Generation and Infection of Airway Organoids

An airway organoid (AO) model was generated according to our previous report^{2 (link),78 (link)}. Briefly, normal human bronchial epithelial cells (NHBEs, Cat# CC-2540, Lonza) were used to generate AOs. NHBEs were suspended in 10 mg/ml cold Matrigel growth factor reduced basement membrane matrix (Corning, Cat# 354230). Fifty microliters of cell suspension were solidified on prewarmed cell culture-treated multiple dishes (24-well plates; Thermo Fisher Scientific, Cat# 142475) at 37°C for 10 min, and then, 500 μl of expansion medium was added to each well. AOs were cultured with AO expansion medium for 10 days. For maturation of the AOs, expanded AOs were cultured with AO differentiation medium for 5 days.
The AO-ALI model (Fig. 5f) was generated according to our previous report^{2 (link),78 (link)}. For generation of AO-ALI, expanding AOs were dissociated into single cells, and then were seeded into Transwell inserts (Corning, Cat# 3413) in a 24-well plate. AO-ALI was cultured with AO differentiation medium for 5 days to promote their maturation. AO-ALI was infected with SARS-CoV-2 from the apical side.