Cholangiocyte organoids to study drug-induced injury.

BACKGROUND: Drug induced bile duct injury is a frequently observed clinical problem leading to a wide range of pathological features. During the past decades, several agents have been identified with various postulated mechanisms of bile duct damage, however, mostly still poorly understood. METHODS: Here, we investigated the mechanisms of chlorpromazine (CPZ) induced bile duct injury using advanced in vitro cholangiocyte cultures. Intrahepatic cholangiocyte organoids (ICOs) were driven into mature cholangiocyte like cells (CLCs), which were exposed to CPZ under cholestatic or non-cholestatic conditions through the addition of a bile acid cocktail. RESULTS: CPZ caused loss of monolayer integrity by reducing expression levels of tight junction protein 1 (TJP1), E-cadherin 1 (CDH1) and lysyl oxidase homolog 2 (LOXL2). Loss of zonula occuludens-1 (ZO-1) and E-cadherin was confirmed by immunostaining after exposure to CPZ and rhodamine-123 leakage further confirmed disruption of the cholangiocyte barrier function. Furthermore, oxidative stress seemed to play a major role in the early damage response by CPZ. The drug also decreased expression of three main basolateral bile acid transporters, ABCC3 (ATP binding cassette subfamily C member 3), SLC51A/B (solute carrier family 51 subunit alpha/beta) and multidrug resistance transporter ABCB1 (ATP binding cassette subfamily B member 1), thereby contributing to bile acid accumulation. CPZ did not induce an inflammatory response by itself, but addition of TNFα revealed a synergistic effect. CONCLUSION: These results show that ICOs present a model to identify toxic drugs affecting the bile ducts while providing mechanistic insights into hepatotoxicity.

Gelatin-Based Hybrid Hydrogels as Matrices for Organoid Culture.

The application of liver organoids is very promising in the field of liver tissue engineering; however, it is still facing some limitations. One of the current major limitations is the matrix in which they are cultured. The mainly undefined and murine-originated tumor matrices derived from Engelbreth-Holm-Swarm (EHS) sarcoma, such as Matrigel, are still the standard culturing matrices for expansion and differentiation of organoids toward hepatocyte-like cells, which will obstruct its future clinical application potential. In this study, we exploited the use of newly developed highly defined hydrogels as potential matrices for the culture of liver organoids and compared them to Matrigel and two hydrogels that were already researched in the field of organoid research [i.e., polyisocyanopeptides, enriched with laminin-entactin complex (PIC-LEC) and gelatin methacryloyl (GelMA)]. The newly developed hydrogels are materials that have a physicochemical resemblance with native liver tissue. Norbornene-modified dextran cross-linked with thiolated gelatin (DexNB-GelSH) has a swelling ratio and macro- and microscale properties that highly mimic liver tissue. Norbornene-modified chondroitin sulfate cross-linked with thiolated gelatin (CSNB-GelSH) contains chondroitin sulfate, which is a glycosaminoglycan (GAG) that is present in the liver ECM. Furthermore, CSNB-GelSH hydrogels with different mechanical properties were evaluated. Bipotent intrahepatic cholangiocyte organoids (ICOs) were applied in this work and encapsulated in these materials. This research revealed that the newly developed materials outperformed Matrigel, PIC-LEC, and GelMA in the differentiation of ICOs toward hepatocyte-like cells. Furthermore, some trends indicate that an interplay of both the chemical composition and the mechanical properties has an influence on the relative expression of certain hepatocyte markers. Both DexNB-GelSH and CSNB-GelSH showed promising results for the expansion and differentiation of intrahepatic cholangiocyte organoids. The stiffest CSNB-GelSH hydrogel even significantly outperformed Matrigel based on ALB, BSEP, and CYP3A4 gene expression, being three important hepatocyte markers.

Locational memory of macrovessel vascular cells is transcriptionally imprinted.

Vascular pathologies show locational predisposition throughout the body; further insights into the transcriptomics basis of this vascular heterogeneity are needed. We analyzed transcriptomes from cultured endothelial cells and vascular smooth muscle cells from nine adult canine macrovessels: the aorta, coronary artery, vena cava, portal vein, femoral artery, femoral vein, saphenous vein, pulmonary vein, and pulmonary artery. We observed that organ-specific expression patterns persist in vitro, indicating that these genes are not regulated by blood flow or surrounding cell types but are likely fixed in the epigenetic memory. We further demonstrated the preserved location-specific expression of GATA4 protein in cultured cells and in the primary adult vessel. On a functional level, arterial and venous endothelial cells differed in vascular network morphology as the arterial networks maintained a higher complexity. Our findings prompt the rethinking of the extrapolation of results from single-origin endothelial cell systems.

Are slaughterhouse-obtained livers suitable for use in ex vivo perfusion research?

OBJECTIVES: The success of the ex vivo machine perfusion of pig livers used for preclinical research depends on organ quality and availability. In this study, we investigated whether livers obtained from slaughterhouses are suitable and equivalent to livers obtained from laboratory pigs. METHODS: Livers were obtained from slaughterhouse pigs stunned by electrocution or CO2 inhalation and from laboratory pigs. For the latter group, 45 minutes of warm ischemia was mimicked for a subgroup, ensuring a valid comparison with slaughterhouse-derived livers. RESULTS: Livers from CO2-stunned pigs showed lower indocyanine green clearance and bile production, higher blood lactate and potassium concentrations, and higher alanine aminotransferase activities than electrically stunned pigs. Furthermore, livers from electrically stunned pigs, and livers from laboratory pigs, subjected or not to warm ischemia, showed similar performance in terms of perfusion and metabolism. CONCLUSION: For an ex vivo liver model generated using slaughterhouse pigs, electrical stunning is preferable to CO2 stunning. Livers from electrically stunned slaughterhouse pigs performed similarly to laboratory pig livers. These findings support the use of livers from electrically stunned slaughterhouse pigs, which may therefore provide an alternative to livers obtained from laboratory pigs, consistent with the principle of the 3Rs.

The application of organ-on-chip models for the prediction of human pharmacokinetic profiles during drug development.

Organ-on-chip (OoC) technology has led to in vitro models with many new possibilities compared to conventional in vitro and in vivo models. In this review, the potential of OoC models to improve the prediction of human oral bioavailability and intrinsic clearance is discussed, with a focus on the functionality of the models and the application in current drug development practice. Multi-OoC models demonstrating the application for pharmacokinetic (PK) studies are summarized and existing challenges are identified. Physiological parameters for a minimal viable platform of a multi-OoC model to study PK are provided, together with PK specific read-outs and recommendations for relevant reference compounds to validate the model. Finally, the translation to in vivo PK profiles is discussed, which will be required to routinely apply OoC models during drug development.

Drug Metabolism of Hepatocyte-like Organoids and Their Applicability in In Vitro Toxicity Testing.

Emerging advances in the field of in vitro toxicity testing attempt to meet the need for reliable human-based safety assessment in drug development. Intrahepatic cholangiocyte organoids (ICOs) are described as a donor-derived in vitro model for disease modelling and regenerative medicine. Here, we explored the potential of hepatocyte-like ICOs (HL-ICOs) in in vitro toxicity testing by exploring the expression and activity of genes involved in drug metabolism, a key determinant in drug-induced toxicity, and the exposure of HL-ICOs to well-known hepatotoxicants. The current state of drug metabolism in HL-ICOs showed levels comparable to those of PHHs and HepaRGs for CYP3A4; however, other enzymes, such as CYP2B6 and CYP2D6, were expressed at lower levels. Additionally, EC50 values were determined in HL-ICOs for acetaminophen (24.0−26.8 mM), diclofenac (475.5−>500 µM), perhexiline (9.7−>31.5 µM), troglitazone (23.1−90.8 µM), and valproic acid (>10 mM). Exposure to the hepatotoxicants showed EC50s in HL-ICOs comparable to those in PHHs and HepaRGs; however, for acetaminophen exposure, HL-ICOs were less sensitive. Further elucidation of enzyme and transporter activity in drug metabolism in HL-ICOs and exposure to a more extensive compound set are needed to accurately define the potential of HL-ICOs in in vitro toxicity testing.

A human kidney and liver organoid-based multi-organ-on-a-chip model to study the therapeutic effects and biodistribution of mesenchymal stromal cell-derived extracellular vesicles.

Mesenchymal stromal cell (MSC)-derived small extracellular vesicles (sEVs) show therapeutic potential in multiple disease models, including kidney injury. Clinical translation of sEVs requires further preclinical and regulatory developments, including elucidation of the biodistribution and mode of action (MoA). Biodistribution can be determined using labelled sEVs in animal models which come with ethical concerns, are time-consuming and expensive, and may not well represent human physiology. We hypothesised that, based on developments in microfluidics and human organoid technology, in vitro multi-organ-on-a-chip (MOC) models allow us to study effects of sEVs in modelled human organs like kidney and liver in a semi-systemic manner. Human kidney- and liver organoids combined by microfluidic channels maintained physiological functions, and a kidney injury model was established using hydrogenperoxide. MSC-sEVs were isolated, and their size, density and potential contamination were analysed. These sEVs stimulated recovery of the renal epithelium after injury. Microscopic analysis shows increased accumulation of PKH67-labelled sEVs not only in injured kidney cells, but also in the unharmed liver organoids, compared to healthy control conditions. In conclusion, this new MOC model recapitulates therapeutic efficacy and biodistribution of MSC-sEVs as observed in animal models. Its human background allows for in-depth analysis of the MoA and identification of potential side effects.

Normothermic Ex Vivo Liver Platform Using Porcine Slaughterhouse Livers for Disease Modeling.

Metabolic and toxic liver disorders, such as fatty liver disease (steatosis) and drug-induced liver injury, are highly prevalent and potentially life-threatening. To allow for the study of these disorders from the early stages onward, without using experimental animals, we collected porcine livers in a slaughterhouse and perfused these livers normothermically. With our simplified protocol, the perfused slaughterhouse livers remained viable and functional over five hours of perfusion, as shown by hemodynamics, bile production, indocyanine green clearance, ammonia metabolism, gene expression and histology. As a proof-of-concept to study liver disorders, we show that an infusion of free fatty acids and acetaminophen results in early biochemical signs of liver damage, including reduced functionality. In conclusion, the present platform offers an accessible system to perform research in a functional, relevant large animal model while avoiding using experimental animals. With further improvements to the model, prolonged exposure could make this model a versatile tool for studying liver diseases and potential treatments.

High level of polarized engraftment of porcine intrahepatic cholangiocyte organoids in decellularized liver scaffolds.

In Europe alone, each year 5500 people require a life-saving liver transplantation, but 18% die before receiving one due to the shortage of donor organs. Whole organ engineering, utilizing decellularized liver scaffolds repopulated with autologous cells, is an attractive alternative to increase the pool of available organs for transplantation. The development of this technology is hampered by a lack of a suitable large-animal model representative of the human physiology and a reliable and continuous cell source. We have generated porcine intrahepatic cholangiocyte organoids from adult stem cells and demonstrate that these cultures remained stable over multiple passages whilst retaining the ability to differentiate into hepatocyte- and cholangiocyte-like cells. Recellularization onto porcine scaffolds was efficient and the organoids homogeneously differentiated, even showing polarization. Our porcine intrahepatic cholangiocyte system, combined with porcine liver scaffold paves the way for developing whole liver engineering in a relevant large-animal model.

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.

Hepatic bile acid synthesis and secretion: Comparison of in vitro methods.

Reliable hepatic in vitro systems are crucial for the safety assessment of xenobiotics. Certain xenobiotics decrease the hepatic bile efflux, which can ultimately result in cholestasis. Preclinical animal models and the currently available in vitro systems poorly predict a xenobiotic's cholestatic potential. Here, we compared the phenotype and capacity of three liver derived in vitro systems to emulate human functionality to synthesize and secrete bile acids (BAs). To this end, basal BA production of sandwich cultured human hepatocytes (SCHHs), HepaRG cells (HepaRGs) and hepatocyte-like intrahepatic cholangiocyte organoids (ICO-heps) were analysed, and the effect of the known BSEP (Bile Salt Export Pump)-inhibitors bosentan and lopinavir on BA disposition in SCHHs and HepaRGs was quantified. RT-qPCR of selected target genes involved in maturation status, synthesis, transport and conjugation of BAs was performed to mechanistically underpin the observed differences in BA homeostasis. ICO-heps produced a (very) low amount of BAs. SCHHs are a powerful tool in cholestasis-testing due to their high basal BA production and high transporter expression compared to the other models tested. HepaRGs were responsive to both selected BSEP-inhibitors and produced a BA profile that is most similar to the human in vivo situation, making them a suitable and practical candidate for cholestasis-testing.

Hydrogels derived from decellularized liver tissue support the growth and differentiation of cholangiocyte organoids.

Human cholangiocyte organoids are promising for regenerative medicine applications, such as repair of damaged bile ducts. However, organoids are typically cultured in mouse tumor-derived basement membrane extracts (BME), which is poorly defined, highly variable and limits the direct clinical applications of organoids in patients. Extracellular matrix (ECM)-derived hydrogels prepared from decellularized human or porcine livers are attractive alternative culture substrates. Here, the culture and expansion of human cholangiocyte organoids in liver ECM(LECM)-derived hydrogels is described. These hydrogels support proliferation of cholangiocyte organoids and maintain the cholangiocyte-like phenotype. The use of LECM hydrogels does not significantly alter the expression of selected genes or proteins, such as the cholangiocyte marker cytokeratin-7, and no species-specific effect is found between human or porcine LECM hydrogels. Proliferation rates of organoids cultured in LECM hydrogels are lower, but the differentiation capacity of the cholangiocyte organoids towards hepatocyte-like cells is not altered by the presence of tissue-specific ECM components. Moreover, human LECM extracts support the expansion of ICO in a dynamic culture set up without the need for laborious static culture of organoids in hydrogel domes. Liver ECM hydrogels can successfully replace tumor-derived BME and can potentially unlock the full clinical potential of human cholangiocyte organoids.

Volumetric Bioprinting of Organoids and Optically Tuned Hydrogels to Build Liver-Like Metabolic Biofactories.

Organ- and tissue-level biological functions are intimately linked to microscale cell-cell interactions and to the overarching tissue architecture. Together, biofabrication and organoid technologies offer the unique potential to engineer multi-scale living constructs, with cellular microenvironments formed by stem cell self-assembled structures embedded in customizable bioprinted geometries. This study introduces the volumetric bioprinting of complex organoid-laden constructs, which capture key functions of the human liver. Volumetric bioprinting via optical tomography shapes organoid-laden gelatin hydrogels into complex centimeter-scale 3D structures in under 20 s. Optically tuned bioresins enable refractive index matching of specific intracellular structures, countering the disruptive impact of cell-mediated light scattering on printing resolution. This layerless, nozzle-free technique poses no harmful mechanical stresses on organoids, resulting in superior viability and morphology preservation post-printing. Bioprinted organoids undergo hepatocytic differentiation showing albumin synthesis, liver-specific enzyme activity, and remarkably acquired native-like polarization. Organoids embedded within low stiffness gelatins (<2 kPa) are bioprinted into mathematically defined lattices with varying degrees of pore network tortuosity, and cultured under perfusion. These structures act as metabolic biofactories in which liver-specific ammonia detoxification can be enhanced by the architectural profile of the constructs. This technology opens up new possibilities for regenerative medicine and personalized drug testing.

The potential and limitations of intrahepatic cholangiocyte organoids to study inborn errors of metabolism.

Inborn errors of metabolism (IEMs) comprise a diverse group of individually rare monogenic disorders that affect metabolic pathways. Mutations lead to enzymatic deficiency or dysfunction, which results in intermediate metabolite accumulation or deficit leading to disease phenotypes. Currently, treatment options for many IEMs are insufficient. Rarity of individual IEMs hampers therapy development and phenotypic and genetic heterogeneity suggest beneficial effects of personalized approaches. Recently, cultures of patient-own liver-derived intrahepatic cholangiocyte organoids (ICOs) have been established. Since most metabolic genes are expressed in the liver, patient-derived ICOs represent exciting possibilities for in vitro modeling and personalized drug testing for IEMs. However, the exact application range of ICOs remains unclear. To address this, we examined which metabolic pathways can be studied with ICOs and what the potential and limitations of patient-derived ICOs are to model metabolic functions. We present functional assays in patient ICOs with defects in branched-chain amino acid metabolism (methylmalonic acidemia), copper metabolism (Wilson disease), and transporter defects (cystic fibrosis). We discuss the broad range of functional assays that can be applied to ICOs, but also address the limitations of these patient-specific cell models. In doing so, we aim to guide the selection of the appropriate cell model for studies of a specific disease or metabolic process.

Bioprinting of Human Liver-Derived Epithelial Organoids for Toxicity Studies.

There is a need for long-lived hepatic in vitro models to better predict drug induced liver injury (DILI). Human liver-derived epithelial organoids are a promising cell source for advanced in vitro models. Here, organoid technology is combined with biofabrication techniques, which holds great potential for the design of in vitro models with complex and customizable architectures. Here, porous constructs with human hepatocyte-like cells derived from organoids are generated using extrusion-based printing technology. Cell viability of bioprinted organoids remains stable for up to ten days (88-107% cell viability compared to the day of printing). The expression of hepatic markers, transporters, and phase I enzymes increased compared to undifferentiated controls, and is comparable to non-printed controls. Exposure to acetaminophen, a well-known hepatotoxic compound, decreases cell viability of bioprinted liver organoids to 21-51% (p < 0.05) compared to the start of exposure, and elevated levels of damage marker miR-122 are observed in the culture medium, indicating the potential use of the bioprinted constructs for toxicity testing. In conclusion, human liver-derived epithelial organoids can be combined with a biofabrication approach, thereby paving the way to create perfusable, complex constructs which can be used as toxicology- and disease-models.

Building consensus on definition and nomenclature of hepatic, pancreatic, and biliary organoids.

Hepatic, pancreatic, and biliary (HPB) organoids are powerful tools for studying development, disease, and regeneration. As organoid research expands, the need for clear definitions and nomenclature describing these systems also grows. To facilitate scientific communication and consistent interpretation, we revisit the concept of an organoid and introduce an intuitive classification system and nomenclature for describing these 3D structures through the consensus of experts in the field. To promote the standardization and validation of HPB organoids, we propose guidelines for establishing, characterizing, and benchmarking future systems. Finally, we address some of the major challenges to the clinical application of organoids.

Tissue-Engineered Bile Ducts for Disease Modeling and Therapy.

Recent biotechnical advances in the in vitro culture of cholangiocytes and generation of bioengineered biliary tissue have a high potential for creating biliary tissue to be used for disease modeling, drug screening, and transplantation. For the past few decades, scientists have searched for a source of cholangiocytes, focused on primary cholangiocytes or cholangiocytes derived from hepatocytes or stem cells. At the same time, the development of scaffolds for biliary tissue engineering for transplantation and modeling of cholangiopathies has been explored. In this review, we provide an overview on the current understanding of cholangiocytes sources, the effect of signaling molecules, and transcription factors on cell differentiation, along with the effects of extracellular matrix molecules and scaffolds on bioengineered biliary tissues, and their application in disease modeling and drug screening. Impact statement Over the past few decades, biliary tissue engineering has acquired significant attention, but currently a number of factors hinder this field to eventually generate bioengineered bile ducts that mimic in vivo physiology and are suitable for transplantation. In this review, we present the latest advances with respect to cell source selection, influence of growth factors and scaffolds, and functional characterization, as well as applications in cholangiopathy modeling and drug screening. This review is suited for a broad spectrum of readers, including fundamental liver researchers and clinicians with interest in the current state and application of bile duct engineering and disease modeling.

Human extrahepatic and intrahepatic cholangiocyte organoids show region-specific differentiation potential and model cystic fibrosis-related bile duct disease.

The development, homeostasis, and repair of intrahepatic and extrahepatic bile ducts are thought to involve distinct mechanisms including proliferation and maturation of cholangiocyte and progenitor cells. This study aimed to characterize human extrahepatic cholangiocyte organoids (ECO) using canonical Wnt-stimulated culture medium previously developed for intrahepatic cholangiocyte organoids (ICO). Paired ECO and ICO were derived from common bile duct and liver tissue, respectively. Characterization showed both organoid types were highly similar, though some differences in size and gene expression were observed. Both ECO and ICO have cholangiocyte fate differentiation capacity. However, unlike ICO, ECO lack the potential for differentiation towards a hepatocyte-like fate. Importantly, ECO derived from a cystic fibrosis patient showed no CFTR channel activity but normal chloride channel and MDR1 transporter activity. In conclusion, this study shows that ECO and ICO have distinct lineage fate and that ECO provide a competent model to study extrahepatic bile duct diseases like cystic fibrosis.

From fatty hepatocytes to impaired bile flow: Matching model systems for liver biology and disease.

A large variety of model systems are used in hepatobiliary research. In this review, we aim to provide an overview of established and emerging models for specific research questions. We specifically discuss the value and limitations of these models for research on metabolic associated fatty liver disease (MAFLD), (previously named non-alcoholic fatty liver diseases/non-alcoholic steatohepatitis (NAFLD/NASH)) and cholestasis-related diseases such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). The entire range of models is discussed varying from immortalized cell lines, mature or pluripotent stem cell-based models including organoids/spheroids, to animal models and human ex vivo models such as normothermic machine perfusion of livers and living liver slices. Finally, the pros and cons of each model are discussed as well as the need in the scientific community for continuous innovation in model development to better mimic the human (patho)physiology.

New approach methodologies (NAMs) for human-relevant biokinetics predictions. Meeting the paradigm shift in toxicology towards an animal-free chemical risk assessment

For almost fifteen years, the availability and regulatory acceptance of new approach methodologies (NAMs) to assess the absorption, distribution, metabolism and excretion (ADME/biokinetics) in chemical risk evaluations are a bottleneck. To enhance the field, a team of 24 experts from science, industry, and regulatory bodies, including new generation toxicologists, met at the Lorentz Centre in Leiden, The Netherlands. A range of possibilities for the use of NAMs for biokinetics in risk evaluations were formulated (for example to define species differences and human variation or to perform quantitative in vitro-in vivo extrapolations). To increase the regulatory use and acceptance of NAMs for biokinetics for these ADME considerations within risk evaluations, the development of test guidelines (protocols) and of overarching guidance documents is considered a critical step. To this end, a need for an expert group on biokinetics within the Organisation of Economic Cooperation and Development (OECD) to supervise this process was formulated. The workshop discussions revealed that method development is still required, particularly to adequately capture transporter mediated processes as well as to obtain cell models that reflect the physiology and kinetic characteristics of relevant organs. Developments in the fields of stem cells, organoids and organ-on-a-chip models provide promising tools to meet these research needs in the future.