Chondrogenic Differentiation Protocol

For chondrogenic differentiation, 1 × 10⁶ cells were imbedded in 100 µL alginate.^{15 (link)} Chondrogenic differentiation was induced by DMEM/F12 (Gibco) supplemented with 2.8 g/L glucose, 1 mM sodium pyruvate (Gibco), 0.1 mM ascorbic acid-2-phosphate, 0.1 µM dexamethasone, 1% ITS (insulin 25 µg/mL, transferrin 25 µg/mL, and sodium selenite 25 ng/mL), 1.25 mg/mL human serum albumin (Octapharma, Jessheim, Norway), 500 ng/mL bone morphogenic protein-2 (Wyeth Pharmaceuticals, Taplow, UK), 25 ng/mL recombinant human transforming growth factor-β1 (R&D Systems, Minneapolis, MN, USA), and 200 mg/mL insulin-like growth factor 1 (IGF1; Sigma–Aldrich, St. Louis, MO, USA). Medium was changed every 3–4 days. Cells were harvested after 21 days of culture for use in qRT-PCR. The qRT-PCR was done for SOX9, COL2A1, and ACAN (Table 1).