Isolation and Culture of Endothelial Progenitor Cells

4-week-old male Sprague-Dawley (SD) rats provided by the Centers for Disease Control of Hubei Province were anesthetized with 3% pentobarbital sodium at 30 mg/kg and then killed, isolated the tibia and femur of the rats aseptically, and rinsed their bone marrow cavities with PBS buffer repeatedly to collect fresh bone marrow. Mononuclear cells were isolated by density gradient centrifugation using the Ficoll-Paque solution (d = 1.077, Sigma, USA). Then, they were inoculated in a T25 culture flask with endothelial cells basal medium-2 (EBM-2, Lonza, Switzerland) and placed in a cell incubator with 5% CO₂ atmosphere at 37°C. Changing the media every 72 hours and the growth state of the cells were observed every day. In our previous experiments [27 (link)], we identified these cells by the property of uptaking acetylated LDL (ac-LDL) and Ulex europaeus agglutinin-1 (UEA-1) by EPCs, and cells cultured by this method have been identified as EPCs.