Chondrogenic Induction of Cells

Two-dimensional chondrogenic induction was performed as previously described [32 (link)]. Briefly, cells (1.5 x10⁵) were suspended in 5 μl of chondrogenic medium (DMEM/F12 (Life Technologies), 1% (v/v) ITS1 mix (BD), 0.17 mM AA2P (Sigma), 0.35 mM Proline (Sigma), 0.1 μM dexamethasone (WAKO), 0.15% (v/v) glucose (Sigma), 1 mM Na-pyruvate (Sigma), and 2 mM GlutaMax (Life Technologies) supplemented with 40 ng/ml PDGF-BB (R&D System) and 1% (v/v) FBS (Nichirei, Inc., Tokyo, Japan)). They were subsequently transferred to fibronectin-coated 24-well plates (Corning, Inc., NY, USA). One milliliter of the chondrogenic medium was added after 1 h. TGFb3 (R&D System) was subsequently added at 10 ng/ml on days 6 to 10. Differentiation was confirmed on day 10 using Alcian Blue staining.