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 2D Differentiated AOs with Transwell Culture. Transwell culture of AOs was performed as described elsewhere (24, 25) with modifications. Briefly, the organoids were dissociated into single-cell suspensions after digested with 10× TrypLE Select Enzyme (Invitrogen) for 1-5 min at 37°C, sheared using a Pasteur pipette, and strained over a 40-μm filter. Approximately 3.5 × 10 5 cells were seeded into each Transwell insert (product no. 3494; Corning). The cells were cultured in AO medium at 37°C in a humidified incubator with 5% CO 2 for 1-2 d. When cells reached >90% confluence, the AO medium was changed to PD medium in both the apical and basal chambers. The medium was changed every other day, and the cells were maintained for 14 d. TEER was measured every other day using a Millicell ERS-2 Volt-Ohm Meter (EMD Millipore). To assess the integrity of the 2D organoid monolayer as an epithelial barrier, at day 12 after seeding, FITC-dextran with an average molecular weight of 10,000 (Sigma Aldrich) was added to the medium in the upper chamber at a concentration of 1 mg/mL followed by incubation at 37°C for 4 h. Subsequently, the culture media were harvested from the upper and bottom chambers to detect the fluorescence intensity using the Victor XIII Multilabel Reader (PerkinElmer). eggs at 37°C for 36 h. The eggs were chilled overnight at 4°C; then the virus-containing allantoic fluid was harvested, aliquoted, and stored at −80°C. Virus titer was determined by plaque assay.