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Conventional static culture of organoids necessitates weekly manual passaging and results in nonhomogeneous exposure of organoids to nutrients, oxygen, and toxic metabolites. Here, we developed a miniaturized spinning bioreactor, RPMotion, specifically optimized for accelerated and cost-effective culture of epithelial organoids under homogeneous conditions. We established tissue-specific RPMotion settings and standard operating protocols for the expansion of human epithelial organoids derived from the liver, intestine, and pancreas. All organoid types proliferated faster in the bioreactor (5.2-fold, 3-fold, and 4-fold, respectively) compared to static culture while keeping their organ-specific phenotypes. We confirmed that the bioreactor is suitable for organoid establishment directly from biopsies and for long-term expansion of liver organoids. Furthermore, we showed that after accelerated expansion, liver organoids can be differentiated into hepatocyte-like cells in the RPMotion bioreactor. In conclusion, this miniaturized bioreactor enables work-, time-, and cost-efficient organoid culture, holding great promise for organoid-based fundamental and translational research and development., Competing Interests: Declaration of interests S.Y., G.S.v.T., B.S., and K.S. hold a Dutch patent, NL2029095B1, and a PCT patent application, WO2023031329A1, on the organoid bioreactor with royalties paid. K.S. and B.S. are founders, employees, and shareholders of the Utrecht University spin-off company Orgonex B.V., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Autofluorescence is an intrinsic feature of cells, caused by the natural emission of light by photo-excitatory molecular content, which can complicate analysis of flow cytometry data. Different cell types have different autofluorescence spectra and, even within one cell type, heterogeneity of autofluorescence spectra can be present, for example, as a consequence of activation status or metabolic changes. By using full spectrum flow cytometry, the emission spectrum of a fluorochrome is captured by a set of photo detectors across a range of wavelengths, creating an unique signature for that fluorochrome. This signature is then used to identify, or unmix, that fluorochrome's unique spectrum from a multicolor sample containing different fluorescent molecules. Importantly, this means that this technology can also be used to identify intrinsic autofluorescence signal of an unstained sample, which can be used for unmixing purposes and to separate the autofluorescence signal from the fluorophore signals. However, this only works if the sample has a singular, relatively homogeneous and bright autofluorescence spectrum. To analyze samples with heterogeneous autofluorescence spectral profiles, we setup an unbiased workflow to more quickly identify differing autofluorescence spectra present in a sample to include as "autofluorescence signatures" during the unmixing of the full stained samples. First, clusters of cells with similar autofluorescence spectra are identified by unbiased dimensional reduction and clustering of unstained cells. Then, unique autofluorescence clusters are determined and are used to improve the unmixing accuracy of the full stained sample. Independent of the intensity of the autofluorescence and immunophenotyping of cell subsets, this unbiased method allows for the identification of most of the distinct autofluorescence spectra present in a sample, leading to less confounding autofluorescence spillover and spread into extrinsic phenotyping markers. Furthermore, this method is equally useful for spectral analysis of different biological samples, including tissue cell suspensions, peripheral blood mononuclear cells, and in vitro cultures of (primary) cells., (© 2024 The Author(s). Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.)

Cholangiocyte organoids provide a powerful platform for applications ranging from in vitro modeling to tissue engineering for regenerative medicine. However, their expansion and differentiation are typically conducted in animal-derived hydrogels, which impede the full maturation of organoids into functional cholangiocytes. In addition, these hydrogels are poorly defined and complex, limiting the clinical applicability of organoids. In this study, a novel medium composition combined with synthetic polyisocyanopeptide (PIC) hydrogels to enhance the maturation of intrahepatic cholangiocyte organoids (ICOs) into functional cholangiocytes is utilized. ICOs cultured in the presence of sodium butyrate and valproic acid, a histone deacetylase inhibitor, and a Notch signaling activator, respectively, in PIC hydrogel exhibit a more mature phenotype, as evidenced by increased expression of key cholangiocyte markers, crucial for biliary function. Notably, mature cholangiocyte organoids in PIC hydrogel display apical-out polarity, in contrast to the traditional basal-out polarization of ICOs cultured in Matrigel. Moreover, these mature cholangiocyte organoids effectively model the biliary pro-fibrotic response induced by transforming growth factor beta. Taken together, an animal-free, chemically defined culture system that promotes the ICOs into mature cholangiocytes with apical-out polarity, facilitating regenerative medicine applications and in vitro studies that require access to the apical membrane, is developed., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Purpose: In an effort to reduce waitlist mortality, extended criteria donor organs, including those from donation after circulatory death (DCD), are being used with increasing frequency. These donors carry an increased risk for postoperative complications, and balancing donor-recipient risks is currently based on generalized nomograms. Abdominal normothermic regional perfusion (aNRP) enables individual evaluation of DCD organs, but a gold standard to determine suitability for transplantation is lacking. This study aimed to incorporate individualized and predictive measurements of the liver maximum capacity (LiMAx) test to objectively grade liver function during aNRP and prevent post-op complications., Methods: aNRP was performed to salvage 18 DCD liver grafts, otherwise discarded. Continuous variables were presented as the median with the interquartile range., Results: The liver function maximum capacity (LiMAx) test was successfully performed within the aNRP circuit in 17 aNRPs (94%). Donor livers with good lactate clearance during aNRP demonstrated significantly higher LiMAx scores (396 (301-451) µg/kg/h versus those who did not 105 (70-158) µg/kg/h; P  = 0.006). This was also true for manifesting stress hyperglycemia > 20 mmol/l ( P  = 0.032). LiMAx score correlated with alanine aminotransferase (ALT; R  =  - 0.755) and aspartate transaminase (AST; R  =  - 0.800) levels during perfusion and distinguished livers that were selected for transplantation (397 (346-453) µg/kg/h) from those who were discarded (155 (87-206) µg/kg/h; P  < 0.001). Twelve livers were accepted for transplantation, blinded for LiMAx results, and all had LiMAx scores of > 241 µg/kg/h. Postoperatively, LiMAx during aNRP displayed correlation with 24-h lactate levels., Conclusions: This study shows for the first time the feasibility to assess liver function during aNRP in individual donor livers. LiMAx presents an objective tool to predict donor liver function and risk of complications in the recipient, thus enabling individualized matching of donor livers for an individual recipient. The LiMAx test may present a valuable test for the prediction of donor liver function, preventing post-transplant complication, and personalizing the selection of donor livers for individual recipients., Supplementary Information: The online version contains supplementary material available at 10.1007/s13167-024-00371-7., Competing Interests: Competing interestsThe authors have no conflict of interest., (© The Author(s) 2024.)

Fibroblastic reticular cells (FRCs) are mesenchymal stromal cells in human lymph nodes (LNs) playing a pivotal role in adaptive immunity. Several FRC subsets have been identified, yet it remains to be elucidated if their heterogeneity is maintained upon culture. Here, we established a protocol to preserve and culture FRCs from human LNs and characterized their phenotypic profile in fresh LN suspensions and upon culture using multispectral flow cytometry. We found nine FRC subsets in fresh human LNs, independent of donor, of which four persisted in culture throughout several passages. Interestingly, the historically FRC-defining marker podoplanin (PDPN) was not present on all FRC subsets. Therefore, we propose that CD45 neg CD31 neg human FRCs are not restricted by PDPN expression, as we found CD90, BST1, and CD146/MCAM to be more widely expressed. Together, our data provide insight into FRC heterogeneity in human LNs, enabling further investigation into the function of individual FRC subsets., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Background: Human lymph node (HuLN) models have emerged with invaluable potential for immunological research and therapeutic application given their fundamental role in human health and disease. While fibroblastic reticular cells (FRCs) are instrumental to HuLN functioning, their inclusion and recognition of importance for organotypic in vitro lymphoid models remain limited., Methods: Here, we established an in vitro three-dimensional (3D) model in a collagen-fibrin hydrogel with primary FRCs and a dendritic cell (DC) cell line (MUTZ-3 DC). To study and characterise the cellular interactions seen in this 3D FRC-DC organotypic model compared to the native HuLN; flow cytometry, immunohistochemistry, immunofluorescence and cytokine/chemokine analysis were performed., Results: FRCs were pivotal for survival, proliferation and localisation of MUTZ-3 DCs. Additionally, we found that CD1a expression was absent on MUTZ-3 DCs that developed in the presence of FRCs during cytokine-induced MUTZ-3 DC differentiation, which was also seen with primary monocyte-derived DCs (moDCs). This phenotype resembled HuLN-resident DCs, which we detected in primary HuLNs, and these CD1a - MUTZ-3 DCs induced T cell proliferation within a mixed leukocyte reaction (MLR), indicating a functional DC status. FRCs expressed podoplanin (PDPN), CD90 (Thy-1), CD146 (MCAM) and Gremlin-1, thereby resembling the DC supporting stromal cell subset identified in HuLNs., Conclusion: This 3D FRC-DC organotypic model highlights the influence and importance of FRCs for DC functioning in a more realistic HuLN microenvironment. As such, this work provides a starting point for the development of an in vitro HuLN., (© 2023. Korean Tissue Engineering and Regenerative Medicine Society.)

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., (© 2024. The Author(s).)

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.

Hepatitis E virus (HEV) infection can cause severe complications and high mortality, particularly in pregnant women, organ transplant recipients, individuals with pre-existing liver disease and immunosuppressed patients. However, there are still unmet needs for treating chronic HEV infections. Herein, we screened a best-in-class drug repurposing library consisting of 262 drugs/compounds. Upon screening, we identified vidofludimus calcium and pyrazofurin as novel anti-HEV entities. Vidofludimus calcium is the next-generation dihydroorotate dehydrogenase (DHODH) inhibitor in the phase 3 pipeline to treat autoimmune diseases or SARS-CoV-2 infection. Pyrazofurin selectively targets uridine monophosphate synthetase (UMPS). Their anti-HEV effects were further investigated in a range of cell culture models and human liver organoids models with wild type HEV strains and ribavirin treatment failure-associated HEV strains. Encouragingly, both drugs exhibited a sizeable therapeutic window against HEV. For instance, the IC 50 value of vidofludimus calcium is 4.6-7.6-fold lower than the current therapeutic doses in patients. Mechanistically, their anti-HEV mode of action depends on the blockage of pyrimidine synthesis. Notably, two drugs robustly inhibited ribavirin treatment failure-associated HEV mutants (Y1320H, G1634R). Their combination with IFN-α resulted in synergistic antiviral activity. In conclusion, we identified vidofludimus calcium and pyrazofurin as potent candidates for the treatment of HEV infections. Based on their antiviral potency, and also the favorable safety profile identified in clinical studies, our study supports the initiation of clinical studies to repurpose these drugs for treating chronic hepatitis E., Competing Interests: Conflict of interest The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript., (Copyright © 2023 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

The extracellular matrix (ECM) is essential for cell support during homeostasis and plays a critical role in cancer. Although research often concentrates on the tumor's cellular aspect, attention is growing for the importance of the cancer-associated ECM. Biochemical and physical ECM signals affect tumor formation, invasion, metastasis, and therapy resistance. Examining the tumor microenvironment uncovers intricate ECM dysregulation and interactions with cancer and stromal cells. Anticancer therapies targeting ECM sensors and remodelers, including integrins and matrix metalloproteinases, and ECM-remodeling cells, have seen limited success. This review explores the ECM's role in cancer and discusses potential therapeutic strategies for cell-ECM interactions.

Allogeneic transplant organs are potentially highly immunogenic. The endothelial cells (ECs) located within the vascular system serve as the primary interface between the recipient's immune system and the donor organ, playing a key role in the alloimmune response. In this study, we investigated the potential use of recipient-derived ECs in a vein recellularization model. In this study, human iliac veins underwent complete decellularization using a Triton X-100 protocol. We demonstrated the feasibility of re-endothelializing acellular blood vessels using either human umbilical cord vein endothelial cell or human venous-derived ECs, with this re- endothelialization being sustainable for up to 28 days in vitro. The re-endothelialized veins exhibited the restoration of vascular barrier function, along with the restoration of innate immunoregulatory capabilities, evident through the facilitation of monocytic cell transmigration and their polarization toward a macrophage phenotype following transendothelial extravasation. Finally, we explored whether recellularization with EC of a different donor could prevent antibody-mediated rejection. We demonstrated that in chimeric vessels, allogeneic EC became a target of the humoral anti-donor response after activation of the classical immune complement pathway whereas autologous EC were spared, emphasizing their potential utility before transplantation. In conclusion, our study demonstrates that replacement of EC in transplants could reduce the immunological challenges associated with allogeneic grafts.

Bile ducts are essential for bile transport and consist of complex branching tubular networks. Human patient-derived cholangiocyte develops a cystic rather than branching duct morphology. Here, we present a protocol to establish branching morphogenesis in cholangiocyte and cholangiocarcinoma organoids. We describe steps for the initiation, maintenance, and expansion of intrahepatic cholangiocyte organoids branching morphology. This protocol enables the study of organ-specific and mesenchymal-independent branching morphogenesis and provides an improved model to study biliary function and diseases. For complete details on the use and execution of this protocol, please refer to Roos et al. (2022). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Primary sclerosing cholangitis (PSC) is a chronic progressive pathological process, related to inflammatory bowel disease and subsequent bacterial translocation. Liver transplantation (LT) is the only curative therapy, but outcomes are compromised by recurrence of PSC (rPSC). The aim of the study was to investigate a potential link between intestinal bacteremia, fucosyltransferase-2 (FUT2), and rPSC after LT., Methods: LT recipients with PSC (n = 81) or without PSC (n = 271) were analyzed for clinical outcomes and positive bacterial blood cultures. A link between bacteremia and the genetic variant of the FUT2 gene was investigated., Results: The incidence of inflammatory bowel disease was significantly higher in PSC recipients but not associated with rPSC. Bacteremia occurred in 31% of PSC recipients. The incidence of rPSC was 37% and was significantly more common in patients with intestinal bacteremia versus no bacteremia (82% versus 30%; P = 0.003). The nonsecretor polymorphism of the FUT2 gene was identified as a genetic risk factor for both intestinal bacteremia and rPSC. Combined FUT2 genotype and intestinal bacteremia in recipients resulted in the highest risk for rPSC (hazard ratio, 15.3; P < 0.001)., Conclusions: Thus, in this article, we showed that bacterial translocation is associated with rPSC after LT and related to the FUT2 nonsecretor status., Competing Interests: The authors declare no funding or conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Liver disease is a common cause of morbidity and mortality, and many patients would benefit from liver transplantation. However, because of a shortage of suitable donor livers, even of those patients who are placed on the donor liver waiting list, many do not survive the waiting time for transplantation. Therefore, alternative treatments for end-stage liver disease need to be explored. Recent advances in organoid technology might serve as a solution to overcome the donor liver shortage in the future. In this overview, we highlight the potential of organoid technology for cell therapy and tissue engineering approaches. Both organoid-based approaches could be used as treatment for end-stage liver disease patients. Additionally, organoid-based cell therapy can also be used to repair liver grafts ex vivo to increase the supply of transplantable liver tissue. The potential of both approaches to become clinically available is carefully assessed, including their clinical, ethical, and economic implications. We provide insight into what aspects should be considered further to allow alternatives to donor liver transplantation to be successfully clinically implemented., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Cholangiocarcinoma (CCA) is a rare malignancy that develops at any point along the biliary tree. CCA has a poor prognosis, its clinical management remains challenging, and effective treatments are lacking. Therefore, preclinical research is of pivotal importance and necessary to acquire a deeper understanding of CCA and improve therapeutic outcomes. Preclinical research involves developing and managing complementary experimental models, from in vitro assays using primary cells or cell lines cultured in 2D or 3D to in vivo models with engrafted material, chemically induced CCA or genetically engineered models. All are valuable tools with well-defined advantages and limitations. The choice of a preclinical model is guided by the question(s) to be addressed; ideally, results should be recapitulated in independent approaches. In this Consensus Statement, a task force of 45 experts in CCA molecular and cellular biology and clinicians, including pathologists, from ten countries provides recommendations on the minimal criteria for preclinical models to provide a uniform approach. These recommendations are based on two rounds of questionnaires completed by 35 (first round) and 45 (second round) experts to reach a consensus with 13 statements. An agreement was defined when at least 90% of the participants voting anonymously agreed with a statement. The ultimate goal was to transfer basic laboratory research to the clinics through increased disease understanding and to develop clinical biomarkers and innovative therapies for patients with CCA., (© 2023. Springer Nature Limited.)

Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease of the biliary tree and a risk factor for development of cholangiocarcinoma (CCA). The pathogenesis of PSC-related CCA is largely unclear, although it is assumed that chronic inflammatory environment plays a pivotal role. We aimed to investigate the effect of inflammation-related cytokines in PSC on the proliferation rate of cancer cells. For this, the proliferation index in PSC-CCA and sporadic CCA was determined by Ki-67 immunohistochemistry. The percentage of Ki-67 positivity in cancer cells was significantly higher in PSC-CCA than in sporadic CCA (41.3% ± 5.7% vs 25.8% ± 4.1%; P = .038). To assess which cytokines in the inflammatory environment have the potential to stimulate cancer cell proliferation, patient-derived CCA organoids (CCAOs) were exposed to five cytokines related to PSC (Interleukin (IL)-1β, IL-6, IL-17A, interferon gamma and tumor necrosis factor alpha). Only IL-17A showed a significant stimulatory effect on cell proliferation in CCAOs, increasing organoid size by 45.9% ± 16.4% (P < .01) and proliferation rate by 38% ± 16% (P < .05). IL-17A immunohistochemistry demonstrated that PSC-CCA might express more IL-17A than sporadic CCA. Moreover, correlation analysis in sporadic CCA and PSC-CCA found a significant correlation between IL-17A expression and proliferation. In conclusion, tumor cell proliferation is increased in PSC-CCA cells compared with sporadic CCA cells. IL-17A increases CCA cell proliferation in vitro and may contribute to the high proliferation rate in PSC-CCA in situ. Therefore, IL-17A represents a new potential therapeutic target in (PSC-)CCA, to be tested in future trials., (© 2022 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

In the past decade, treatments targeting the immune system have revolutionized the cancer treatment field. Therapies such as immune checkpoint inhibitors have been approved as first-line treatment in a variety of solid tumors such as melanoma and non-small cell lung cancer while other therapies, for instance, chimeric antigen receptor (CAR) lymphocyte transfer therapies, are still in development. Although promising results are obtained in a small subset of patients, overall clinical efficacy of most immunotherapeutics is limited due to intertumoral heterogeneity and therapy resistance. Therefore, prediction of patient-specific responses would be of great value for efficient use of costly immunotherapeutic drugs as well as better outcomes. Because many immunotherapeutics operate by enhancing the interaction and/or recognition of malignant target cells by T cells, in vitro cultures using the combination of these cells derived from the same patient hold great promise to predict drug efficacy in a personalized fashion. The use of two-dimensional cancer cell lines for such cultures is unreliable due to altered phenotypical behavior of cells when compared with the in vivo situation. Three-dimensional tumor-derived organoids, better mimic in vivo tissue and are deemed a more realistic approach to study the complex tumor-immune interactions. In this review, we present an overview of the development of patient-specific tumor organoid-immune co-culture models to study the tumor-specific immune interactions and their possible therapeutic infringement. We also discuss applications of these models which advance personalized therapy efficacy and understanding the tumor microenvironment such as: (1) Screening for efficacy of immune checkpoint inhibition and CAR therapy screening in a personalized manner. (2) Generation of tumor reactive lymphocytes for adoptive cell transfer therapies. (3) Studying tumor-immune interactions to detect cell-specific roles in tumor progression and remission. Overall, these onco-immune co-cultures might hold a promising future toward developing patient-specific therapeutic approaches as well as increase our understanding of tumor-immune interactions., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Tumor initiation and progression are critically dependent on interaction of cancer cells with their cellular and extracellular microenvironment. Alterations in the composition, integrity, and mechanical properties of the extracellular matrix (ECM) dictate tumor processes including cell proliferation, migration, and invasion. Also in primary liver cancer, consisting of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), the dysregulation of the extracellular environment by liver fibrosis and tumor desmoplasia is pertinent. Yet, the exact changes occurring in liver cancer ECM remain uncharacterized and underlying tumor-promoting mechanisms remain largely unknown. Herein, an integrative molecular and mechanical approach is used to extensively characterize the ECM of HCC and CCA tumors by utilizing an optimized decellularization technique. We identified a myriad of proteins in both tumor and adjacent liver tissue, uncovering distinct malignancy-related ECM signatures. The resolution of this approach unveiled additional ECM-related proteins compared to large liver cancer transcriptomic datasets. The differences in ECM protein composition resulted in divergent mechanical properties on a macro- and micro-scale that are tumor-type specific. Furthermore, the decellularized tumor ECM was employed to create a tumor-specific hydrogel that supports patient-derived tumor organoids, which provides a new avenue for personalized medicine applications. Taken together, this study contributes to a better understanding of alterations to composition, stiffness, and collagen alignment of the tumor ECM that occur during liver cancer development., Competing Interests: Declaration of competing interest Authors declare that they have no competing interests., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Ischemia of the bile duct is a common feature in liver disease and transplantation, which represents a major cause of morbidity and mortality, especially after liver transplantation. Detailed knowledge of its pathogenesis remains incomplete due to the lack of appropriate in vitro models., Methods: To recapitulate biliary damage induced by ischemia and reperfusion in vitro, human intrahepatic cholangiocyte organoids (ICOs) were grown at low oxygen levels of 1% up to 72 h, followed by re-oxygenation at normal levels., Findings: ICOs stressed by ischemia and subsequent re-oxygenation represented the dynamic change in biliary cell proliferation, upregulation of epithelial-mesenchymal transition (EMT)-associated markers, and the evocation of phase-dependent cell death programs similar to what is described in patients. Clinical-grade alpha-1 antitrypsin was identified as a potent inhibitor of both ischemia-induced apoptosis and necroptosis., Interpretation: These findings demonstrate that ICOs recapitulate ischemic cholangiopathy in vitro and enable drug assessment studies for the discovery of new therapeutics for ischemic cholangiopathies., Funding: Dutch Digestive FoundationMLDS D16-26; TKI-LSH (Topconsortium Kennis en Innovatie-Life Sciences & Health) grant RELOAD, EMC-LSH19002; Medical Delta program "Regenerative Medicine 4D"; China Scholarship Council No. 201706230252., Competing Interests: Declaration of interests The authors declared there are no conflicts of interest., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

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.

Background: Statin use could benefit patients with non-alcoholic fatty liver disease (NAFLD), but the evidence is segmented and inconclusive. This multidimensional study comprehensively investigated the potential benefits and mechanism-of-action of statins in NAFLD., Methods: A cross-sectional investigation was performed within the Rotterdam Study (general population; n = 4.576) and the PERSONS cohort (biopsy-proven NAFLD patients; n = 569). Exclusion criteria were secondary causes for steatosis and insufficient data on alcohol, dyslipidemia or statin use. Associations of statin use with NAFLD (among entire general population), fibrosis and NASH (among NAFLD individuals and patients) were quantified. These results were pooled with available literature in meta-analysis. Last, we assessed statins' anti-lipid and anti-inflammatory effects in 3D cultured human liver organoids and THP-1 macrophages, respectively., Findings: Statin use was inversely associated with NAFLD in the Rotterdam study compared to participants with untreated dyslipidemia. In the PERSONS cohort, statin use was inversely associated with NASH, but not with fibrosis. The meta-analysis included 7 studies and indicated a not significant inverse association for statin use with NAFLD (pooled-Odds Ratio: 0.69, 95% Confidence Interval: 0.46-1.01) and significant inverse associations with NASH (pooled-OR: 0.59, 95% CI: 0.44-0.79) and fibrosis (pooled-OR: 0.48, 95% CI: 0.33-0.70). In vitro, statins significantly reduced lipid droplet accumulation in human liver organoids and downregulated expression of pro-inflammatory cytokines in macrophages., Interpretation: Pooled results demonstrated that statin use was associated with a lower prevalence of NASH and fibrosis and might prevent NAFLD. This may be partially attributed to the anti-lipid and anti-inflammatory characteristics of statins. Given their under-prescription, adequate prescription of statins may limit the disease burden of NAFLD., Funding: ZonMw, KWF, NWO, SLO, DGXII, RIDE, National and regional government, Erasmus MC and Erasmus University., Competing Interests: Declaration of interests RdK received grants from Echosens, Abbvie, Gilead and Inventiva and received consulting fees from Abbvie, Gilead and Echosens. HJ received grants and consulting fees from Gilead, Janssen, GLaxoSmithKLine, Roche and Vir Biotechnology Inc. HJ received consulting fees from Eiger, Antios, Aligos. MP received consulting fees from Pfizer and Walvax Inc. The remaining authors reported no relevant conflicts., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

The use of extended criteria donor grafts is a promising strategy to increase the number of organ transplantations and reduce waitlist mortality. However, these organs are often compromised and/or damaged, are more susceptible to preservation injury, and are at risk for developing post-transplant complications. Ex vivo organ perfusion is a novel technology to preserve donor organs while providing oxygen and nutrients at distinct perfusion temperatures. This preservation method allows to resuscitate grafts and optimize function with therapeutic interventions prior to solid organ transplantation. Stem cell-based therapies are increasingly explored for their ability to promote regeneration and reduce the inflammatory response associated with in vivo reperfusion. The aim of this review is to describe the current state of stem cell-based therapies during ex vivo organ perfusion for the kidney, liver, lung, and heart. We discuss different strategies, including type of cells, route of administration, mechanisms of action, efficacy, and safety. The progress made within lung transplantation justifies the initiation of clinical trials, whereas more research is likely required for the kidney, liver, and heart to progress into clinical application. We emphasize the need for standardization of methodology to increase comparability between future (clinical) studies., (© 2022 The Authors. American Journal of Transplantation published by Wiley Periodicals LLC on behalf of The American Society of Transplantation and the American Society of Transplant Surgeons.)

Advances in biomaterials, particularly in combination with encapsulation strategies, have provided excellent opportunities to increase reproducibility and standardization for cell culture applications. Herein, hybrid microcapsules are produced in a flow-focusing microfluidic droplet generator combined with enzymatic outside-in crosslinking of dextran-tyramine, enriched with human liver extracellular matrix (ECM). The microcapsules provide a physiologically relevant microenvironment for the culture of intrahepatic cholangiocyte organoids (ICO) and patient-derived cholangiocarcinoma organoids (CCAO). Micro-encapsulation allowed for the scalable and size-standardized production of organoids with sustained proliferation for at least 21 days in vitro. Healthy ICO ( n = 5) expressed cholangiocyte markers, including KRT7 and KRT19, similar to standard basement membrane extract cultures. The CCAO microcapsules ( n = 3) showed retention of stem cell phenotype and expressed LGR5 and PROM1. Furthermore, ITGB1 was upregulated, indicative of increased cell adhesion to ECM in microcapsules. Encapsulated CCAO were amendable to drug screening assays, showing a dose-response response to the clinically relevant anti-cancer drugs gemcitabine and cisplatin. High-throughput drug testing identified both pan-effective drugs as well as patient-specific resistance patterns. The results described herein show the feasibility of this one-step encapsulation approach to create size-standardized organoids for scalable production. The liver extracellular matrix-containing microcapsules can provide a powerful platform to build mini healthy and tumor tissues for potential future transplantation or personalized medicine applications.

Monitoring tumor growth dynamics is crucial for understanding cancer. To establish an in vitro method for the continuous assessment of patient-specific tumor growth, tumor organoids were generated from patients with intrahepatic CCA (iCCA). Organoid growth was monitored for 48 h by label-free live brightfield imaging. Growth kinetics were calculated and validated by MTS assay as well as immunohistochemistry of Ki67 to determine proliferation rates. We exposed iCCA organoids (iCCAOs) and non-tumor intrahepatic cholangiocyte organoids (ICOs) to sub-therapeutic concentrations of sorafenib. Monitoring the expansion rate of iCCAOs and ICOs revealed that iCCAO growth was inhibited by sorafenib in a time- and dose-dependent fashion, while ICOs were unaffected. Quantification of the proliferation marker Ki67 confirmed inhibition of iCCAO growth by roughly 50% after 48 h of treatment with 4 µM sorafenib. We established a robust analysis pipeline combining brightfield microscopy and a straightforward image processing approach for the label-free growth monitoring of patient-derived iCCAOs. Combined with bioanalytical validation, this approach is suitable for a fast and efficient high-throughput drug screening in tumor organoids to develop patient-specific systemic treatment options.

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., (© 2022 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Background: Immunotherapy with immune checkpoint inhibitors (ICIs) is being explored to improve cholangiocarcinoma (CCA) therapy. However, it remains difficult to predict which ICI will be effective for individual patients. Therefore, the aim of this study is to develop a co-culture method with patient-derived CCA organoids and immune cells, which could represent anti-cancer immunity in vitro., Methods: CCA organoids were co-cultured with peripheral blood mononuclear cells or T cells. Flow cytometry, time-lapse confocal imaging for apoptosis, and quantification of cytokeratin 19 fragment (CYFRA) release were applied to analyse organoid and immune cell behaviour. CCA organoids were also cultured in immune cell-conditioned media to analyse the effect of soluble factors., Results: The co-culture system demonstrated an effective anti-tumour organoid immune response by a decrease in live organoid cells and an increase in apoptosis and CYFRA release. Interpatient heterogeneity was observed. The cytotoxic effects could be mediated by direct cell-cell contact and by release of soluble factors, although soluble factors only decreased viability in one organoid line., Conclusions: In this proof-of-concept study, a novel CCA organoid and immune cell co-culture method was established. This can be the first step towards personalised immunotherapy for CCA by predicting which ICIs are most effective for individual patients., (© 2022. The Author(s).)

For liver transplantations, human leukocyte antigen (HLA) matching is not routinely performed because observed effects have been inconsistent. Nevertheless, long-term liver transplantation outcomes remain suboptimal. The availability of a more precise HLA-matching algorithm, Predicted Indirectly Recognizable HLA Epitopes II (PIRCHE-II), now enables robust assessment of the association between HLA matching and liver transplantation outcomes. We performed a single-center retrospective cohort study of 736 liver transplantation patients. Associations between PIRCHE-II and HLAMatchmaker scores and mortality, graft loss, acute and chronic rejection, ischemic cholangiopathy, and disease recurrence were evaluated with Cox proportional hazards models. Associations between PIRCHE-II with 1-year, 2-year, and 5-year outcomes and severity of acute rejection were assessed with logistic and linear regression analyses, respectively. Subgroup analyses were performed for autoimmune and nonautoimmune indications, and patients aged 30 years and younger, and older than 30 years. PIRCHE-II and HLAMatchmaker scores were not associated with any of the outcomes. However, patients who received transplants for autoimmune disease showed more acute rejection and graft loss, and these risks negatively associated with age. Rhesus mismatch more than doubled the risk of disease recurrence. Moreover, PIRCHE-II was inversely associated with graft loss in the subgroup of patients aged 30 years and younger with autoimmune indications. The absence of associations between PIRCHE-II and HLAMatchmaker scores and the studied outcomes refutes the need for HLA matching for liver (stem cell) transplantations for nonautoimmune disease. For autoimmune disease, the activated immune system seems to increase risks of acute rejection and graft loss. Our results may suggest the benefits of transplantations with rhesus matched but PIRCHE-II mismatched donor livers., (© 2022 The Authors. Liver Transplantation published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Background: Cholangiocarcinoma is a rare but lethal cancer of the biliary tract. Its first-line treatment is currently restricted to chemotherapy, which provides limited clinical benefit. Kinase inhibitors targeting oncogenic intracellular signaling have changed the treatment paradigm of cancer over the last decades. However, they are yet to be widely applied in cholangiocarcinoma therapy. Cholangiocarcinoma has marked molecular heterogeneity, which complicates the discovery of new treatments and requires patient stratification. Therefore, we investigated whether a commercial kinome profiling platform could predict druggable targets in cholangiocarcinoma., Methods: Kinase activity in patient-derived cholangiocarcinoma organoids, non-tumorous adjacent tissue-derived and healthy donor-derived intrahepatic cholangiocyte organoids was determined using the PamChip® phosphotyrosine kinase microarray platform. Kinome profiles were compared and correlated with RNA sequencing and (multi-)kinase inhibitor screening of the cholangiocarcinoma organoids., Results: Kinase activity profiles of individual cholangiocarcinoma organoids are different and do not cluster together. However, growth factor signaling (EGFR, PDGFRβ) and downstream effectors (MAPK pathway) are more active in cholangiocarcinoma organoids and could provide potential druggable targets. Screening of 31 kinase inhibitors revealed several promising pan-effective inhibitors and compounds that show patient-specific efficacy. Kinase inhibitor sensitivity correlated to the activity of its target kinases for several inhibitors, signifying them as potential predictors of response. Moreover, we identified correlations between drug response and kinases not directly targeted by those drugs., Conclusions: In conclusion, kinome profiling is a feasible method to identify druggable targets for cholangiocarcinoma. Future studies should confirm the potential of kinase activity profiles as biomarkers for patient stratification and precision medicine., (© 2022. The Author(s).)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Faria, van der Laan, Penning, Masereeuw and Spee.)

Background: Early allograft dysfunction (EAD) following liver transplantation (LT) remains a major threat to the survival of liver grafts and recipients. In animal models, it is shown that hepatic ischemia-reperfusion injury (IRI) triggers phosphorylation of Mixed Lineage Kinase domain-like protein (pMLKL) inducing necroptotic cell death. However, the clinical implication of pMLKL-mediated cell death in human hepatic IRI remains largely unexplored. In this study, we aimed to investigate the expression of pMLKL in human liver grafts and its association with EAD after LT., Methods: The expression of pMLKL was determined by immunohistochemistry in liver biopsies obtained from both human and rat LT. Human liver biopsies were obtained at the end of preservation (T0) and ~1 hour after reperfusion (T1). The positivity of pMLKL was quantified electronically and compared in rat and human livers and post-LT outcomes. Multiplex immunofluorescence staining was performed to characterize the pMLKL-expressing cells., Results: In the rat LT model, significant pMLKL expression was observed in livers after IRI as compared to livers of sham-operation animals. Similarly, the pMLKL score was highest after IRI in human liver grafts (in T1 biopsies). Both in rats and humans, the pMLKL expression is mostly observed in the portal triads. In grafts who developed EAD after LT (n=24), the pMLKL score at T1 was significantly higher as compared to non-EAD grafts (n=40). ROC curve revealed a high predictive value of pMLKL score at T1 (AUC 0.70) and the ratio of pMLKL score at T1 and T0 (pMLKL-index, AUC 0.82) for EAD. Liver grafts with a high pMLKL index (>1.64) had significantly higher levels of serum ALT, AST, and LDH 24 hours after LT compared to grafts with a low pMLKL index. Multivariate logistical regression analysis identified the pMLKL-index (Odds ratio=1.3, 95% CI 1.1-1.7) as a predictor of EAD development. Immunohistochemistry on serial sections and multiplex staining identified the periportal pMLKL-positive cells as portal fibroblasts, fibrocytes, and a minority of cholangiocytes., Conclusion: Periportal pMLKL expression increased significantly after IRI in both rat and human LT. The histological score of pMLKL is predictive of post-transplant EAD and is associated with early liver injury after LT. Periportal non-parenchymal cells (i.e. fibroblasts) appear most susceptible to pMLKL-mediated cell death during hepatic IRI., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shi, Bonaccorsi-Riani, Schurink, van den Bosch, Doukas, Lila, Roest, Xhema, Gianello, de Jonge, Verstegen and van der Laan.)

Human cholangiocyte organoids show great promise for regenerative therapies and in vitro modeling of bile duct development and diseases. However, the cystic organoids lack the branching morphology of intrahepatic bile ducts (IHBDs). Here, we report establishing human branching cholangiocyte organoid (BRCO) cultures. BRCOs self-organize into complex tubular structures resembling the IHBD architecture. Single-cell transcriptomics and functional analysis showed high similarity to primary cholangiocytes, and importantly, the branching growth mimics aspects of tubular development and is dependent on JAG1/NOTCH2 signaling. When applied to cholangiocarcinoma tumor organoids, the morphology changes to an in vitro morphology like primary tumors. Moreover, these branching cholangiocarcinoma organoids (BRCCAOs) better match the transcriptomic profile of primary tumors and showed increased chemoresistance to gemcitabine and cisplatin. In conclusion, BRCOs recapitulate a complex process of branching morphogenesis in vitro. This provides an improved model to study tubular formation, bile duct functionality, and associated biliary diseases., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: In cystic fibrosis (CF), loss of CF transmembrane conductance regulator (CFTR)-dependent bicarbonate secretion precipitates the accumulation of viscous mucus in the lumen of respiratory and gastrointestinal epithelial tissues. We investigated whether the combination of elexacaftor (ELX), ivacaftor (IVA) and tezacaftor (TEZ), apart from its well-documented effect on chloride transport, also restores Phe508del-CFTR-mediated bicarbonate transport., Methods: Epithelial monolayers were cultured from intestinal and biliary (cholangiocyte) organoids of homozygous Phe508del-CFTR patients and controls. Transcriptome sequencing was performed, and bicarbonate and chloride transport were assessed in the presence or absence of ELX/IVA/TEZ, using the intestinal current measurement technique., Results: ELX/IVA/TEZ markedly enhanced bicarbonate and chloride transport across intestinal epithelium. In biliary epithelium, it failed to enhance CFTR-mediated bicarbonate transport but effectively rescued CFTR-mediated chloride transport, known to be requisite for bicarbonate secretion through the chloride-bicarbonate exchanger AE2 (SLC4A2), which was highly expressed by cholangiocytes. Biliary but not intestinal epithelial cells expressed an alternative anion channel, anoctamin-1/TMEM16A (ANO1), and secreted bicarbonate and chloride upon purinergic receptor stimulation., Conclusions: ELX/IVA/TEZ has the potential to restore both chloride and bicarbonate secretion across CF intestinal and biliary epithelia and may counter luminal hyper-acidification in these tissues., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., (© 2022 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

Reliable establishment of tumor organoids is paramount to advance applications of organoid technology for personalized medicine. Here, we share our multi-center experience on initiation and tumorigenic confirmation of hepatobiliary cancer organoids. We discuss current concerns, propose potential solutions, and provide future perspectives for improvements in hepatobiliary cancer organoid establishment., Competing Interests: Declaration of interests M.H. is an inventor in a patent on liver organoids. F.M.S. is an inventor in a patent for liver cancer treatment (PCT/US2021/016849, 17/623,060, 16/479,652). The other authors declare no conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Organoid technology holds great promise for regenerative medicine. Recent studies show feasibility for bile duct tissue repair in humans by successfully transplanting cholangiocyte organoids in liver grafts during perfusion. Large-scale expansion of cholangiocytes is essential for extending these regenerative medicine applications. Human cholangiocyte organoids have a high and stable proliferation capacity, making them an attractive source of cholangiocytes. Commercially available basement membrane extract (BME) is used to expand the organoids. BME allows the cells to self-organize into 3D structures and stimulates cell proliferation. However, the use of BME is limiting the clinical applications of the organoids. There is a need for alternative tissue-specific and clinically relevant culture substrates capable of supporting organoid proliferation. Hydrogels prepared from decellularized and solubilized native livers are an attractive alternative for BME. These hydrogels can be used for the culture and expansion of cholangiocyte organoids in a clinically relevant manner. Moreover, the liver-derived hydrogels retain tissue-specific aspects of the extracellular microenvironment. They are composed of a complex mixture of bioactive and biodegradable extracellular matrix (ECM) components and can support the growth of various hepatobiliary cells. In this review, we provide an overview of the clinical potential of native liver ECM-based hydrogels for applications with human cholangiocyte organoids. We discuss the current limitations of BME for the clinical applications of organoids and how native ECM hydrogels can potentially overcome these problems in an effort to unlock the full regenerative clinical potential of the organoids.

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., (© 2021 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Fatty liver disease has grown into a major global health burden, attributed to multi-factors including sedentary lifestyle, obesogenic diet and prevalence of metabolic disorders. The lack of robust experimental models is hampering the research and therapeutic development for fatty liver disease. This study aims to develop an organoid-based 3D culture model to recapitulate key features of fatty liver disease focusing on intracellular lipid accumulation and metabolic dysregulation. We used human liver-derived intrahepatic cholangiocyte organoids and hepatocyte differentiated organoids. These organoids were exposed to lactate, pyruvate, and octanoic acid (LPO) for inducing lipid accumulation and mitochondrial impairment. Lipid accumulation resulted in alternations of gene transcription with major effects on metabolic pathways, including triglyceride and glucose level increase, which is consistent with metabolic changes in fatty liver disease patients. Interestingly, lipid accumulation affected mitochondria as shown by morphological transitions, alternations in expression of mitochondrial encoded genes, and reduction of ATP production. Meanwhile, we found treatment with obeticholic acid and metformin can alleviate fat accumulation in organoids. This study demonstrated that LPO exposure can induce lipid accumulation and associated metabolic dysregulation in human liver-derived organoids. This provides an innovative model for studying fatty liver disease and testing potential therapeutics. KEY MESSAGES: Lactate, pyruvate, and octanoic acid induce lipid accumulation in liver organoids. Organoids of human compared to mouse origin are more efficient in lipid accumulation. Lipid accumulation dysregulates metabolic pathway and impairs mitochondrial function. Demonstrating a proof-of-concept for testing medications in organoids., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Hepatotropic viruses naturally have narrow host and tissue tropisms, challenging the development of robust experimental models. The advent of organoid technology provides a unique opportunity for moving the field forward. Here, we demonstrate that three-dimensional cultured organoids from fetal and adult human liver with cholangiocyte or hepatocyte phenotype support hepatitis E virus (HEV) replication. Inoculation with infectious HEV particles demonstrates that human liver–derived organoids support the full life cycle of HEV infection. By directing organoids toward polarized monolayers in a transwell system, we observed predominantly apical secretion of HEV particles. Genome-wide transcriptomic and tRNAome analyses revealed robust host responses triggered by viral replication. Drug screening in organoids identified brequinar and homoharringtonine as potent HEV inhibitors, which are also effective against the ribavirin resistance variant harboring G1634R mutation. Thus, successful recapitulation of HEV infection in liver-derived organoids shall facilitate the study of virus-host interactions and development of antiviral therapies.