P-351 Optimisation of an in vitro patient-derived multicellular model to recapitulate superficial endometriotic lesion formation

Abstract Study question Can an in vitro patient-derived multicellular model of superficial endometriosis be generated to support studies exploring associated fibrosis? Summary answer The three-dimensional (3D) co-culture of primary peritoneal cells and endometrial spheroids is a promising mimic of superficial endometriosis to support studies exploring early endometriosis formation. What is known already Peritoneal endometriosis causes adhesions and scars that can interfere with ovulation and conception, resulting in infertility. However, the underlying mechanisms that drive endometriosis-associated scar formation are not fully understood. Endometriotic lesions are proposed to arise from menstrual debris attachment to the peritoneum during retrograde menstruation, creating a new endometrial-peritoneal microenvironment. However, it is difficult to explore the process of early endometriosis formation in situ from patient biopsies due to the typically late diagnosis of endometriosis. A 3D co-culture approach provides unique opportunities to explore endometriosis lesion initiation and progression. Study design, size, duration For primary cell isolation, peritoneal washes, fallopian tube mesentery and endometrial tissue biopsies were collected over a two-year period from 10 patients undergoing laparoscopic surgery. Primary peritoneal mesothelial cells and fibroblast were co-cultured for 3 days to model the peritoneum before incorporation of an endometrium-derived construct to mimic superficial lesion formation. The superficial endometriosis model was co-cultured for a maximum of 10-days. Participants/materials, setting, methods A 3D peritoneal model was assembled by embedding human peritoneal fibroblasts (HPFs) in a bipartite Matrigel-collagen I matrix (2,000 cells/µl) before seeding human peritoneal mesothelial cells (HPMCs) (125 cells/µl). Following endometrial digestion, endometrial epithelial organoids (EEOs) were expanded in Matrigel, then co-cultured with endometrial stromal cells (ESCc) in rat-tail collagen I (4,000 cells/µl) to form endometrial spheroids. The 3D peritoneal model and endometrial spheroid were co-cultured and were characterised using immunostaining and ELISA measurements. Main results and the role of chance HPMCs were successfully isolated from peritoneal washes and exhibited a polygonal morphology up to passage 5, expressing dual positivity for pan-cytokeratin (pCK) and vimentin with 98.2% purity (n = 6). HPFs isolated from fallopian tube mesentery explants grew in a spindle shape appearance up to passage 4, with localisation of vimentin and lack of pCK that supported fibroblast characteristics and showed 81.4% purity (n = 3). Optimisation of hydrogel matrices demonstrated that a Matrigel-collagen I matrix at 1:1 ratio (v/v) supported the establishment of a 3D peritoneal in vitro model with minimal contraction. Histological investigation confirmed successful establishment of a simple peritoneum structure comprising a mesothelial monolayer with basal secretion of a collagen IV basement membrane and underlying fibroblasts. Secretion of tissue plasminogen activator (tPA) following 3- to 10-day culture demonstrated physiological functioning of the mesothelial element. Co-culture of EEOs and ESCs formed a compact endometrial spheroid comparable to in vivo endometrial tissue architecture. Endometrial spheroids adhered to the peritoneal 3D model, thus mimicking early endometriotic lesion formation. Histological analysis demonstrated direct cell-cell contacts between HPMC, HPF and ESC at the endometrial-peritoneal interface, suggesting the involvement of those cell types in lesion initiation and early endometriosis scar formation. Limitations, reasons for caution Characterisation of HPMC and HPF was performed using relatively few donors. Other important cell types present in endometriotic lesions, such as endothelial and immune cells, have not yet been incorporated into the model. Wider implications of the findings Our modifiable 3D endometriosis model enables further studies to explore the involvement of other cells and substances, such as the impact of immune cells and ovarian hormones, on superficial endometriosis initiation. This platform shows promising features to explore the underlying molecular mechanism involved in endometriotic lesion formation and scarring. Trial registration number No