Expansion and Differentiation of Primary Human Airway Epithelial Cells

The pHAECs from healthy adult patients were kindly provided by Dr. C.U. Cotton (Case Western Reserve University), expanded, and cultured as described [34 (link),35 (link)]. Briefly, cells were plated on a layer of mitomycin C (Sigma) treated 3T3 mouse fibroblast feeder cells and grown until 70% confluency in the 1:3 mixture of Ham’s F12/DMEM media (HyClone) supplemented with 5% FBS (Sigma), 24 µg/mL adenine (Sigma), 0.4 µg/mL hydrocortisone (Sigma), 5 µg/mL Insulin (Sigma), 10 ng/mL EGF (Sigma), 8.4 ng/mL Cholera toxin, and 10 µM ROCK1 inhibitor Y-2763 (Selleck Chemical LLC). After expansion, cells (passage 3 or 4) were plated on Costar transwell inserts (Corning Inc., Corning, NY), grown until confluency, then transferred to air-liquid interface where cells were maintained in 1:1 mixture of Ham’s F12/DMEM media supplemented with 2% Ultroser G (Pall Biosepra, SA, Cergy-Sainte-Christophe, France) for 3–4 weeks until they were differentiated. Differentiation was confirmed by transepithelial resistance measurements >500 ohms, and flow cytometry staining for FoxJ1 (eBioscience) and acetylated α-tubulin (Life Technologies).