Optimized culture of primary human alveolar type II cell–derived 3D organoids from fibrotic lung tissue with phenotypic and metabolic profiling

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-associated features, enable cryopreservation, and capture disease-associated metabolic alterations. HT-II-280+ AT-II cells were isolated by magnetic bead sorting from 63 lung tissues (15 idiopathic pulmonary fibrosis, 26 secondary fibrosis, 22 tumor-distant controls). Cells were expanded as organoids in 3D culture from initial passage 0 up to passage 3. AT-II-associated features were assessed by immunofluorescence, flow cytometry, and transmission electron microscopy. Cryopreserved cells were recovered after ≥ 28 days and tested for viability and organoid-forming capacity. Metabolic profiling was performed using extracellular flux assays. 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. Primary tissue-derived lung organoids display heterogeneous morphologies and sizes, most prominently in fibrotic-derived cultures, as indicated by histology and microcomputed tomography. Culture conditions were optimized to minimize differentiation towards AT-I cells or dedifferentiated epithelial states with partial basaloid features. Expression of key AT-II-associated markers (proSP-C, HT-II-280), and the presence of lamellar bodies were maintained across passages at the population level. Cryopreservation maintained high viability, organoid-forming capacity, and metabolic activity, enabling long-term storage. Fibrotic organoids exhibited disease-associated metabolic reprogramming characterized by a pronounced glycolytic shift with increased ATP production. We established a reproducible cell-line-free 3D culture system from primary human AT-II cells of end-stage ILD lungs to generate patient-derived lung organoids. These organoids maintain AT-II-associated features across passages, remain viable after cryostorage, and capture disease-associated metabolic reprogramming. Fibrotic-derived AT-II cells consistently demonstrated a Warburg-like glycolytic phenotype, reflecting increased energy demand. This scalable model in vitro provides a defined resource for mechanistic studies of epithelial dysfunction in pulmonary diseases and supports biobanking for future precision medicine applications.