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

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.