Arrangement into layers and mechanobiology of multi-cell co-culture models of the uterine wall

• Published in: Human reproduction (Oxford) 2024-08, Vol.39 (8), p.1767-1777
• Main Authors: Shlomo, Yael, Gavriel, Mark, Jaffa, Ariel J, Grisaru, Dan, Elad, David
• Format: Article
• Language: English
• Subjects: Actins - metabolism; Cell Line; Coculture Techniques; Endometrium - cytology; Endometrium - metabolism; Endometrium - physiology; Epithelial Cells - cytology; Epithelial Cells - metabolism; Epithelial Cells - physiology; Female; Humans; Myocytes, Smooth Muscle - metabolism; Myocytes, Smooth Muscle - physiology; Myometrium - cytology; Myometrium - metabolism; Myometrium - physiology; Stress, Mechanical; Stromal Cells - cytology; Stromal Cells - metabolism; Stromal Cells - physiology; Uterus - cytology; Uterus - metabolism; Uterus - physiology
• ISSN: 0268-1161; 1460-2350; 1460-2350
• DOI: 10.1093/humrep/deae130

Can a co-culture of three cell types mimic the in vivo layers of the uterine wall? Three protocols tested for co-culture of endometrial epithelial cells (EEC), endometrial stromal cells (ESC), and myometrial smooth muscle cells (MSMC) led to formation of the distinct layers that are characteristic of the structure of the uterine wall in vivo. We previously showed that a layer-by-layer co-culture of EEC and MSMC responded to peristaltic wall shear stresses (WSS) by increasing the polymerization of F-actin in both layers. Other studies showed that WSS induced significant cellular alterations in epithelial and endothelial cells. Human EEC and ESC cell lines and primary MSMC were co-cultured on a collagen-coated synthetic membrane in custom-designed wells. The co-culture model, created by seeding a mixture of all cells at once, was exposed to steady WSS of 0.5 dyne/cm2 for 10 and 30 min. The co-culture of the three different cells was seeded either layer-by-layer or as a mixture of all cells at once. Validation of the models was by specific immunofluorescence staining and confocal microscopy. Alterations of the cytoskeletal F-actin in response to WSS were analyzed from the 2-dimensional confocal images through the Z-stacks following a previously published algorithm. We generated three multi-cell in vitro models of the uterine wall with distinct layers of EEC, ESC, and MSMC that mimic the in vivo morphology. Exposure of the mixed seeding model to WSS induced increased polymerization of F-actin in all the three layers relative to the unexposed controls. Moreover, the increased polymerization of F-actin was higher (P-value