Figure 6.
Promotion of salivary organoid functionality by 3D microwell cultures. (A) Effect of isoproterenol and epinephrine on amylase secretion in SGSCs. The amylase activities per wells containing 1 × 105 SGSCs were examined using an assay kit. The data are presented as means ± standard errors of the mean. The data from three independent experiments were analyzed and presented as a mean ± standard errors of the mean (n = 3). Two-way ANOVA, Bonferroni post hoc test. *, compared with the floating culture method; #, compared with the Matrigel group; $, compared with CON in each group. *P < 0.05, ***P < 0.001, ###P < 0.001, $$$P < 0.001. (B) Amylase expression in the cell supernatants was tested by Western blotting analysis in 2D plastic and 3D spheroid at 3 days. (C) The measurement of intracellular pH level. SGSCs were loaded with BCECF-AM and pretreated Cl–/free HCO–3 medium. After the addition of Cl–/HCO–3 medium, the intensities of [pH] in baseline (Cl–/free HCO–3) medium increased significantly, with greater increases being observed in the 3D spheroids in microwells. The data from three independent experiments were analyzed and presented as the mean fluorescent intensity ± standard errors of the mean (n = 3). Two-way ANOVA, Bonferroni post hoc test. *, compared with the floating group; #, compared with the Matrigel group; $, compared between HCO–3 (−) and HCO–3 (+) in each group. ***P < 0.001, ###P < 0.001, $$$P < 0.001. (C) Measurement of intracellular calcium level after stimulation with agonists. The hPECs were loaded with Fluo4 and treated with adenosine triphosphate (ATP), carbachol, or thapsigargin for 30 s in the media. The data from three independent experiments were analyzed and presented as the mean fluorescent intensity ± standard errors of the mean (n = 3). Two-way ANOVA, Bonferroni post hoc test. *, compared with floating group; #, compared with Matrigel group; $, compared between the baseline and after-stimulation in each group. ***P < 0.001, ###P < 0.001, $$$P < 0.001.