Dulbecco s modified eagle medium dmem f12 medium

In this study, we used nontumorigenic immortalized human granulosa cells (SVOG cells) previously produced by transfection of the simian virus 40 large T antigen into early luteal phase human granulosa cells obtained from women undergoing in vitro fertilization (IVF) [31 (link)]. These cells have been proven to display biological responses to a number of various treatments that are similar to the responses of human granulosa cells under physiological conditions [11 (link),32 (link),33 (link),34 (link),35 (link)]. The SVOG cells were seeded and cultured in Dulbecco’s modified Eagle medium (DMEM)/F12 medium (Sigma, Oakville, ON, Canada) supplemented with 10% charcoal/dextran-treated fetal bovine serum (FBS; HyClone Laboratories, Logan, UT, USA), 1× GlutaMAX (Thermo Fisher Scientific/Invitrogen), 100 mg/mL streptomycin sulfate (Thermo Fisher Scientific/Invitrogen), and 100 U/mL penicillin (Thermo Fisher Scientific/Invitrogen). The cells were maintained at humidified atmosphere with 5% CO₂ at 37 °C. The medium was changed every other day, and the cells were maintained in serum-free medium for 24 h before treatment.

hESCs were used according to the guidelines provided by the ethical committee of Kyoto University (Approved# ES3-9). WA09 (H9) (RRID:CVCL_9773, hPSCreg Name WAe009-A) hESCs used in this study were purchased from WiCell Research Institute (Madison, WI, USA). Before culturing, hESC-certified Matrigel (Corning, Corning, NY, USA) was diluted with Dulbecco’s modified Eagle medium (DMEM)/F12 medium (Sigma-Aldrich, St. Louis, MO, USA) at a 1:75 (v/v) ratio and coated onto a culture dish. The Matrigel was incubated in the culture dish for 24 h at 4 °C. Then, excess Matrigel was removed, and the coated dish was washed with fresh DMEM/F12 medium. We used mTeSR-1-defined medium (Stem Cell Technologies, Vancouver, Canada) for daily culturing of hPSCs. For passaging, cells were dissociated with TryPLE Express (Thermo Fisher Scientific, Tokyo, Japan) for 3 min at 37 °C and then harvested. A cell strainer was used to remove undesired cell aggregates from the cell suspension, and cells were then centrifuged at 200×g for 3 min and resuspended in mTeSR-1 medium. Live/dead cells were counted using a NucleoCounter NC-200 (Chemetec, Baton Rouge, LA, USA). We used mTeSR-1 medium containing 10 µM of the ROCK inhibitor Y-27632 (Wako, Osaka, Japan) to prevent apoptosis of dissociated hPSCs on day 1. On subsequent days, we used mTeSR-1 medium without the ROCK inhibitor with daily medium changes.