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However, preserving mature hepatobiliary characteristics and concurrently incorporating fibrosis-producing hepatic stellate cells (HSCs) remains a significant challenge, often hindering the large-scale production of organoids capable of replicating key liver functions. Here, we report a robust 3D organoid culture system that enables simultaneous isolation and long-term propagation of primary hepatocytes, cholangiocytes, and HSCs from a single source of mouse liver tissue. By supplementing the Hep-Med with Notch signaling inhibitor and dexamethasone, we achieved sustained organoid maturity, including stable albumin production, metabolic activity, and liver-specific gene expression, over multiple passages in culture. Quiescent HSCs within the system retained lipid droplets and could be activated into a myofibroblast-like phenotype, also called activated HSCs, via TGF\u03b2 stimulation. Activated HSCs impaired the proliferation and stemness, and induced epithelial-mesenchymal transition (EMT) of Hep-Orgs and Cho-Orgs, enabling in vitro liver fibrosis modeling. Optimized for minimal tissue input, this platform maximizes tissue utilization efficiency while preserving the liver\u2019s heterogeneous cellular architecture. Its versatility supports diverse applications in liver disease modeling, drug discovery, and regenerative medicine.<\/jats:p>","DOI":"10.1038\/s41598-026-42990-2","type":"journal-article","created":{"date-parts":[[2026,3,19]],"date-time":"2026-03-19T04:25:00Z","timestamp":1773894300000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A robust mouse liver organoid platform enables sustained multicellular maturation and fibrosis modeling from a single tissue sample"],"prefix":"10.1038","volume":"16","author":[{"given":"Yingyu","family":"Liang","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yongqin","family":"Ye","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hua","family":"Xie","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vincent Chi Hang","family":"Lui","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yan","family":"Chen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Paul Kwong Hang","family":"Tam","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2026,3,19]]},"reference":[{"key":"42990_CR1","doi-asserted-by":"publisher","first-page":"151","DOI":"10.1016\/j.jhep.2018.09.014","volume":"70","author":"SK Asrani","year":"2019","unstructured":"Asrani, S. 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