Airway Epithelial Cell Culture Differentiation

Primary NHBE cells were purchased from Lonza (catalog no. CC-2541; Basel, Switzerland). NHBE cells in a submerged condition were cultured on 6.5-mm Transwell (Corning, NT, USA) using commercially available bronchial epithelial growth medium (BEGM; Lonza) and incubated at 37 °C in a humidified atmosphere with 5% carbon dioxide. When the NHBE cells reached full confluency in an immersed culture condition, the cells were transferred to an ALI culture condition using ALI culture medium (HBTEC Air–Liquid Interface Differentiation Medium; Lifeline Cell Technology, Frederick, MD, USA), as previously described with slight modifications [3 (link), 4 (link), 23 (link), 24 (link)]. The cells were then cultured for 4 weeks in ALI conditions to facilitate polarization and ciliary differentiation for subsequent experiments [25 (link), 26 (link)].