Mesencult chondrogenic differentiation kit

Tri-lineage differentiation was assessed for cells seeded directly from cryopreservation. Adipogenic differentiation was performed using the MesenCult Adipogenic Differentiation Kit (Stemcell Technologies) according to the manufacturer’s instructions. Similarly, chondrogenic differentiation was done using the MesenCult Chondrogenic Differentiation Kit (Stemcell Technologies) according to the manufacturer’s instructions. The osteogenic differentiation protocol consisted of 25–28 day culture on hMSC medium with the addition of β-glycerolphosphate (0.01 M) and dexamethasone (10⁻⁸M). For adipogenic differentiation, presence of lipid vacuoles was demonstrated using Oil Red O staining. For chondrogenic differentiation, presence of aggrecan was demonstrated using Alcian Blue staining. For osteogenic differentiation, calcium deposits were stained with Alizarin Red.

For chondrogenic differentiation, cells were cultured as cell aggregates on ultra-low attachment plates (Corning), as previously reported [11 (link)]. Cells were maintained in a chondrogenic differentiation medium (MesenCult™ Chondrogenic Differentiation Kit, Stemcell Technologies, Vancouver, BC, Canada) for 21 days, and chondrogenic differentiation was confirmed with Alcian Blue staining (Sigma-Aldrich) [11 (link)].

MSCs were plated on a cell culture dish and cultured in a specific MSC culture medium supplemented with 10% FBS and 2% penicillin‒streptomycin (Cyagen Biosciences Inc). The cells were incubated for 24 to 48 h to facilitate attachment before the induction of adipogenic, osteogenic or chondrogenic differentiation. Adipogenic differentiation was carried out with the human adipose-derived stem cell adipogenic differentiation kit (Cyagen Biosciences Inc, GUXMX-90031) for 21 days according to the manufacturer’s protocol. Osteogenic differentiation was performed by using the MesenCult Osteogenic Differentiation Kit (Stem Cell Technologies cat: 05465). The medium was changed every 3 days until bone matrix formation occurred (10–15 days). Chondrogenic differentiation was performed by using the MesenCult Chondrogenic Differentiation Kit (Stem Cell Technologies, Cat: 05455). The medium was replaced with 0.5 mL medium every 3 days for a total of 21 days.