Development and Application of a Humanized Lung Fibrosis Organoid Model with Stromal and Macrophage Niches for Pharmaceutical Evaluation

Development and Application of a Humanized Lung Fibrosis Organoid Model with Stromal and Macrophage Niches for Pharmaceutical Evaluation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development and Application of a Humanized Lung Fibrosis Organoid Model with Stromal and Macrophage Niches for Pharmaceutical Evaluation Huan Zou, Ze-Xin Chen, Yu Zhu, Suo-Ri-Ya Meng, Dian-Qi Wu, Yi Gao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8303567/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background The preclinical development of therapies for pulmonary fibrosis (PF) faces a fundamental roadblock: existing models fail to simultaneously capture the complex multicellular pathophysiology of the human disease and provide a scalable platform for drug screening. This trade-off between physiological complexity and experimental scalability has severely hampered therapeutic progress. Methods To bridge this critical gap, we established a fully humanized in vitro platform by co-differentiating human induced pluripotent stem cells (hiPSCs) into lung organoids (hiLOs), functional fibroblasts (HF), and macrophages. This tri-culture system (hiLOFM) recapitulates the core functional units of the human lung-epithelium, stromal and immune components (macrophages) – within a controlled and reproducible microenvironment. Results Upon LPS and TGF-β1 challenge, the hiLOFM model robustly recapitulated hallmark fibrotic features, including myofibroblast activation (α-SMA, Fibronectin and Vimentin), extracellular matrix (ECM) deposition (Collagen I), and pro-inflammatory cytokines secretion (IL-11 and IL-1β). The integration of macrophages specifically amplified the fibrotic response, underscoring the critical role of stromal-immune crosstalk. The platform demonstrated high translational utility: it confirmed the efficacy of standard-of-care antifibrotics (pirfenidone and nintedanib), revealed a superior synergistic effect of their combination, and successfully identified the profibrotic toxicity of the anticancer agent DS-8201 at clinically relevant concentrations. Conclusions We present a human-based tri-culture system that effectively resolves the long-standing dilemma between complexity and scalability in PF modeling. This versatile platform provides a powerful and transformative tool for de-risking drug discovery by enabling simultaneous evaluation of efficacy and safety in a physiologically relevant context. Lung organoids Fibroblasts Macrophages hiPSCs Pulmonary fibrosis (PF) Pharmaceutical Efficacy and Toxicity Evaluation Full Text Additional Declarations No competing interests reported. Supplementary Files Video1HLOs.mp4 Video2HLOFFibrosis.mp4 Video3HLOFMFibrosis.mp4 Video4.1hiLOsD50.mp4 Video4.2hiLOsD50.mp4 Video5HiLOs.mp4 Video6HiLOFFibrosis.mp4 Video7HiLOFMFibrosis.mp4 ARRIVE2.0Checklistb14a8060b4b74759bd7784e8c4bca090.xlsx supplementaryfigure.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 01 Apr, 2026 Reviews received at journal 08 Mar, 2026 Reviews received at journal 07 Mar, 2026 Reviewers agreed at journal 14 Feb, 2026 Reviewers agreed at journal 11 Feb, 2026 Reviewers invited by journal 09 Feb, 2026 Editor assigned by journal 28 Jan, 2026 Submission checks completed at journal 08 Jan, 2026 First submitted to journal 08 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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This trade-off between physiological complexity and experimental scalability has severely hampered therapeutic progress.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTo bridge this critical gap, we established a fully humanized \u003cem\u003ein vitro\u003c/em\u003e platform by co-differentiating human induced pluripotent stem cells (hiPSCs) into lung organoids (hiLOs), functional fibroblasts (HF), and macrophages. This tri-culture system (hiLOFM) recapitulates the core functional units of the human lung-epithelium, stromal and immune components (macrophages) \u0026ndash; within a controlled and reproducible microenvironment.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eUpon LPS and TGF-β1 challenge, the hiLOFM model robustly recapitulated hallmark fibrotic features, including myofibroblast activation (α-SMA, Fibronectin and Vimentin), extracellular matrix (ECM) deposition (Collagen I), and pro-inflammatory cytokines secretion (IL-11 and IL-1β). 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