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

IAV Infection in Human AOs. The 3D AOs were sheared mechanically to expose the apical surface to the virus inoculum. The sheared organoids were then incubated with viruses at a multiplicity of infection (MOI) of 0.01 for 2 h at 37°C. After washing, the inoculated organoids were re-embedded in Matrigel and then were cultured in the PD medium. In the H7N9/Ah and H7N2 infection in the 3D PD organoids, one set of H7N9/Ah-inoculated organoids was treated with the serine protease inhibitor AEBSF (0.125 mM; Merck Millipore) during inoculation and after inoculation. At the indicated times, organoids, dissolved Matrigel, and culture medium were harvested for detection of viral load. The cell-free Matrigel and the culture medium from each droplet were pooled together as one sample, referred as the "supernatant." The supernatant samples were also used for viral titration. The 2D PD AOs were inoculated with the indicated viruses at an MOI of 0.001 from the apical side by adding the virus inoculum to the apical chamber followed by incubation for 2 h at 37°C. At the indicated times, cell-free media were collected from the apical and basolateral chambers for viral titration. The membranes seeded with 2D organoids were incised from the Transwell inserts and fixed, and immunofluorescence staining was applied as described previously (13) .