Stempro chondrogenesis differentiation kit

Chondrogenesis was induced by using the StemPro Chondrogenesis Differentiation Kit (A1007101, Thermofisher). Briefly, cells were seeded in micromasses (80,000 cells/each) and incubated for 2 h in a humidified chamber in the incubator before differentiation medium was added. After 28 days, the chondrogenic micromasses were either harvested into Trizol for gene expression measurements or fixed in paraformaldehyde (4%) for 45 min and directly stained overnight with Alcian Blue (1%; Sigma). Pellets were imaged in a Zeiss Axiovert 25 Inverted Phase microscope and radius measured with Zen 2 to calculate the spherical area (A = 4πr², where A is the area and r the radius).

Isolated BMSCs were further cultured and passaged twice using standard growth media of DMEM +10% FBS in order to enrich the cell number. A cell solution of 1.6 × 10⁷ cells/ml was generated using StemPro Chondrogenesis Differentiation Kit (Thermo Fisher), and 5 µl droplets were applied in the center of 48-well plate wells for micro-mass culture. 3 hr later, warmed chondrogenic medium was overlayed over the micro-mass and the formation of osteogenic pellets was observed after 3 d of culture.

Differentiation ability of transplanted MSC was evaluated by their differentiation into adipo-, osteo- and chondro-lineage. MSC were seeded onto 12-well cultivation dishes (TPP) with a seeding density of 3.8 × 104 cells/well for adipogenic and chondrogenic differentiation and 1.9 × 104 cells/well for osteogenic differentiation in culture media. After a 24-h attachment period, the media was discarded and replaced with 3 mL of specific differentiation media: StemPro^® Adipogenesis Differentiation Kit (Thermo Fisher Scientific, Waltham, MA, USA) for adipogenic differentiation, StemPro^® Chondrogenesis Differentiation Kit (Thermo Fisher Scientific, Waltham, MA, USA) for chondrogenic differentiation and StemPro^® Osteogenesis Differentiation Kit (Thermo Fisher Scientific, Waltham, MA, USA) for osteogenic differentiation. After the differentiation period of 21 days, oil red O (Sigma Aldrich, St. Louis, MO, USA) staining for lipid droplet visualization in adipogenesis, alcian blue (Sigma Aldrich, St. Louis, MO, USA) staining for glycoprotein visualization in chondrogenesis and alizarin red S (Sigma Aldrich, St. Louis, MO, USA) staining for calcium ion visualization in osteogenesis were performed.

OASC2 and BMSC were cultured at 37 °C in 5% CO₂ in their respective growth medium for 1 week. After the cells reached confluency, they were sub-cultured at 1.25 × 10⁵ cells/well in 12-well plates. For each group at each time point, cells were treated with StemPro® Chondrogenesis Differentiation kit (Thermo Scientific, Waltham, MA USA) to induce chondrogenic differentiation or with stem cell growth medium (DMEM, 1 mM glutamine, 100 mM sodium pyruvate, 100 mg/mL ascorbic acid) as control. Medium was changed every 4 days. On days 3, 7, and 14 of chondrogenic differentiation, outcome parameters were assessed by qPCR, western blot, and histological staining (Alcian blue staining and Alizarin red staining). Alcian blue- and Alizarin red-stained wells were scanned and analyzed using ImageJ software. OASC2 and BMSC were transfected with Lipofectamine 3000 (Thermo Scientific, Waltham, MA USA) in 12-well plates. Forty-eight hours after transfection, cells were lysed in QIAzol lysis reagent (Qiagen, Hilden, Germany) for RNA isolation and real-time qPCR analysis.

The chondrogenic differentiation was achieved by means of the StemPro™ Chondrogenesis Differentiation kit (Thermofisher, A1007101, Waltham, MA, USA). On day 21, culture-wells were rinsed 3 times in PBS and fixed with 4% formaldehyde at RT for 20 min. After 3 new washings, the AB staining (1%, Sigma-Aldrich, A3157-10G, Saint Louis, MO, USA) was added to the wells for 30 min to stain proteoglycans. The last three washes were performed with HCl (0.1N, Sigma-Aldrich, 1.09057.1000, Saint Louis, MO, USA).

Equine MSCs were assessed for chondrogenic differentiation potential following culture for 72 h in either 10% FBS or autologous or allogeneic equine serum by Alcian blue staining (ThermoFisher Scientific StemPro® Chondrogenesis Differentiation Kit, Waltham, MA). Briefly, following culture in respective serum sources for 72 h, cells were trypsinized and washed in phosphate-buffered saline, and the cell pellets resuspended in chondrogenesis medium [StemPro® osteocyte/chondrocyte differentiation basal medium and StemPro® chondrogenesis supplement (9:1), penicillin (100 U/mL) and streptomycin (100 μg/mL)] to generate a cell solution of 1.6 × 10⁶ cells/100 μL and then seeded in 5-μL droplets on 6-well plates. Cells were maintained on plates for 2 h in 37°C incubator at 5% CO₂, and then chondrogenesis media was added to the plates to cover the pellets. Media was changed every 3 days, and pellets maintained in culture in the 37°C incubator at 5% CO₂ for a total of 28 days and then evaluated for Alcian blue staining visually by microscopy (Olympus SC30 microscope, Tokyo, Japan).