Isolation and Culture of Human Endometrial Stromal Cells

HESCs were obtained at the time of hysterectomy for uterine fibroids from normally cycling premenopausal women. Patients were not undergoing hormonal treatment at the time of surgery. All the samples were collected during the proliferative phase of the cycle. HESCs were isolated and cultured as previously described30 (link)31 (link)32 (link). Briefly, endometrial samples were collected in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 containing Antibiotic-Antimycotic solution (Invitrogen). After enzymatic digestion by DNase I (Sigma) and Collagenase I a (Sigma), the stromal cells were separated from epithelial cells and passed into culture. Proliferating HESCs were cultured in maintenance medium of DMEM/F-12 containing 10% dextran-coated charcoal-treated FBS and 1% antibiotic–antimycotic solution (Life technologies). Confluent monolayers of HESCs were treated with or without 0.5 mM 8-bromo-cyclic adenosine monophosphate (8-br-cAMP; Sigma) and 10⁻⁶ M medroxyprogesterone acetate (MPA; Sigma). All experiments were conducted before the third passage of the cultures.