Generating Human Airway Organoids from Lung Tissue

Human airway organoids were generated from human lung tissue as previously described [37 (link)]. Briefly, the lung tissues were obtained from the Matthay lab (UCSF) and underwent enzymatic digestion to obtain single cells, which were then resuspended in Basement Membrane Extract (BME, R&D Systems). This single-cell mixture was plated into droplets, which were submerged in a specialized human airway organoid (HAO) medium consisting of 1 mM HEPES (Corning), 1x GlutaMAX (Gibco), 1x Penicillin-Streptomycin (Corning), 10% R-spondin1 conditioned medium, 1% B27 (Gibco), 25 ng/mL noggin (Peprotech), 1.25 mM N-acetylcysteine (Sigma-Aldrich), 10 mM nicotinamide (Sigma-Aldrich), 5 nM heregulin-β1 (Peprotech), and 100 μg/mL Primocin (InvivoGen) in DMEM. This HAO medium was also supplemented with 5 μM Y-27632, 500 nM A83-01, 500 nM SB202190, 25 ng/mL FGF7, and 100 ng/mL FGF10 (all obtained from Stem Cell Technologies). The HAO medium was refreshed bi-weekly, and the droplets were transferred into a single-cell suspension every two weeks to facilitate growth. To induce differentiation, the HAO medium was substituted with a mixture of HAO medium and PneumaCult-ALI Medium (Stem Cell Technologies) at a 1:1 ratio.