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

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 en...

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Autores principales: Hewitt, Sylvia C., Dickson, Mackenzie J., Edwards, Nicole, Hampton, Kathleen, Garantziotis, Stavros, DeMayo, Francesco J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10552259/
https://www.ncbi.nlm.nih.gov/pubmed/37810883
http://dx.doi.org/10.3389/fendo.2023.1220622
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author Hewitt, Sylvia C.
Dickson, Mackenzie J.
Edwards, Nicole
Hampton, Kathleen
Garantziotis, Stavros
DeMayo, Francesco J.
author_facet Hewitt, Sylvia C.
Dickson, Mackenzie J.
Edwards, Nicole
Hampton, Kathleen
Garantziotis, Stavros
DeMayo, Francesco J.
author_sort Hewitt, Sylvia C.
collection PubMed
description 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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spelling pubmed-105522592023-10-06 From cup to dish: how to make and use endometrial organoid and stromal cultures derived from menstrual fluid Hewitt, Sylvia C. Dickson, Mackenzie J. Edwards, Nicole Hampton, Kathleen Garantziotis, Stavros DeMayo, Francesco J. Front Endocrinol (Lausanne) Endocrinology 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. Frontiers Media S.A. 2023-09-21 /pmc/articles/PMC10552259/ /pubmed/37810883 http://dx.doi.org/10.3389/fendo.2023.1220622 Text en Copyright © 2023 Hewitt, Dickson, Edwards, Hampton, Garantziotis and DeMayo https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Endocrinology
Hewitt, Sylvia C.
Dickson, Mackenzie J.
Edwards, Nicole
Hampton, Kathleen
Garantziotis, Stavros
DeMayo, Francesco J.
From cup to dish: how to make and use endometrial organoid and stromal cultures derived from menstrual fluid
title From cup to dish: how to make and use endometrial organoid and stromal cultures derived from menstrual fluid
title_full From cup to dish: how to make and use endometrial organoid and stromal cultures derived from menstrual fluid
title_fullStr From cup to dish: how to make and use endometrial organoid and stromal cultures derived from menstrual fluid
title_full_unstemmed From cup to dish: how to make and use endometrial organoid and stromal cultures derived from menstrual fluid
title_short From cup to dish: how to make and use endometrial organoid and stromal cultures derived from menstrual fluid
title_sort from cup to dish: how to make and use endometrial organoid and stromal cultures derived from menstrual fluid
topic Endocrinology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10552259/
https://www.ncbi.nlm.nih.gov/pubmed/37810883
http://dx.doi.org/10.3389/fendo.2023.1220622
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