Human fibroblast growth factor 2

The human ovarian cancer cell lines SKOV3 and A2780 were obtained from the Cell Bank of the Shanghai Institute of Biochemistry & Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai, China (http://www.cellbank.org.cn), and were cultured in RPMI-1640 medium supplemented with 10 % fetal bovine serum (Gibco, Grand Island, NY, USA). All cell lines were maintained in a humidified atmosphere at 37 °C with 5 % CO₂. For tumor spheres culture in vitro, cells were plated in ultralow attachment six-well plates (Corning Inc., Corning, NY, USA) at a density of 10,000 cells per well in serum-free endothelial basal medium-2 (Lonza, Basel, Switzerland) with 10 ng/ml leukemia inhibitory factor (Sigma. St. Louis, MO, USA), 20 ng/ml human fibroblast growth factor-2 (Sigma. St. Louis, MO, USA), or 20 ng/ml epidermal growth factor (Gibco, Grand Island, NY, USA). Sphere cultures were passaged every 7–10 days. To passage spheres, media were removed and spheres were incubated at room temperature for 5 minutes in 0.05 % trypsin (Gibco, Grand Island, NY, USA). Spheres were observed under the microscope to verify dissociation. Cells were then washed with Hanks’ buffered salt solution (HBSS; Gibco) and filtered through a 40 nm strainer before replanting.