A Simplified and Robust Protocol for the Isolation and Long-term Expansion of Primary Human Endometrial Epithelial Cells

Research on endometrial disorders, including tamoxifen-associated endometrial hyperplasia, has been limited by the lack of robust in vitro epithelial models. Conventional methods for isolating human endometrial epithelial cells (HEECs) are often inefficient, technically demanding, and poorly scalable. Here, a simplified, feeder-free protocol is described that integrates mixed-enzyme digestion, erythrocyte lysis, and sustained ROCK inhibitor (Y-27632) treatment to enable efficient isolation and expansion of primary HEECs. The inclusion of Y-27632 is critical for mitigating dissociation-induced apoptosis and enhancing early-stage cell adhesion. This approach supports serial passaging while maintaining epithelial identity for at least 3 passages, with morphology preserved beyond 10 passages. Characterization by Western blot and immunofluorescence confirms high expression of Cytokeratin 18 and E-cadherin, with a progressive reduction in vimentin-positive stromal contaminants under selective culture conditions. By addressing key technical limitations, this method provides a reliable, scalable source of epithelial cells for mechanistic studies, disease modeling, drug screening, and regenerative applications in endometrial research. The standardized workflow offers an alternative to complex co-culture systems, facilitating broader access to high-quality primary cell models.