Effect of Physiological Oxygen on Primary Human Corneal Endothelial Cell Cultures

Sangita P. Patel; Brayan Calle Gonzalez; Nataliia Paone; Christian Mueller; Jamie C. Floss; Maria E. Sousa; Michael Y. Shi

doi:10.1167/tvst.11.2.33

Effect of Physiological Oxygen on Primary Human Corneal Endothelial Cell Cultures

Sangita P. Patel
, Brayan Calle Gonzalez
, Nataliia Paone
, Christian Mueller
, Jamie C. Floss
, Maria E. Sousa
, Michael Y. Shi

Department of Ophthalmology

SUNY Buffalo
Department of Veterans Affairs

Research output: Contribution to journal › Article › peer-review

9 Scopus citations

Abstract

Purpose: Primary human corneal endothelial cells (HCEnCs) cultured in room air are exposed to significantly higher O₂ concentrations [O₂ ]thanwhatisnormallypresent in the eye. We evaluated the growth and metabolism of HCEnCs cultured under physiological [O₂ ] (2.5%; [O₂ ]_2.5 ) and room air ([O₂ ]_A). Methods: Primary cultures of HCEnCs from normal donors and donors with Fuchs dystrophy were grown at [O₂ ]_2.5 and [O₂ ]_A. Growth and morphology were compared using phase-contrast microscopy, zonula occludens (ZO-1) localization, cell density measurements, and senescence marker staining. CD44 (cell quality) and HIF-1α (hypoxia-inducible factor-1α) levels were evaluated by Western blotting. Cell adaptability to a reversal of [O₂ ] growth conditions was measured with cell viability assays, and cell metabolism was assessed via oxygen consumption and extracellular acidification rates. Results: HCEnCs grown at [O₂ ]_A and [O₂ ]_2.5 displayed similar morphologies, ZO-1 localization, CD44 expression, and senescence. Cells from donors with Fuchs dystrophy grew better at [O₂ ]_2.5 than at [O₂ ]_A.HIF-1α was undetectable. Cells displayed greater viability at [O₂ ]_2.5 than at [O₂ ]_A. HCEnCs showed significantly greater proton leak (P < 0.01), nonmitochondrial oxygen consumption (P < 0.01), and spare capacity (P < 0.05) for oxygen consumption rates, and greater basal glycolysis (P < 0.05) with a decreased glycolytic reserve capacity (P < 0.05) for extracellular acidification rates. Conclusions: Primary HCEnCs show unique metabolic characteristics at physiologic [O₂ ]. The effect of [O₂ ] for optimization of HCEnC culture conditions should be considered. Translational Relevance: With the advance of cell-based therapeutics for corneal endothelial diseases, [O₂ ] should be considered an important variable in the optimization of HCEnC culture conditions.

Original language	English
Article number	33
Journal	Translational Vision Science and Technology
Volume	11
Issue number	2
DOIs	https://doi.org/10.1167/tvst.11.2.33
State	Published - Feb 2022

Keywords

cell culture
corneal endothelial cells
Fuchs endothelial corneal dystrophy
oxidative damage

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10.1167/tvst.11.2.33

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@article{e713d51df7c74017b6308f0d237fccdf,

title = "Effect of Physiological Oxygen on Primary Human Corneal Endothelial Cell Cultures",

abstract = "Purpose: Primary human corneal endothelial cells (HCEnCs) cultured in room air are exposed to significantly higher O2 concentrations [O2 ]thanwhatisnormallypresent in the eye. We evaluated the growth and metabolism of HCEnCs cultured under physiological [O2 ] (2.5\%; [O2 ]2.5 ) and room air ([O2 ]A). Methods: Primary cultures of HCEnCs from normal donors and donors with Fuchs dystrophy were grown at [O2 ]2.5 and [O2 ]A. Growth and morphology were compared using phase-contrast microscopy, zonula occludens (ZO-1) localization, cell density measurements, and senescence marker staining. CD44 (cell quality) and HIF-1α (hypoxia-inducible factor-1α) levels were evaluated by Western blotting. Cell adaptability to a reversal of [O2 ] growth conditions was measured with cell viability assays, and cell metabolism was assessed via oxygen consumption and extracellular acidification rates. Results: HCEnCs grown at [O2 ]A and [O2 ]2.5 displayed similar morphologies, ZO-1 localization, CD44 expression, and senescence. Cells from donors with Fuchs dystrophy grew better at [O2 ]2.5 than at [O2 ]A.HIF-1α was undetectable. Cells displayed greater viability at [O2 ]2.5 than at [O2 ]A. HCEnCs showed significantly greater proton leak (P < 0.01), nonmitochondrial oxygen consumption (P < 0.01), and spare capacity (P < 0.05) for oxygen consumption rates, and greater basal glycolysis (P < 0.05) with a decreased glycolytic reserve capacity (P < 0.05) for extracellular acidification rates. Conclusions: Primary HCEnCs show unique metabolic characteristics at physiologic [O2 ]. The effect of [O2 ] for optimization of HCEnC culture conditions should be considered. Translational Relevance: With the advance of cell-based therapeutics for corneal endothelial diseases, [O2 ] should be considered an important variable in the optimization of HCEnC culture conditions.",

keywords = "cell culture, corneal endothelial cells, Fuchs endothelial corneal dystrophy, oxidative damage",

author = "Patel, \{Sangita P.\} and Gonzalez, \{Brayan Calle\} and Nataliia Paone and Christian Mueller and Floss, \{Jamie C.\} and Sousa, \{Maria E.\} and Shi, \{Michael Y.\}",

note = "Publisher Copyright: {\textcopyright} 2022 The Authors.",

year = "2022",

month = feb,

doi = "10.1167/tvst.11.2.33",

language = "English",

volume = "11",

journal = "Translational Vision Science and Technology",

issn = "2164-2591",

publisher = "Association for Research in Vision and Ophthalmology Inc.",

number = "2",

}

TY - JOUR

T1 - Effect of Physiological Oxygen on Primary Human Corneal Endothelial Cell Cultures

AU - Patel, Sangita P.

AU - Gonzalez, Brayan Calle

AU - Paone, Nataliia

AU - Mueller, Christian

AU - Floss, Jamie C.

AU - Sousa, Maria E.

AU - Shi, Michael Y.

PY - 2022/2

Y1 - 2022/2

N2 - Purpose: Primary human corneal endothelial cells (HCEnCs) cultured in room air are exposed to significantly higher O2 concentrations [O2 ]thanwhatisnormallypresent in the eye. We evaluated the growth and metabolism of HCEnCs cultured under physiological [O2 ] (2.5%; [O2 ]2.5 ) and room air ([O2 ]A). Methods: Primary cultures of HCEnCs from normal donors and donors with Fuchs dystrophy were grown at [O2 ]2.5 and [O2 ]A. Growth and morphology were compared using phase-contrast microscopy, zonula occludens (ZO-1) localization, cell density measurements, and senescence marker staining. CD44 (cell quality) and HIF-1α (hypoxia-inducible factor-1α) levels were evaluated by Western blotting. Cell adaptability to a reversal of [O2 ] growth conditions was measured with cell viability assays, and cell metabolism was assessed via oxygen consumption and extracellular acidification rates. Results: HCEnCs grown at [O2 ]A and [O2 ]2.5 displayed similar morphologies, ZO-1 localization, CD44 expression, and senescence. Cells from donors with Fuchs dystrophy grew better at [O2 ]2.5 than at [O2 ]A.HIF-1α was undetectable. Cells displayed greater viability at [O2 ]2.5 than at [O2 ]A. HCEnCs showed significantly greater proton leak (P < 0.01), nonmitochondrial oxygen consumption (P < 0.01), and spare capacity (P < 0.05) for oxygen consumption rates, and greater basal glycolysis (P < 0.05) with a decreased glycolytic reserve capacity (P < 0.05) for extracellular acidification rates. Conclusions: Primary HCEnCs show unique metabolic characteristics at physiologic [O2 ]. The effect of [O2 ] for optimization of HCEnC culture conditions should be considered. Translational Relevance: With the advance of cell-based therapeutics for corneal endothelial diseases, [O2 ] should be considered an important variable in the optimization of HCEnC culture conditions.

AB - Purpose: Primary human corneal endothelial cells (HCEnCs) cultured in room air are exposed to significantly higher O2 concentrations [O2 ]thanwhatisnormallypresent in the eye. We evaluated the growth and metabolism of HCEnCs cultured under physiological [O2 ] (2.5%; [O2 ]2.5 ) and room air ([O2 ]A). Methods: Primary cultures of HCEnCs from normal donors and donors with Fuchs dystrophy were grown at [O2 ]2.5 and [O2 ]A. Growth and morphology were compared using phase-contrast microscopy, zonula occludens (ZO-1) localization, cell density measurements, and senescence marker staining. CD44 (cell quality) and HIF-1α (hypoxia-inducible factor-1α) levels were evaluated by Western blotting. Cell adaptability to a reversal of [O2 ] growth conditions was measured with cell viability assays, and cell metabolism was assessed via oxygen consumption and extracellular acidification rates. Results: HCEnCs grown at [O2 ]A and [O2 ]2.5 displayed similar morphologies, ZO-1 localization, CD44 expression, and senescence. Cells from donors with Fuchs dystrophy grew better at [O2 ]2.5 than at [O2 ]A.HIF-1α was undetectable. Cells displayed greater viability at [O2 ]2.5 than at [O2 ]A. HCEnCs showed significantly greater proton leak (P < 0.01), nonmitochondrial oxygen consumption (P < 0.01), and spare capacity (P < 0.05) for oxygen consumption rates, and greater basal glycolysis (P < 0.05) with a decreased glycolytic reserve capacity (P < 0.05) for extracellular acidification rates. Conclusions: Primary HCEnCs show unique metabolic characteristics at physiologic [O2 ]. The effect of [O2 ] for optimization of HCEnC culture conditions should be considered. Translational Relevance: With the advance of cell-based therapeutics for corneal endothelial diseases, [O2 ] should be considered an important variable in the optimization of HCEnC culture conditions.

KW - cell culture

KW - corneal endothelial cells

KW - Fuchs endothelial corneal dystrophy

KW - oxidative damage

UR - https://www.scopus.com/pages/publications/85125020311

U2 - 10.1167/tvst.11.2.33

DO - 10.1167/tvst.11.2.33

M3 - Article

C2 - 35191961

AN - SCOPUS:85125020311

SN - 2164-2591

VL - 11

JO - Translational Vision Science and Technology

JF - Translational Vision Science and Technology

IS - 2

M1 - 33

ER -

Effect of Physiological Oxygen on Primary Human Corneal Endothelial Cell Cultures

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Keywords

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