Time-dependent morphological alterations and viability of cultured human trabecular cells after exposure to Trypan blue

Background To determine the time-dependent toxicity of Trypan blue at 0.06% concentration in cultured human trabecular meshwork cells. Methods Human trabecular meshwork cells cultured in vitro were exposed to Trypan blue and acute toxicity was evaluated. Cells were exposed for 5, 30, 60s and for 5, 15, 30, 60min to a commercially available Trypan blue preparation (Vision Blue; DORC International Zuidland, The Netherlands). Morphology was observed by phase-contrast microscopy and cell viability was measured using Hoechst 33342 (Intergen; Purchase, NY, USA) and propidium iodide assays to determine the percentage of living and dead cells. Results Morphological changes occurred mainly after 5min of exposure to Trypan blue. Viability was 96.0%+/- 3.6%, 94.8%+/- 3.5% and 92.5%+/- 4.4% after 5, 30 and 60s of exposure, respectively; a significant toxic effect of Trypan blue was observed after 5, 15, 30 and 60min of exposure with a viability of 85.0%+/- 3.7%, 77.0%+/- 6.7%, 70.8%+/- 5.9% and 68.3%+/- 8.7%, respectively. Conclusions Trypan blue is not toxic, in terms of cell viability, over an exposure time of up to 60s; however, further exposure results in a gradual increase in damage of cultured human trabecular meshwork cells.