From cup to dish: how to make and use endometrial organoid and stromal cultures derived from menstrual fluid

Sylvia C. Hewitt, Mackenzie J. Dickson, N EDWARDS, Kathleen Hampton, Stavros Garantziotis, Francesco J. DeMayo
In: Frontiers in Endocrinology · 2023 · vol. 14 , pp. 1220622 · doi:10.3389/fendo.2023.1220622 · PMID:37810883 · PMC10552259 · W4386915992
article OA: gold CC0 ⤵ 4 in-corpus citations
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⚙ AI-generated summary by claude@2026-06, 2026-06-09 ⓘ

This study details a non-invasive protocol for isolating paired endometrial epithelial organoids and stromal cells from menstrual fluid for in vitro study.

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⚙ AI-generated deep summary by claude@2026-06, 2026-06-09 ⓘ

This paper develops a non-invasive protocol to isolate paired endometrial epithelial organoids and endometrial stromal (HESC) cells from individual women’s menstrual fluid collected using a menstrual cup. Using washes, enzymatic digestion, filtration to separate epithelial fragments from stromal cells, and Ficoll-based separation of residual red blood cells, the authors establish organoids in Matrigel and stromal cultures in flasks, then measure epithelial estrogen responsiveness and stromal decidualization by treating cultures and performing qPCR for hormone-induced genes. A key limitation is that the protocol involves inter-individual variability and establishes primary cultures from menstrual fluid fragments, which may not fully standardize starting material. This paper is centrally about endometriosis—endometrial organoid and stromal culture methods enabling investigation of epithelial–stromal biology relevant to endometriosis.

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Abstract

Diseases impacting the female reproductive tract pose a critical health concern. The establishment of in vitro models to study primary endometrial cells is crucial to understanding the mechanisms that contribute to normal endometrial function and the origins of diseases. Established protocols for endometrial stromal cell culture have been in use for decades but recent advances in endometrial organoid culture have paved the way to allowing study of the roles of both epithelial and stromal endometrial cells in vitro . Due to inter-individual variability, primary cell cultures must be established from numerous persons. Generally, endometrial epithelial and stromal cells can be isolated from an endometrial biopsy, however, this is collected in a clinical setting by an invasive transcervical procedure. Our goal was to develop a non-invasive method for the isolation of paired endometrial epithelial organoids and stromal cells from menstrual fluid collected from individual women, based on recent reports describing the isolation of endometrial epithelial organoids or endometrial stromal cells from menstrual fluid. Participants recruited by the NIEHS Clinical Research Unit were provided with a menstrual cup and instructed to collect on the heaviest day of their menstrual period. Endometrial tissue fragments in the menstrual fluid samples were washed to remove blood, minced, and digested with proteinases. Following digestion, the solution was strained to separate epithelial fragments from stromal cells. Epithelial fragments were washed, resuspended in Matrigel, and plated for organoid formation. Stromal cells were separated from residual red blood cells using a Ficoll gradient and then plated in a flask. Once established, estrogen responsiveness of endometrial epithelial organoids was assessed and the decidual response of stromal cells was evaluated. Following treatments, qPCR was performed on organoids for genes induced by estradiol and on stromal cells for genes induced by decidualization. In this manner, the relative responsiveness of paired organoid and stroma cell cultures isolated from each woman could be assessed. In conclusion, we can isolate both epithelial and stromal cells from a single menstrual fluid sample, allowing us to establish organoids and cells in a paired manner. This protocol can greatly enhance our knowledge of the role of epithelial and stromal cells alone and in coordination.

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