Hmsc chondrogenic singlequots

hADSC chondrogenic differentiation was achieved as previously described (Calabrese et al., 2015 (link)). Briefly, 2.5 × 10⁵ cells were centrifuged to form a three-dimensional aggregate and resuspended in complete chondrogenic medium containing differentiation basal medium (Chondrogenic Basal Medium, Lonza) supplemented with chondrogenic differentiation inducing factors (hMSC Chondrogenic SingleQuots containing Dexamethasone, Ascorbate, ITS+supplement, GA-1000 Sodium Pyruvate, Proline and L-Glutamine, Lonza) and TGF-β3 (Lonza). Pellets were incubated at 37°C in a humidified atmosphere of 5% CO₂. The growth medium was replaced every 2–3 days. The chondrogenic differentiation was completed on day 28 after induction. Pellets were fixed in formalin at three different time points along differentiation (1, 2, and 4 weeks). Subsequently, pellets were paraffin embedded and cut into 3 μm-thick sections for immunohistological processing.

The adipogenic, osteogenic and chondrogenic differentiation potential of group 1 and group 2 UC-MSCs and BM-MSCs (group 3) was tested after expansion in the particular medium type used for the primary isolation (adipogenic differentiation: passage = 2, osteogenic and chondrogenic differentiation: passage = 3). For osteogenic and adipogenic differentiation, 1000 BM- or UC-MSCs/cm² were seeded. After 24 h, medium was replaced by differentiation medium as described [36 (link)]. At day 21, cells were fixed using 4 % paraformaldehyd (PFA, Sigma Aldrich) and stained with either 0.5 % Alizarin Red (Sigma Aldrich) or 1 % Sudan III (Sigma Aldrich). Chondrogenic differentiation was induced using 250,000 cells per pellet cultivated in hMSC chondrogenic SingleQuots (Lonza, Basel, Switzerland) in the presence of TGF-β3 (20 µg/mL) for 21 days. Pellets were harvested by centrifugation (1500×g for 5 min), fixed in 4 % PBS buffered formalin and paraffin-embedded. After deparaffination of 2 µm sections in graded alcohols, 1 % Alcian Blue staining solution (8GS, Gatt-Koller, Absam, Austria) and Nuclear Fast Red solution (Sigma Aldrich) was applied for 15 min (Multistainer platform, Leica, Wetzlar, Germany). Photographs were taken using a PrimoVert Light microscope equipped with an AxioCam ERc5 s digital camera (both from Zeiss, Oberkochen, Germany).