Fibronectin collagen

The primary tissue samples and OESC and CESC cells analyzed in this study are in S1 Table. Cryopreserved endometrial tissue obtained by diagnostic laparoscopy was minced and incubated with collagenase (Sigma-Aldrich, St. Louis, MO) at 37°C for 10 min., followed by filtration, as previously described [22 (link)]. This method produces 95–99% pure stromal cells. The purity of these stromal cells at passage one was evaluated by immunofluorescence analysis using antibodies against vimentin (stromal cell marker), cytokeratin7 (epithelial cell marker) and CD45 (leukocyte marker), which showed that all cultures were 98–99% pure stromal mesenchymal cells. The cells were then cultured as previously described [23 (link)]. Briefly, the cells were cultured on fibronectin-collagen (Gibco, Grand Island, NY) coated dishes in DMEM-F12 without phenol red (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 2 mM L-glutamine (Gibco) and 1% antibiotics–antimycotic (Gibco) up to passage 3. To exclude influences of the serum derived steroid hormones on DNA methylation and expression in the cultures, the cells were grown in culture medium containing 10% charcoal stripped fetal bovine serum (CS-FBS; Gibco).

Endometrial tissue obtained from control patients by curettage or from ectopic lesions from endometriosis patients by laparoscopic surgery was used to make primary endometrial stromal cells as previously described [57 (link)]. Briefly, the tissue was minced and incubated with collagenase (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 10 min, filtered and then cultured further, as previously documented [58 (link)]. The cells were cultured on fibronectin–collagen-(Gibco, Grand Island, NY, USA) coated dishes in DMEM-F12 without phenol red (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 2 mM L-glutamine (Gibco) and 1% antibiotics–antimycotic (Gibco) up until a maximum of passage 7 in a 37 °C CO₂-humified incubator. The purity of these stromal cells was evaluated using immunofluorescence analysis with antibodies against vimentin (stromal cell marker), CD10 (endometrial stromal cell marker) and by qPCR using PECAM1 (endothelial marker) and EPCAM (epithelial marker). This method produces 95–99% pure stromal cells.