{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,29]],"date-time":"2026-05-29T07:04:47Z","timestamp":1780038287534,"version":"3.53.1"},"reference-count":43,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,5,29]],"date-time":"2026-05-29T00:00:00Z","timestamp":1780012800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004587","name":"Instituto de Salud Carlos III","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100004587","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell Dev. Biol."],"abstract":"<jats:p>Bile represents a clinically accessible biological fluid that can mitigate major limitations associated with tissue-based sampling for the generation of organoid models to study hepatobiliary disease, including biliary tract cancers, where tissue availability is often limited. Importantly, bile can also enable the generation of non-malignant cholangiocyte organoids that are otherwise difficult to obtain. Here, we describe an operator-oriented, step-by-step protocol to generate organoids from fresh bile collected during endoscopic retrograde cholangiopancreatography (ERCP), together with two complementary workflows for siRNA delivery in 3D cultures. We detail critical control points that are often under-reported, yet considerably influence success and reproducibility. The protocol was optimized and applied in a real-world cohort of 21 patients undergoing ERCP, including benign biliary obstruction due to choledocholithiasis (n = 5) and malignant strictures (n = 16: cholangiocarcinoma; n = 13: gallbladder adenocarcinoma; n = 1: ampullary tumors; n = 2). Expandable organoids were established in 17\/21 cases (81%), with establishment rates of 60% for choledocholithiasis and 85%\u2013100% across malignant entities. The anticipated results include organoid outgrowth within \u223c2\u20133 weeks and morphological heterogeneity in cultures derived from malignant strictures, where normal-like and tumor-like populations may initially coexist and can drift toward a cystic phenotype under routine expansion, motivating optional manual handpicking when tumor-enriched lines are required. As downstream readouts, we show the feasibility of DNA-based profiling in selected paired bile\u2013organoid samples (targeted sequencing and ULP-WGS copy-number analysis) and demonstrate proof-of-concept gene silencing via siRNA in both dissociated cells prior to re-embedding and intact fully formed organoids while preserving the 3D architecture. Collectively, this workflow provides a practical and reproducible framework to establish, expand, characterize, and functionally perturb bile-derived organoids from routine clinical procedures, facilitating standardized implementation across laboratories.<\/jats:p>","DOI":"10.3389\/fcell.2026.1812445","type":"journal-article","created":{"date-parts":[[2026,5,29]],"date-time":"2026-05-29T06:38:13Z","timestamp":1780036693000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["A guide for establishing patient-derived organoids from bile samples obtained during endoscopic procedures and performing gene expression knockdown"],"prefix":"10.3389","volume":"14","author":[{"given":"Carla","family":"Rojo","sequence":"first","affiliation":[{"name":"Hepatology Laboratory, Solid Tumors Program, CIMA, CCUN, University of Navarra","place":["Pamplona, Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juan 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Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vanesa","family":"Jusu\u00e9-Irurita","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology and Hepatology, Navarra University Hospital","place":["Pamplona, Spain"]},{"name":"IdiSNA, Navarra Institute for Health Research","place":["Pamplona, Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juan","family":"Carrascosa-Gil","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology and Hepatology, Navarra University Hospital","place":["Pamplona, Spain"]},{"name":"IdiSNA, Navarra Institute for Health Research","place":["Pamplona, Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mar\u00eda","family":"Rullan","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology and Hepatology, Navarra University Hospital","place":["Pamplona, Spain"]},{"name":"IdiSNA, Navarra Institute for Health Research","place":["Pamplona, Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Javier","family":"R\u00e1ndez-Garbayo","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology and Hepatology, Navarra University Hospital","place":["Pamplona, Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maite G.","family":"Fern\u00e1ndez-Barrena","sequence":"additional","affiliation":[{"name":"Hepatology Laboratory, Solid Tumors Program, CIMA, CCUN, University of Navarra","place":["Pamplona, Spain"]},{"name":"IdiSNA, Navarra Institute for Health Research","place":["Pamplona, Spain"]},{"name":"National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd, Carlos III Health Institute)","place":["Madrid, Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Meritxell","family":"Huch","sequence":"additional","affiliation":[{"name":"Max Planck Institute of Molecular Cell Biology and Genetics","place":["Dresden, Germany"]},{"name":"Center for Systems Biology (CSBD)","place":["Dresden, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jes\u00fas","family":"Urman","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology and Hepatology, Navarra University Hospital","place":["Pamplona, Spain"]},{"name":"IdiSNA, Navarra Institute for Health Research","place":["Pamplona, Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mat\u00edas A.","family":"\u00c1vila","sequence":"additional","affiliation":[{"name":"Hepatology Laboratory, Solid Tumors Program, CIMA, CCUN, University of Navarra","place":["Pamplona, Spain"]},{"name":"IdiSNA, Navarra Institute for Health Research","place":["Pamplona, Spain"]},{"name":"National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd, Carlos III Health Institute)","place":["Madrid, Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carmen","family":"Berasain","sequence":"additional","affiliation":[{"name":"Hepatology Laboratory, Solid Tumors Program, CIMA, CCUN, University of Navarra","place":["Pamplona, Spain"]},{"name":"National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd, Carlos III Health Institute)","place":["Madrid, Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria","family":"Arechederra","sequence":"additional","affiliation":[{"name":"Hepatology Laboratory, Solid Tumors Program, CIMA, CCUN, University of Navarra","place":["Pamplona, Spain"]},{"name":"IdiSNA, Navarra Institute for Health Research","place":["Pamplona, Spain"]},{"name":"National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd, Carlos III Health Institute)","place":["Madrid, 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