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Solange Landreville, Laurence Trudel-Vandal, Constance Barry-Marcheterre, Renée Paradis, Stephanie Proulx; Impact of physiological oxygen level on the growth and the transcriptome of melanocytes and fibroblasts from the choroid. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4457.
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© ARVO (1962-2015); The Authors (2016-present)
Cell culture is traditionally performed in CO2 incubators maintained at atmospheric oxygen level (21%), while oxygen is transported by the blood and delivered to the cells at levels lower than 14%. Some oxygen derivatives such as the reactive oxygen species (ROS) can induce replicative senescence of cells when produced in excess, presumably because of irreversible damages to nucleic acids, lipids and proteins. The choroid is an interesting model for understanding the molecular effects of oxygen because the cells are exposed to oxygen levels varying between 4.5-11%. The goal of this study was to recreate the physiological oxygen conditions of the native choroid to study the growth and the transcriptome of melanocytes and fibroblasts.
Melanocytes and fibroblasts were isolated from human choroids (n=3) by successive digestions in trypsin and collagenase. The cells were then exposed to physiological (<5%) or atmospheric (21%) oxygen levels. Cell viability and proliferation under both oxygen levels were measured by a colorimetric assay using the MTS tetrazolium salt and by phospho-histone H3 immunostaining, respectively. Then, gene expression profiling by microarray was conducted using RNA prepared from melanocytes and fibroblasts grown under both oxygen levels. Presence of oxidative DNA damage was studied with the CellROX Green reagent that binds oxidized DNA.
A significant increase in the percentage of viability was measured in melanocytes cultured at low oxygen level, compared to cells grown at 21% oxygen (increases of 65%, 31% and 50%, respectively). An opposite effect was observed with the fibroblasts (decreases of 4%, 35% and 23%, respectively). Scatter plot analysis of the microarray data indicated very few modifications in gene expression between choroidal cells exposed to both oxygen levels (R2=0.9715 and R2=0.9777, respectively). Finally, the level of oxidative stress was reduced at physiological oxygen level.
This study demonstrated that choroidal cells could grow in physiological oxygen conditions that more closely replicate the native tissue environment. Choroidal cells exposed to different oxygen levels shared larger similarities in their transcriptome than we had predicted. Extensive work is warranted to understand the discrepancy between adaptive responses of choroidal melanocytes and fibroblasts to oxygen level.
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