Passive siRNA transfection method for gene knockdown in air-liquid interface airway epithelial cell cultures

Differentiation of human bronchial epithelial cells (HBEs) in air-liquid interface (ALI) cultures recapitulates organotypic modeling of the in vivo environment. Although ALI cultures are invaluable for studying the respiratory epithelial barrier, loss-of-function studies are limited by potentially cytotoxic reagents in classical transfection methods, the length of the differentiation protocol, and the number of primary epithelial cell passages. Here, we present the efficacy and use of a simple method for small interfering RNA (siRNA) transfection of normal HBEs (NHBEs) in ALI cultures that does not require potentially cytotoxic transfection reagents and does not detrimentally alter the physiology or morphology of NHBEs during the differentiation process. This transfection protocol introduces a reproducible and efficient method for loss-of-function studies in HBE ALI cultures that can be leveraged for modeling the respiratory system and airway diseases.