Stemmacs adipodiff media differentiation medium

For differentiation experiments, E-MSCs were cultured at the third passage in osteogenic, adipogenic, and chondrogenic mediums, according to the manufacturer’s instructions. Briefly, for osteogenic induction, 45,000 cells were plated in each well of six well plates and cultivated in StemMACS OsteoDiff Media (Miltenyi, Bergisch Gladbach, Germany). After 21 days, osteogenic differentiation was demonstrated by the accumulation of calcium (crystalline hydroxyapatite detection by Von Kossa staining). For adipogenic differentiation, 75,000 cells were cultured in StemMACS AdipoDiff Media differentiation medium (Miltenyi, Bergisch Gladbach, Germany) for 21 days, after which the presence of intracellular lipid vesicles was assessed after fixation with paraformaldehyde vapors and Oil Red O staining. For chondrogenic differentiation, an aliquot of 250,000 cells were cultured in StemMACS ChondroDiff Media differentiation medium (Miltenyi, Bergisch Gladbach, Germany) for 21 days in 15 mL polypropylene culture tubes. During chondrogenic differentiation, cellular growth occurred as cellular aggregates floated freely in suspension. The pellet was included in paraffin and stained with Alcian Blue to identify the presence of hyaluronic acid and sialomucin.

Four aliquots of 75 × 10³ cells were cultured in 35 mm Petri dishes: two plates were set up with StemMACS.
AdipoDiff Media differentiation medium (Miltenyi, Germany) and the other two were used as control. The changing of the medium was performed three times a week, and, after 21 days, cells were fixed with paraformaldehyde (PAF) vapours for 10 min and stained with oil red O dye. They were morphologically observed using an optical microscope to evaluate the presence of red lipid vacuoles, the differentiated and coloured cells having red lipid granules and blue nuclei.