Cytotoxicity Evaluation of EDTA-Vanadium Conjugates

The glioma cell line CNS-1 (obtained from Mariano S. Viapiano [23 (link)]) was grown in 96 well plates in DMEM containing 10% fetal calf serum; 2 mM L-glutamine, penicillin (100 units/mL), and streptomycin (0.1 mg/mL) under humidified atmosphere containing 5% CO₂. Cells were seeded at 1000 cells/well. Twenty-four hours later, the EDTA and vanadium containing conjugates were added to each plate to give concentrations as indicated in the text. Control experiments using non-cancer cells were conducted with primary bovine brain pericytes and CD34+ human endothelial cells (both obtained and characterized at the Artois University, France [24 (link),25 (link),26 (link)]) treated with the HSA-EDTA-VO⁺⁺ conjugate. These cells were seeded at 15,000 cells/well in ECM medium (Sciencell, Carlsbad, CA, USA), which was composed as follows: 5% fetal calf serum (Gibco, Gaithersburg, MD, USA), ECGS supplements, and 50 mg/mL gentamicin (Biological industries, Beit-Haemek, Israel). Cells were treated the day after. Cell viability was measured after 72 h using a standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay as described before [27 (link)]. Experiments were repeated at least 3 times in quadruplicate. IC₅₀ values were calculated from the dose response curves using a median-effect plot.