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William A Eddington, Radha Pertaub, Marc D Friedman, David Muller; Evaluation of Cytotoxicity of Riboflavin/UVA Cross-Linking Applied to Human Corneal Endothelium Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2022.
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To measure the cytotoxic level of Riboflavin (RF) and UVA on human corneal endothelial cells for various RF concentrations and UVA irradiances while mimicking an in vivo environment.
A custom chamber was designed to hold two 96-well cell culture plates in which cells can be irradiated from below using two UVA sources (KXL™, Avedro, Waltham, MA). Gas composition is controlled by adjusting nitrogen and oxygen gas flow rates into the chamber. Oxygen level is maintained at 10% (±20%) using a calibrated oxygen meter (GAXT-X-DL-2, Honeywell, Calgary, Canada). Bubbling the inlet gas mixture through a pre-inlet in-line water chamber helps maintain a high humidity (>75%) in the chamber. A thin heating sheet is adhered to an aluminum plate that serves as a lid and covers the plates while maintaining the cells at a temperature of 37oC (± 20%). This lid is lined with a black flocked material that serves the dual purpose of absorbing any UVA out of the wells as well as acting as a black body radiator for a homogeneous heat source. Black walled cell culture plates are seeded with cells in every other column and row to minimize UVA cross-contamination. Human corneal endothelium cells were obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, in Braunschweig, Germany. Treatment cells were dosed with 0.01%, 0.02% or 0.04% RF and irradiated with either 3 mW/cm2 or 30 mW/cm2 for varying lengths of time for an increasing energy dosage. The optical density of the cells with 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromid (MTT) is measured and normalized to RF soaked controls. Dose response curves were plotted against exposure time for low and high irradiances.
LD50 thresholds were found for each irradiance and RF concentration combination. Higher irradiance allowed for a higher total UVA dose while differences in RF concentration did not significantly affect the LD50 threshold.
Higher irradiance produces less cytotoxic effect on human endothelial cells. This may have interesting implications for the safety thresholds of new corneal riboflavin/UVA cross-linking protocols especially for thin keratoconic corneas.
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