazzabosco M.
      • LaRusso N.F.
      • Gores G.J.
      Cholangiocyte pathobiology.
      of which the latter is absent in small cholangiocytes. More specifically, secretin-mediated Cl secretion leads to bicarbonate transport in the duct lumen via AE2, followed by passive influx of water through aquaporin 1.
      • Banales J.M.
      • Arenas F.
      • Rodríguez-Ortigosa C.M.
      • Sáez E.
      • Uriarte I.
      • Doctor R.B.
      • et al.
      Bicarbonate-rich choleresis induced by secretin in normal rat is taurocholate-dependent and involves AE2 anion exchanger.
      ,
      • Hohenester S.
      • Maillette de Buy Wenniger L.
      • Paulusma C.C.
      • van Vliet S.J.
      • Jefferson D.M.
      • Oude Elferink R.P.
      • et al.
      A biliary HCO 3- umbrella constitutes a protective mechanism against bile acid-induced injury in human cholangiocytes.
      This secreted bicarbonate forms an alkaline layer over the apical surface of cholangiocytes, known as the “bicarbonate umbrella”. The bicarbonate umbrella deprotonates apolar bile acids, making them impermeable to the cell membrane and allowing cholangiocytes to tolerate otherwise toxic concentrations of bile acids.
      • Beuers U.
      • Hohenester S.
      • de Buy Wenniger L.J.M.
      • Kremer A.E.
      • Jansen P.L.M.
      • Elferink R.P.J.O.
      The biliary HCO3− umbrella: a unifying hypothesis on pathogenetic and therapeutic aspects of fibrosing cholangiopathies.
      Consequently, modelling luminal secretion and reabsorption is crucial to study cholangiocyte's resistance to bile and response to bile toxicity, which, as outlined in the next section, plays an important role in biliary physiology and the pathogenesis of various cholangiopathies.
      • Trauner M.
      • Fickert P.
      • Halilbasic E.
      • Moustafa T.
      Lessons from the toxic bile concept for the pathogenesis and treatment of cholestatic liver diseases.
      ,
      • Fickert P.
      • Wagner M.
      Biliary bile acids in hepatobiliary injury – what is the link?.
      Figure thumbnail gr1
      Fig. 1Anatomy of the biliary tree and physiological functions.
      Schematic representation of the biliary tree highlighting 3 regions with differing physiological roles: the small ducts, the large ducts and the gallbladder. An overview of the morphological and functional features of the cells lining these regions is provided. The distinction between intra- and extrahepatic biliary tract is also shown.

      Pathophysiology of cholestasis

      Cholestasis is defined as the impairment of bile flow from the liver to the duodenum, which can result from impaired bile secretion across the canalicular membrane of hepatocytes (hepatocellular cholestasis) or impaired bile flow secondary to bile duct pathology.
      European Association for the Study of the Liver
      EASL Clinical Practice Guidelines: management of cholestatic liver diseases.
      Herein, we will focus on cholestasis secondary to bile duct disorders (Box 1).
      Cholangiocellular causes of cholestasis.
      The bile duct epithelium plays a key role in the pathogenesis of cholestasis. Bile duct injury leads to impaired barrier function, bile penetration and cytotoxic damage in the periductal tissue.
      • Jansen P.L.M.
      • Ghallab A.
      • Vartak N.
      • Reif R.
      • Schaap F.G.
      • Hampe J.
      • et al.
      The ascending pathophysiology of cholestatic liver disease.
      In parallel, it prompts secretion of pro-inflammatory and pro-fibrogenic mediators, angiogenetic factors and chemokines from cholangiocytes, known as cholangiocyte activation.
      • Banales J.M.
      • Huebert R.C.
      • Karlsen T.
      • Strazzabosco M.
      • LaRusso N.F.
      • Gores G.J.
      Cholangiocyte pathobiology.
      Activation results in the recruitment of inflammatory, endothelial and mesenchymal cells, which physiologically repair the epithelium and restore homeostasis.
      • Pinto C.
      • Giordano D.M.
      • Maroni L.
      • Marzioni M.
      Role of inflammation and proinflammatory cytokines in cholangiocyte pathophysiology.
      However, in cholestatic disorders, these compensatory mechanisms are likely overwhelmed, resulting in immune recruitment, excessive deposition of scar tissue, liver damage and eventually carcinogenesis.
      • Blechacz B.
      • Gores G.J.
      Cholangiocarcinoma: advances in pathogenesis, diagnosis, and treatment.
      Therefore, cholestasis represents a complex pathology encompassing multiple cell types, matrix changes and intricate signalling pathways. Recapitulating this complexity remains one of the main challenges in modelling cholestatic disorders.
      Multiple tissue engineering platforms exist, each with unique features that can recapitulate specific characteristics of cholestatic liver disease.

      Cholestatic disorders

      The disease mechanisms leading to bile duct disruption, epithelial damage and cholestasis are diverse and remain poorly understood.
      • Lazaridis K.N.
      • Larusso N.F.
      The cholangiopathies.
      Adult cholangiopathies range from immune-mediated disease affecting small to medium size ducts, such as primary biliary cholangitis (PBC),
      • Gulamhusein A.F.
      • Hirschfield G.M.
      Primary biliary cholangitis: pathogenesis and therapeutic opportunities.
      to fibro-inflammatory disease causing multifocal strictures in medium to large size bile ducts, such as primary sclerosing cholangitis (PSC).
      • Karlsen T.H.
      • Folseraas T.
      • Thorburn D.
      • Vesterhus M.
      Primary sclerosing cholangitis – a comprehensive review.
      Although the pathogenesis of disorders such as PSC remains largely unknown, multiple factors play a role in the disease evolution including the immune system, changes in the composition of bile, the gut and its microbiome.
      • Hov J.R.
      • Karlsen T.H.
      The microbiome in primary sclerosing cholangitis: current evidence and potential concepts.
      ,
      • Pollheimer M.J.
      • Trauner M.
      • Fickert P.
      Will we ever model PSC? - ‘It's hard to be a PSC model!’.
      Capturing the interplay between this variety of contributing factors is a key requirement for understanding multifactorial adult cholangiopathies.
      Infantile cholangiopathies result predominantly from defects in the development of the biliary tree. Physiologically, the intra- and extrahepatic bile ducts develop from different embryologic origins. The extrahepatic ducts originate from bipotent Sox17+/PDX1+ pancreatobiliary progenitors in a HES1-dependent manner, while intrahepatic ducts originate from bipotent hepatobiliary progenitors known as hepatoblasts. Notch signalling from the portal vein mesenchyme instructs hepatoblasts to give rise to a monolayer of cholangiocytes surrounding the portal vein, known as the ductal plate, which subsequently duplicates and remodels into intrahepatic bile ducts.
      • Ober E.A.
      • Lemaigre F.P.
      Development of the liver: insights into organ and tissue morphogenesis.
      Disorders affecting bile duct development range from defects in ductal plate remodelling,
      • Raynaud P.
      • Tate J.
      • Callens C.
      • Cordi S.
      • Vandersmissen P.
      • Carpentier R.
      • et al.
      A classification of ductal plate malformations based on distinct pathogenic mechanisms of biliary dysmorphogenesis.
      such as Alagille syndrome (ALGS)
      • Gilbert M.A.
      • Spinner N.B.
      Alagille syndrome: genetics and functional models.
      and ductal plate malformations (DPMs) to multifactorial fibro-obliterative disease affecting predominantly the extrahepatic ducts, such as biliary atresia.
      • Asai A.
      • Miethke A.
      • Bezerra J.A.
      Pathogenesis of biliary atresia: defining biology to understand clinical phenotypes.
      In addition to developmental disease, infantile cholangiopathies also encompass monogenic disorders affecting specific cholangiocyte functions. Characteristic examples include cystic fibrosis-related liver disease which is characterised by impaired chloride, water and bicarbonate transfer in the bile duct lumen,
      • Fiorotto R.
      • Strazzabosco M.
      Pathophysiology of cystic fibrosis liver disease: a channelopathy leading to alterations in innate immunity and in microbiota.
      or polycystic liver disease (PCLD) which is characterised by abnormal cholangiocyte proliferation and/or secretion due to defects in cilia proteins.
      • Perugorria M.J.
      • Masyuk T.V.
      • Marin J.J.
      • Marzioni M.
      • Bujanda L.
      • Larusso N.F.
      • et al.
      Polycystic liver diseases: advanced insights into the molecular mechanisms.
      Cholangiocyte cilia act as mechanosensors activated by the flow of bile, amongst other stimuli.
      • Masyuk A.I.
      • Masyuk T.V.
      • Splinter P.L.
      • Huang B.Q.
      • Stroope A.J.
      • LaRusso N.F.
      Cholangiocyte cilia detect changes in luminal fluid flow and transmit them into intracellular Ca2+ and cAMP signaling.
      Therefore, understanding the crosstalk between the developing cholangiocyte, its surrounding cells and its microenvironment is important for improving our understanding of the pathogenesis of infantile cholangiopathies.

      Modelling cholestatic liver disease

      The multifactorial aetiology, disease heterogeneity and complex microenvironment of cholangiopathies pose significant modelling challenges.
      • Banales J.M.
      • Huebert R.C.
      • Karlsen T.
      • Strazzabosco M.
      • LaRusso N.F.
      • Gores G.J.
      Cholangiocyte pathobiology.
      These challenges could be addressed by using multiple complementary platforms, capturing different pathophysiological features of the disease. Multicellular systems could be optimal for recapitulating the pathogenesis of developmental, inflammatory and immune-mediated cholangiopathies, which involve interactions between cholangiocytes and various other cell types including endothelial, immune and stellate cells. Platforms enabling cholangiocytes to polarise around accessible lumens and maintain luminal homeostasis could represent optimal systems for studying cholangiocyte-to-bile interactions, including the role of tight junctions in maintaining barrier function and the protective role of the bicarbonate umbrella, which play a pivotal role in cholestasis. Biliary fibrosis is a shared feature of many cholangiopathies,
      • Pinzani M.
      • Luong T.V.
      Pathogenesis of biliary fibrosis.
      thus models encompassing physiologically relevant extracellular matrices (ECMs) are indispensable for dissecting fibro-obliterative diseases, such as PSC. Systems allowing self-organisation of cholangiocytes into tubular structures would be ideal for studying ductal plate remodelling and tubulogenesis, which are crucial for developmental cholangiopathies, such as ALGS and DPMs. Models incorporating mechanical stimuli such as flow are optimal for studying mechanosensing organelles such as cilia and interrogating their role in health and ciliopathies. Platforms recapitulating the crosstalk between the bile ducts and other organs, such as the intestine, would be ideal for studying aspects of the pathogenesis of cholestatic disorders extending beyond the liver, such as the gut-liver axis and the role of the intestinal microbiome.
      • Jia W.
      • Xie G.
      • Jia W.
      Bile acid–microbiota crosstalk in gastrointestinal inflammation and carcinogenesis.
      In addition to these requirements, ideal models should provide easy access to the cells of interest, minimal variability and ease of characterisation of experimental outputs. Combining all these features into 1 model may not be feasible with current technology. However, a combination of several models, each accurately reproducing a different aspect of the disease could prove an equally powerful approach.

      Experimental models of cholestasis and cholestatic disorders

      This section focuses on existing models of cholestatic disorders, their advantages, limitations and how they address different modelling requirements for cholangiopathies.

      In vivo models

      Animal models provide an excellent platform for capturing the complexity of biliary disease and studying these disorders in the context of an organism. They incorporate the full spectrum of cell types and different systems involved in cholestasis (cholangiocytes, stroma, immune cells) and provide an excellent system for recapitulating their interactions in a physiological environment; they reproduce architectural and matrix changes, such as fibrosis
      • Fickert P.
      • Fuchsbichler A.
      • Wagner M.
      • Zollner G.
      • Kaser A.
      • Tilg H.
      • et al.
      Regurgitation of bile acids from leaky bile ducts causes sclerosing cholangitis in Mdr2 (Abcb4) knockout mice.
      and stricture formation,
      • Goetz M.
      • Lehr H.A.
      • Neurath M.F.
      • Galle P.R.
      • Orth T.
      Long-term evaluation of a rat model of chronic cholangitis resembling human primary sclerosing cholangitis.
      which are challenging to recapitulate in vitro and they capture the contribution of environmental stimuli to the disease phenotype, such as the microbiome or the composition of bile.
      • Schrumpf E.
      • Kummen M.
      • Valestrand L.
      • Greiner T.U.
      • Holm K.
      • Arulampalam V.
      • et al.
      The gut microbiota contributes to a mouse model of spontaneous bile duct inflammation.
      Consequently, multiple models have been developed for different disorders, including surgical ligation of the bile duct, chemically induced bile duct injury (3,5-diethoxycarbonyl-1,4-dihydrocollidine, α-naphthylisothiocyanate, and litocholic acid), viral infections (Rhesus rotavirus type A), and genetic manipulation (Mdr2, Bsep, IL2R, AE2, Pkd1/2, Cftr, Jag1), which have been thoroughly reviewed elsewhere.
      • Mariottia V.
      • Strazzabosco M.
      • Fabris L.
      • Calvisi D.F.
      Animal models of biliary injury and altered bile acid metabolism.
      ,
      • Mariotti V.
      • Cadamuro M.
      • Spirli C.
      • Fiorotto R.
      • Strazzabosco M.
      • Fabris L.
      Animal models of cholestasis: an update on inflammatory cholangiopathies.
      Although these models have significantly improved our understanding of the basic mechanisms of cholestatic disease, limitations remain. Indeed, the phenotype of various human cholangiopathies is only partially reproduced in animals due to inter-species variation.
      • Rosen B.H.
      • Chanson M.
      • Gawenis L.R.
      • Liu J.
      • Sofoluwe A.
      • Zoso A.
      • et al.
      Animal and model systems for studying cystic fibrosis.
      ,
      • Pollheimer M.J.
      • Fickert P.
      Animal models in primary biliary cirrhosis and primary sclerosing cholangitis.
      Furthermore, differences in the disease environment, such as the composition of bile or the microbiome render studies on multifactorial disease challenging.
      • Hofmann A.F.
      • Hagey L.R.
      • Krasowski M.D.
      Bile salts of vertebrates: structural variation and possible evolutionary signifi cance.
      ,
      • Woolbright B.L.
      • Dorko K.
      • Antoine D.J.
      • Clarke J.I.
      • Gholami P.
      • Li F.
      • et al.
      Bile acid-induced necrosis in primary human hepatocytes and in patients with obstructive cholestasis.
      Finally, cost and animal numbers preclude large scale, high-throughput studies, while the complexity of interactions in vivo make it challenging to dissect the contribution of each cell type to the disease phenotype.

      In vitro models

      In vitro systems also hold great potential for dissecting the pathogenesis of complex disorders, such as cholangiopathies. They are more cost-effective than animal models, allow high-throughput, large scale experiments and facilitate mechanistic studies by enabling exposure of a single cell type to well-defined stimuli. Primary cholangiocytes remain the gold standard for in vitro studies on cholestasis. However, their use has been hampered by poor access to healthy human tissue and challenges in long-term (beyond 10–15 passages) 2D culture of functional cholangiocytes.
      • Rodrigues M.A.
      • Gomes D.A.
      • Nathanson M.H.
      Calcium signaling in cholangiocytes: methods, mechanisms, and effects.
      ,
      • Sampaziotis F.
      • Segeritz C.P.
      • Vallier L.
      Potential of human induced pluripotent stem cells in studies of liver disease.
      3D culture has improved both cell viability and function, resulting in cells recapitulating key features of native tissue.
      • Günther C.
      • Brevini T.
      • Sampaziotis F.
      • Neurath M.F.
      What gastroenterologists and hepatologists should know about organoids in 2019.
      Under these conditions cholangiocytes spontaneously organise into 3D structures encompassing a central lumen, which have been described as organoids.
      • Sampaziotis F.
      • Justin A.W.
      • Tysoe O.C.
      • Sawiak S.
      • Godfrey E.M.
      • Upponi S.S.
      • et al.
      Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids.
      • Huch M.
      • Gehart H.
      • Van Boxtel R.
      • Hamer K.
      • Blokzijl F.
      • Verstegen M.M.A.
      • et al.
      Long-term culture of genome-stable bipotent stem cells from adult human liver.
      • Hu H.
      • Gehart H.
      • Artegiani B.
      • LÖpez-Iglesias C.
      • Dekkers F.
      • Basak O.
      • et al.
      Long-term expansion of functional mouse and human hepatocytes as 3D organoids.
      Importantly, while the term “organoid” can be used broadly in different contexts,
      • Rossi G.
      • Manfrin A.
      • Lutolf M.P.
      Progress and potential in organoid research.
      herein, it refers to any self-organising 3D cellular structure suspended within an ECM, which can be derived from primary tissue, adult stem cells or embryonic/induced pluripotent stem cells (iPSCs).
      • Rossi G.
      • Manfrin A.
      • Lutolf M.P.
      Progress and potential in organoid research.
      • Drost J.
      • Clevers H.
      Translational applications of adult stem cell-derived organoids.
      • Yin X.
      • Mead B.E.
      • Safaee H.
      • Langer R.
      • Karp J.M.
      • Levy O.
      Engineering stem cell organoids.
      Despite their advantages, these systems are not optimally suited for studies on epithelial permeability, barrier function or cholangiocyte-to-bile interactions requiring regular sampling of the organoid lumen, which remains relatively inaccessible, while access to primary tissue remains a limitation.
      iPSC-derived cholangiocytes could address access to tissue challenges and provide an alternative to primary cholangiocyte culture.
      • Sampaziotis F.
      • Segeritz C.P.
      • Vallier L.
      Potential of human induced pluripotent stem cells in studies of liver disease.
      Indeed, iPSCs can be derived through minimally invasive procedures, such as blood or skin biopsies, resulting in a highly scalable system and patient-specific genetic backgrounds. Furthermore, protocols for the differentiation of iPSCs into hepatocytes and cholangiocytes mimic hepatobiliary development in vitro which renders them well suited for studying developmental disorders of the bile ducts, such as ALGS or other infantile cholangiopathies.
      • Sampaziotis F.
      • De Brito M.C.
      • Madrigal P.
      • Bertero A.
      Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and drug validation.
      • Sampaziotis F.
      • De Brito M.C.
      • Geti I.
      • Bertero A.
      • Hannan N.R.F.
      • Vallier L.
      Directed differentiation of human induced pluripotent stem cells into functional cholangiocyte-like cells.
      • Ogawa M.
      • Ogawa S.
      • Bear C.E.
      • Ahmadi S.
      • Chin S.
      • Li B.
      • et al.
      Directed differentiation of cholangiocytes from human pluripotent stem cells.
      Conversely, the cells generated by iPSC differentiation protocols retain a foetal phenotype, which can pose a challenge when studying adult cholangiopathies.
      • Baxter M.
      • Withey S.
      • Harrison S.
      • Segeritz C.P.
      • Zhang F.
      • Atkinson-Dell R.
      • et al.
      Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes.
      Finally, established cholangiocyte cell lines, such as H69, are available.
      • Maruyama M.
      • Kobayashi N.
      • Westerman K.A.
      • Sakaguchi M.
      • Allain J.E.
      • Totsugawa T.
      • et al.
      Establishment of a highly differentiated immortalized human cholangiocyte cell line with SV40T and hTERT.
      These provide an alternative to iPSCs and primary cells, with high expansion potential and lower requirements in terms of cost and expertise; however, they are restricted by their non-physiological genetic background, which represents a common limitation for all immortalised cell lines.
      Despite their individual advantages and drawbacks, in vitro systems allow larger-scale, in-depth mechanistic studies, at the cost of not being able to recapitulate all aspects of the complex milieu of cholestasis ex vivo. Consequently, they are limited to reproducing the disease phenotype only partially, usually focusing on cell-autonomous features.

      Tissue engineering to model cholestatic disorders

      Despite their advantages, animal models remain limited by intraspecies variation, while laboratory platforms are restricted in their ability to capture the complexity of in vivo organs. Tissue engineering could provide an optimal solution to overcome such challenges and bridge the gap between in vitro and in vivo systems.
      • Healy K.
      Tissue-engineered disease models.
      Tissue engineering refers to the combination of cells, biomaterials, and biologically active molecules into functional structures resembling tissue.
      • Langer R.
      • Vacanti J.
      Advances in tissue engineering.
      Tissue engineered systems allow the assembly of cells in 3D structures, mimicking the microarchitecture of human tissue.
      • Griffith L.
      Emerging design principles in biomaterials and scaffolds for tissue engineering.
      These structures can be embedded into a wide range of matrices resembling the properties of human ECM, such as stiffness and porosity.
      • Eberli D.
      • Filho L.F.
      • Atala A.
      • Yoo J.J.
      Composite scaffolds for the engineering of hollow organs and tissues.
      For tissues with different compartments, such as a lumen or a vascular bed, each compartment can be maintained in different conditions; luminal flow can be modelled through perfusion with media at different rates,
      • Teresa Raimondi M.
      Engineered tissue as a model to study cell and tissue function from a Biophysical perspective.
      while diffusion of nutrients or oxygen can be captured using chemical gradients.
      • Kang Y.B.
      • Eo J.
      • Bulutoglu B.
      • Yarmush M.L.
      • Usta O.B.
      Progressive hypoxia-on-a-chip: an in vitro oxygen gradient model for capturing the effects of hypoxia on primary hepatocytes in health and disease.
      Consequently, tissue engineering enables the transition from single cell, static culture platforms to dynamic model systems, providing the complexity required to reproduce the salient aspects of disease in vitro. Conversely, tissue engineered platforms are simpler than in vivo organs and animal models. However, contrary to animals, each component of a tissue engineered model can be modified in isolation. This approach enables mechanistic studies that interrogate the impact of a single parameter on a complex phenotype, while their simplicity minimises signal-to-noise ratio.
      The main limitation for modelling cholestatic disorders using tissue engineering has been the relative lack of appropriate bioengineered biliary tissue platforms. Nonetheless, recent advances in engineered matrices, 3D-printing and biomaterials have overcome this issue and provided multiple iterations of bioengineered biliary tissue.
      • Tysoe O.C.
      • Justin A.W.
      • Brevini T.
      • Chen S.E.
      • Mahbubani K.T.
      • Frank A.K.
      • et al.
      Isolation and propagation of primary human cholangiocyte organoids for the generation of bioengineered biliary tissue.
      We provide an overview of these platforms, their unique advantages and limitations and examples of their application in disease modelling.
      Hydrogels allow self-organisation of cells into 3D matrices, which is ideal for studying developmental disorders.

      Tissue engineering approaches

      Hydrogels

      Disruption of early bile duct development, including ductal plate remodelling, lumen formation and polarisation play a key role in the pathogenesis of infantile cholangiopathies such as ALGS and biliary atresia. However, modelling tubulogenesis requires systems that enable the rearrangement of cholangiocytes in 3D, which cannot be captured by conventional monolayer culture. This challenge could be addressed by hydrogels. Hydrogels are biocompatible 3D hydrophilic matrices with physiological stiffness, which can be degraded via hydrolysis or cell-mediated degradation by matrix metalloproteases (MMPs), allowing for matrix remodelling.
      • Stevens K.R.
      • Miller J.S.
      • Blakely B.L.
      • Chen C.S.
      • Bhatia S.N.
      Degradable hydrogels derived from PEG-diacrylamide for hepatic tissue engineering.
      ,
      • Burdick J.A.
      • Murphy W.L.
      Moving from static to dynamic complexity in hydrogel design.
      These properties allow free movement of cells in the hydrogel
      • Gjorevski N.
      • Sachs N.
      • Manfrin A.
      • Giger S.
      • Bragina M.E.
      • Ordóñez-Morán P.
      • et al.
      Designer matrices for intestinal stem cell and organoid culture.
      and self-organisation into complex 3D structures, which is ideal for studies on tissue morphogenesis and remodelling.
      • Lutolf M.P.
      • Hubbell J.A.
      Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering.
      • Kleinman H.K.
      • Philip D.
      • Hoffman M.P.
      Role of the extracellular matrix in morphogenesis.
      • Handorf A.M.
      • Zhou Y.
      • Halanski M.A.
      • Li W.J.
      Tissue stiffness dictates development, homeostasis, and disease progression.
      Hydrogels consist of depolymerised proteins in solution. In their liquid form these matrices can be mixed with cells and solidified by heat or chemical crosslinking for tissue culture applications
      • Caliari S.R.
      • Burdick J.A.
      A practical guide to hydrogels for cell culture.
      (Fig. 2); or they can be used as ‘bioinks’ for 3D printing (see 3D bioprinting section). Furthermore, they can be composed of synthetic polymers or physiological ECM components. The composition of synthetic hydrogels is user-defined and can be customised to adjust their mechanical and chemical properties and degradation profile, which renders them optimal platforms for mechanistic studies on the impact of matrix attributes on tissue morphogenesis.
      • Stevens K.R.
      • Miller J.S.
      • Blakely B.L.
      • Chen C.S.
      • Bhatia S.N.
      Degradable hydrogels derived from PEG-diacrylamide for hepatic tissue engineering.
      ,
      • Burdick J.A.
      • Murphy W.L.
      Moving from static to dynamic complexity in hydrogel design.
      ,
      • Handorf A.M.
      • Zhou Y.
      • Halanski M.A.
      • Li W.J.
      Tissue stiffness dictates development, homeostasis, and disease progression.
      ,
      • Rape A.D.
      • Zibinsky M.
      • Murthy N.
      • Kumar S.
      A synthetic hydrogel for the high-throughput study of cell-ECM interactions.
      Indeed, cholangiocytes embedded in polyethylene glycol hydrogels have been used to investigate the role of matrix stiffness, integrin ligand density and MMP-mediated matrix degradation in tubulogenesis and lumen formation.
      • Funfak A.
      • Bouzhir L.
      • Gontran E.
      • Minier N.
      • Dupuis-Williams P.
      • Gobaa S.
      Biophysical control of bile duct epithelial morphogenesis in natural and synthetic scaffolds.
      A similar approach using synthetic hydrogels containing MMP-sensitive peptides could be used to dissect the role of cell-mediated matrix degradation and MMPs in the natural history of cholangiopathies, which have, been associated with disease progression in disorders such as PCLD
      • Urribarri A.D.
      • Munoz-garrido P.
      • Erice O.
      • Arbelaiz A.
      • Lozano E.
      • Hijona E.
      • et al.
      Inhibition of metalloprotease hyperactivity in cystic cholangiocytes halts the development of polycystic liver diseases.
      and biliary atresia.
      • Lertudomphonwanit C.
      • Mourya R.
      • Fei L.
      • Zhang Y.
      • Gutta S.
      • Yang L.
      • et al.
      Large-scale proteomics identifies MMP-7 as a sentinel of epithelial injury and of biliary atresia.
      Conversely, biological hydrogels encompassing bioactive components of the native bile duct ECM, such as collagen I, III and IV, fibronectin, laminin and elastin provide a more physiological environment compared to their synthetic counterparts.
      • Khandekar G.
      • Llewellyn J.
      • Kriegermeier A.
      • Waisbourd-Zinman O.
      • Johnson N.
      • Du Y.
      • et al.
      Coordinated development of the mouse extrahepatic bile duct: implications for neonatal susceptibility to biliary injury.
      • Tanimizu N.
      • Kikkawa Y.
      • Mitaka T.
      • Miyajima A.
      α1- and α5-containing laminins regulate the development of bile ducts via β1 integrin signals.
      • Vestentoft P.S.
      • Jelnes P.
      • Andersen J.B.
      • Tran T.A.T.
      • Jørgensen T.
      • Rasmussen M.
      • et al.
      Molecular constituents of the extracellular matrix in rat liver mounting a hepatic progenitor cell response for tissue repair.
      Furthermore, hydrogels combining multiple components recapitulate the natural ECM more closely than single component matrices. Accordingly, cholangiocytes in hydrogels generated from digested whole liver ECM enable studies on branching morphogenesis,
      • Lewis P.L.
      • Su J.
      • Yan M.
      • Meng F.
      • Glaser S.S.
      • Alpini G.D.
      • et al.
      Complex bile duct network formation within liver decellularized extracellular matrix hydrogels.
      which are not possible with single component hydrogels, such as collagen I gels, because of the lack of biochemical cues provided by other ECM components.
      3D printing can be used to recapitulate tissue microarchitecture.
      Figure thumbnail gr2
      Fig. 2Hydrogels allow self-organisation of cells in 3D.
      (A) Schematic representation of the process of embedding cells in hydrogel. (B) Cells are embedded in liquid hydrogel. Polymerisation is induced by heat or chemical crosslinking resulting in hydrogel solidification. Cells subsequently reorganise into complex 3D structures through cell movement and matrix degradation. MMP-sensitive sites can be added to the protein backbone to allow cell-mediated remodelling of the surrounding matrix. MMP, matrix metalloprotease.
      Despite their advantages, single-cell hydrogel systems do not capture the interactions between cholangiocytes and different cell types, such as the vascular endothelium or immune cells, which play a key role in cholangiopathies and biliary development. To address this limitation, Matrigel (a widely applied ECM hydrogel produced by the mouse Englebreth-Holm-Swarm sarcoma cell line), has been used to generate multicellular systems combining iPSC-derived hepatic progenitors, mesenchymal and endothelial cells. This system allows the cells to spontaneously organise into vascularised organoid structures with bile canaliculi resembling embryonic liver buds,
      • Takebe T.
      • Sekine K.
      • Enomura M.
      • Koike H.
      • Kimura M.
      • Ogaeri T.
      • et al.
      Vascularized and functional human liver from an iPSC-derived organ bud transplant.
      thus providing new insights into the inter-lineage communication between endothelial and hepatic cells during early development.
      • Camp J.G.
      • Sekine K.
      • Gerber T.
      • Loeffler-Wirth H.
      • Binder H.
      • Gac M.
      • et al.
      Multilineage communication regulates human liver bud development from pluripotency.
      Despite the presence of bile canaliculi, most liver organoid systems lack bona-fide bile ducts, limiting their application for studies in cholangiopathies or bile duct development. Nonetheless, early biliary tree morphogenesis has been modelled using a mixture of foregut and midgut organoids. These organoids spontaneously organise into complex structures incorporating liver, pancreas, intestinal and biliary tissue through a process of invagination and branching that resembles early development of the extrahepatic ducts at the foregut-midgut boundary.
      • Koike H.
      • Iwasawa K.
      • Ouchi R.
      • Maezawa M.
      • Giesbrecht K.
      • Saiki N.
      • et al.
      Modelling human hepato-biliary-pancreatic organogenesis from the foregut–midgut boundary.
      This system could still provide interesting insights into infantile cholangiopathies such as biliary atresia.
      Overall, hydrogels provide an ideal platform for studying complex cell-to-cell interactions and mechanisms of tissue morphogenesis in vitro, which can provide insights into infantile cholangiopathies and bile duct development. However, limitations remain. Most systems are based on non-chemically defined hydrogels, such as Matrigel, which results in batch-to-batch variability, complicating mechanistic studies and reducing reproducibility, while downstream analysis remains challenging in multicellular systems.

      3D bioprinting

      Tissue microarchitecture plays a key role in the pathogenesis of cholangiopathies. Characteristic examples include the impact of the portal triad anatomy for disorders such as infantile or ischaemic cholangiopathies and the difference in the order of the affected ducts between PBC or PSC. Hydrogel organoid systems do not provide an optimal platform for recapitulating the microanatomy of primary tissue, because organoid formation is based on spontaneous organisation. Consequently, the spatial relationship of the cells cannot be predetermined, and the resulting structures differ from the complex architecture of primary tissue. 3D bioprinting could provide a solution to this challenge. The term bioprinting refers to the generation of complex 3D structures through a layer-by-layer positioning technique, using a 3D printer.
      • Murphy S.V.
      • Atala A.
      3D bioprinting of tissues and organs.
      ,
      • Moroni L.
      • Burdick J.A.
      • Highley C.
      • Lee S.J.
      • Morimoto Y.
      • Takeuchi S.
      • et al.
      Biofabrication strategies for 3D in vitro models and regenerative medicine.
      The printer head starts by depositing droplets of a biomaterial (e.g. hydrogel) to generate a cross-section of the desired structure and continues by stacking each section on top of the previous one until the structure is complete (Fig. 3). A wide variety of bioactive inks ranging from hydrogels with tuneable properties to decellularised ECM (dECM) gels can be used.
      • Murphy S.V.
      • De Coppi P.
      • Atala A.
      Opportunities and challenges of translational 3D bioprinting.
      Cells can be mixed with the bioink to enable printing of tissue-like structures, while different crosslinking methods can be used to solidify the bioink after printing and adjust the stiffness of the resulting tissue.
      • Hölzl K.
      • Lin S.
      • Tytgat L.
      • Van Vlierberghe S.
      • Gu L.
      • Ovsianikov A.
      Bioink properties before, during and after 3D bioprinting.
      Using these approaches hepatocytes mixed with ECM have been used to 3D print liver-like tissue.
      • Lee H.
      • Han W.
      • Kim H.
      • Ha D.H.
      • Jang J.
      • Kim B.S.
      • et al.
      Development of liver decellularized extracellular matrix bioink for three-dimensional cell printing-based liver tissue engineering.
      • Hiller T.
      • Berg J.
      • Elomaa L.
      • Röhrs V.
      • Ullah I.
      • Schaar K.
      • et al.
      Generation of a 3D liver model comprising human extracellular matrix in an alginate/gelatin-based bioink by extrusion bioprinting for infection and transduction studies.
      • Ma X.
      • Yu C.
      • Wang P.
      • Xu W.
      • Wan X.
      • Lai C.S.E.
      • et al.
      Rapid 3D bioprinting of decellularized extracellular matrix with regionally varied mechanical properties and biomimetic microarchitecture.
      Furthermore, light processing and photo-crosslinking have been used to tailor the mechanical properties of dECM hydrogels, in order to mimic in vitro pathological changes in tissue stiffness corresponding to fibrosis. Bioprinting of tubular networks, such as the biliary tree, is more challenging due to the mechanical properties of hydrogels, which lack the strength to maintain the patency of cell-laden lumens during printing. This limitation can be overcome by printing inside a template made by a sacrificial material. The mould can be removed after the structures have been fully printed and other cell types can be printed in the surrounding space. This process is known as dual step bioprinting. Using this technique, immortalised mouse cholangiocytes have been 3D printed in dECM bioink around a pluronic F-127 sacrificial mould to generate bioprinted bile ducts.
      • Lewis P.L.
      • Yan M.
      • Su J.
      • Shah R.N.
      Directing the growth and alignment of biliary epithelium within extracellular matrix hydrogels.
      Following mould removal, hepatocytes were seeded around bile duct branches in structures reminiscent of the canals of Hering. Although such platforms have not yet been used for disease modelling, they show great potential for studying ductular reaction or cholangiopathies affecting the terminal branches of the biliary tree.
      Figure thumbnail gr3
      Fig. 33D bioprinting of liver-like tissue via layer-by-layer positioning.
      (A) Schematic representation of the process of 3D bioprinting. The printer head extrudes droplets of bioink, a liquid solution of cells in hydrogel. Each droplet is positioned to generate the cross-section of the desired tissue; the bioprinter then proceeds via layer-by-layer positioning fabricating the desired 3D structure.
      In summary, 3D printed tissue provides an optimal platform for studying the impact of tissue microarchitecture and its disruption on the pathogenesis of cholangiopathies. However, technical limitations remain, such as cytotoxicity secondary to sheer stress on printed cells, and the requirement for sacrificial mould bioprinting due to the lack of bioinks with optimal mechanical properties.
      Organ-on-a-chip allows dynamic culture incorporating flow and captures interactions between bile ducts and other systems.

      Organ-on-a-chip

      Bile flow and its disruption play a key role in cholestasis. Systems incorporating flow enable studies on the function of mechanosensing organelles, such as cilia, which are pivotal for the pathogenesis of ciliopathies; furthermore, physiological mechanical stimuli – such as fluid shear stress – induce strengthening of cadherin-based cell junctions that promote the formation of tight junctions,
      • Citi S.
      The mechanobiology of tight junctions.
      ,
      • Pinheiro D.
      • Bellaïche Y.
      Mechanical force-driven adherens junction remodeling and epithelial dynamics.
      resulting in enhanced cell polarisation and increased epithelial barrier function.
      • Rao R.
      • Samak G.
      Bile duct epithelial tight junctions and barrier function.
      However, both multicellular organoids and 3D printing generate bioengineered tissue which is maintained in static culture. Organ-on-chip technology could address this limitation. A chip is a microfluidic device that consists of a network of microchannels lined with cells.
      • Mittal R.
      • Woo F.W.
      • Castro C.S.
      • Cohen M.A.
      • Karanxha J.
      • Mittal J.
      • et al.
      Organ-on-chip models: implications in drug discovery and clinical applications.
      ,
      • Zhang B.
      • Korolj A.
      • Lai B.F.L.
      • Radisic M.
      Advances in organ-on-a-chip engineering.
      Different channels correspond to distinct tissue compartments separated by a cell barrier, such as the luminal and basolateral area of a vessel or hollow organ. The presence of multiple channels allows independent sampling, perfusion
      • Kaarj K.
      • Yoon J.Y.
      Methods of delivering mechanical stimuli to organ-on-a-chip.
      • Coppeta J.
      • Mescher M.J.
      • Isenberg B.C.
      • Spencer A.J.
      • Kim E.S.
      • Lever A.R.
      • et al.
      A portable and reconfigurable multi-organ platform for drug development with onboard microfluidic flow control.
      • Wang Y.I.
      • Shuler M.L.
      UniChip enables long-term recirculating unidirectional perfusion with gravity-driven flow for microphysiological systems.
      and manipulation of each compartment through the application of distinct environmental, mechanical and/or biochemical stimuli,
      • Kaarj K.
      • Yoon J.Y.
      Methods of delivering mechanical stimuli to organ-on-a-chip.
      such as perfusion with different media at varying flow rates (Fig. 4). This layout recapitulates key features of primary tissue, such as spatial organisation around a lumen, polarisation and flow. Each chip corresponds to a single functional unit of an organ, such as a bile duct, while chips with different cell types can be integrated into more complex circuits to capture interactions across multiple organs.
      • Miller P.G.
      • Shuler M.L.
      Design and demonstration of a pumpless 14 compartment microphysiological system.
      This design provides a modular customisable system which is ideal for studying crosstalk between different systems.
      Figure thumbnail gr4
      Fig. 4Key features of organ-on-a-chip.
      (A) Single-channel chips consist of a cell-populated conduit. Intraluminal flow connects the inlet/outlet to a pump. The intra/extraluminal compartments can be sampled independently, enabling studies on barrier function. (B) Multi-channel chips allow co-culture of different cells types. Semi-permeable membranes allow communication between different compartments corresponding to different organs, e.g. gut and liver. (C) Chips promote cell polarisation through cell-to-matrix (integrins) and cell-to-cell interaction (cadherins) combined with fluid shear stress stimulating junction formation and polarisation. (D) Different chips are connected to create multi-organ-on-chip platforms. (E) Flow can be applied through external or integrated pumps or pumpless rocking systems.
      In the context of cholestasis and cholangiopathies, organ-on-a-chip technology has been used to elucidate the pathogenesis of biliary atresia through toxicity, barrier function and permeability studies. This was achieved using bile-ducts-on-chip generated by seeding mouse cholangiocytes into microfluidic devices.
      • Du Y.
      • Khandekar G.
      • Llewellyn J.
      • Polacheck W.
      • Chen C.S.
      • Wells R.G.
      A bile duct-on-a-chip with organ-level functions.
      The resulting ducts exhibited polarisation, tight junction formation, and physiological responses to flow perturbation resulting in increased calcium signalling. Treatment with the toxin biliatresone, shown to cause biliary atresia in animal models,
      • Lorent K.
      • Gong W.
      • Koo K.A.
      • Waisbourd-Zinman O.
      • Karjoo S.
      • Zhao X.
      • et al.
      Identification of a plant isoflavonoid that causes biliary atresia.
      ,
      • Waisbourd-Zinman O.
      • Koh H.
      • Tsai S.
      • Lavrut P.M.
      • Dang C.
      • Zhao X.
      • et al.
      The toxin biliatresone causes mouse extrahepatic cholangiocyte damage and fibrosis through decreased glutathione and SOX17.
      reproduced the disease phenotype in vitro by increasing the permeability of the cholangiocyte membrane and disrupting barrier function. In addition to shedding light on the pathogenesis of biliary atresia, this study highlights the potential of ducts-on-chip as a platform for studies on cholangiocyte bicarbonate secretion and barrier function.
      In addition to studies focusing on bile ducts, organ-on-chip technology provides a unique system for studying interactions between the biliary tree and other systems, such as the gut-liver axis. The gut-liver axis has a critical role in the pathogenesis of cholangiopathies such as PSC; however, capturing multiple organs in a single culture system has not been possible with conventional technology. To address this limitation, microfluidic circuits incorporating liver and intestinal chips have been developed, which reproduce key features of the interaction between the liver and the intestine, including modelling the kinetics of bile acid synthesis and recycling through the entero-hepatic circulation.
      • Chen W.L.K.
      • Edington C.
      • Suter E.
      • Yu J.
      • Velazquez J.J.
      • Velazquez J.G.
      • et al.
      Integrated gut/liver microphysiological systems elucidates inflammatory inter-tissue crosstalk.
      • Choe A.
      • Ha S.K.
      • Choi I.
      • Choi N.
      • Sung J.H.
      Microfluidic gut-liver chip for reproducing the first pass metabolism.
      • Esch M.B.
      • Ueno H.
      • Applegate D.R.
      • Shuler M.L.
      Modular, pumpless body-on-a-chip platform for the co-culture of GI tract epithelium and 3D primary liver tissue.
      Furthermore, intestine-on-a-chip devices have been colonised with bacteria to model the gut microbiome and study host-to-microbe interactions.
      • Jalili-Firoozinezhad S.
      • Gazzaniga F.S.
      • Calamari E.L.
      • Camacho D.M.
      • Fadel C.W.
      • Bein A.
      • et al.
      A complex human gut microbiome cultured in an anaerobic intestine-on-a-chip.
      Although multi-organ chip systems incorporating liver, intestine and bile duct chips have not been developed, the platforms described herein provide proof-of-principle. Multiple other features of the organ-on-chip technology could prove useful for studies in cholangiopathies, such as the generation of small molecule gradients, including oxygen, which could be used to model the effects of ischaemia on bile ducts.
      Overall, organ-on-chip technology provides a unique platform for recapitulating flow dynamics, barrier function and solute gradients through the control of mechano-chemical stimuli, as well as modelling cell-to-cell and tissue-to-tissue signalling by combining different types of chips. Moreover, due to the low media volume and small number of cells required per chip, this technology is optimally suited for high-throughput screening and drug testing. Conversely, wide application of this technology remains restricted by technical limitations. Indeed, fabrication strategies can be operator-dependent, introducing batch-to-batch variation and complicating large scale studies. Furthermore, the small volume of circulating fluid is often below the detection threshold of existing analysers,
      • Capulli A.K.
      • Tian K.
      • Mehandru N.
      • Bukhta A.
      • Choudhury S.F.
      • Suchyta M.
      • et al.
      Approaching the in vitro clinical trial: engineering organs on chips.
      preventing in-depth characterisation of the perfusate and requiring the development of new miniaturised sensors. Finally, chips incorporate cells embedded in hydrogels arranged around a microfluidic channel. This design fails to capture the complex microarchitecture of primary tissue and may not be optimally suited for studies where tissue architecture plays a key role.

      Scaffolded bioengineered tissue

      Cell-to-matrix interactions represent a key aspect of the pathogenesis of fibro-obliterative or fibro-inflammatory cholangiopathies, such as biliary atresia and PSC. The bioengineered systems described in previous sections focus on the role of different cell types in cholangiopathies by interrogating their crosstalk, spatial relationship and interaction with environmental stimuli. However, they are not optimal for exploring the contribution of the cells' ECM to disease pathogenesis. Indeed, the majority of these systems are based on hydrogels which fail to capture the microarchitecture of the matrix, such as the presence of a vascular niche or peribiliary glands, while the tertiary structure and spatial distribution of matrix components, such as the basal lamina proteins, is lost due to digestion and re-polymerisation of these elements. To address these limitations, cholangiocytes have been grown on a variety of synthetic, biological or decellularised scaffolds. Scaffolded biliary tissue is generated using a 2-step process. First the ductal ECM is generated using a variety of fabrication methods, such as rolling of a polymeric sheet, moulding, 3D printing, electrospinning and freeze-drying from synthetic or biological materials or through tissue decellularisation.
      • Justin A.W.
      • Saeb-Parsy K.
      • Markaki A.E.
      • Vallier L.
      • Sampaziotis F.
      Advances in the generation of bioengineered bile ducts.
      Subsequently, the matrices are populated with cells, generating bioengineered bile ducts. These ducts can be connected to bioreactors, which allow independent access to the luminal and extraluminal side of the construct and enable luminal flow of media without the challenge of small volume perfusates (Fig. 5). Consequently, scaffolded biliary tissue allows for the incorporation of physiological or tuneable matrices, while maintaining many of the advantages of ducts-on-chip, such as the capacity to study barrier function and flow dynamics.
      Scaffolded tissue provides advantages of organ-on-a-chip, while allowing studies on cell-to-matrix interaction.
      Figure thumbnail gr5
      Fig. 5Generation and culture of bioengineered bile ducts.
      (A) Schematic representation of different scaffold matrices used for bile duct engineering. Synthetic, biological and decellularised matrices are depicted including their interaction with cells. (B) Scaffolds are seeded with cholangiocytes to generate bioengineered bile ducts. (C) Bioengineered bile ducts are cultured in bioreactors. Perfusion systems (outlined in E) provide intraluminal flow, while different reservoirs enable independent sampling of the intra and extraluminal content.
      Decellularised scaffolds are generated through physical, chemical and/or enzymatic removal of cells from a tissue, resulting in an acellular scaffold that maintains the composition and structure of native ECM. Consequently, decellularised scaffolds closely recapitulate topological cues and physicochemical properties of primary tissue.
      • Mazza G.
      • Rombouts K.
      • Rennie Hall A.
      • Urbani L.
      • Vinh Luong T.
      • Al-Akkad W.
      • et al.
      Decellularized human liver as a natural 3D-scaffold for liver bioengineering and transplantation.
      The feasibility of generating bile ducts from decellularised scaffolds has been demonstrated in rat livers. Decellularised rat livers were repopulated with cholangiocytes via retrograde injection in the extrahepatic biliary tree followed by repopulation with hepatocytes through portal vein perfusion.
      • Chen Y.
      • Devalliere J.
      • Bulutoglu B.
      • Yarmush M.L.
      • Uygun B.E.
      Repopulation of intrahepatic bile ducts in engineered rat liver grafts.
      This resulted in the generation of a bioengineered liver recapitulating the complex 3D interactions between hepatocytes and cholangiocytes in a whole organ setting. The same principle could be applied to healthy or diseased human livers or isolated ducts to unravel the impact of a pathological ECM on cholangiocytes or other liver cell types involved in cholangiopathies and their role in matrix deposition, remodelling and epithelial-to-mesenchymal transition.
      • Mazza G.
      • Al-Akkad W.
      • Rombouts K.
      Engineering in vitro models of hepatofibrogenesis.
      However, the composition of decellularised scaffolds is not defined and their properties are not tuneable.
      To address this limitation, scaffolds have been fabricated from defined synthetic or biological components.
      • Tysoe O.C.
      • Justin A.W.
      • Brevini T.
      • Chen S.E.
      • Mahbubani K.T.
      • Frank A.K.
      • et al.
      Isolation and propagation of primary human cholangiocyte organoids for the generation of bioengineered biliary tissue.
      Synthetic scaffolds are made of materials such as polystyrene, poly-l-lactic acid, polyglycolic acid, poly-ethersulfone and expanded polytetra-fluoroethylene which are not generated by cells or found in human ECM. Synthetic scaffolds are chemically defined and can be tuned to incorporate bioactive matrix molecules
      • Tallawi M.
      • Rosellini E.
      • Barbani N.
      • Grazia Cascone M.
      • Rai R.
      • Saint-Pierre G.
      • et al.
      Strategies for the chemical and biological functionalization of scaffolds for cardiac tissue engineering: a review.
      ,
      • Carmagnola I.
      • Ranzato E.
      • Chiono V.
      Scaffold functionalization to support a tissue biocompatibility.
      – such as laminins, integrins or growth factors – and interrogate their effect on cholangiocytes.
      • Tanimizu N.
      • Kikkawa Y.
      • Mitaka T.
      • Miyajima A.
      α1- and α5-containing laminins regulate the development of bile ducts via β1 integrin signals.
      ,
      • Peng Z.W.
      • Ikenaga N.
      • Liu S.B.
      • Sverdlov D.Y.
      • Vaid K.A.
      • Dixit R.
      • et al.
      Integrin αvβ6 critically regulates hepatic progenitor cell function and promotes ductular reaction, fibrosis, and tumorigenesis.
      Synthetic bile ducts have been generated using collagen-coated polyethersulfone hollow fibres seeded with cholangiocyte-like cells, derived from murine Lgr5+ liver stem cells.
      • Chen C.
      • Jochems P.G.M.
      • Salz L.
      • Schneeberger K.
      • Penning L.C.
      • Van De Graaf S.F.J.
      • et al.
      Bioengineered bile ducts recapitulate key cholangiocyte functions.
      The resulting structures captured basic features of native bile ducts, including expression of appropriate genes, tight junction formation and bile acid transport. Like organ-on-chip, polyethersulfone hollow fibres are compatible with dynamic cultures, providing flow stimuli to the cells, while they still allow independent access to the apical and basolateral compartments, enabling studies on membrane transporters and barrier function. Furthermore, their larger dimensions address challenges associated with low perfusate volume in chips, while the fibres can be coated with matrix components, such as collagen, to allow studies on specific matrix components.
      Conversely, biological scaffolds are fabricated from bioactive molecules – such as collagen, fibronectin, proteoglycans or alginate substrates – which are components of the ECM.
      • Hussey G.S.
      • Dziki J.L.
      • Badylak S.F.
      Extracellular matrix-based materials for regenerative medicine.
      Biological scaffolds are amenable to modification and remodelling by cells. Therefore, they provide insight not only on the impact of the matrix on the cells, but also on the effect of the cells on the matrix.
      • Brown B.N.
      • Badylak S.F.
      Extracellular matrix as an inductive scaffold for functional tissue reconstruction.
      Collagen-based bioengineered bile ducts have been generated using densified collagen seeded with cholangiocyte organoids to generate bioengineered bile ducts recapitulating key features of primary tissue such as expression of cholangiocyte markers, enzymatic activity (alkaline phosphatase and gamma-glutamyltransferase), polarisation, barrier function and luminal transfer of substrates.
      • Sampaziotis F.
      • Justin A.W.
      • Tysoe O.C.
      • Sawiak S.
      • Godfrey E.M.
      • Upponi S.S.
      • et al.
      Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids.
      These bioengineered ducts were used to reconstruct the biliary tree in small animal models, providing evidence of scaffold remodelling in vivo; this model also demonstrated the feasibility of generating animal models with humanised bile ducts, with potential applications for modelling stricturing disease. Despite their advantages, fabricated synthetic and biological scaffolds remain limited to structures with simple geometry and composition, which fail to fully reproduce the complex architecture of in vivo matrices.

      Challenges and future directions

      Overall, the different tissue engineering approaches described provide a series of complementary systems for the study of cholangiopathies, each with its unique advantages (Table 1). Organoids in hydrogels are optimal for studying developmental cholangiopathies and bile duct remodelling, 3D printed tissue provides an excellent platform for recapitulating tissue patterning, bile ducts-on-chip are ideal for investigating barrier function and the interaction between the biliary tree and other organs, while scaffolded tissue is best suited for studies on cell-to-matrix interactions. Despite the advantages of each system, universal challenges remain. Matrices with poorly defined composition, such as Matrigel, remain the gold standard for embedding cells because of their higher biocompatibility compared to chemically defined biomaterials. Furthermore, matrix properties cannot be modified after fabrication of the bioengineered construct or tissue, restricting real-time mechanistic studies on cell-to-matrix interactions. Experimental readouts are restricted by technical limitations. In particular, reclaiming and characterising small numbers and different types of cells and matrices making up bioengineered tissue is technically challenging; visualising live cells is not possible due to increased thickness of the constructs, while the minimal volume of media used in microfluidics systems is below the threshold of detection of conventional sensors. Finally, bioengineered constructs remain in vitro platforms which, despite their advantages, still fail to capture the full complexity of in vivo systems.
      Table 1Characteristics of different tissue engineering approaches.
      MethodMaterialAdvantagesDisadvantages
      HydrogelsSynthetic

      Biological
      Cell self-organisation/remodelling

      Customisable properties

      Reproducible chemical and mechanical properties (synthetic hydrogels)

      Recapitulate ECM (biological hydrogels)
      Challenging downstream analysis (multicellular systems)

      Non-physiological materials (synthetic hydrogels)

      Batch-to-batch variability (biological hydrogels)

      Static culture
      3D printingSynthetic

      Biological
      Customisable structures

      Controlled cell positioning
      Cytotoxicity of the printing process

      Challenging downstream analysis

      Structural limit due to poor mechanical strength

      Static culture
      Organ-on-a-chipVariousDynamic culture

      Trans-epithelial transport

      Multi-organ systems
      Challenging downstream analysis

      Low volume of perfusate

      Small cell numbers
      ScaffoldsSyntheticChemically defined

      Dynamic culture

      Trans-epithelial transport

      Customisable perfusate volume

      Customisable cell numbers
      Limited biocompatibility

      Non-physiological materials
      BiologicalChemically defined

      Dynamic culture

      Trans-epithelial transport

      Customisable perfusate volume

      Customisable cell numbers

      High biocompatibility
      Poor mechanical strength

      Simple architecture
      DecellularisedPhysiological ECM composition

      Physiological mechanical properties

      Physiological tissue microarchitecture

      Dynamic culture

      Trans-epithelial transport

      Customisable perfusate volume

      Customisable cell numbers
      Batch-to-batch variability

      Lack of customisation
      ECM, extracellular matrix.
      Emerging technologies could address these challenges, enabling the development of tissue engineered models able to better recapitulate key pathophysiological features of cholestasis (Fig. 6). Indeed, novel chemically defined materials with high biocompatibility have recently been developed. Furthermore, ‘smart’ hydrogels allow modification of the matrix composition and properties at any point following fabrication using stimuli, such as light, which do not affect the viability of cells.
      • Tong X.
      • Jiang J.
      • Zhu D.
      • Yang F.
      Hydrogels with dual gradients of mechanical and biochemical cues for Deciphering cell-niche interactions.
      • Mohamed M.A.
      • Fallahi A.
      • El-Sokkary A.M.A.
      • Salehi S.
      • Akl M.A.
      • Jafari A.
      • et al.
      Stimuli-responsive hydrogels for manipulation of cell microenvironment: from chemistry to biofabrication technology.
      • Mantha S.
      • Pillai S.
      • Khayambashi P.
      • Upadhyay A.
      • Zhang Y.
      • Tao O.
      • et al.
      Smart hydrogels in tissue engineering and regenerative medicine.
      These systems pave the way for future studies interrogating cell-to-matrix crosstalk.
      • Schrumpf E.
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      • Holm K.
      • Arulampalam V.
      • et al.
      The gut microbiota contributes to a mouse model of spontaneous bile duct inflammation.
      Omics technologies such as proteomics or single-cell transcriptomics address issues with regards to characterisation of cells and matrices.
      • Maarten Altelaar A.F.
      • Munoz J.
      • Heck A.J.R.
      Next-generation proteomics: towards an integrative view of proteome dynamics.
      • Henning N.F.
      • LeDuc R.D.
      • Even K.A.
      • Laronda M.M.
      Proteomic analyses of decellularized porcine ovaries identified new matrisome proteins and spatial differences across and within ovarian compartments.
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      • Mazza G.
      • Telese A.
      • Al-akkad W.
      • Frenguelli L.
      • Levi A.
      • Marrali M.
      • et al.
      Cirrhotic human liver extracellular matrix 3D scaffolds promote smad-dependent TGF-β1 epithelial mesenchymal transition.
      Novel non-destructive imaging techniques, such as micro-CT, optimal coherence tomography or 2-photon microscopy promise to overcome the barrier of increased tissue thickness.
      • Dmitriev R.I.
      Multi-parametric live cell microscopy of 3D tissue models.
      • Teodori L.
      • Crupi A.
      • Costa A.
      • Diaspro A.
      • Melzer S.
      • Tarnok A.
      Three-dimensional imaging technologies: a priority for the advancement of tissue engineering and a challenge for the imaging community.
      • Wang L.
      • Xu M.E.
      • Luo L.
      • Zhou Y.
      • Si P.
      Iterative feedback bio-printing-derived cell-laden hydrogel scaffolds with optimal geometrical fidelity and cellular controllability.
      The use of second-harmonic generation or modified amino acids, such as azide-labelled proline, provides an ideal system for visualising matrix modifications.
      • Bardsley K.
      • Yang Y.
      • El Haj A.J.
      Fluorescent labeling of collagen production by cells for noninvasive imaging of extracellular matrix deposition.
      • Perentes J.Y.
      • Mckee T.D.
      • Ley C.D.
      • Mathiew H.
      • Dawson M.
      • Padera T.P.
      • et al.
      In vivo imaging of extracellular matrix remodeling by tumor-associated fibroblasts.
      • Chen X.
      • Nadiarynkh O.
      • Plotnikov S.
      • Campagnola P.J.
      Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure.
      The development of microsensors which can be embedded directly in microfluidic channels will overcome issues related to minimal volume detection thresholds.
      • Neužil P.
      • Giselbrecht S.
      • Länge K.
      • Huang T.J.
      • Manz A.
      Revisiting lab-on-a-chip technology for drug discovery.
      ,
      • Henry O.Y.F.
      • Villenave R.
      • Cronce M.J.
      • Leineweber W.D.
      • Benz M.A.
      • Ingber D.E.
      Organs-on-chips with integrated electrodes for trans-epithelial electrical resistance (TEER) measurements of human epithelial barrier function.
      Finally, the generation of animals with humanised bile ducts
      • Sampaziotis F.
      • Justin A.W.
      • Tysoe O.C.
      • Sawiak S.
      • Godfrey E.M.
      • Upponi S.S.
      • et al.
      Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids.
      following transplantation of bioengineered constructs could limit the gap between engineering platforms and animal models even further.
      Figure thumbnail gr6
      Fig. 6Tissue engineering for cholestatic disorders: model requirements, current platforms and future perspectives.
      The key pathophysiological features of cholestasis that need to be captured by experimental models are represented in the inner circle. Tissue engineering platforms recapitulating these aspects are depicted in different coloured arcs. Emerging technologies and future perspectives for tissue engineering and modelling of cholestatic disorders are depicted in the outer circle.

      Conclusion

      Cholestatic diseases are multifactorial disorders which are difficult to capture with a single modelling platform. Nonetheless, this challenge can be addressed using a series of complementary platforms. Tissue engineering plays a unique role in achieving this goal by bridging the gap between single-cell systems and animal studies. Multiple engineering platforms have been developed each with unique advantages. Although modelling applications of many of these systems remain restricted by technical limitations, emerging technologies promise to overcome these issues, making tissue engineered models an essential tool for future studies in cholestatic liver disease.

      Abbreviations

      ALGS, Alagille syndrome; dECM, decellularised ECM; DPMs, ductal plate malformations; ECM, extracellular matrix; iPSC, induced pluripotent stem cell; MMPs, matrix metalloproteases; PBC, primary biliary cholangitis; PCLD, polycystic liver disease; PSC, primary sclerosing cholangitis.

      Financial support

      TB is supported by an EASL Juan Rodès Fellowship (PSAG/208) . OCT is supported by an MRC-Sackler Doctoral Training Partnership (REAG/168) and by Rosetrees Trust ( REAG/240 & NMZG/233 ). FS is supported by an Addenbrooke's Charitable Trust Grant , the Academy of Medical Sciences Clinical Lecturer Starter Grant (SGL019/1071) , an NIHR Clinical Lectureship and the Rosetrees Trust ( REAG/240 & NMZG/233 ).

      Authors' contributions

      TB contributed to conceptualization and writing of the manuscript. OCT provided critical revision of the manuscript and proof-reading. FS contributed to conceptualization, critical revision and writing of the manuscript.

      Conflict of interest

      FS is founder and shareholder of Bilitech. The other authors have no conflict of interest.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Acknowledgements

      The authors would like to thank Prof Ludovic Vallier for his constructive input and critical comments and Costanza Brevini for proof-reading the manuscript.

      Supplementary data

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