Fig. 2

Morphology observation of USC-organoids and cell density optimization via proliferation assay. a Morphology of 3D USC-organoids generated from different cell density was observed. The size of the organoids grew larger with the increasing initial cell density (scale bar 200 μm). b ATP measurement for proliferation was performed in USC-organoids from different cell density. There was a non-linear relationship between proliferative ability and cell density. The proliferative ability increased with the ascent of initial USC concentration and reached a peak at 5000 cells/well, after which the proliferative ability began to descend. These indicated that suitable size was helpful for the balance of live cells and nutrition supply. Therefore, the 5000 cells/well was the optimal cell density for organoid culture in our study (six replicated wells for each cell density, data presented as mean ± SD)