April 2014
Volume 55, Issue 13
ARVO Annual Meeting Abstract  |   April 2014
Evaluation of Cytotoxicity of Riboflavin/UVA Cross-Linking Applied to Human Corneal Endothelium Cells
Author Affiliations & Notes
  • William A Eddington
    R&D, Avedro, Waltham, MA
  • Radha Pertaub
    R&D, Avedro, Waltham, MA
  • Marc D Friedman
    R&D, Avedro, Waltham, MA
  • David Muller
    R&D, Avedro, Waltham, MA
  • Footnotes
    Commercial Relationships William Eddington, Avedro Inc. (E); Radha Pertaub, Avedro Inc. (E); Marc Friedman, Avedro Inc. (E); David Muller, Avedro Inc. (E)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 2022. doi:
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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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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: 449 cell survival • 574 keratoconus • 647 photodynamic therapy  

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