Supplemental data for: 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 air-liquid
interface, and we demonstrate proof of principle for differentiation of
BAL-derived alveolar organoids in 3D 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.