Two-Dimensional Chondrogenic Induction

Two-dimensional chondrogenic induction was performed as previously described [30] (link). Briefly, cells (1.5×10⁵) that induced hMSCs were suspended in 5 µl of chondrogenic medium (DMEM: F12 (Invitrogen), 1% (v/v) ITS1 mix (BD), 0.17 mM AA2P, 0.35 mM Proline (Sigma), 0.1 mM dexamethasone (Sigma), 0.15% (v/v) glucose (Sigma), 1 mM Na-pyruvate (Invitrogen), 2 mM GlutaMax, and 0.05 mM MTG supplemented with 40 ng/ml PDGF-BB and 1% (v/v) FBS (Nichirei)), and were subsequently transferred to fibronectin-coated 24-well plates (BD). A total of 1 ml of the chondrogenic medium was added after 1 hour. TGFβ3 (R&D) was subsequently added at 10 ng/ml on days 3 to 6, and BMP4 was added to a concentration of 50 ng/ml on day 10. Micromass cultures were maintained at 37°C under 5% CO₂ and 5% O₂ for 16 days. Differentiation properties were confirmed by Alcian Blue staining. Briefly, induced cells were fixed for 30 minutes with 10% formalin (Sigma) and rinsed with PBS. These cells were then stained overnight with Alcian Blue solution (1% Alcian Blue (MUTO PURE CHEMICAL CO., LTD, Tokyo, Japan) in 3% glacial acetic and 1% HCl, pH 1) and destained with the acetic acid solution.