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

Summary

The primary culture of human endometrial epithelial cells (HEECs) is technically challenging. This protocol combines mixed-enzyme digestion with ROCK inhibitor (Y-27632) to improve cell survival and expansion while maintaining epithelial identity, providing a robust platform for endometrial research and regenerative applications.

Abstract

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.

Introduction

Reliable in-vitro models are essential for advancing the study of endometrial disorders, particularly those associated with tamoxifen (TAM)¹. Although TAM is widely used as endocrine therapy for breast cancer, its partial estrogen-agonist activity in the endometrium markedly increases the incidence of endometrial hyperplasia, polyps, and, in some cases, malignant transformation². Current clinical management of tamoxifen-associated endometrial pathology relies mainly on progesterone therapy or surgical intervention, both of which have significant limitations. Progesterone treatment is frequently associated with a....

Protocol

Fresh endometrial tissue samples were obtained from uterine polyp specimens surgically resected from patients at the First Affiliated Hospital of Zhejiang Chinese Medical University. All procedures were conducted in accordance with the guidelines approved by the Medical Ethics Committee of the First Affiliated Hospital of Zhejiang Chinese Medical University (Protocol No. 2025-KLS-569-01; approved September 14, 2025). Handling of primary human samples was performed in a Class II biological safety cabinet using appropriate personal protective equipment, including laboratory coats, gloves, and eye protection. All enzymatic reagents were handled in accordance with institu....

Results

Successful establishment of the primary HEEC culture system was confirmed through morphological, proliferative, and molecular assessments, as outlined in Figure 1. By combining mixed-enzyme digestion, erythrocyte lysis, and a ROCK inhibitor, this protocol enabled efficient isolation and stable expansion of epithelial cells from human endometrial tissue.

Morphological observations demonstrated the essential role of Y-27632 during early culture. HEECs cultured with .......

Discussion

Establishing a reliable in vitro model of human endometrial epithelial cells (HEECs) is essential for investigating endometrial biology and pathology¹¹. Although several methods have been developed for HEEC isolation, most remain limited by technical complexity, low efficiency, and restricted expansion potential^4,5. Many protocols also rely on feeder layers, which introduce variability and hinder scalability¹²

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dispase	Solarbio	CAS:42613-33-2	For HEECs isolation
Collagenase Type I	Merck	SKU:SCR103	For HEECs isolation
100 µm mesh filter	Solarbio	431752	For HEECs filtration
rabbit anti CK18	abcam	ab133263	For immunofluorescence staining to check differentiation marker of HEECs
rabbit anti CDH1	abcam	ab40772	For immunofluorescence staining to check differentiation marker of HEECs
rabbit anti VIM	abcam	ab16700	For immunofluorescence staining to check differentiation marker of HEECs
4% paraformaldehyde	solarbio	P0099	For immunofluorescence staining to check differentiation marker of HEECs
CO2 Incubator	Thermo Scientific	42820906	For cell incubation
Centrifuge	Eppendorf	5404HN133048	Cell centrifuge
100mm Cell Culture Dish	Corning	430167	For cell culture
50 ml Centrifuge Tube	Corning	430829	For cell centrifugation
1.5 ml microcentrifuge Tubes	NEST	081722CK01	For cell digestion
Cell Strainer	Solarbio	431752	Cell filtration
Phosphate buffered solution	Tecono	20201033	Washing solution
DMEM	Gibco	8122622	Component of neutralization medium
Penicillin/Streptomycin	Thermo Scientific	15140-122	Antibiotics
Fetal Bovine Serum	Gibco	2556132P	Component of neutralization medium
0.05% Trypsin	Basalmedia	K431010	For HEECs  dissociation
Y-27632	Solarbio	IY0040	ROCK inhibitor
Red Blood Cell Lysis Buffer	Solarbio	R1010	Red blood cell lysis
1% Triton X-100	Thermo Scientific	HFH10	Membrane permeabilization
Donkey anti-Rabbit IgG	Thermo Scientific	A32790TR	Secondary antibody
DAPI	Thermo Scientific	62248	DNA staining
1 x PBST	Solarbio	P1031	Washing buffer
Dedicated Medium for Primary HEECs	iCell	iCell-f004-002h	For HEECs culture
Inverted Confocal Microscope	Zeiss	LSM880	Used for acquiring immunofluorescence images; equipped with 40x
MRC-5 cell line	iCell	iCell-h146	Human endometrial adenocarcinoma cell line; STR profiled.
Ishikawa cell line	iCell	iCell-h113	Human endometrial adenocarcinoma cell line; STR profiled.