Generation of apical-out airway organoids from human primary airway epithelial cells

Airway organoids can be successfully generated from primary airway epithelial cells, recapitulating the architecture, polar organization, and key functions of the in vivo tissue. However, the closed organoid structure limits the accessibility to the luminal apical cell surface, which is exposed to the environment in vivo. This inaccessibility hinders the utilization of airway organoids in assays examining host-pathogen interactions and responses to environmental stimuli, both of which occur primarily at the apical surface. To address this limitation, we have developed PneumaCult™ Apical-Out Airway Organoid (AOAO) Medium, which supports the fast and efficient generation of airway organoids exposing their apical side to the environment in the absence of an extracellular matrix hydrogel. To generate AOAOs, two-dimensional expanded human bronchial epithelial cells (HBECs) were seeded in PneumaCult™ AOAO Medium in micropatterned [AggreWell™400] plates to promote cell aggregation. The aggregates were then transferred to suspension culture in fresh PneumaCult™ AOAO, and incubated at 37°C until they differentiated into AOAOs. These organoids can be generated from HBECs from a multitude of different passages and display high culture homogeneity, with low inter-donor size variability (average diameter 74 ± 4 μm at p3, n=3). The organoids are mainly composed of ciliated cells (61 ± 16% of all cells at p3) that display outward-facing, beating cilia and KRT5-positive basal cells (n=3). The easy access to the apical surface of the epithelium and scalability of this culture system offer a powerful in vitro model suitable for studying host-pathogen interactions and high-throughput antiviral drug screening.