Corneal endothelial cytotoxicity of riboflavin/UVA treatment in vitro

Ophthalmic Res. 2003 Nov-Dec;35(6):324-8. doi: 10.1159/000074071.


Recently, we have developed collagen crosslinking induced by combined riboflavin/UVA treatment, thus increasing the biomechanical rigidity of the cornea to treat progressive keratoconus. The present safety study was performed to evaluate possible cytotoxic effects of combined riboflavin/UVA treatment on the corneal endothelium in vitro. Endothelial cell cultures from porcine corneas were treated with 500 microM riboflavin solution, exposed to various endothelial UVA irradiances (370 nm) ranging from 0.1 to 1.6 mW/cm2 for 30 min and evaluated 24 h later using trypan blue staining and Yopro fluorescence staining. The effect of either treatment alone (UVA irradiation ranging from 0.2 to 6 mW/cm2) was also tested. An abrupt cytotoxic threshold irradiance level was found at 0.35 mW/cm2 after combined treatment with riboflavin plus UVA irradiation and at 4 mW/cm2 with UVA irradiation alone. Riboflavin alone was not toxic. A cytotoxic effect of the combined riboflavin/UVA treatment on corneal endothelial cells is to be expected with a corneal thickness of less than 400 microm. Therefore, pachymetry should be routinely performed before riboflavin/UVA treatment to exclude patients at risk.

MeSH terms

  • Animals
  • Cell Survival
  • Cells, Cultured
  • Combined Modality Therapy
  • Endothelium, Corneal / drug effects*
  • Endothelium, Corneal / pathology
  • Endothelium, Corneal / radiation effects*
  • Photosensitizing Agents / toxicity*
  • Riboflavin / toxicity*
  • Staining and Labeling
  • Swine
  • Trypan Blue
  • Ultraviolet Rays*


  • Photosensitizing Agents
  • Trypan Blue
  • Riboflavin