Development of three-dimensional (3D) engineered tissue model for in vitro study of human endometriosis.

DEL VALLE S; RUIZ I; OPPENHEIMER F; LEIROS G; RICCI A; MERESMAN G

Congreso; LXVIII reunión anual de la sociedad argentina de investigación clínica (SAIC),; 2023

EDT is a gynecological disease characterized by the presence ofendometrial tissue outside the uterine cavity affecting 10-15% ofmenstruating people. The available in vitro models for studying EDTare insufficient to clarify the complex epithelium-stroma interactionswithin tissues. Therefore, our aim was to develop and characterize3D endometrial constructs that could be used as experimental modelsfor studying this disease. We generated 3D constructs using typeI collagen (5 mg/ml) as extracellular matrix, and genipin (120 μM), across-linking reagent. First, 1x106 t-HESC endometrial stromal cellswere embedded in the hydrogel, then 5x105 ECC-1 endometrial epithelialcells were seeded onto the collagen scaffold. They were incubatedfor 9 days at 37 °C with 5% CO2 in DMEM-F12 medium. Weexamined how the construct’s percentage of contraction changeddepending on whether genipin was present or not, as well as howtoxic it was using the LIVE/DEAD Viability/Cytotoxicity kit. We alsoperformed histological analysis by using Periodic Acid-Schiff (PAS)and hematoxylin-eosin (H&E) staining and characterized the cellularorganization by immunodetection of epithelial and stromal cells withcytokeratin and vimentin, respectively. 3D constructs without genipinformed glandular structures positive for cytokeratin which sharedmorphological similarities with endometrial tissue. These structureswere also positive for PAS staining, suggesting active secretory activity.The stromal phenotype was confirmed with positive immunodetectionfor vimentin. Genipin did not cause cytotoxicity since cellviability was equivalent to basal (95%). Furthermore, it significantlyreduced collagen contraction improving the mechanical handling ofthe constructs. H&E staining revealed the formation of a partiallycolumnar continuous epithelium over the stroma. This innovativemodel will allow us to study the complex interactions of different celltypes within a relevant biological microenvironment.