Establishment of Airway Organoids

Organoids were established as described previously (15 (link)). In brief, cells were counted under a microscope and centrifuged at 500 × g for 5 minutes. Then, cells were resuspended in ice-cold 500 μL Matrigel (Corning) and 20 μL drops of Matrigel cell suspension were seeded on prewarmed 48-well culture plates (Corning) at a density of ∼2 × 10³ cells per 20 μL Matrigel/well. The Matrigel was solidified for 15 minutes at 37°C and overlaid with 250 μL airway organoid medium [AO; AdDF+++, 20% conditioned R-spondin1 medium supplemented with B27 (Invitrogen), 1.25 mmol/L N-acetylcystein (Sigma-Aldrich), 5 mmol/L nicotinamide (Sigma), 25 ng/mL human fibroblast growth factor 7 (Peprotech), 100 ng/mL human noggin (Peprotech), 100 ng/mL human FGF 10 (Peprotech), 500 nmol/L A83–01 (Tocris), and 500 nmol/L SB202190 (Sigma)]. AdDF+++ medium is advanced DMEM/F12 medium (Invitrogen) supplemented with 10 nmol/L HEPES (Invitrogen), 1× GlutaMax (Invitrogen), and 1× antibiotic–antimycotic (Invitrogen). Y-27632 (10 μmol/L; Enzo Life Science) was added for the first 2 days. Cultures were kept at 37°C, 5% CO₂ in a humidified incubator. Medium was replenished every 2 to 3 days.