25 cm3 culture flasks

The GCs, suspended in follicular fluid, were washed twice by centrifugation at 200 × g for 10 min at RT. Medium consisted of Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich Co.; Merck KGaA), 2% fetal bovine serum FBS (FBS; Sigma-Aldrich; Merck KGaA), 4 mM L-glutamine (Invitrogen; Thermo Fisher Scientific, Inc.), 10 mg/ml gentamycin (Invitrogen; Thermo Fisher Scientific, Inc.), 10,000 U/ml penicillin, and 10,000 µg/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were cultivated at 37°C under aerobic conditions (5% CO₂) in 25 cm³ culture flasks (Corning Inc., Corning, NY, USA). The cells were passaged upon reaching 90% confluence; they were detached with 0.05% trypsin-EDTA (Invitrogen; Thermo Fisher Scientific, Inc.) for 1–2 min and counted using an ADAM Cell Counter and Viability Analyzer (Bulldog Bio). After counting, the cells were seeded onto a number of flasks appropriate for total cell number (2–3×10⁶ cells per 25 cm³ flask). GCs were then cultivated for 30 days, the morphology was checked daily and photographed using an Olympus inverted microscope (Olympus). The medium was changed twice a week. Finally, total RNA was isolated from GCs after 1, 7, 15 and 30 days. The viability of each collected sample was tested using the ADAM CCVA, with only samples containing 95% or more viable cells used for subsequent molecular analyses.