Msc basal medium

MSC were plated at 20,000 cells/cm² in 12-well plates (Costar Corning Incorporated, Corning, NY, USA) for induction of adipogenic differentiation as described by provider (Thermo Fisher Scientific). In brief, after 48-hour incubation in MSC Basal Medium (Thermo Fisher Scientific), the medium was replaced by adipogenic differentiation medium (StemPro^® Adipocyte Differentiation Basal Medium 1×, StemPro^® Adipogenesis supplement 1×, and gentamicin (10 mg/mL). This medium was renewed twice a week over the incubation period of 11–14 days. Adipogenesis was studied by staining using the oil red standard stain procedure with some modifications to achieve costaining for iron. Twelve-well plates were washed 3× with PBS and cells were fixed for 15 minutes at RT with zinc (1:10) in dH₂O,63 (link) washed 2× with dH₂O and processed for Oil Red O staining. For staining, wells were rinsed with 60% isopropanol and 0.5% (w/v) Oil Red O (Sigma-Aldrich Co) stock solution was prepared. Fat deposits were stained by incubation with Oil Red O for 15 minutes. Finally, the cells were rinsed with 60% isopropanol and stained for iron as described earlier.

Labeled MSC in monolayer were trypsinized and transferred into 15 mL Falcon tubes at a concentration of 20,000 cells/2 mL to stimulate micromass formation by centrifugation for 4 minutes at 800× g. Micromasses were incubated at 37°C and 5% CO₂ in MSC Basal Medium (Thermo Fisher Scientific), followed by detachment from the bottom by gentle flicking. Chondrogenic differentiation was then induced by incubation with Chondrogenic Differentiation Medium as described by supplier (StemPro^® differentiation kits; Thermo Fisher Scientific) for 20 days with medium change twice a week. Condensates were collected, gently washed in PBS, and frozen in cryomolds with OCT Cryomedium (Tissue Tek Sakura Finetek Europe B.V, Alphen aan den Rijn, the Netherlands) and stored at −20°C. Cryosections of 5–10 µm thickness were used to stain glycosaminoglycans with a modified Alcian Blue protocol (www.ihcworld.com). Cryosections were dried at RT and fixed with 4% paraformaldehyde for 10 minutes, followed by several washes. Then, the sections were stained with Alcian Blue, pH 1.0 (1 N HCl) for 20 minutes and counterstained with filtered 1% Neutral Red (Applichem GmbH, Darmstadt Germany) in glacial acetic acid.