Sw1353

ATDC5 and SW1353 cells were purchased from RIKEN Cell Bank (Ibaraki, Japan) and cultured in Dulbecco's Modified Eagle Medium (Thermo Fisher Scientific, MA, USA) containing 5% fetal bovine serum (Life Technologies, Carlsbad, CA, USA) at 37 °C in a 5% CO₂ humidified atmosphere.
The medium was replaced with a chondrogenic-induced medium additionally supplemented with 1 × insulin-transferrin-selenium (ITS, Corning, NY, USA) [32 (link),33 ]. The chondrogenic culture medium was replaced daily. After differentiation for 14 days, ATDC5 cells were washed with phosphate buffer solution (PBS) and fixed with 4% paraformaldehyde solution for 10 min. Subsequently, stained with 1% alcian blue 8GX (Sigma-Aldrich, Saint Louis, MO, USA). Shanghai, China) dissolved in 0.1 M HCl overnight. Cells were washed with PBS twice and photographed.

The human OS cell line MG‐63 and CS cell line SW‐1353 were purchased from the RIKEN Cell Bank. The lines were cultured in D‐MEM medium (FUJIFILM Wako Pure Chemical) supplemented with 10% fetal bovine serum (Sigma‐Aldrich Japan) and penicillin (100 units/mL)/streptomycin (100 μg/mL) mixture (FUJIFILM Wako Pure Chemical). All cells were cultured in a humidified atmosphere containing 5% CO₂ in air at 37°C. Flat‐bottomed dishes and plates (Corning) were used in the two‐dimensional (2D) cell culture. The PrimeSurface^® system (Sumitomo Bakelite) was used for 3D spheroid formation. Cell suspensions of MG‐63 and SW‐1353 were seeded onto a PrimeSurface 96 U plate at 10⁴ cells/well and then cultured for 3‐4 d.