Figure 4.

Histological analysis of cell viability revealed no significant differences between spheroids of different diameters. (a) Live (green)/dead (red) staining revealed that MSCs in the 15 000-, 30 000- and 60 000-cell spheroids are equally viable. A 60 000-cell spheroid was incubated in methanol as a positive control for propidium iodide (far right). (b) Annexin V (red) and DAPI (blue) staining further verified the absence of apoptosis, as the 15 000-, 30 000- and 60 000-cell spheroids did not stain positively in either the spheroid core or the periphery. A 500 000-cell spheroid served as a positive control for Annexin V. Scale bar represents 500 µm; images were captured using a 20× objective. (c) Quantification of apoptotic index, calculated by the area of positive Annexin V staining in the core relative to the periphery (n = 3). Groups with no significance are linked by the same letters, while groups with significance do not share the same letters.