Figure 1. Conversion of differentiated air–liquid interface (ALI) human nasal epithelial cell (HNEC) cultures into nasal-airway organoids (NAOs) and use in forskolin-induced swelling (FIS) assays.

(A) Graphic illustration showing workflow of culturing NAOs from 2D differentiated HNECs and use in FIS assays. (B) Brightfield images of HNEC. (C) IF staining of HNEC with basal-cell markers p63 (red) and cytokeratin 5 (KRT5, green). (D) Sections of nasal tissue (top) and 18 d differentiated ALI-cultured HNEC (bottom), demonstrating IF staining of MUC5AC (goblet cells, red), β-tubulin IV (ciliated cells, green), p63, and KRT5 (basal-cell markers, green and red, respectively). (E) Time course showing self-organization of differentiated ALI-HNEC–derived epithelial fragments into organoids. (F) Confocal images showing in the top panels staining with DAPI (blue), β-tubulin IV (green), and MUC5AC (red). The bottom panels show staining with DAPI (blue), p63 (green), and KRT5 (red). (G) Representative brightfield images (top) and images of calcein green AM esters–stained (bottom) organoids from a HC subject, which were unstimulated (control) or treated with forskolin (Fsk, 5 μM). Images were taken at t = 0, 60, and 120 min after stimulation. (H, I) NAOs from HC subjects (n = 5 independent donors) were stimulated with different concentrations of Fsk (0–10 μM), followed by quantification of FIS. (C, D, F) Data information: DAPI (blue) was used as nuclear staining (C, D, F). (H, I) Quantification of FIS is depicted as the percentage change in surface area relative to t = 0 (normalized area) measured at 15-min time intervals for 2 h (means ± SD) (H), and area-under-the-curve (AUC) plots (t = 120 min, means ± SD, datapoints represent individual donors) (I). (I) Analysis of differences was determined with a one-way ANOVA and Bonferroni post hoc test (I). **P < 0.01, ****P < 0.0001.