FIGURE 2.

Characterisation of the epithelial nature of cells in the bronchioid model. a) Dot plots representing epithelial cell adhesion molecule (EpCAM)-peridinin chlorophyll protein complex (PerCP)-cyanine 5.5 fluorescence (y-axis) versus pan cytokeratin-fluorescein isothiocyanate fluorescence (x-axis) of cells dissociated from two-dimensional (2D) air–liquid interface (ALI) culture and bronchioids at day (D) 1. The gating strategy is shown in the left panels. The percentages of EpCAM⁺ cytokeratin⁺ cells are shown in pink. b) Expression of the genes CDH1 and EPCAM in bronchioids over time and in cells dissociated from 2D culture at day 9 after ALI introduction. The bronchioid and ALI culture samples were obtained from six and three different donors, respectively, and data are presented as mean±sd. Gene expression was normalised to that of the housekeeping genes PPIA, RPL13 and GusB and expressed relative to that of basal epithelial cells in 2D submerged culture. c, d) Longitudinal and transverse sections and three-dimensional (3D) views of 3D reconstructions obtained from Z-stack confocal images of a 2-day-old bronchioid stained for F-actin (phalloidin, magenta) and nuclei (DAPI, white) (c) and F-actin (magenta), zonula occludens-1 (ZO-1) (cyan) and nuclei (white) (d). Scale bars: 100 µm and 50 µm in the magnified lower panel.