Human Alveolar Type II Organoids from Fibrotic Lungs Capture Disease-Specific Metabolic Reprogramming and Provide a Platform for Personalized Medicine

Background Alveolar type II (AT-II) epithelial cells are essential for alveolar repair, immune regulation, and surfactant secretion. Despite their promise for pulmonary disease modeling, limited access and culture methods hinder translational use. We established a patient-derived 3D AT-II organoid system from fibrotic and non-fibrotic lung tissue to maintain AT-II identity, enable cryopreservation, and capture disease-specific metabolic alterations. Methods HT-II-280 ⁺ AT-II cells were isolated by magnetic bead sorting from 62 lung tissues (15 idiopathic pulmonary fibrosis, 26 secondary fibrosis, 21 tumor-distant controls). Cells were expanded as organoids in 3D culture from initial passage 0 up to passage 3. AT-II identity was verified by immunofluorescence, flow cytometry, and transmission electron microscopy. Cryopreserved cells were recovered after ≥ 28 days and tested for viability. Metabolic profiling was performed using extracellular flux assays. Results AT-II cells were successfully (~ 80%) isolated and combined with a serum- free feeder-free culturing approach to reproducibly generated alveolospheres with highly efficient colony formation (> 90% in P1), especially in AT-II cells from fibrotic explants. Interestingly, primary tissue-derived lung organoids display heterogeneous morphologies and sizes, particularly in fibrotic-derived cultures indicated by histology and microcomputed tomography. Culturing conditions were optimized to avoid differentiation towards AT-I cells or aberrant basaloid cells. Lineage fidelity was preserved across passages, with stable expression of proSP-C, HT-II-280, and pronounced presence of lamellar bodies. Cryopreservation maintained high viability, organoid-forming capacity, and metabolic activity, highlighting possibility for on demand long-term storage. Fibrotic organoids exhibited metabolic reprogramming illustrated by a pronounced glycolytic shift with increased ATP production. Conclusion We established a robust and reproducible cell-line-free 3D platform from primary human AT-II cells of end-stage ILD lungs to generate personalized lung organoids. These organoids retain AT-II identity across passages, remain viable after cryostorage, and recapitulate patient-specific metabolic reprogramming. Fibrotic-derived AT-II cells consistently demonstrated a Warburg-like glycolytic phenotype, reflecting possible mitochondrial dysfunction and high energy demand. This reproducible scalable model provides a transferable resource for mechanistic studies of epithelial dysfunction in pulmonary diseases and supports biobanking for precision medicine.