Chondrocyte Isolation and 3D Agarose Culture

Articular cartilage was harvested from the stifle of adult dogs (N=4 joints) euthanatized for unrelated purposes. Briefly, chondrocytes were isolated via collagenase digestion and cultured in high glucose Dulbecco’s Modified Eagle’s Medium (hgDMEM; ThermoFisher) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals), 1 ng/ml transforming growth factor beta 1 (TGF-β1; ThermoFisher), 5 ng/ml fibroblast growth factor-2 (FGF-2; ThermoFisher), and 1% antibiotic/antimycotic (ThermoFisher) for two passages (P2), or approximately 8 population doublings (Alegre-Aguarón et al., 2014 (link); Ng et al., 2010a (link)). P2 cells were encapsulated in agarose (2% (w/v) Type VII Agarose, Sigma) to form cylindrical constructs with an initial composition of 30 × 10⁶ cells/ml (Ng et al., 2010a (link)). Constructs were cultured in serum-free chondrogenic medium consisting of hgDMEM supplemented with 1% insulin transferrin selenium (ITS+) premix (Sigma), 50 μg/ml L-proline (Sigma), 0.9 mM sodium pyruvate (Sigma), 10 ng/ml TGF-β3 (Life Technologies), 100 nM dexamethasone (Sigma), and 50 μg/ml ascorbic acid-2-phosphate (Sigma).