Abstract
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