Fig 2: Establishment of human salivary gland organoids from PG, SMG, and SLG.a Schematic image of experiments using three major human salivary gland organoids, including human parotid gland (hPG), sublingual gland (hSLG), and submandibular gland (hSMG) organoids. b hSMG organoids were maintained in the GEM. The growth of a single organoid was tracked in the time-lapse of images obtained at each time point. Scale bars indicate 50 µm. c hSMG organoids were maintained in the GEM for 2 weeks and incubated in the DAM for another 3 days. Harvested organoids were subjected to H&E staining or IF staining for duct (KRT5; green, KRT7; red), acinar (AQP5; green, MIST1; red), and myoepithelial (ACTA2; green, KRT14; red) markers. Nuclei were stained with Hoechst 33342 (blue). Scale bars indicate 50 µm. d hSMG organoids were maintained for 3 months in the GEM, followed by differentiation in the DAM for another 3 days. Single-cell suspensions were prepared, and calcium influx was assessed using Fluo 4-AM in stimulation with either ATP (top) or carbachol (CCh; bottom) at different dosages. Red arrows indicate time points at which cells were treated with stimulants. e–g hPG, hSMG, and hSLG organoids were maintained for 1 month in the GEM, followed by differentiation in the DAM for another 3 days. e Differentiated hSMG organoids were stimulated with vehicle (DMSO), CCh, isoproterenol (IPR), or vasoactive intestinal peptide (VIP) for 1 h. Then, organoid swelling was observed under a brightfield microscope. Red arrows indicate swollen organoids. Scale bars indicate 500 µm. f Sections from hPG (top), hSMG (middle), and hSLG (bottom) tissues (left) and differentiated organoids (right) were subjected to PAS staining. Nuclei were counterstained with hematoxylin. Scale bars indicate 50 µm. g hPG, hSMG, and hSLG tissues (left) and differentiated organoids (right) were subjected to IF staining for mucous (MUC7, green) and serous (AMY1, red) acinar cells. Scale bars indicate 50 µm. All data were representative of three independent experiments. Source Data are provided as a Source data file.