Stempro differentiation media

Approximately 3–5 mL cell pellets were obtained from 10–30 mL adipose tissue after tissue dissociation. Following the cell isolation, cells adhered to the culture flasks and displayed the characteristic shape of ADSCs and achieved confluency in 1–2 weeks. Furthermore, mesenchymal stem cell phenotype with capacity for tri‐linage development was confirmed by culturing obtained cells in StemPro differentiation media (Gibco) and subsequent staining for adipogenesis (lipids) with Oil red O, chondrogenesis (proteoglycans) with Alcian blue and osteogenesis (alkaline phosphatase activity) with Alizarin Red.

For all differentiation assays, hUC-MSCs were first cultured in microcryogels for 0, 3, and 5 d before being digested, and were subsequently seeded in six-well plates. Cells were cultured in growth medium for 12 h followed by changing to differentiation medium. The trilineage differentiation ability of hUC-MSCs was assessed by culturing the cells in StemPro^® differentiation media (Gibco, Life Technologies, USA) to induce osteogenesis, chondrogenesis, and adipogenesis. Osteogenesis was assessed at 14 d by staining the cells for alkaline phosphatase with the BCIP/NBT Kit (Sigma-Aldrich, USA) according to the manufacturer’s instructions. Chondrogenesis was assessed at 14 d by staining for sulfated proteoglycan deposits with Alcian-Blue Staining Solution (Sigma-Aldrich). Adipogenesis was assessed at 21 d by staining for lipid accumulation with an Oil Red O Kit (Sigma-Aldrich).