Protocol for generation of mouse and human endometrial assembloids

Area of Science:

• Reproductive biology
• Developmental biology
• Cell biology

Background:

• Endometrial epithelial and stromal cell interactions are crucial for uterine function and disease.
• Existing organoid models using basement membrane extracts (BME) create cystic structures, limiting stromal contact and luminal access.
• These limitations hinder the study of physiological endometrial crosstalk.

Purpose of the Study:

• To develop a BME-free protocol for generating endometrial assembloids.
• To create a model system that facilitates direct investigation of epithelial-stromal interactions in the uterus.
• To enable research into uterine development and endometrial pathologies.

Main Methods:

• Enzymatic isolation of primary endometrial epithelial and stromal cells from mouse and human tissues.
• Expansion of isolated primary cells.
• Formation of assembloids using a rotational culture system without BME.
• Incorporation of quality control benchmarks for reproducibility.

Main Results:

• Successful generation of BME-free endometrial assembloids.
• Assembloids self-organize into a stromal core surrounded by a polarized epithelial monolayer.
• The model allows for direct investigation of stromal-epithelial crosstalk.
• Established quality control metrics ensure reliable and reproducible results.

Conclusions:

• The developed protocol provides a BME-free platform for creating functional endometrial assembloids.
• This novel model system overcomes limitations of previous organoid methods.
• It offers a tractable approach for studying uterine stromal-epithelial crosstalk in development and disease.