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 compo-sition via wholemount staining and assess cilia motility via 3D AOAO rotation upon Matrigel embedding. The protocol offers an effective model for investi-gating human airway pathophysiology.For complete details on the use and execution of this protocol, please refer to Wijesekara et al. (2022).1