Background: Endometriosis is an estrogen-dependent, inflammatory gynaecological disease that affects about 10% of women of reproductive age. Literature suggests that lesion establishment and progression are driven not only by displacement of the viable endometriotic tissue but also by dynamic interactions with the surrounding microenvironment. While recent endometriosis models have provided valuable insights into epithelial cell biology, they often fail to recapitulate the complex stromal, immune and endothelial interactions that regulate inflammation, angiogenesis, proliferation and fibrosis within the microenvironment.
We aimed to establish a widely accessible and physiologically relevant in vitro 3D co-culture model using commercially available human cell lines to reconstruct the cellular complexity of the endometriotic microenvironment.
Methods: Human ectopic endometriotic 12Z epithelial cells were cultured with endometrial stromal cells, uterine endothelial cells and monocyte-derived macrophages to generate multicellular spheroids. The model was then validated using immunofluorescence with the specific cell-identification markers Pan cytokeratin, Vimentin, CD31, CD14, and CD206.
Results: Spheroid co-cultures formed reproducibly and retained distinct epithelial, stromal, endothelial and macrophage populations with 72.5% increase in spheroid area day6 to day12. CD206 expression indicated M2-polarised macrophage activation within the co-culture environment, while CD31-positive endothelial structures demonstrated pseudo-vascular network formation. We confirmed the expression of inflammatory markers including IL1B, IL8, NF-kB, and SDF-1, fibrotic and ECM-remodelling genes such as COL1A1, COL3A1, ACTA2, FN1, MMP2, and MMP9 in our co-culture model. Additionally, we confirmed the expression of angiogenic genes like ANGPT2 and VEGFR2, oxidative stress markers NOX4 and NOX5, as well as proliferative markers MAPK1, MAPK3, and PI3K.
Conclusions & significance: Our findings demonstrate the successful reconstruction of a multicellular endometriotic microenvironment in-vitro. This model provides a physiologically relevant platform for investigating disease mechanisms inducing pathways involved in macrophage activation and angiogenesis in endometriosis, identifying therapeutic targets, and accelerating translational research in endometriosis.