In vitro characterization of a novel primary ovarian endometriosis epithelial cell line, EEC16. (A) EEC16 cells have a mesenchymal-type epithelial morphology in vitro at (i) low cell density and (ii) high seeding density. (B) Western blot analysis of marker expression. The EEC16 line expressed cytokeratin and vimentin. EEC16 does not express ERα, E-, P-, or N-Cadherin. Beta-actin was used as a loading control. Control lysates used were breast and ovarian cancer cell lines: MCF7, for cytokeratin; T47D, for ERα and P-Cadherin; MDA-MB-231 for vimentin; BT549 for N-Cadherin; and IGROV for E-Cadherin. The difference in ovarian surface epithelial cell (OSEC181) and EEC16 profiles indicates that the EEC16 line is unlikely to be contaminated with normal OSECs. (C) Real-time PCR analysis of primary endometrioma and endometrial tissues, CDH1 is downregulated in endometrioma tissues compared to eutopic endometrium. Expression of CDH1 in endometrium of women without endometriosis is independent on the stage of the menstrual cycle (Additional file 2: Figure S1). (D) The EEC16 line has a normal, female karyotype. (E) Growth curves. The EEC16 and OSEC10 lines have a finite in vitro lifespan. In comparison, the SV40T-expressing EEC12Z endometriosis line, which has spontaneously acquired the ability to evade replicative crisis, did not show any signs of senescence after extended time in culture . (F) In anchorage-independent growth assays the EEC12Z line forms significantly more colonies than EEC16. EEC16 formed colonies in agar up until passage 11. OSEC10 fails to form colonies in soft agar. EEC16 cells are significantly more (G) migratory and (H) invasive than OSEC10. Each assay was performed three times. * P > 0.05, two-tailed unpaired T-test.