Endothelial basal medium

UtMECs and EECs were isolated and characterized as previously described by Bulla et al. [17 (link)]. Both ECs were positively selected with Dynabeads M-450 (Life Technologies, Milan, Italy) coated with Ulex europaeus 1 lectin (Sigma-Aldrich), seeded on 12,5 cm² flask precoated with 2 μg/cm² fibronectin (Roche, Milan, Italy), and maintained in serum-free endothelial basal medium (Life Technologies, Monza, Italy) supplemented with 20 ng/mL bFGF (basic Fibroblast Growth Factor), 10 ng/mL EGF (Epidermal Growth Factor), 10% FCS (all from Life Technologies), and 10% human serum and incubated at 37°C, 5% CO₂. The purity of the resulting EC populations was more than 98% as verified by staining with antibodies to VWF, CD105, VE cadherin (Dako, Milano, Italy), and CD31/PECAM-1 kindly provided by M. R. Zocchi (San Raffaele Hospital, Milan, Italy).

Primary endometriotic cells were isolated from EM patient lesions. Briefly, tissues were digested overnight (ON) at 4°C with 0.25% trypsin (Sigma-Aldrich), 50 μg/mL DNase 1 (Roche, Milan, Italy) in PBS and then treated with collagenase type I (3 mg/mL; Worthington Biochemical) for 30 min at 37°C. As described earlier (24 (link)), endothelial cells (EECs) were positively selected with Dynabeads M-450 (Life Technologies, Milan, Italy) coated with Ulex europaeus 1 lectin (Sigma-Aldrich), seeded on 12,5 cm² flask precoated with 2 µg/cm² fibronectin (Roche). Cells were maintained in serum-free endothelial basal medium (Life Technologies, Monza, Italy), supplemented with 20 ng/mL bFGF (basic Fibroblast Growth Factor), 10 ng/mL EGF (Epidermal Growth Factor), 10% v/v FBS (all from Life Technologies), and 10% v/v heat inactivated human serum and incubated at 37°C, 5% CO₂. Endometriotic epithelial/stromal cells (EM cells), obtained via the negative selection, were cultured in the same medium containing only 10% FBS.
Moreover, uterine microvascular endothelial cells (UtMECs) were isolated from normal uterus of healthy women, following the same procedure.