Fig. 4: SARS-CoV-2 replication in 3D airway organoids and alveolar organoids.

a At the indicated hours post-infection, culture media were harvested from three lines of 3D AwOs infected with WT SARS-CoV-2 (MOI = 1) and applied to viral titration by TCID₅₀ assay. Data represent means ± SD in three organoid lines, n = 3 for each line. The dashed line indicates the detection limit. b 3D AwOs inoculated with SARS-CoV-2 (MOI = 2) were fixed at 8 or 24 hpi and immunostained to identify viral nucleoprotein (NP, green) positive cells. Nuclei and actin filaments were counterstained with DAPI (blue) and Phalloidin-647 (white), respectively. Scale bar, 20 µm. c After fixation at 24 hpi, SARS-CoV-2-infected AwOs were co-stained with α-NP (green) and α-FOXJ1 (red, top) or α-P63 (red, bottom). Scale bar, 20 µm. d–g Representative microphotographs of transmission electron microscopy. d Ciliated cells. Longitudinal section (left, scale bar 1 µm) and cross-section (right, scale bar 200 nm) of cilia showing microtubules with a 9 + 2 arrangement. e Goblet cells. Scale bar, 4 µm. f A virion particle in the cytoplasm of SARS-CoV-2-infected (MOI = 5) airway organoids. Scale bar, 50 nm. g Virion particles in a secretory vesicle in a cell of infected organoids. Scale bar, 400 nm. h At the indicated hours post-infection, culture media were harvested from three lines of AlvOs infected with WT SARS-CoV-2 (MOI = 1) and applied to viral titration by TCID₅₀ assay. Data represent means ± SD in three organoid lines, n = 3 for each line. i Representative confocal images of SARS-CoV-2-infected SFTPB⁺ AT2 cells in an AlvO (MOI = 2). Scale bar, 20 µm.