Dataset Information

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] than what is normally present 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.

SUBMITTER: Patel SP

PROVIDER: S-EPMC8883143 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Effect of Physiological Oxygen on Primary Human Corneal Endothelial Cell Cultures.

Patel Sangita P SP Calle Gonzalez Brayan B Paone Nataliia N Mueller Christian C Floss Jamie C JC Sousa Maria E ME Shi Michael Y MY

Translational vision science & technology 20220201 2

<h4>Purpose</h4>Primary human corneal endothelial cells (HCEnCs) cultured in room air are exposed to significantly higher O2 concentrations [O2] than what is normally present 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).<h4>Methods</h4>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 microscop ...[more]

PMID: 35191961

Similar Datasets

Project description:BackgroundGlobal shortage of donor corneas greatly restricts the numbers of corneal transplantations performed yearly. Limited ex vivo expansion of primary human corneal endothelial cells is possible, and a considerable clinical interest exists for development of tissue-engineered constructs using cultivated corneal endothelial cells. The objective of this study was to investigate the density-dependent growth of human corneal endothelial cells isolated from paired donor corneas and to elucidate an optimal seeding density for their extended expansion in vitro whilst maintaining their unique cellular morphology.ResultsEstablished primary human corneal endothelial cells were propagated to the second passage (P2) before they were utilized for this study. Confluent P2 cells were dissociated and seeded at four seeding densities: 2,500 cells per cm2 ('LOW'); 5,000 cells per cm2 ('MID'); 10,000 cells per cm2 ('HIGH'); and 20,000 cells per cm2 ('HIGH(×2)'), and subsequently analyzed for their propensity to proliferate. They were also subjected to morphometric analyses comparing cell sizes, coefficient of variance, as well as cell circularity when each culture became confluent. At the two lower densities, proliferation rates were higher than cells seeded at higher densities, though not statistically significant. However, corneal endothelial cells seeded at lower densities were significantly larger in size, heterogeneous in shape and less circular (fibroblastic-like), and remained hypertrophic after one month in culture. Comparatively, cells seeded at higher densities were significantly homogeneous, compact and circular at confluence. Potentially, at an optimal seeding density of 10,000 cells per cm2, it is possible to obtain between 10 million to 25 million cells at the third passage. More importantly, these expanded human corneal endothelial cells retained their unique cellular morphology.ConclusionsOur results demonstrated a density dependency in the culture of primary human corneal endothelial cells. Sub-optimal seeding density results in a decrease in cell saturation density, as well as a loss in their proliferative potential. As such, we propose a seeding density of not less than 10,000 cells per cm2 for regular passage of primary human corneal endothelial cells.

Project description:PURPOSE: To identify genes whose expressions in primary human trabecular meshwork (TM) cell cultures are affected by the transcription factor pituitary homeobox 2 (PITX2) and to identify genes that may have roles in glaucoma. Known glaucoma causing genes account for disease in a small fraction of patients, and we aimed at identification of other genes that may have subtle and accumulative effects not easily identifiable by a genetic approach. METHODS: Expression profiles derived using microarrays were compared between TM control cells and cells treated with PITX2 siRNAs using three protocols so as to minimize false positive and negative results. The first protocol was based on the commonly used B statistic. The second and third protocols were based on fold change in expression. The second protocol used a threshold of at least 2 fold change in expression, whereas the third protocol used ranking in fold change without setting a threshold. The likelihood of a selected gene being a true positive was considered to correlate with the number of protocols by which it was selected. By considering all genes that were selected by at least one protocol, the likelihood of false negatives was expected to decrease. Effects on a subset of selected genes were verified by real time PCR, western blots, and immunocytochemistry. Effects on ALDH1A1, were further pursued because its protein product, aldehyde dehydrogenase 1 family, member A1, has roles in oxidative stress and because oxidative stress is known to be relevant to the etiology of glaucoma. RESULTS: The expression level of 41 genes was assessed by to be possibly affected by PITX2 knockdown. Twenty one genes were down-regulated and twenty were upregulated. The expression of five genes was assessed to be altered by all three analysis protocols. The five genes were DIRAS3 (DIRAS family, GTP-binding RAS-like 3), CXCL6 (chemokine (C-X-C motif) ligand 6), SAMD5 (sterile alpha motif domain containing 5), CBFB (core-binding factor, beta subunit), and MEIS2 (meis homeobox 2). Real time PCR experiments verified results on a subset of genes tested. Notably, the results were also confirmed in two independent TMs. Effects on CXCL6 and ALDH1A1 were also confirmed by western blots, and effects on ALDH1A1 were further shown by immunocytochemistry. Data consistent with PITX2 involvement in ALDH1A1 mediated response to oxidative stress were presented. CONCLUSIONS: Bioinformatics tools revealed that the genes identified affect functions and pathways relevant to glaucoma. Involvement of PITX2 in expression of some of the genes and in some of the pathways is being reported here for the first time. As many of the genes identified have not been studied vis-à-vis glaucoma, we feel they introduce new candidates for understanding this devastating disease.

Project description:PurposeTo investigate effects of magnetic microparticles on movement of magnet controlled human corneal endothelial cells (HCECs).MethodsImmortalized HCEC line (B4G12) and primary culture of HCECs were exposed to two commercially available magnetic micro- or nanoparticles, SiMAG (average size 100 nm) and fluidMAG (average size <1000 nm). Cell viability assays and reactive oxygen species production assays were performed. Cellular structural changes, intracellular distribution of microparticles, and expression levels of proteins related to cellular survival were analyzed. Ex vivo human corneas were exposed to microparticles to further evaluate their effects. Magnetic particle-laden HCECs were cultured under the influence of a neodymium magnet.ResultsNo significant decrease of viability was found in HCECs after exposure to both magnetic particles at concentrations up to 20 µg/mL for 48 hours. However, high concentrations (40 µg/mL and 80 µg/mL) of SiMAG and FluidMAG significantly decreased viability in immortalized HCECs, and only 80 µg/mL of SiMAG and FluidMAG decreased viability in primary HCECs after 48 hours of exposure. There was relative stability of viability at various concentrations of magnetic particles, despite a dose-dependent increase of reactive oxygen species, lactate dehydrogenase, and markers of apoptosis. Ex vivo human cornea study further revealed that exposure to 20 µg/mL of SiMAG and fluidMAG for 72 hours was tolerable. Endocytosed magnetic particles were mainly localized in the cytoplasm. The application of a magnetic field during cell culture successfully demonstrated that magnetic particle-loaded HCECs moved toward the magnet area and that the population density of HCECs was significantly increased.ConclusionsWe verified short-term effects of SiMAG and fluidMAG on HCECs and their ability to control movement of HCECs by an external magnetic field.Translational relevanceA technology of applying magnetic particles to a human corneal endothelial cell culture and controlling the movement of cells to a desired area using a magnetic field could be used to increase cell density during cell culture or improve the localization of corneal endothelial cells injected into the anterior chamber to the back of the cornea.

Dataset Information

Effect of Physiological Oxygen on Primary Human Corneal Endothelial Cell Cultures.

Purpose

Methods

Results

Conclusions

Translational relevance

Publications

Effect of Physiological Oxygen on Primary Human Corneal Endothelial Cell Cultures.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets