Objective: To quantify the toxicity of trypan blue on human corneal cells according to exposure time and concentration.
Methods: Three in vitro experiments were performed. (1) We exposed cultured human corneal fibroblasts to trypan blue (0.0001% to 0.1%) in Eagle modified minimum essential medium (EMEM) or phosphate-buffered saline (PBS) for 15 minutes to 24 hours. Cytotoxicity was evaluated by Mosmann's colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MMT) assay. (2) We exposed human corneas in EMEM for 24 hours to trypan blue (0.001% to 0.1%). Fellow donor corneas served as controls. Endothelial survival was evaluated morphologically and by cell density assessment. (3) We morphologically compared the endothelial viability of human donor corneas after exposure to 0.1% trypan blue for 5 to 30 minutes with control corneas.
Results: In experiment 1, trypan blue in EMEM was not significantly toxic at concentrations of 0.005% or lower. Higher concentrations were toxic only after exposure to trypan blue for at least 6 hours. In PBS, significant toxicity was found after exposure to 0.1% trypan blue for 30 minutes or longer. Lower concentrations were toxic after longer exposures. In experiment 2, exposure to 0.01% and 0.1% trypan blue for 24 hours resulted in significant loss in cell density. At lower concentrations, the endothelium was affected only morphologically. In experiment 3, endothelial morphology changed in control corneas and after exposure to 0.1% trypan blue for as little as 5 minutes. After 30-minute exposure, morphologic deterioration was more pronounced.
Conclusions: Trypan blue was toxic in vitro to corneal endothelium and corneal fibroblasts at higher concentrations and notably longer exposure times. Toxicity was less in EMEM than in PBS. Clinical Relevance At commonly used concentrations, both during cataract surgery and in the cornea bank, trypan blue is safe for corneal cells. At higher concentrations or longer exposures, however, caution is warranted.