Stemmacs chondrodiff 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.

Aliquots of 25 × 10⁴ cells were resuspended in tubes (it is handy to use 15 mL) in 1 mL of StemMACS ChondroDiff Media differentiation medium (Miltenyi, Germany) and were grown as a three-dimensional cell aggregation for 21 days by changing the culture medium three times a week. At the end of the third week the differentiation was evaluated by Alcian Blue staining.