Chondrogenic Differentiation of MSCs

Chondrogenesis was induced in micromass pellet cultures as described.25 Micromass-pellet cultures were prepared from 1.0–2.5×10⁵ MSCs (passage 2) in 15-mL polypropylene tubes that were centrifuged at 150 g for 10 min in complete medium. Cell pellets were incubated with induction medium (AdvanceSTEM chondrogenic differentiation medium, ThermoScientific) for 3 weeks. Each pellet was parafinized after dehydration and cut into thin sections (4–5 μm). The sections were analyzed for chondrogenic differentiation using a commercially available Alcian blue kit (IHC World, Woodstock). For staining, sections were fixed with 4% PFA and washed with distilled water followed by treatment with Alcian blue for 20 min. Alcian blue uptake was analyzed using a colorimetric assay as described.^{26 (link)} After 21 days, the micromass cultures were fixed with methanol and whole mount stained with Alcian blue. Alcian blue was extracted with 6M guanidine HCl and absorbance was read at 620 nm. All experiments were performed in triplicate.