2.1. Cell Culture

Epithelial ovarian cancer cell line A2780 and A2780-SP cells were gifted by Jae Ho Kim (Pusan National University, Republic of Korea). A2780 cells were cultured in RPMI-1640 medium (Hyclone, Logan, UT, USA) supplemented with 10% FBS (Hyclone, Logan, UT, USA) and 1% penicillin/streptomycin (Hyclone, Logan, UT, USA). Cells were detached using trypsin/EDTA solution (Hyclone, Logan, UT, USA). SKOV3 cells (ATCC, Manassas, VA, USA) were cultured in McCoy’s 5A medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and 1% penicillin/streptomycin (Hyclone, Logan, UT, USA). OVCAR3 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). OVCAR3 cells were cultured in RPMI-1640 medium (Gibco, Gaithersburg, USA) supplemented with 20% fetal bovine serum (Hyclone, Logan, UT, USA), 1% penicillin/streptomycin (Hyclone, Logan, UT, USA), and 0.01 mg/ml bovine insulin (Sigma-Aldrich, St Louis, MO, USA). A2780-SP, SKOV3-SP, OVCAR3-SP, and FACS sorted (ALDH⁺ or CD133⁺ or CD117⁺) cells were cultured in complete medium (CM) composed of Neurobasal medium (NBM, Gibco, Gaithersburg, MD, USA) supplemented with B27 (Gibco, Gaithersburg, MD, USA), HEPES (Sigma-Aldrich, St Louis, MO, USA), Glutamax (Gibco, Gaithersburg, MD, USA), 2.5 µg/mL amphotericin B (Gibco, Gaithersburg, MD, USA), 10 ng/mL basic fibroblast growth factor (bFGF) (R&D system, Minneapolis, MN, USA), 20 ng/mL human epidermal growth factor (hEGF) (R&D system, Minneapolis, MN, USA) in Ultra-Low Attachment 100 mm² plate (Corning, NY, USA). Complete medium was changed every 2 to 3 days. Spheres were dissociated into single cells by treatment with Accutase (Gibco, Gaithersburg, MD, USA).