Differentiated human airway organoids to assess infectivity of emerging influenza virus

Zhou, Jie; Li, Cun; Sachs, Norman; Chiu, Man Chun; Wong, Bosco Ho-Yin; Chu, Hin; Poon, Vincent Kwok-Man; Wang, Dong; Zhao, Xiaoyu; Wen, Lei; Song, Wenjun; Yuan, Shuofeng; Wong, Kenneth Kak-Yuen; Chan, Jasper Fuk-Woo; To, Kelvin Kai-Wang; Chen, Honglin; Clevers, Hans; Yuen, Kwok-Yung

Establishing a 2D Airway Monolayer from AOs to Assess the Infectivity of IAVs. A limitation of 3D organoids for studying microbial infections is the inaccessibility of the apical surface to pathogens, since most organoids are orientated inwards, while receptors for most respiratory viruses are distributed in the apical surface. For virus inoculation, organoids must be sheared to enable sufficient apical exposure to virus inoculum (21) . As reported previously, primary bronchial epithelial cells cultured in Transwell inserts with the defined medium undergo mucociliary differentiation (22) . This prompted us to transform 3D AOs into a 2D monolayer using Transwell culture. To this end, 3D AOs were enzymatically dissociated into a single-cell suspension, seeded in Transwell inserts, and then cultured in PD medium. The transepithelial electrical resistance (TEER) in the 2D monolayers stabilized in the second week after seeding (SI Appendix, Fig. S1 ). In addition, the dextran penetration assay performed at day 12 indicated that an intact epithelial barrier was formed across the 2D monolayers (SI Appendix, Fig. S1 ). The intense signal of ACCTUB indicated that the 2D monolayers developed appreciable PD (Fig. 5A) . The productive infection of H7N9/Ah was clearly shown by the virus nucleoprotein-positive (NP + ) cells at 8 h post infection (hpi) in the 2D PD organoids (Fig. 5A) .