2.2. Preparation of Concentrated HUVEC-CM and Analysis of Cytokine Expression

HUVECs were cultured to passage 3. Upon the cells reaching approximately 80% to 90% confluence, serum-free cell culture medium was added. After 24 hours, the supernatant of the HUVEC culture was aspirated and centrifuged for 5 minutes at 1000 r/min; the supernatant was then collected and frozen at -20°C. The frozen endothelial cell supernatant was placed in a vacuum freeze-drying centrifuge and freeze-dried for 20 hours to obtain a frozen sample of 10-fold concentrated supernatant. Cytokines are released by HUVECs. The levels of PDGF, bFGF, EGF, and VEGF were measured using ELISA kits (R&D Systems, Minneapolis, MN) [12].