Rhtgf β3

Pellet cultures were performed in accordance with the protocol published previously.^{19 (link)} HACs and STRO-1⁺ SSCs were harvested at confluence from monolayer cultures and suspended in serum-free chondrogenic media at final concentrations of 0.6 × 10⁵, 1 × 10⁵, 2 × 10⁵, 3 × 10⁵ and 5 × 10⁵ cells/ml. 1 ml of cell suspension was added to each sterile 25 ml polycarbonate universal tube and centrifuged at 400 × g for 5 minutes at 4℃. The resulting cell pellet was cultured in humidified atmosphere at 37℃, 5% CO₂ and 21% O₂ for 21 days. The serum-free chondrogenic medium was made up of α-MEM supplemented with 10 ng/ml rhTGF-β3 (PeproTech, London, UK), 100 μM ascorbate-2-phosphate, 10 nM dexamethasone and 1X ITS liquid supplement (10 µg/ml insulin, 5.5 µg/ml transferrin and 5 ng/ml selenite premix), and media changes were carried out every two days over the 21-day culture period. For each cell type, 2–5 pellets were generated using each of the above mentioned cell numbers. At the end of the culture period, three pellets (generated using 3 × 10⁵ HACs and STRO-1⁺ SSCs) were harvested for analysis of chondrogenic gene expression, while two pellets from each group were fixed in 4% paraformaldehyde (PFA) and used for histological examination.