Protocol for generation of mouse and human endometrial assembloids

Interactions between the endometrial epithelium and stroma are fundamental to development, physiology, and diseases of the uterus. Current endometrial organoid methods frequently rely on basement membrane extracts (BME), which support epithelial organoid growth in the absence of physiological mesenchyme/stroma. These systems typically generate cystic epithelial structures with apical-in polarity, limiting access to the luminal surface and preventing direct contact with the stroma. To overcome these limitations, we developed a protocol for generating BME-free assembloids composed of endometrial epithelial and stromal cells. This protocol details enzymatic isolation of primary cells from neonatal and adult mouse uteri and human endometrial biopsies, expansion of primary cells, and assembloid formation using a rotational culture system. Quality control benchmarks, including formation efficiency and cell type composition, were incorporated to ensure reproducibility. The resulting assembloids self-organize into structures consisting of a stroma core surrounded by a polarized epithelial monolayer and provide a tractable platform for investigating stromal-epithelial crosstalk during uterine development and endometrial disease.