To evaluate the adipogenic and osteogenic potential of ADSCs, cells at 90% confluence were cultured with adipogenic or osteogenic differentiation medium (STEMCELL Technologies) for 10–15 days. The differentiation medium was replaced every 2 days. Adipogenic and osteogenic differentiation were assessed by Oil Red O staining and alizarin red staining, respectively.
For chondrogenic differentiation, a previously described protocol was followed [31 (link)]. Briefly, ADSCs were cultured in a normal cell growth medium at 37 °C with 5% CO2 for 24 h, then the cells were cultured with chondrogenic differentiation medium (STEMCELL Technologies), which was replaced every 3 days. After 28–30 days, chondrogenic pellets were fixed with 4% PFA and embedded in Tissue-Tek O.C.T.™ Compound. Chondrogenic pellets were sliced into 7 μm thick sections using a freezing microtome (Leica), and then the sections were stained with 0.1% alcian blue.
For chondrogenic differentiation, a previously described protocol was followed [31 (link)]. Briefly, ADSCs were cultured in a normal cell growth medium at 37 °C with 5% CO2 for 24 h, then the cells were cultured with chondrogenic differentiation medium (STEMCELL Technologies), which was replaced every 3 days. After 28–30 days, chondrogenic pellets were fixed with 4% PFA and embedded in Tissue-Tek O.C.T.™ Compound. Chondrogenic pellets were sliced into 7 μm thick sections using a freezing microtome (Leica), and then the sections were stained with 0.1% alcian blue.
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