Endogro vegf complete culture media

Human umbilical vein endothelial cells (HUVECs) were cultured in EndoGRO-VEGF Complete Culture Media (S. Rafii et al., 1994 (link)) (SCME 002-5, Millipore). HEK293T cells were cultured in DMEM full medium (C11995500, Gibico) with 10% FBS at 37°C in a humidified atmosphere of 5% CO₂.

To evaluate the formation of capillary-like structures, HMEC were seeded at 20,000 cells/well into 24-well plate precoated with growth factor-reduced Matrigel (Corning, Tewksbury, MA, USA). Cells were treated with DMEM 0% FBS as negative control for untreated cells (NT), DMEM 0% FBS with 50,000 EVs/cell for BMSC or ADSC-EVs (BMSC or ADSC-EV) and EndoGRO-VEGF Complete Culture Media (Millipore, St Louis, MO, USA) (CTR1+) or DMEM 0% FBS plus 0.1 µg/mL EGF (Sigma-Aldrich, St Louis, MO, USA) (CTR2+) as positive controls. After 24 h, cells were observed with a Nikon-inverted microscope (10×), and photographed with a Leica-digital camera (five photos for each well, in triplicate). Image analysis was performed automatically with the Angiogenesis Analyzer tool by ImageJ 1.49v software (NIH). Three experiments were performed in triplicate and results are expressed as ratio in comparison to the untreated cells (NT).