Fig. 3

Differentiation of CTOs at ALI and their characterization. A Method for the seeding and differentiation of CTOs. Briefly, 100,000 cells from CTOs (passage 3–10) were seeded on a fibronectin coated Transwell (TW). The cells were cultured submerged in airway organoid (AO) medium for 2 days. The medium was then replaced by 1:1 AO and Pneumacult-air–liquid-interface (PC-ALI) medium. When confluent, the cells were brought to ALI and the medium replaced by PC-ALI medium. The medium was refreshed every 2 days until day 14, time during which the cells differentiated. Created with BioRender.com. B Permeability of the cell layer during the first 4 days post-seeding, indicated as leakage of 4 kDa dextran through the epithelial cell layer on TW membranes expressed as percentage of maximum leakage through an empty TW. Bars represent the mean and error bars the SD of two technical replicates performed on the same day. C Transepithelial electrical resistance (TEER) measurements of two independent seeding experiments. Points represent the mean and the shading the SD of six (repeat #1) and three (repeat #2) individual TWs. The star icon indicates the moment when cells were confluent and placed at ALI. D Hematoxylin and eosin (H&E) and Periodic Acid Schiff (PAS), indicating polysaccharide-rich mucus-containing areas, stainings of CTO-derived 2D cultures on day 14 post-seeding. The arrows mark PAS-positive goblet cells. (E–H) Immunofluorescent stainings of CTO-derived 2D cultures on day 14 post-seeding. E Cross section of CTO-derived 2D cultures stained for ciliated cell-marker acetylated-α-tubulin (ac-α-tub; white), goblet cell-marker muc5AC (green), basal cell-marker p63 (pink), and nuclear marker Hoechst (blue). F–H Maximum intensity projections of Z-stacks of CTO-derived 2D cultures stained with Hoechst (F–H), muc5AC (F), ac-α-tub (G), and tight junction-markers zonula occludens-1 (ZO-1; red; in (F–H)), claudin 5 (green; in (H)), and occludin (green; in (H))