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ALI culture allows us to manipulate the environmental variables in the assay independently for the apical side and basal side of the epithelium. Traditionally, sphere culture was carried out on inserts and supported clonal bronchosphere generation to facilitate enumeration of stem cells. The insert-dependent nature of this assay, however, makes adaptation of this system to a high-throughput format exceedingly challenging. Recently, insert-independent sphere culture techniques have been developed. This method is described in the recent Cool Tools post describing 3D airway epithelial models also published by Danahay et al.. This insert-independent sphere culture method is amenable for high-throughput studies.