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Charlene Choo, Doug Chung, Anthony J Aldave; Investigation of Oxidative Stress in Ex Vivo Corneal Endothelial Cells During Cell Culture and Expansion. Invest. Ophthalmol. Vis. Sci. 2021;62(8):814.
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© ARVO (1962-2015); The Authors (2016-present)
Ex vivo expansion of corneal endothelial cells (CEnC) has the potential to alleviate the global shortage of donor corneal tissue that limits access to corneal transplantation. However, successful expansion of ex vivo corneal endothelial cells (evCEnC) is limited by early cellular senescence and loss of CEnC-like morphology. To determine whether or not oxidative stress plays a role in the expansion capacity of evCEnC, we cultured evCEnC with and without SkQ1, a mitochondria-targeting antioxidant, and measured the level of intracellular free radical (IFR) levels.
To determine the optimal SkQ1 dose range that will lead to minimal cell toxicity and maximal oxidative stress protection in CEnC, HCEnC-21T, a CEnC line, was cultured in media supplemented with 0nM (untreated), 50nM, 250nM, 500nM or 750nM of SkQ1 for 3-5 days. Then the cells were treated with or without tBH, an oxidative stress inducing agent, and cell viability was assessed by MTT assay. To determine the impact of SkQ1 treatment on IFR levels, evCEnC were isolated from donor corneas and then split into two culture (SKQ1-treated and untreated), which were each grown to confluence. DCFH-DA assay was performed to measure differences in IFR levels.
When compared to untreated cells, HCEnC-21T treated with 50nM or 250nM SkQ1 retained 98.8% and 96.9% cell viability, respectively, while HCEnC-21T treated with 500nM or 750nM SkQ1 retained 65.8% and 22.7% cell viability, respectively. Under 100 µM tBH oxidative stress induction, cell viability protection was observed in a SkQ1 dose-dependent manner with 250nM and 50nM SkQ1 treatment leading to 36.3% and 18.2% cell viability, respectively, while 0nM SkQ1 treatment yielded 14.8% viability. Compared to their tBH-untreated counterparts, evCEnC treated with 50nM and 250nM SkQ1 demonstrated a ~7.5% and ~22.5% decrease, respectively, in IFR concentrations at passage 0. Additional evCEnC isolations are being assessed in order to perform statistical analyses.
Supplementing culture media with SkQ1 provides a CEnC line with tBH-induced oxidative stress protection and decreases IFR concentrations in cultured evCEnC. The preliminary findings of this study suggest antioxidants, such as SkQ1, may increase the expansion potential of ex vivo CEnC by reducing oxidative stress levels.
This is a 2021 ARVO Annual Meeting abstract.
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