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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: Rinkoski, Tommy A., Bahler, Cindy K., Pacheco, Johann M., Khanna, Maya L., Holmes, David M., Roy Chowdhury, Uttio, Baratz, Keith H., Patel, Sanjay V., Maguire, Leo J., Wieben, Eric D., Fautsch, Michael P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8480743/
https://www.ncbi.nlm.nih.gov/pubmed/34587219
http://dx.doi.org/10.1371/journal.pone.0258006
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author Rinkoski, Tommy A.
Bahler, Cindy K.
Pacheco, Johann M.
Khanna, Maya L.
Holmes, David M.
Roy Chowdhury, Uttio
Baratz, Keith H.
Patel, Sanjay V.
Maguire, Leo J.
Wieben, Eric D.
Fautsch, Michael P.
author_facet Rinkoski, Tommy A.
Bahler, Cindy K.
Pacheco, Johann M.
Khanna, Maya L.
Holmes, David M.
Roy Chowdhury, Uttio
Baratz, Keith H.
Patel, Sanjay V.
Maguire, Leo J.
Wieben, Eric D.
Fautsch, Michael P.
author_sort Rinkoski, Tommy A.
collection PubMed
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.
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spelling pubmed-84807432021-09-30 Characterization of a dual media system for culturing primary normal and Fuchs endothelial corneal dystrophy (FECD) endothelial cells Rinkoski, Tommy A. Bahler, Cindy K. Pacheco, Johann M. Khanna, Maya L. Holmes, David M. Roy Chowdhury, Uttio Baratz, Keith H. Patel, Sanjay V. Maguire, Leo J. Wieben, Eric D. Fautsch, Michael P. PLoS One Research Article 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. Public Library of Science 2021-09-29 /pmc/articles/PMC8480743/ /pubmed/34587219 http://dx.doi.org/10.1371/journal.pone.0258006 Text en © 2021 Rinkoski et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Rinkoski, Tommy A.
Bahler, Cindy K.
Pacheco, Johann M.
Khanna, Maya L.
Holmes, David M.
Roy Chowdhury, Uttio
Baratz, Keith H.
Patel, Sanjay V.
Maguire, Leo J.
Wieben, Eric D.
Fautsch, Michael P.
Characterization of a dual media system for culturing primary normal and Fuchs endothelial corneal dystrophy (FECD) endothelial cells
title 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_short 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
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8480743/
https://www.ncbi.nlm.nih.gov/pubmed/34587219
http://dx.doi.org/10.1371/journal.pone.0258006
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