Dmem hg media

Normal human articular chondrocytes (NHAC-kn, #CC-2250) were obtained from Lonza (Walkersville Inc).²⁷ (link) The cells were cultured in monolayer at 37 °C in a humidified atmosphere with 5% CO₂ in chondrocyte growth media consisting of DMEM/F12 supplemented with 10% FBS, 25ug/mL ascorbic acid and 1% penicillin/streptomycin solution. NHAC-kn cells (2.5 × 10⁵ cells) were sedimented (300 × g, 5 min) in polypropylene tubes to generated high-density pellets. Re-differentiation was induced by culturing the pellets in serum-free media consisting of DMEM-HG media (Gibco) supplemented with 1% ITS⁺ premix (containing human recombinant insulin, human transferrin, seleneous acid, BSA and linoleic acid) (BD Biosciences), 40 μg/mL l-proline, 1 mM sodium pyruvate, 1% non-essential amino acids, 2 mM Glutamax, 50 μg/mL ascorbic acid 2-phosphate, 10⁻⁷ M dexamethasone, penicillin/streptomycin and 100 ng/mL human recombinant BMP-2 (Medtronic).27 (link), 28 (link) Media and growth factor were replenished every other day for up to 21 days of differentiation.

MCF-7 cells (Shanghai, ChineseAcademy of Sciences Cell Repository) were cultured in DMEM/H-G media (USA, GIBCO) containing 10% fetal bovine serum (FBS) (GIBCO, USA). MCF-7/TAM cells (TRC, Canada) were cultured in complete DMEM/H-G containing 1.0 × 10⁻⁷ mol/L TAM for 6 months41 . After drug resistance was established, the cells were cultured in DMEM/H-G media containing 10% FBS. The MCF-7/TAM cells were treated with 10 μg/mL or 20 μg/mL Gefitinib (AstraZeneca, USA) for 48 h. The experiments consisted of the following groups: the MCF-7 group (M0), the MCF-7/TAM group (M/T), and the MCF-7/TAM-Gefitinib (10 μg/ml) group (G10). Cell culture was performed at 37°C and 5% CO₂, and the cells were passaged every 3–4 days.