Establishment of primary transgenic human airway epithelial cell cultures to study respiratory virus – host interactions

Hulda R. Jonsdottir; Sabrina Marti; Dirk Geerts; Regulo Rodriguez; Volker Thiel; Ronald Dijkman

Due to the extended culture time required for hAEC culture establishment and differentiation, 76 and the fact that well-differentiated cultures can be maintained for months [19] , stable integration of 77 any genes to be expressed post-differentiation is required. Therefore, we established our protocol 78 using lentiviral vectors, where the proviral genomic material is integrated directly into the cellular 79 genome. Furthermore, such a system allows for the integration of both transgene and shRNA 80 scaffolds for over expression and/or knockdown of both host and viral factors [20] [21] [22] . Although The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/694380 doi: bioRxiv preprint were cultured as previously described [19] , with the following modifications. Monolayer cultures of Once cells reached confluency, the apical and basolateral medium was changed to air-liquid 111 interphase (ALI) medium, and one day later the apical side medium was aspirated, establishing ALI.