Culture of primary endometrial stromal cell and Ishikawa cell lines

Endometrium was finely minced and cells were dispersed by incubation in Hanks’ balanced salt solution containing HEPES (25mm), 1% penicillin/streptomycin, collagenase (1 mg/ml, 15 U/mg), and deoxyribonuclease (0.1 mg/ml, 1500 U/mg) for 60 min at 37°C with agitation and pipetting. The cells were pelleted; washed; suspended in Ham’s F12:DMEM (1:1) containing 10% fetal bovine serum, 1%penicillin/streptomycin, and 1% amphotericin B; passed through a 40-m cell strainer (Falcon, Franklin Lakes, NJ, USA); and plated into 75 cm² Falcon tissue culture flasks (BD Biosciences, Franklin Lakes, NJ). Cultured HESC at 3–5 passages were used for further analysis. Ishikawa cells were maintained in MEM (Invitrogen, Carlsbad, CA) containing 2.0 mM l-glutamine and Earl’s salts, supplemented with 10% fetal bovine serum (FBS), 1% sodium pyruvate, and 1% penicillin/streptomycin.