Primary human small airway epithelial cells (SAEC; CC-2547S, Lonza, Walkersville, MD) of a healthy 11-year-old donor were grown and differentiated in a humidified atmosphere (5% CO2, 37°C) as described previously. Briefly, reaching 75–80% confluence in T75 tissue culture flasks fed with small airway growth medium (SAGM; CC-3281, Lonza) supplemented with growth factors and hormones (ALI SingleQuots Kit; CC-4538, Lonza), the cells were dissociated with trypsin/EDTA and seeded on the semipermeable membrane of transwell culture inserts (6.5 mm diameter, 0.4 µm pore size; Corning-Costar, Lowell, MA) coated with rat tail collagen type 1 (BD Biosciences, Bedford, MA). When confluence was reached, an air-liquid interface (ALI) was created to trigger differentiation by removing the growth medium from the apical compartment of the culture inserts set in 24-well plates and replacing growth medium in the basal compartment with differentiation medium (CC-3281, Lonza) supplemented with the differentiation inducer (ALI SingleQuots Kit; CC-4538, Lonza). Thereafter, the differentiation medium was changed in the basal compartment every other day, and the apical compartment was gently washed with the culture medium once or twice a week to remove accumulated debris and mucus. The cells fully differentiate in 21 to 25 days of ALI culture into ciliated cells, goblet cells, basal cells, and non-ciliated columnar cells.