Ectopic endometrial cell isolation

To isolate cells from ectopic tissue, the protocol derived from Ryan et al. was used (Ryan et al., 1994). Endometriosis cultures were prepared from biopsies of ovarian endometrioma cyst linings. All biopsies were collected in the operating room under sterile conditions. The tissue was rinsed with phosphate-buffered saline (PBS), and cyst lining was dissected free from underlying parenchyma, minced and digested with collagenase 0.1% at 37°C in 5% CO₂ for 90 min. At the end of the incubation, cell clumps were mechanically disaggregated by aspiration through a Pasteur pipette and allowed to adhere selectively to tissue culture plates. Glands and stroma were not further separated. They were cultured as monolayers in 1 ml of Ham’s F-10 with 10% foetal calf serum, 2 mmol/l L-glutamine, antibiotics and 2.5 μg/ml fungizone in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. When the cell culture reached 60–80% confluence, cells were resuspended and fixed with 100% cold methanol.