2.10. Treatment of Human Chondrocytes with SBOs-Derived Exosomes

Macroscopically healthy cartilage was collected and washed with PBS for three times. After that, cartilage tissues were digested in 1 g/L of Type II Collagenase (Thermo Fisher) in DMEM to isolate ACCs for 6–8 h at 37 °C. After centrifugation (1200 rpm, 10 min), the supernatant was removed, and ACCs were cultured with complete culture medium at 37 °C with 5% CO₂. Cells were grown to confluence before they were used for 3-dimensional pellet cultures in accordance with our published protocols (Figure 2A) [6]. A total of 20 µg/mL of exosomes were cocultured with 3-dimensional pellet and the chondrogenic culture medium (DMEM, High (4.5 g/L) Glucose (DMEM-HG, Invitrogen) supplemented with 1% Inulin-transferrin-selenium (Sigma), 1.25 mg/mL Bovine serum albumin (Sigma), 0.1 µM dexamethasone (Sigma), 0.1 mM ascorbic acid (Sigma), 1% PS (Gibco, Thermo Fisher), 10mM HEPES (Sigma), 0.1 mM L-proline (Sigma), 0.1 mM MEM Nonessential Amino Acids (Gibco, Thermo Fisher) and 10 ng/mL transforming growth factor-beta 1 (TGF-β1, Gibco, Thermo Fisher)) was changed twice a week. After coculturing for 14days, total RNA was extracted for gene expression test according to the protocol we published and some group of pellets from each group were embedded by paraffin for further staining [6].