6 well ultra low attachment culture plates

Human EOC cell lines SKOV3 and HO8910 were obtained from the China Center for Type Culture Collection (CCTCC, Wuhan University, China). Both cell lines were maintained in DMEM/F12 medium supplemented with 10% (v/v) fetal bovine serum. Adherent cells were maintained at 37°C in 5% CO₂ and detached using trypsin/ethylenediaminetetraacetic acid (EDTA) solution. Spheroids were generated from both SKOV3 and HO8910 cells after plating at a density of 500 cells/mL into ultra‐low attachment 6‐well culture plates (Corning, NY, USA). Spontaneously generated spheroids were cultured in a serum‐free DMEM/F12 medium supplemented with 2% B‐27 Supplement without vitamin A (Invitrogen, Carlsbad, CA, USA), 20 ng/mL basic fibroblast growth factor (FGF, Peprotech, Rocky Hill, NJ, USA), 20 ng/mL epidermal growth factor (EGF, Peprotech), 10 ng/mL leukemia inhibitory factor (LIF, Peprotech) and insulin‐transferrin‐selenium (ITS, Invitrogen). Fresh medium was added every 3 days, and spheroids were cultured for approximately 2 weeks before they reached a diameter of approximately 150 μm. The spheres were collected by gentle centrifugation, then dissociated with accutase (Invitrogen) and mechanically disrupted with a pipette. The resulting single cell suspension was then centrifuged and re‐suspended in serum‐free medium to allow for the re‐forming of spheres.

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.