FIGURE 1.

The formation of an alginate tube filled with primary basal epithelial cells is called a “bronchioid”. a) Schematic of the device used to generate bronchioids. Created with BioRender.com. The three solutions were injected simultaneously by a computer-controlled pump inside a three-dimensional-printed device soaked in a 100 mM calcium bath. b) Left panel: brightfield images of bronchioids over time, with (+) or without (−) 10 µM Y-27632 (Y). The green and pink dotted lines indicate the external (alginate tube) and internal (epithelial tube) limits, respectively. Scale bars: 100 µm. Right panel: quantification of external and internal diameters (at least 10 measurements along the same tube for each condition, n=3 experiments). The medians are represented as horizontal lines. c) Immunoblots and analysis of myosin light chain (MLC) II and double-phospho-MLC (PP-MLC) (Thr18, Ser19) expression on day 2 in bronchioid tissue from three different donors (n=3). Total protein was used as a loading control. d) Upper panels: brightfield images of bronchioids cultured with 10 µM Y-27632. Scale bars: 100 µm. Lower panels: dot plots showing propidium iodide fluorescence (y-axis) versus annexin V-allophycocyanin fluorescence (x-axis) in cells dissociated from bronchioids and analysed by flow cytometry at the indicated time points. The percentages of propidium iodide⁻ annexin V⁻ cells are shown in pink. IS: intermediate d-sorbitol solution; AS: alginate solution.