Hypothermia protects cultured human retinal pigment epithelial cells against trypan blue toxicity

Purpose: To determine whether trypan blue (TB) is toxic to cultured human retinal pigment epithelial (ARPE-19) cells, and whether hypothermia can protect ARPE-19 cells against TB toxicity. Methods: ARPE-19 cells were cultured and exposed to balanced salt solution as a control, while other cells were exposed to 0.05, 0.2, and 0.5% TB dye at 37 and 4°C for 5 and 30 min. The percentage of ARPE-19 cells that survived was determined by resazurin 1 day after the exposure. Results: A statistically significant decrease in the percentage of ARPE-19 cells surviving was found after exposure to 0.2 and 0.5% TB at any temperature or for any exposure duration (p < 0.01). The percentage of RPE cells surviving at 0.05% was not significantly different from that of controls except for a 30-min exposure at 37°C. The percentage of cells surviving at 4°C for a 5-min exposure to 0.5% TB and a 30-min exposure to 0.2 and 0.5% was significantly higher than that at 37°C under each condition (p < 0.01 for all). Conclusions: These results indicate that TB is toxic to human RPE cells, and the toxicity is dose- and exposure duration-dependent. Exposing the cells at 4°C had a protective effect against higher concentrations or longer exposure durations of TB compared to exposure at 37°C.