Determining Ciliary Function and Membrane Impermeability of the Pseudostratified Lung Airway Epithelium

Differentiating primary lung airway epithelial cells in the air-liquid interface (ALI) is a popular technique to develop a multi-cellular pseudostratified airway epithelium that mimics the apical side of the lung airway. While the differentiation of primary lung airway cells is expected, the assessment of biophysical properties like ciliary function and membrane impermeability provides a quality assessment of the airway epithelium and ensures the reliability of the experiment. Here, we describe a straightforward protocol for the development of multi-cellular pseudostratified airway epithelium in ALI culture and assess two important biophysical properties: ciliary function and membrane impermeability. To determine ciliary function, we captured the ciliary movement of a 4-week differentiated airway epithelium using a high-speed camera attached to an inverted microscope, followed by quantifying ciliary beat frequency (CBF) using the Simon Amon Video Analysis (SAVA) system. We also measured transepithelial electrical resistance (TEER) using a volt-ohm meter to determine the epithelial barrier integrity of the airway epithelium.