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

Autor: Sanjay V. Patel, Keith H. Baratz, Johann M. Pacheco, Cindy K. Bahler, Leo J. Maguire, David M. Holmes, Eric D. Wieben, Uttio Roy Chowdhury, Tommy A. Rinkoski, Michael P. Fautsch, Maya L. Khanna
Rok vydání: 2021
Předmět:
Aging
Eye Diseases
Physiology
Cell
Cell Culture Techniques
Cell morphology
Epithelium
Cornea
Medical Conditions
Spectrum Analysis Techniques
Animal Cells
Medicine and Health Sciences
Staining
Multidisciplinary
medicine.diagnostic_test
Chemistry
Endothelium
Corneal
Flow Cytometry
Cell biology
Corneal Disorder
medicine.anatomical_structure
Spectrophotometry
Corneal Disorders
Medicine
Cytophotometry
Anatomy
Cellular Types
Research Article
Cell Physiology
Corneal endothelium
Science
Ocular Anatomy
Research and Analysis Methods
Flow cytometry
Ocular System
In vivo
medicine
Humans
Cell Proliferation
Fuchs' Endothelial Dystrophy
DAPI staining
Biology and Life Sciences
Endothelial Cells
Epithelial Cells
Cell Biology
eye diseases
Culture Media
Nuclear Staining
Ophthalmology
Biological Tissue
Specimen Preparation and Treatment
Cell culture
sense organs
Cell Immortalization
Physiological Processes
Organism Development
Developmental Biology
Zdroj: PLoS ONE
PLoS ONE, Vol 16, Iss 9, p e0258006 (2021)
ISSN: 1932-6203
DOI: 10.1371/journal.pone.0258006
Popis: 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.
Databáze: OpenAIRE
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