Protocol for establishing primary human lung organoid- derived air-liquid interface cultures from cryopreserved human lung tissue

Primary human lung organoid-derived air-liquid interface (ALI) cultures serve as a physiologically relevant model to study human airway epithelium in vitro. Here, we present a protocol for establishing these cultures from cryopreserved human lung tissue. We describe steps for lung tissue cryostorage, tissue dissociation, lung epithelial organoid generation, and ALI culture differentiation. We also include quality control steps and technical readouts for monitoring virus response. This protocol demonstrates severe acute respiratory syndrome coro-navirus 2 infection in these cultures as an example of their utility. For complete details on the use and execution of this protocol, please refer to Diana Cadena Castaneda et al. (2023).1