Characterization of human endometrial cells in a 3D system: the model and its study possibilities

Author(s)

Bárbara Stefany da Silva Souza

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2024-01-19

Examining board members

Estela Maris Andrade Forell Bevilacqua; José Maria Soares Júnior; Glaucia Maria Machado Santelli; Sandra Maria Miraglia Valdeolivas

• Advisor: Estela Maris Andrade Forell Bevilacqua

Abstract

Introduction: The in vitro modeling of a three-dimensional (3D) coculture system, using human endometrial cells and eventually embryonic cells, is an innovative area that can increase knowledge of endometrial physiology and embryo implantation. Objectives: Characterize a 3D study model with endometrial cells that allows studies of uterine physiology. Material and methods: Endometrial cells were isolated from uterine biopsies (n=3) collected according to human research ethics protocols (IC-USP, CONEP). Biopsies were digested with collagenase II/DNAse I and filtered to retain trypsinized endometrial glands to obtain isolated epithelial cells. Anti-CD105 magnetic beads (MACS) were used for the positive and negative selection of endothelial and stromal cells, respectively. The purity of uterine samples was assessed by immunofluorescence/immunohistochemistry for the following proteins: vimentin, cytokeratin, glycodelin, IGFBP1 and CD105/VEGFR2. The 3D coculture system included the preparation of an extracellular matrix composed of a mixture of fibronectin, hyaluronic acid, collagen V, collagen I and collagen III in a Transwell system containing stromal cells, onto which endothelial and epithelial cells were plated. As a preliminary test, trophectoderm from aneuploid embryos (n=3) were collected and inserted into endometrial cells cultured in a 3D system, where they remained for 48 hours and were then processed for histological analysis. The support matrix\'s isolation, cultivation and preparation were standardized, and the cultures were characterized. Results: The reconstituted endometrium was characterized morphologically by H&E staining and immunohistochemistry, showing a typical organization in which vimentin-positive cells remained viable and embedded in the extracellular matrix. Endothelial and epithelial cells deposited on this matrix organized themselves as a monolayer. Only one embryonic fragment made up of trophectoderm showed adherence to endometrial cells. Conclusion: The results show a coculture system with viable endothelial, epithelial and stromal cells, with characteristics similar to those observed in vivo, regarding their tissue organization and the expression of typical biomarkers. The adhesion of the trophectoderm to this layer suggests that mechanisms associated with embryonic implantation can be studied in this model. (AU)

• FAPESP's process: 20/14982-3 - Trophoblast-endothelial cells co-culture in a 3D system mimicking the embryo implantation to study the trophoblast's role in inducing apoptosis
• Grantee: Bárbara Stefany da Silva Souza
• Support Opportunities: Scholarships in Brazil - Master