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Solange Landreville, Juliane Guay, Florence Pagé-Larivière; Effects of Oxygen Level on In Vitro Culture of Human Choroidal Melanocytes. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3047.
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Most cells in the human body are exposed to oxygen levels varying from 2-14% (physiological oxygen levels). The choroidal cells for instance, are exposed to 5% oxygen. Traditional cell culture at 21% oxygen thus induces an hyperoxic state in these cells, which reduces the validity of the in vitro models. Our goal was to study the differentiation and proliferation of choroidal melanocytes grown under physiological (5%) and atmospheric (21%) oxygen levels.
Melanocytes were isolated from human choroids by successive digestions in trypsin and collagenase, then the cells were exposed to 5% or 21% oxygen. Melanocyte morphology and pigmentation were assessed by phase-contrast microscopy. Culture purity was verified by immunostaining with melanocyte and retinal pigment epithelium (RPE) markers. Proliferation was measured using MTS assay.
Reduced oxygen conditions did not change the morphology and pigmentation of melanocytes. Compared to the melanocytes grown in 5% oxygen, the cultures exposed to 21% oxygen contained contaminant RPE cells, as confirmed by HMB45, ZO-1 and cytokeratins 8/18 immunostaining. In addition, an increased percentage of proliferation was measured in the 5% oxygen group (+30-64%).
This study demonstrated that pure long-term cultures of choroidal melanocytes could be established using physiological oxygen conditions that more closely replicates the native tissue environment.
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