Standardized pipeline for establishing, expanding, and differentiating airway and alveolar organoids from human BAL fluid.

Lung organoids are versatile experimental models, but their broader use in studying human disease is limited by the scarcity of starting material and the complexity of current methods. To align organoid technology with common clinical practice, we developed airway and alveolar organoids using cells obtained from patients' bronchoalveolar lavage (BAL) fluid. Building on existing techniques, we showed that BAL is a reliable, accessible source of primary human epithelial cells, yielding airway and alveolar organoids within 10 days. Organoids can then be expanded over many passages for downstream analysis. Our streamlined methods do not require cell sorting or other complex procedures; all cells are derived from a single patient, and media are based on serum-free, chemically defined formulations. Here, we present detailed protocols for organoid establishment, standardized passaging and phenotyping, and differentiation of both airway and alveolar models. We provide a time course of BAL-derived airway organoid differentiation at the air-liquid interface, and we demonstrate proof of principle for the differentiation of BAL-derived alveolar organoids in three-dimensional culture. These methods can be readily adapted to generate and characterize organoids from lung tissue, tracheobronchial specimens, or other primary cells from humans or mice, expanding the potential to use lung organoids for disease modeling.NEW & NOTEWORTHY We provide streamlined protocols to generate both airway and alveolar epithelial organoids from a single, clinical BAL specimen. From standardized specimen collection to organoid plating to passaging and differentiation, we show that rare, primary epithelial cells in BAL can give rise to all the major airway and alveolar cell types. Our serum-free, feeder-free, and sorting-free methods offer a simplified starting point for using patient-derived organoids to model lung disease.