Purpose
To investigate the feasibility of using a custom-designed chamber that controls the temperature and oxygen levels to mimic an in vivo environment when measuring the cytotoxic level of Riboflavin (RF) and UVA on human corneal endothelial cells.
Methods
A custom chamber is 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) under the plates. 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 lid and covers the plates while maintaining the cells at a temperature of 37°C (± 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 are obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, in Braunschweig, Germany. Treatment cells are dosed with 0.01%, 0.02% or 0.04% RF in Phosphate Buffered Saline (PBS). They are then irradiated with either 3 mW/cm2 or 30 mW/cm2 for varying lengths of time for an increasing energy dosage.
Results
The optical density of the cells is measured via an MTT assay and compared to controls (cells in PBS alone) to obtain the cell viability after treatment with UVA-RF combination. Dose response curves are plotted against exposure time for low and high irradiances and the time required to get 50% cell viability (=EC50) is estimated using a non-linear regression model and compared for high and low irradiance treatments. Results show cell viability decreasing as a function of increasing energy dose applied.
Conclusions
This custom chamber allows for the comparison of cytotoxicity levels of UVA-RF combination on human corneal endothelial cells while controlling the gaseous composition and temperature of the chamber close to in vivo conditions.
Keywords: 481 cornea: endothelium •
503 drug toxicity/drug effects •
480 cornea: basic science