4.5. MTT Viability Assay

HUVECs were seeded at 2 × 10⁵ cells/mL in a 24-well plate. CMs (1 mg/mL) and endothelial cell growth media with 5% FBS (as positive controls) were added to triplicate wells and media were replaced every 3 days. Cell growth of HUVECs was evaluated at day 1, 3, 5, and 7 using a 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St. Louis, MO, USA) assay. After several washes with PBS, cells were immersed in freshly prepared MTT reaction solution and incubated at 37 °C for 3 h in a CO₂ incubator. The absorbance of each solution was detected at 570 nm using a Tecan Sunrise spectrophotometer (Tecan, Switzerland). Experiments were repeated three times (technical repeats).