Original article
Comparison of the In vitro Safety of Intraocular Dyes Using Two Retinal Cell Lines: A Focus on Brilliant Blue G and Indocyanine Green

https://doi.org/10.1016/j.ajo.2008.08.031Get rights and content

Purpose

To compare the in vitro toxicity of brilliant blue G (BBG), indocyanine green (ICG), Trypan blue (TB), and Evans blue (EB) in a human retinal pigment epithelial cell line (ARPE-19) and a murine retinal ganglion/Muller glial (RGC) primary cell culture.

Design

In vitro cell biology experimental study.

Methods

The dose-dependent toxicity of the dyes was determined by exposing each dye at four different concentrations to the two cell cultures for a short exposure (three minutes) and a medium exposure (30 minutes). The time-dependent toxicity of the dyes was also determined. All four dyes, each diluted to 1/500th of stock concentration, were applied only to the ARPE-19 cells for a prolonged exposure of two, 24, 48, and 72 hours. Cell viability was measured via a mitochondrial dehydrogenase assay.

Results

BBG was the only dye to cause toxicity in the ARPE-19 cell line at short exposure times. BBG and TB demonstrated toxicity at medium exposure times. BBG and ICG demonstrated toxicity at long exposure times and dilute concentrations. At short exposure times, none of the dyes caused toxicity in the RGC mixed primary cultures. In contrast, at medium exposure times, all dyes except ICG demonstrated toxicity that lessened with lower concentrations.

Conclusions

All dyes demonstrated relatively safe viability profiles in both cell lines at surgically relevant concentrations and times. Cell toxicity could be elicited at higher concentrations and longer exposure times. ICG had a favorable viability profile at almost all of the concentrations and times tested.

Section snippets

Reagents

All cell culture reagents were purchased from Invitrogen Canada Inc (Burlington, Ontario, Canada) unless otherwise indicated. Cell viability assay and all dyes were purchased from Sigma-Aldrich Co (Oakville, Ontario, Canada) unless otherwise indicated.

Retinal Pigment Epithelial Cell Culture

The human retinal pigment epithelial cell line (ARPE-19) was purchased from American Type Culture Collection (Manassas, Virginia, USA). Western blot and immunostaining were done using anti-RPE65 to verify the cell line. ARPE-19 expresses RPE65,

ARPE-19 Cell Cultures

BBG was the only dye to cause significant toxicity after three minutes of exposure. Cell viability differed significantly when compared with control for BBG stock concentration (10 mg/ml, cell viability = 73%) and first dilution (2.5 mg/ml, cell viability = 75%) (Figure 1).

After 30 minutes of exposure, there was no toxicity with ICG and EB. With BBG 30 minute exposure, cell viability differed significantly when compared with control for stock concentration (10 mg/ml, cell viability = 64%),

Discussion

The in vitro cytotoxicity of four dyes was compared in both ARPE-19 and mixed primary RGC/glial cell cultures. This is the first study to examine BBG and EB toxicity under these conditions. Experimental parameters were chosen primarily to mimic intraoperative conditions during macular hole surgery. Short- and medium-exposure times approximated typical intraoperative uses and a prolonged exposure time simulated the possible long-lasting persistence of dilute dye in the vitreous cavity

Darana Yuen, MD, completed medical training at the University of Alberta, Canada. She is currently in her final year of residency in Ophthalmology at the University of Western Ontario. In July 2009, upon completion of residency, she will be pursuing a one year glaucoma fellowship at the University of Toronto under the mentorship of Dr Trope and Dr Buys. Dr Yuen's research interests lie in both clinical and basic science.

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    Darana Yuen, MD, completed medical training at the University of Alberta, Canada. She is currently in her final year of residency in Ophthalmology at the University of Western Ontario. In July 2009, upon completion of residency, she will be pursuing a one year glaucoma fellowship at the University of Toronto under the mentorship of Dr Trope and Dr Buys. Dr Yuen's research interests lie in both clinical and basic science.

    Cindy Hutnik, MD, PhD, is an Associate Professor in the Departments of Ophthalmology and Pathology at the University of Western Ontario, and an Associate Scientist at the Lawson Health Research Institute. Dr Hutnik has a full-time clinical practice at the Ivey Eye Institute in London Ontario and is active in both basic science and clinical research.

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