Stempro chondrogenic differentiation kit

To perform chondrogenic differentiation, MSCs were induced to differentiate into chondrocytes after twenty-one days of culture in a chondrogenic induction medium supplemented with growth factors.
In a 12-well plate, the cells were seeded at the same concentration in triplicate (5 × 10⁴ cells). After 24 hours of culture in a basal culture medium, the culture medium was changed to the specific chondrogenic differentiation medium supplemented with growth factors (StemPro® Chondrogenic Differentiation Kit; Gibco Invitrogen, Grand Island, NY).
For the evaluation of chondrogenic differentiation, we performed staining with alcian blue after 21 days in differentiation conditions to identify the proteoglycan (extracellular matrix) released by the chondrocytes.
The induction medium was removed from the cell cultures, which were washed twice with PBS (Gibco Invitrogen, Grand Island, NY) and fixed with 4% formaldehyde (Sigma Aldrich, St. Louis, MO) for 20 minutes at room temperature. After fixation, the cells were stained with 1 mg/ml of alcian blue (Sigma Aldrich, St. Louis, MO) for two hours in the dark at room temperature. For the final wash, hydrochloric acid (0.1 M) was used once and with PBS twice.

All three MSCs strains (fourth or fifth passage) were introduced in vitro into osteogenic (21 days), chondrogenic (21 days), and adipogenic (18 days) differentiation with respective specific media using StemPro^® Osteogenesis Differentiation Kit, StemPro Chondrogenic Differentiation Kit, and StemPro Adipogenic Differentiation Kit media (Gibco; Invitrogen, Grand Island, NY) and a 12-well cell culture plate (Corning^® Costar^®; Sigma‐Aldrich, St. Louis, MO) (5 × 10³ cells/well). The three types of media were prepared according to manufacturer data sheets.

ASCs were seeded in a 96-well V-shaped tissue culture plate (Greiner Bio-one, Frickenhausen, Germany) at 80,000 cells per well, centrifuged at 500× g for 5 min, and incubated for 3 weeks with the STEMPRO^® Chondrogenic-differentiation kit (ThermoFisher Scientific, Life Technologies, Roskilde, Denmark). The control pellets were maintained in a growth medium. Hereafter, the pellets were embedded in 5 µm paraffin sections, the sulfated glycosaminoglycans (GAGs) stained with Alcian Blue 8GX (Sigma-Aldrich, Søborg, Denmark) for 30 min, and the degree of differentiation was evaluated by bright field microscopy (Axio Observer).