Fig. 3: Monitoring of the differentiation profile based on spheroid deformability across adipogenic, chondrogenic, and osteogenic lineages.

a Differentiation status of spheroids into adipogenic, chondrogenic, and osteogenic lineages evaluated by immunofluorescence at differentiation culture periods (Control, days 7, 14, and 21). Correlation between deformability and gene expression fold change in b adipogenic, c chondrogenic, d and osteogenic lineages over 21 days. e Representative images of adipogenic spheroid deformability. Blue outlines and red areas indicate spheroid boundaries before and during ultrasound exposure, respectively. f Comparison of deformability across differentiation periods within each lineage. g Statistical comparison of deformability among lineages on days 5, 7, and 13. h Deformability of each lineage under different acoustic pressures on day 21. Origin-specific correlation between deformability and gene expression for fate estimation: i adipogenic, j chondrogenic, and k osteogenic spheroids differentiated for 21 days. In c–h, deformability measurements were performed using n = 10 spheroids per donor, with three independent donors (total n = 30). For gene expression analyses, the number of biological replicates was n = 3 per donor, and each measurement was performed with n = 3 technical replicates. In i–k, deformability measurements were performed using n = 10 spheroids per donor. All bar graphs represent mean ± S.D. All scale bars indicate 100 μm