Characterization of a dual media system for culturing primary normal and Fuchs endothelial corneal dystrophy (FECD) endothelial cells.

Primary cultures of human corneal endothelial cells (HCECs) are an important model system for studying the pathophysiology of corneal endothelium. The purpose of this study was to identify and validate an optimal primary culture model of normal and Fuchs endothelial corneal dystrophy (FECD) endothel...

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Autores principales:	Tommy A Rinkoski, Cindy K Bahler, Johann M Pacheco, Maya L Khanna, David M Holmes, Uttio Roy Chowdhury, Keith H Baratz, Sanjay V Patel, Leo J Maguire, Eric D Wieben, Michael P Fautsch
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/80d4ea72a60048aca716b4c0b6854e4d
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spelling	oai:doaj.org-article:80d4ea72a60048aca716b4c0b6854e4d2021-12-02T20:14:00ZCharacterization of a dual media system for culturing primary normal and Fuchs endothelial corneal dystrophy (FECD) endothelial cells.1932-620310.1371/journal.pone.0258006https://doaj.org/article/80d4ea72a60048aca716b4c0b6854e4d2021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0258006https://doaj.org/toc/1932-6203Primary cultures of human corneal endothelial cells (HCECs) are an important model system for studying the pathophysiology of corneal endothelium. The purpose of this study was to identify and validate an optimal primary culture model of normal and Fuchs endothelial corneal dystrophy (FECD) endothelial cells by comparing cell morphology and marker expression under different media conditions to in vivo donor tissues. Primary and immortalized HCECs, isolated from normal and FECD donors, were cultured in proliferation media (Joyce, M4, Bartakova) alone or sequentially with maturation media (F99, Stabilization 1, M5). CD56, CD73 and CD166 expressions were quantified in confluent and matured cell lines by flow cytometry. HCECs that were allowed to proliferate in Joyce's medium followed by maturation in low-mitogen containing media yielded cells with similar morphology to corneal endothelial tissues. Elevated expression of CD56 and CD166 and low expression of CD73 correlated with regular, hexagonal-like HCEC morphology. CD56:CD73 > 2.5 was most consistent with normal HCEC morphology and mimicked corneal endothelial tissue. Immortalization of normal HCECs by hTERT transduction showed morphology and CD56:CD73 ratios similar to parental cell lines. HCECs established from FECD donors showed reduced CD56:CD73 ratios compared to normal HCECs which coincided with reduced uniformity and regularity of cell monolayers. Overall, a dual media system with Joyce's medium for proliferation and a low-mitogen media for maturation, provided normal cultures with regular, hexagonal-like cell morphologies consistent with corneal endothelial cells in vivo. CD56:CD73 expression ratio >2.5 was predictive of in vivo-like cellular morphology.Tommy A RinkoskiCindy K BahlerJohann M PachecoMaya L KhannaDavid M HolmesUttio Roy ChowdhuryKeith H BaratzSanjay V PatelLeo J MaguireEric D WiebenMichael P FautschPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 9, p e0258006 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Tommy A Rinkoski Cindy K Bahler Johann M Pacheco Maya L Khanna David M Holmes Uttio Roy Chowdhury Keith H Baratz Sanjay V Patel Leo J Maguire Eric D Wieben Michael P Fautsch Characterization of a dual media system for culturing primary normal and Fuchs endothelial corneal dystrophy (FECD) endothelial cells.
description	Primary cultures of human corneal endothelial cells (HCECs) are an important model system for studying the pathophysiology of corneal endothelium. The purpose of this study was to identify and validate an optimal primary culture model of normal and Fuchs endothelial corneal dystrophy (FECD) endothelial cells by comparing cell morphology and marker expression under different media conditions to in vivo donor tissues. Primary and immortalized HCECs, isolated from normal and FECD donors, were cultured in proliferation media (Joyce, M4, Bartakova) alone or sequentially with maturation media (F99, Stabilization 1, M5). CD56, CD73 and CD166 expressions were quantified in confluent and matured cell lines by flow cytometry. HCECs that were allowed to proliferate in Joyce's medium followed by maturation in low-mitogen containing media yielded cells with similar morphology to corneal endothelial tissues. Elevated expression of CD56 and CD166 and low expression of CD73 correlated with regular, hexagonal-like HCEC morphology. CD56:CD73 > 2.5 was most consistent with normal HCEC morphology and mimicked corneal endothelial tissue. Immortalization of normal HCECs by hTERT transduction showed morphology and CD56:CD73 ratios similar to parental cell lines. HCECs established from FECD donors showed reduced CD56:CD73 ratios compared to normal HCECs which coincided with reduced uniformity and regularity of cell monolayers. Overall, a dual media system with Joyce's medium for proliferation and a low-mitogen media for maturation, provided normal cultures with regular, hexagonal-like cell morphologies consistent with corneal endothelial cells in vivo. CD56:CD73 expression ratio >2.5 was predictive of in vivo-like cellular morphology.
format	article
author	Tommy A Rinkoski Cindy K Bahler Johann M Pacheco Maya L Khanna David M Holmes Uttio Roy Chowdhury Keith H Baratz Sanjay V Patel Leo J Maguire Eric D Wieben Michael P Fautsch
author_facet	Tommy A Rinkoski Cindy K Bahler Johann M Pacheco Maya L Khanna David M Holmes Uttio Roy Chowdhury Keith H Baratz Sanjay V Patel Leo J Maguire Eric D Wieben Michael P Fautsch
author_sort	Tommy A Rinkoski
title	Characterization of a dual media system for culturing primary normal and Fuchs endothelial corneal dystrophy (FECD) endothelial cells.
title_short	Characterization of a dual media system for culturing primary normal and Fuchs endothelial corneal dystrophy (FECD) endothelial cells.
title_full	Characterization of a dual media system for culturing primary normal and Fuchs endothelial corneal dystrophy (FECD) endothelial cells.
title_fullStr	Characterization of a dual media system for culturing primary normal and Fuchs endothelial corneal dystrophy (FECD) endothelial cells.
title_full_unstemmed	Characterization of a dual media system for culturing primary normal and Fuchs endothelial corneal dystrophy (FECD) endothelial cells.
title_sort	characterization of a dual media system for culturing primary normal and fuchs endothelial corneal dystrophy (fecd) endothelial cells.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/80d4ea72a60048aca716b4c0b6854e4d
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Characterization of a dual media system for culturing primary normal and Fuchs endothelial corneal dystrophy (FECD) endothelial cells.

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