Ecm culture medium

Both hBMSCs and HUVECs were purchased from Sciencell (Carlsbad, CA, United States). The cells were seeded at 5,000 cells/cm² in culture bottles treated with bovine plasma fibronectin (Sciencell). ECM culture medium (Sciencell) containing 5% fetal bovine serum (Gibco, Grand Island, NY, United States), 1% penicillin mixture, and 1% endothelial cell growth factor (Sciencell) was used for HUVECs. hBMSCs were cultured in mesenchymal stem cell medium (Sciencell) containing 5% fetal bovine serum, 1% penicillin mixture, and 1% mesenchymal stem cell growth factor. The cells were placed in a constant temperature incubator with 5% CO₂ at 37°C to subculture. The third to fifth passages of cells were used in the experiments.

Human umbilical vein endothelial cells (HUVECs) were trypsinized and resuspended in ECM culture medium (Sciencell, USA) containing 0.6 gr/L methylcellulose (Sigma). Cells were seeded (400 cells per well in 100 µl) in U-bottom 96 well plates and cultured for 24 h at 37 °C and 5% CO₂ to allow for the formation of spheroids. The spheroids were collected and resuspended in FBS (Sciencell) containing 2.4 gr/L methylcellulose and mixed 1:1 with collagen I solution containing 3.77 g/L collagen I (Corning, USA), 10% M199 medium (Sigma), 0.018 M HEPES, and 0.2 M NaOH to adjust pH to 7.4. The mixture with the spheroids was allowed to polymerize for 30 min in a 24 well plate. A total of 100 µl of conditioned medium with or without VEGF (50 ng/mL) (Preprotech) was added to the gels. Spheroids were visualized after 24 h using an Olympus IX50 microscope. The cumulative sprout length per spheroid was measured using ImageJ software.