2.5.1. Cell Culture and Treatment

Human chondrocyte cells line C20A4 was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). It was maintained at 37 ◦C in a humidified atmosphere containing 5% CO2 in Dulbecco's modified Eagle's Medium/Nutrient Mixture F-12 (DMEM/F12) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, 50 U/mL penicillin, 50 mg/mL streptomycin, 50 μg/mL ascorbic acid, and 50 μM α-tocopherol (Euroclone, Milan, Italy). Cells were tested for contamination, including *Mycoplasma*, and used within 2–4 months. All experiments were performed with an 80% confluent monolayer. The protective effects of Hyt were studied with the acute toxicity model by pre-treating cells with Hyt@tgel for 24 and 96 h followed by 24 h H2O2 (230 μM) [38] treatment in the absence of hydroxytyrosol. A shorter exposure (4 h) was used to investigate effects on mRNA expression.
