Human Airway Epithelial (HAE) Cell Culture Protocol

Human airway epithelia (HAE) cells (Lonza; cat# CC-2540S) are available in our laboratory and routinely cultured in air–liquid interface (ALI) as described (15 (link), 35 (link)). Briefly, cells were cultured in a T75 flask using Pneuma Cult Ex Plus medium (Stemcell; cat# 05040) supplemented with hydrocortisone (Stemcell; cat# 07980) for 2–4 d until they reached 80% confluence. The cells were dissociated with an Animal Component-Free Cell Dissociation Kit (Stemcell; cat# 05426) and seeded onto collagen-coated 0.33 cm² porous (0.4 µm) polyester membrane inserts with a seeding density of 1  ×  10⁵ cells per Transwell. The cells were grown to near confluence in submerged culture for 2–3 d in Pneuma Cult Ex Plus medium supplemented with hydrocortisone. Cultures were maintained in a humidified atmosphere with 5% CO2 at 37°C and then transferred to ALI culture. The epithelium was expanded and differentiated using Pneuma Cult ALI medium (Stemcell; cat# 05021) supplemented with hydrocortisone and 0.2% heparin solution (Provitro; cat#0863). The number of days in development was designated relative to initiation of ALI culture, corresponding to day 0.