Ovarian Cancer Spheroid Formation

A2780, a human ovarian cancer cell line, was purchased from the American Type Culture Collection (USA). A2780 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO₂. A2780 ovarian cancer spheroids were isolated from A2780 cells as previously described (Choi et al., 2016 (link)). In brief, A2780 cells were seeded at Ultra-Low Attachment 6-well culture plates (Corning, USA) with a density of 2 × 10³ cells/10 cm², followed by culturing in a serum-free Neuro Basal Medium supplemented with B-27 Supplement, 10 ng/ml basic fibroblast growth factor, 20 ng/ml epidermal growth factor, 2.5 μg/ml amphotericin B, 100 U/ml penicillin, and 100 μg/ml streptomycin. To measure the spheroid-forming ability, the cells were seeded in 24-well Corning ultra-low-attachment plates (Corning) at a concentration of 1,000 cells/ml and cultured in spheroid culture media. After the suspension cells were cultured for 20 days, the number of spheres was counted using a microscope.