Air-Liquid Interface Culture of Airway Epithelial Cells

For air-liquid interface (ALI) culture, we used a previously described protocol (12 (link), 57 (link), 58 (link)). Briefly, 6.5-mm Transwells with a 0.4-μm-pore polyester membrane insert (Corning, Inc.) were coated with PureCol for 20 min before cell seeding and then removed. NHBE cells (5 × 10⁴) suspended in 200 μL of AEC medium were seeded on the apical part of a Transwell. Then, 500 μL of AEC medium was added to the basal part of a Transwell. When cells formed a confluent layer on the Transwell, the AEC medium was removed from the apical part of the Transwell and replaced with PneumaCult-ALI basal medium (Stemcell Technologies) with the required supplements (Stemcell Technologies), 2% penicillin-streptomycin, and 1% amphotericin B in the basal part. The ALI medium was changed from the basal medium every other day. Apical surfaces were washed with 1× Dulbecco’s phosphate buffer saline (DPBS) (Thermo Fisher Scientific) once per week initially but washed more frequently when higher levels of mucus were observed in later days. All cells were differentiated for up to 4 weeks (37°C with 5% CO₂) until the cellular and physiological properties of the epithelial layer were obtained.