Three-Dimensional Organoid-like Co-Culture of Human Endometrial Endothelial and Stromal Cells to Study Endometriosis-Associated Responses
Caroline Borgato Guedes, Aline R. Lorenzon, Alexandre U. Borbely, Simone Correa-Silva, Elaine C. Cardoso, Barbara Stefany S. Souza, Elisa Lie Matsumura, Tatiana C. de Souza Bonetti, Thais Sanches Domingues, Selma F. Moreira Tsuji, Beatriz Passaro Biscaro, Renata Fioravanti Schaal, Ana Paula Aquino, Eduardo Leme Alves da Motta, Vanessa Morais Freitas, Lidia Hyung Joo Myung, Mauricio S. Abrao, Estela BevilacquaThree-dimensional (3D) endothelium–stromal co-cultures were established using human endometrial cells from biopsy of healthy women (n = 13) and serum samples from both healthy and endometriotic women (n = 5). For 3D construction, stromal cells were mixed with extracellular matrix components, followed by endothelial cell seeding. Morphological analysis confirmed the organization of tissue-like structures. Immunofluorescence and flow cytometry verified the expression of specific stromal and endothelial markers (Cytokeratin, Vimentin, Insulin-like growth factor-binding protein 1, and von Willebrand factor). Cell viability and proliferation increased over time, with minimal cell death. To test functional responsiveness, these co-cultures were exposed to inflammatory serum from endometriotic patients. After 48 h, cytometric bead array showed elevated levels of IL-1β, IL-6, and IL-8 in cultures treated with inflammatory serum, indicating preserved functional activity and responsiveness. By allowing detailed investigation of functional endometrial states within a physiologically relevant cellular network, this approach provides a valuable organoid-like tool to explore conditions such as implantation failure and infertility and to study the cellular interactions underlying reproductive pathologies.