D95-05 Luminally Accessible Human Pulmonary Aciniform Organoids Enable 3D Dynamic Modeling of Pulmonary Fibrosis

Introduction Conventional organoid models recapitulate three-dimensional morphology but lack apical and functional access. In contrast, flattened two-dimensional cultures permit direct exposure and allow transmembrane analyses, but compromise microanatomical fidelity and biomechanical function, thereby limiting translational relevance. To address these limitations, we developed a hybrid organoid-device system that enables luminal manipulation and quantitative mechanical profiling under physiologically relevant geometry. Methods Human induced pluripotent stem cell (hiPSC)-derived lung progenitors and primary human lung fibroblasts were co-cultured to generate spheroids. These spheroids were automatically seeded into plates and subsequently fused into acinus-like structures, termed pulmonary aciniform organoids (PAcinOs). The organoids were cultured within a custom microengineered platform, the Dynamic Exposure and Infusion Response Observer (DENIRO), which enabled precise intraluminal access. A glass capillary was inserted into the luminal space of the organoids under optical coherence tomography (OCT) guidance, and a programmable pressure-control system applied fluidic pressure to induce organoid expansion. As a fibrosis model, PAcinOs were exposed to bleomycin (BLM) to examine phenotypic alterations. Results PAcinOs formed through fusion of spheroids into structures approximately 1 mm in diameter, larger than conventional lung organoids, and resembling pulmonary acini in plate culture. Live imaging identified SFTPC-GFP positive epithelial cells, corresponding to, alveolar type II epithelial cells. Immunofluorescence staining indicated fusion of luminal spaces and confirmed epithelial polarity, with epithelial cells localized inside and fibroblasts distributed around the periphery. Similar results were obtained in DENIRO culture, where OCT non-invasively visualized continuous luminal connections between fused spheroids. Stable intraluminal access via DENIRO enabled pressure application through the capillary, resulting in expansion of PAcinOs analogous to inspiration, and confirming the feasibility of functional mechanical assessment. In BLM-treated PAcinOs, expansion in response to pressure was markedly reduced compared with untreated controls, resulting in decreased volume change and compliance, consistent with fibrotic stiffening. To characterize the molecular effects of BLM, we performed bulk RNA sequencing of BLM-treated and untreated PAcinOs. Heatmap analysis of fibrosis-associated markers revealed broad upregulation in the BLM group, and gene set enrichment analysis (GSEA) demonstrated significant enrichment of pathways related to pulmonary fibrosis and extracellular matrix organization, supporting the induction of fibrotic change. Conclusion This organoid-device system enables quantitative evaluation of alveolar biomechanics while preserving tissue-relevant architecture, and allows fibrosis modeling in a human-relevant context. Moreover, this approach facilitates patient-specific hiPSC-derived modeling and supports the advancement of precision medicine in interstitial lung diseases.