Dmem 10 fetal calf serum

Each subject (3 PCOS; 3 controls) underwent a SC fat biopsy whereby approximately 0.5–1 g of freshly isolated SC adipose was obtained through a 1 cm lower abdominal incision. SC adipose was washed with DMEM and digested at 37 °C in DMEM (Corning, Tewksbury, MA) containing 0.075% collagenase (Sigma-Aldrich, St Louis, MO) for 45–60 min on a shaker to release all cells present in the adipose tissue including adipocytes and the stromal-vascular fraction (SVF). Digested material was then filtered through a 150-μm mesh, washed with DMEM/10% fetal calf serum (FCS) (Corning, Tewksbury, MA), and centrifuged (200×g, 2 min) to separate adipocytes present in the top layer of the supernatant. Subsequent centrifugation (800×g, 10 min) led to obtaining a cell pellet containing the SVF which was then washed in DMEM/10% FCS and plated in 60 mm dishes containing DMEM/10% FCS, 0.05 U/ml penicillin, 0.05 mg/ml streptomycin, 1.25 mg/ml fungizone and cultured at 37 °C until confluency in 5% CO₂. First- and second-generation ASCs from each patient were frozen and kept in liquid nitrogen for use in subsequent ASC studies.