Protocol to engineer apical-out airway organoids using suspension culture of human airway basal stem cell aggregates

Here we present a protocol to engineer apical-out airway organoids (AOAOs) directly from human airway basal stem cells (hABSCs) using suspension culture of hABSC aggregates on a cell-repellent surface. We describe steps to produce spherical AOAOs with homogenous presentation of exterior-facing motile cilia and of tunable sizes. We then detail procedures to analyze AOAO cellular composition via wholemount staining and assess cilia motility via 3D AOAO rotation upon Matrigel embedding. The protocol offers an effective model for investigating human airway pathophysiology. For complete details on the use and execution of this protocol, please refer to Wijesekara et al. (2022).¹.