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 ASC-Derived Human AOs. The generation of four lines ASCderived human AOs was based on the previous protocol (14) with a success rate of 100%. Briefly, with ethical approval by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (UW 13-364) and informed consent of the patients, small pieces of normal lung tissue (∼0.2-0.6 cm 2 in size) adjacent to the diseased tissues were obtained from patients who underwent resection. The tissues were minced and digested with 2 mg/mL collagenase (Sigma Aldrich) for 1-2 h at 37°C, followed by shearing using a glass Pasteur pipette (Drummond) and straining over a 100-μm cell strainer (FALCON). The resultant single cells were embedded in 60% Matrigel (Growth Factor Reduced Basement Membrane Matrix; Corning) and were seeded in 24-well suspension culture plates (Greiner Bio-One). After solidification, Matrigel droplets were maintained with AO culture medium (SI Appendix, Table S1 ) at 37°C in a humidified incubator with 5% CO 2 . The organoids were passaged every 2-3 wk. The bright-field images of the organoids were acquired using a Nikon Eclipse TS100 Inverted Routine Microscope.