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.