Protocol for primary human lung organoid-derived air-liquid interface in vitro model to study response to SARS-CoV-2

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). HighlightsO_LIHuman lung tissue dissection, embedding in OCT blocks, and tissue cryopreservation. C_LIO_LIThawing & lung tissue dissociation for lung epithelium organoid generation. C_LIO_LIOrganoid-derived air-liquid-interface cultures for the study of viral infection. C_LIO_LIBulk RNA-Seq, flow cytometry, viral titer, and imaging to follow response to virus. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/557067v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@b47fbdorg.highwire.dtl.DTLVardef@2e6e60org.highwire.dtl.DTLVardef@508ac6org.highwire.dtl.DTLVardef@1c701a4_HPS_FORMAT_FIGEXP M_FIG C_FIG