Fully differentiated primary human bronchial epithelial (HBE) cell cultures were derived from lungs removed at the time of lung transplantation from the Center for Organ Recovery and Education. Cells were prepared using previously described methods approved by the University of Pittsburgh IRB49, 50. Briefly, bronchi from the 2nd to 6th generations were collected, rinsed, and incubated overnight in EMEM at 4 °C. The bronchi were then digested in MEM containing protease XIV and DNase (0.2%). The epithelial cells were removed and collected by centrifugation and then resuspended in BronchiaLife medium and plated onto collagen-treated tissue culture flasks. When 80–90% confluence was reached, the passage 0 cells were trypsinized and seeded onto collagen-coated transwell permeable supports (Corning #3450, 105 cells/well). B-ALI Bronchial Air Liquid Interface Medium was replaced three times a week on both apical and basolateal sides of the permeable supports up to 8–10 days. Subsequently the apical medium was removed and the cultures were maintained at air liquid interface (ALI) to promote a further polarization and differentiation of the epithelium. The basolateral medium was changed twice weekly.