Protocol for primary human lung organoid-derived air-liquid interfacein vitromodel to study response to SARS-CoV-2

Summary This article presents a comprehensive protocol for establishing primary human lung organoid-derived air-liquid interface (ALI) cultures from cryopreserved human lung tissue. These cultures serve as a physiologically relevant model to study human airway epithelium in vitro . The protocol encompasses lung tissue cryostorage, tissue dissociation, lung epithelial organoid generation, and ALI culture differentiation. It also demonstrates SARS-CoV-2 infection in these cultures as an example of their utility. Quality control steps, ALI characterization, and technical readouts for monitoring virus response are included in the study. For additional details on the use and execution of this protocol, please refer to Diana Cadena Castaneda et al ( https://doi.org/10.1016/j.isci.2023.107374 ). Highlights Human lung tissue dissection, embedding in OCT blocks, and tissue cryopreservation. Thawing & lung tissue dissociation for lung epithelium organoid generation. Organoid-derived air-liquid-interface cultures for the study of viral infection. Bulk RNA-Seq, flow cytometry, viral titer, and imaging to follow response to virus. Graphical abstract