Safety testing of indocyanine green and trypan blue using retinal pigment epithelium and glial cell cultures

PURPOSE: Indocyanine green (ICG) and trypan blue have been advocated as vital stains for use during macular surgery. The safety of these agents was tested using a cell culture model.

METHODS: Human retinal pigment epithelium (RPE) and Müller cell lines were exposed to ICG over a range of concentrations up to 0.5%, and trypan blue up to 0.2%. Cells were exposed to each dye for 5, 15, or 30 minutes, rinsed, and incubated 24 hours. Cell viability was measured using a mitochondrial dehydrogenase-assay and fluorescent live-dead probe. Experiments were repeated using 0.5% and 1% ICG and 0.06% and 0.12% trypan blue, with follow-up at 0, 1, 5, and 15 days. ICG experiments were repeated in the presence of illumination from a xenon light-source channeled through a surgical endolight, and using reduced osmolarity solutions of 0.1%, 0.5%, and 1% (185 vs. 275 mOsM).

RESULTS: There was no clear relationship between cell viability and the concentration of the agent or duration of follow-up, except in RPE cells exposed to 1% ICG. These showed a linear (R(2) 0.9952) decline in viability with time, with a significant reduction by day 15 (P = 0.016). RPE cells exposed to ICG and illumination were not significantly different from the negative control, but when illumination was combined with low osmolarity, viability was reduced (P = 0.0016). ICG and illumination reduced Müller cell viability (P < 0.0001 for both 185 and 275 mOsM). Müller cells incubated with 185 mOsM 1% ICG showed a significant reduction in viability (P < 0.0001) not seen with the 185 mOsM 0.5% or 0.1% solutions or in the low-osmolarity RPE groups.

CONCLUSIONS: The combination of exposure to 0.5% ICG and the newer endoillumination light-sources can damage cultured Müller cells. Although the preparations of ICG most commonly used clinically did not produce significant damage, relatively small changes in ICG osmolarity and concentration did. This suggests that safety margins are not large. Trypan blue is safe in a cell culture model.