Figure 2. Calu-3 organoids assumed a reversed polarity (inside-out; basal surface facing the central lumen).

On Day 6 of culture, spheroids lacking a lumen (a), containing a developing lumen (b) (white arrowhead), and containing a fully formed lumen (c) (yellow arrowhead; the longest gap of the lumen reached 100 μm) were observed when cultures were co-stained for F-actin (red) and the nuclei (blue). (d) Co-immunostaining of ZO-1 (an apical-domain marker, green), β1 integrin (a basal-domain marker, red), and the nuclei (blue) showing the polarity of one Calu-3 organoid. Scale bars, 20 μm (a, b, c) and 10 μm (d).