Adipogenic differentiation media

3×10⁴ cells/cm² of each diabetic and non-diabetic adipose tissue-derived cells were seeded in a 24-well plate (SPL, Korea). After reaching 80 % confluency, the culture media were replaced with the adipogenic differentiation media (Life Technologies, USA). The differentiation media were changed every 3 days. After 21 days, the cells were washed with PBS and fixed with 10 % formalin. Then, cells were stained with Oil Red O solution and checked by an optical microscope (Olympus, Japan) to observe the fat vacuoles.

The potential of USCs to differentiate into osteogenic, chondrogenic, and adipogenic cells was analyzed as previously described [26 (link)]. Briefly, to induce osteogenic differentiation, cells were cultured with osteogenic differentiation media for 3 weeks (Life Technologies, Gibco). Extracellular accumulation of calcium was examined by Alizarin Red and von Kossa staining. For chondrogenesis induction, cells were pelleted and cultured in the chondrogenic differentiation media (Life Technologies, Gibco) for 4 weeks. Positive induction was detected by Toluidine blue staining after differentiation. For adipogenic induction, passage 5 USCs were seeded at a density of 5,000 cells per centimeter square and cultured with adipogenic differentiation media for 2 weeks (Life Technologies, Gibco). The formation of lipid vacuoles was detected by Oil Red O staining.