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 adverse effects such as breakthrough bleeding and weight gain, while surgical approaches may cause irreversible endometrial injury and compromise fertility³. These limitations underscore the need for robust epithelial models that can support mechanistic studies and aid in the development of improved therapeutic strategies. However, progress in this area has been primarily constrained by technical challenges in obtaining high-purity, expandable human endometrial epithelial cells (HEECs).

Foundational work in this field was reported by Arnold et al. (2001), who isolated stromal and epithelial cell populations through collagenase digestion and sequential sieve filtration, attaining greater than 98% purity as verified by immunostaining⁴. Despite its success, the method depended on single-enzyme digestion, required prolonged processing, and relied on stepwise filtration to remove stromal and erythrocyte contaminants, making the workflow labor-intensive and difficult to standardize⁴. Later, Yokomizo et al. (2002) established a primary HEEC culture system from hysterectomy specimens using a three-dimensional co-culture with stromal feeder cells⁵. While the feeder-based microenvironment partially supported epithelial growth, the system allowed only limited passaging, typically no more than five passages, which restricted its utility for applications requiring large-scale, stable epithelial expansion⁵.

To overcome these limitations, a simplified and highly reproducible protocol for HEEC isolation and long-term culture was developed. Guided by previous work demonstrating that the ROCK inhibitor Y-27632 markedly improves primary culture efficiency for human keratinocytes, gingival epithelial cells, and mammary epithelial cells, the same principle was applied to endometrial tissue^6,7,8. This approach incorporates three key features: mixed-enzyme digestion to promote efficient tissue dissociation, an essential erythrocyte-lysis step to reduce early contamination and facilitate epithelial attachment, and continuous supplementation with Y-27632 to suppress apoptosis, support adhesion, and enhance proliferative capacity. Using this strategy, HEECs can be expanded over multiple passages while maintaining epithelial morphology and high expression of CK18 and E-cadherin^9,10. Notably, serial passaging for at least 3 generations did not result in detectable phenotypic drift, demonstrating the stability and scalability of the culture system.

This method directly addresses longstanding technical challenges in primary HEEC culture, including high tissue input requirements, complex workflows, low reproducibility, and limited expansion potential. By preserving epithelial identity and functional characteristics throughout passaging, the protocol provides a reliable platform for research on endometrial regeneration, pathophysiology, high-throughput drug screening, and cell–biomaterial interactions. By integrating tissue processing with serial expansion, this standardized workflow addresses a longstanding bottleneck in reproductive biology and supports future applications in tissue engineering, regenerative medicine, and personalized therapy.

This protocol requires fresh tissue specimens, preferably processed within 24 h of collection. Cell yield and growth characteristics may vary depending on tissue source, such as endometrial polyps versus normal endometrium. Common sources of failure include incomplete tissue digestion, excessive erythrocyte contamination, or insufficient Y-27632 supplementation. Although the system supports expansion across multiple passages, further optimization may be required to maintain phenotypic stability during extended culture.

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 institutional safety regulations and Material Safety Data Sheets (MSDS) guidelines.

1. Acquisition of tissue

1. Collect fresh endometrial tissue from surgical specimens obtained from adult women. Transfer the tissue into sterile tubes containing 10 mL phosphate-buffered saline (PBS) supplemented with 3% penicillin/streptomycin (P/S).
   NOTE: All tissue should be processed within 24 h of surgical collection.

2. Pretreatment of tissue

1. Agitate the tissue in washing solution (Table 1) for two consecutive washes of 2 min each to remove mucus and blood clots.
2. Briefly rinse the tissue in 5 mL of 75% ethanol for 5 s to disinfect the surface.
3. Immediately transfer the tissue into 20 mL of washing solution and wash for two washes, 5 min each, to remove residual ethanol.
   NOTE: Because ethanol is flammable, perform this step away from open flames and dispose of ethanol-containing waste according to institutional chemical safety guidelines.

3. Tissue digestion and cell harvest

1. Tissue mincing
   1. Mince the endometrial tissue with two sterile blades until fragments (~2 mm³) can be aspirated through a 1 mL pipette tip (about 10 min).
   2. Transfer the minced tissue into a 50 mL centrifuge tube.
2. Enzymatic digestion
   1. Add 10 mL of digestion solution (Table 1) to the tube containing the tissue fragments.
   2. Incubate the tube in a 37 °C water bath for 30 min. Gently shake the tube every 15 min to facilitate digestion.
   3. Continue incubation until the tissue fragments are visibly dissociated and the solution appears cloudy due to released cells.
3. Termination of digestion
   1. Add 10 mL of neutralizing solution to terminate enzymatic digestion.
   2. Mix thoroughly by pipetting up and down approximately 15 times using a P1000 pipette set to 800 µL.
4. Filtration and centrifugation
   1. Filter the digested suspension through a 100 µm mesh filter.
   2. Centrifuge at 156 × g for 5 min at 25 °C.
5. Red blood cell (RBC) lysis
   1. Carefully aspirate the supernatant without disturbing the pellet.
   2. Resuspend the cell pellet in 2 mL of pre-cooled RBC lysis buffer.
   3. Incubate on ice (approximately 4 °C) for 3–5 min with gentle agitation.
6. Termination of lysis
   1. Add ≥10 mL of PBS or complete medium to stop the lysis reaction.
   2. Centrifuge at 156 × g for 5 min at 25 °C.

4. Initiation of primary culture

1. Washing and centrifugation
   1. Carefully aspirate the supernatant without disturbing the pellet.
   2. Resuspend the pellet in 20 mL of neutralizing solution.
   3. Mix by pipetting approximately 15 times.
   4. Centrifuge at 156 × g for 5 min at 25 °C.
2. Resuspension and seeding
   1. Aspirate the supernatant.
   2. Gently resuspend the cell pellet in 10 mL of initial culture medium containing 10 µM Y-27632 by slowly pipetting up and down 8–10 times using a 1 mL pipette.
   3. Plate the cell suspension into a 100 mm culture dish.
      ​NOTE: For optimal initial seeding density, select the culture vessel according to the estimated cell yield. If the expected yield is >1 × 10⁶ cells, use a 100 mm dish, and for lower yields, use a 60 mm dish. Adjust the medium volume accordingly (e.g., 10 mL for a 100 mm dish; 4 mL for a 60 mm dish).
3. Culture conditions
   1. Incubate the dish at 37 °C in a humidified atmosphere containing 5% CO₂.
   2. Replace the medium with fresh epithelial culture medium without Y-27632 every 3 days.
      NOTE: Monitor cell attachment and morphology; refresh medium every 2 days once cells begin to proliferate.

5. Cell passaging

1. Preparation for trypsinization
   1. When cultures reach 80%–90% confluence, remove the 100 mm dish from the incubator.
   2. Discard the spent medium.
   3. Rinse the cells twice with 2 mL PBS.
   4. Carefully aspirate and remove any remaining PBS.
2. Trypsinization
   1. Add 2 mL of 0.05% trypsin to the dish.
   2. Swirl gently to ensure even coverage of the cell layer.
   3. Incubate at 37 °C for 7 min.
   4. Examine the cells under a microscope (40x objective) to confirm detachment.
3. Neutralization and collection
   1. Add 8 mL of neutralizing solution to stop trypsin digestion.
   2. Transfer the cell suspension to a 15 mL centrifuge tube.
   3. Pipette approximately 15 times to obtain a single-cell suspension.
   4. Centrifuge at 156 × g for 5 min at 25 °C.
4. Resuspension and reseeding
   1. Aspirate the supernatant without disturbing the pellet.
   2. Gently resuspend the cell pellet in 10 mL of epithelial cell medium by pipetting up and down approximately 10 times using a 1 mL pipette.
   3. Seed 1 × 10⁶ cells in 10 mL of epithelial cell medium into a new 100 mm dish.
   4. Adjust seeding density to promote isolated, well-spread single cells or small clusters the following day, avoiding overcrowding.
5. Post-passage maintenance
   1. Incubate under standard culture conditions (37 °C, 5% CO₂).
   2. Observe the culture regularly and replace the epithelial cell medium every 2 days.
      NOTE: Cells are typically ready for downstream applications after the first or second passage, once a healthy, adherent monolayer with ≥80% confluence is obtained.
      CAUTION: All materials that came into contact with primary human cells were treated as potential biohazards. Prior to disposal or further processing, these materials were disinfected using 10% bleach in compliance with institutional biosafety guidelines.

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.