Trypan Blue: Effect on Retinal Pigment Epithelial and Neurosensory Retinal Cells

Abstract

purpose. To evaluate the toxicity of trypan blue on retinal cells in vitro.

methods. Human retinal pigment epithelial cells (ARPE-19) and rat neurosensory retinal cells (R28) were grown in tissue culture and treated with four different concentrations of trypan blue (0.1%, 0.05%, 0.025%, and 0.0125%), in combination with surgical light exposure (0, 5, or 10 minutes). Cell viability, mitochondrial function, and DNA synthesis were measured by trypan blue dye-exclusion assay, mitochondrial dehydrogenase assay, and tritiated [³H] thymidine incorporation, respectively.

results. ARPE-19 and R28 cells exposed to trypan blue with or without illumination did not show any significant decrease, either in cell viability by the dye-exclusion assay or in [³H] thymidine incorporation. R28 cells exposed to 0.1% trypan blue with and without light showed a significant reduction of mitochondrial dehydrogenase activity (P < 0.05). ARPE-19 cells exposed to trypan blue, with or without light, did not show any significant decrease in mitochondrial dehydrogenase activity.

conclusions. This study suggests that rat neurosensory retina (R28) cells are more sensitive than human RPE (ARPE-19) cells to trypan blue. ARPE-19 cells showed no evidence of toxicity with any of the three assays, but R28 cells showed evidence of toxicity with the mitochondrial dehydrogenase assay at the higher doses and light-exposure times studied. Clinical studies must be conducted to determine the safety and efficacy of staining of the inner limiting membrane with trypan blue.

Meticulous removal of the inner limiting membrane (ILM) and tractional membranes may influence the outcome of surgery for macular holes and proliferative vitreoretinopathy (PVR). Precise identification and delineation of these membranes facilitates its complete removal. Indocyanine green (ICG) was widely used to stain the ILM in macular hole surgery until the publication of recent reports on its potential toxicity. ^{1 2 3 4} These findings have led to the search for alternative dyes for intraoperative staining of membranes. 

Recently, trypan blue has been used for staining of epiretinal membranes (ERMs) for peeling in surgery for proliferative vitreoretinopathy (PVR) and macular pucker, although it is not approved for surgical use in the United States. ^{5 6 7 8} Trypan blue is a vital dye that has been used mainly in the anterior segment surgery of the eye. ^{9 10} It has been used extensively outside the United States to stain the anterior lens capsule during cataract surgery without any adverse effects. However, trypan blue is known to cause mutagenesis, and there are a few controversial reports on its ocular toxicity. ^{11 12 13} Although trypan blue has been shown to be safe on cultured retinal pigment epithelial cells, it is reported to be toxic to the retina in vivo if exposed for long durations. ^{13 14} There is a paucity of data on the effects of trypan blue on the viability of neurosensory or RPE cells. 

The purpose of this study was to evaluate the effects of trypan blue, in combination with light, on retinal pigment epithelial (ARPE-19) and neurosensory retinal (R28) cells. Various retinal cell lines, including ARPE-19, have been described in the literature in studies of the toxicity of different drugs. ^{15 16} We used three assays (dye exclusion, mitochondrial dehydrogenase activity and tritiated thymidine incorporation) to assess the viability of cells after exposure to four concentrations of trypan blue, with or without simultaneous exposure to light of different durations. Our results identified safe and nontoxic concentrations of trypan blue for retinal cells in vitro. 

Materials and Methods

Cell Culture

A Human retinal pigment epithelial cell line (ARPE-19) was obtained from ATCC (Manassas, VA). Cells were grown in a 1:1 mixture (vol/vol) of Dulbecco’s modified Eagle’s medium and Ham’s nutrient mixture F-12 medium (DMEM F-12; Invitrogen-Gibco, Carlsbad, CA), 1× 10 mM nonessential amino acids, 0.37% sodium bicarbonate, 0.058% l-glutamine, 10% fetal bovine serum, and antibiotics (penicillin G 100 U/mL, streptomycin sulfate 0.1 mg/mL, gentamicin 10 μg/mL, and amphotericin B 2.5 μg/mL). 

R28 cells were derived from postnatal day 6 rat retina in the laboratory of one of the authors (GMS). ¹⁷ R28 cells express genes characteristic of neurons, ¹⁸ as well as functional neuronal properties. ¹⁹ Rat embryonic neurosensory precursor retinal (R28) cells were cultured in DMEM-high glucose (Invitrogen-Gibco) with 10% fetal bovine serum, 1× minimum essential medium (MEM), 1× 10 mM nonessential amino acids, 0.37% sodium bicarbonate, and 10 μg/mL gentamicin. 

The cells were plated onto 96-well ELISA plates or 35-mm tissue culture dishes and incubated at 37°C in 5% CO₂ to reach 70% to 80% confluence before being exposed to dye and illumination. 

Exposure to Dye and Illumination

Trypan blue solution (Sigma-Aldrich, St. Louis, MO) was dissolved in serum-free DMEM (SFDMEM) to achieve concentrations of 0.1%, 0.05%, 0.025%, and 0.0125%. Cells cultured in 35-mm dishes were treated with 2 mL of these concentrations of dye for 2 minutes, and the dye was thereafter rinsed off with culture medium. Cultured cells in 96-well ELISA plates were treated with 100 μL of each concentration of the dye for 2 minutes. After they were rinsed, the cells were exposed to light from a Greishaber surgical halogen light source (Alcon, Fort Worth, TX) for 10 or 5 minutes or were not exposed to light. Cells that were illuminated with only light were exposed for 10 minutes without exposure to trypan blue. The intensity of illumination was maintained at 3000 lux, as measured by a light meter (Lightmeter model 810; AEMC, Boston, MA). The cells were then incubated overnight at 37°C with 5% CO₂ balanced with air. Cells receiving no light served as control samples. 

Dye-Exclusion Assay

Cells were harvested from the 35-mm dishes by treatment with 0.2% trypsin-EDTA and incubating them at 37°C for 5 minutes. The cells were centrifuged at 1000 revolutions per minute (rpm) for 1 minute and then resuspended in 1 mL of culture medium. Automated cell viability analysis was performed (ViCell analyzer; Beckman-Coulter, Inc., Fullerton, CA). The analyzer performs an automated trypan blue dye-exclusion assay and gives the percentage of viable cells. 

Mitochondrial Dehydrogenase Assay

To assess mitochondrial function, mitochondrial dehydrogenase (succinate-tetrazolium-reductase) activity was determined using the WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) colorimetric assay (Roche Diagnostics, Indianapolis, IN). WST-1 is a tetrazolium dye containing an electron coupling reagent that is cleaved by the mitochondrial dehydrogenase enzyme to a formazan dye. The reaction directly correlates to the number of metabolically active cells. Ten microliters of the formazan dye was added to each well containing cells and medium and incubated for 2 hours at 37°C. Absorbance was measured at 490 nm on a multiwell spectrophotometer (PerkinElmer, Wellesley, MA). The data presented are from three separate experiments. 

Tritiated [³H] Thymidine Incorporation Assay

Incorporation of radioactive thymidine into DNA was used to quantify the rate of DNA synthesis. After exposure of ARPE-19 or R28 cells in 96-well ELISA plates to different concentrations of trypan blue, with and without illumination, 0.5 μCu [³H] thymidine was added to each well. The cells were incubated at 37°C overnight in culture medium (2% serum DMEM for the R28 cells and SFDMEM for the ARPE-19 cells). Incorporation of [³H] thymidine was determined with a liquid scintillation counter (Wallac Microbeta Trilux; PerkinElmer). The experiments were performed thrice and each time point and concentration was performed in triplicate in each experiment. 

Statistical Analysis

Data were subjected to statistical analysis by ANOVA (Prism ver. 3.0 statistics program; GraphPad Software Inc., San Diego, CA). The Newman-Keuls multiple-comparison test was used to compare the data within each experiment. P < 0.05 was considered statistically significant. Error bars in the graphs represent the standard error of the mean, with experiments performed in triplicate. 

Results

The cell morphology was not altered in both ARPE-19 and R28 cells after treatment with trypan blue, as seen by phase-contrast microscopy. 

Dye-Exclusion Assay

Human RPE (ARPE-19) and rat neurosensory retina (R28) cells exposed to trypan blue, with or without illumination did not show any significant decrease in cell viability when compared with the control cells by the dye-exclusion assay. The mean cell viability of R28 cells after exposure to light for 10 minutes and trypan blue concentrations 0.1%, 0.05%, 0.025%, and 0.0125% was 85.3%, 89%, 87%, and 85.8%, respectively. Cells not treated with light or trypan blue had a mean cell viability of 87.0% (Fig. 1A) . 

ARPE-19 cells treated with light for 10 minutes and trypan blue concentrations 0.1%, 0.05%, 0.025%, and 0.0125% had a mean cell viability of 93.25%, 95.60%, 92.45%, and 93.60%, respectively. ARPE-19 cells not treated with light or trypan blue had a mean cell viability of 93.8% (Fig. 1B) . These differences were not significant. 

Mitochondrial Dehydrogenase Assay

R28 cells exposed to 0.1% trypan blue with and without light showed a significant reduction of mitochondrial dehydrogenase activity. Cells exposed to lower concentrations (0.0125%, 0.025%, and 0.05%) of trypan blue, with or without light, did not show any significant reduction in the enzyme activity (Fig. 2A) . R28 cells exposed to 0.1% trypan blue, without light and with light for 5 minutes, showed a reduction in mitochondrial dehydrogenase activity of 18% and 20% respectively (P < 0.05), whereas cells exposed to 0.1% trypan blue and light for 10 minutes showed a reduction of 26% (P < 0.01). Cells exposed to 0.05%, 0.025%, and 0.0125% trypan blue, with or without light, did not show any significant reduction of mitochondrial dehydrogenase activity (P > 0.05; Table 1 ). 

ARPE-19 cells exposed to trypan blue alone did not show any significant decrease in mitochondrial dehydrogenase activity. These cells also showed a trend of decreased mitochondrial dehydrogenase activity with increasing duration of light exposure at all concentrations of trypan blue, but these differences were not significant (Fig. 2B ; Table 2 ). 

[³H] Thymidine Incorporation Assay

The counts per minute of β-ray emission were normalized to the control for both R28 and ARPE-19 cells in each of the three replicated set of experiments. R28 cells exposed to trypan blue and light for 5 or 10 minutes showed an increase in [³H] thymidine incorporation compared with those exposed to trypan blue only, but this was not significant (P > 0.05; Fig. 3 ). R28 cells simultaneously exposed to light for 10 minutes and trypan blue 0.1%, 0.05%, 0.025%, and 0.0125% had 0.61 × 10³, 0.61 × 10³, 0.6 × 10³, and 0.58 × 10³ cpm respectively (P > 0.05). Also, there was no significant difference in the [³H] thymidine incorporation between the cells treated with trypan blue without light exposure compared with the control cells. ARPE-19 cells also showed a similar trend and the change in [³H] thymidine incorporation was not significant. ARPE-19 cells exposed to trypan blue concentrations of 0.1%, 0.05%, 0.025%, and 0.0125% and light for 10 minutes had 1.51 × 10³, 1.30 × 10³, 1.29 × 10³, and 1.14 × 10³ cpm, respectively (P > 0.05). Cells treated with trypan blue and light for 5 minutes or trypan blue alone also did not show any significant difference in [³H] thymidine incorporation (P > 0.05). 

Discussion

Both ICG and trypan blue are vital dyes that have been used as adjuvants in ocular surgery to stain and identify transparent tissue intraoperatively. ICG is used to stain the anterior lens capsule in cataract surgery and for staining the ILM in macular hole surgery. ^{10 20 21 22 23 24 25 26} Removal of the ILM and tractional membranes improves the anatomic and functional outcome of surgery for macular hole and PVR. Various reports have described the toxic effects of ICG on the retina, which has led to a search for alternative dyes for macular surgery. ^{1 2 3}  

Trypan blue has been used mainly for staining the anterior capsule of the lens during cataract surgery. ^{9 10} It has recently been described for staining ERMs in PVR surgery, but it is not approved for surgical use in the United States. ^{6 7 8} These reports suggest that trypan blue appears to be safe and effective for staining ERMs. Stalmans et al. ²⁷ have described a technique of double vital staining with trypan blue for staining ERM and infracyanine green for ILM. Infracyanine green does not contain iodine and hence can be dissolved in 5% glucose to form an iso-osmotic solution compared with ICG, which has to be dissolved in distilled water, making it a hypo-osmolar solution. Recent reports have indicated that trypan blue not only stains ERMs but also stains the ILM adequately. ^{5 7}  

There are conflicting reports about the safety of trypan blue. It is reported to have teratogenic and carcinogenic potential. ^{11 12} In an in vivo study on New Zealand White rabbits, trypan blue injected intravitreally in nonvitrectomized eyes at a concentration of 0.2% was shown to be toxic. ¹³ Retinal structure was disintegrated in the inferior retina 4 weeks after surgery, and immunohistochemical staining showed a marked loss of rhodopsin. However, in their study, the trypan blue was not rinsed off at the time of surgery and the retina was also not exposed to light. In a recent study, Tokuda et al., ²⁸ using morphologic examination and lactate dehydrogenase assay, found in an ex vivo model of rat retina tissue that trypan blue was not toxi. Trypan blue at a concentration of 0.02% was found to be safe in a study, but there was disorganization of the inner retinal layers at concentrations of 0.15% and 0.25%. ²⁹  

In our study, we exposed two different retinal cell lines, ARPE-19 and R28, to various concentrations of trypan blue that were higher or lower than the concentrations used clinically (0.06%) and studied its toxicity using three different assays. In addition, some of the cells were exposed to light energy of 3000 lux, which is similar to the light exposure during retinal surgery. The ARPE-19 cells are derived from the retinal pigment epithelium, and the R28 cells represent the neurosensory retinal cells. The three assays used in this study measure the toxicity at both the structural and functional level of cells. The dye-exclusion assay is based on the integrity of the cell membrane and a reduction of cell viability in this assay would suggest severe toxicity of the dye. We found that trypan blue with or without light exposure did not affect cell viability of ARPE-19 and R28 cells by the dye-exclusion method. This is in agreement with the study by Stalmans et al. ¹⁴ who did an in vitro study on the cell viability of trypan blue on cultured human RPE cells by confocal microscopy. They used 0.06%, 0.15%, and 0.30% concentrations of trypan blue, but the cells were not exposed to light. In our study, we used trypan blue concentrations of 0.1%, 0.05%, 0.025%, and 0.0125%. In addition, we exposed the cells to light for different durations to simulate operating conditions. To our knowledge, the effect of trypan blue on cell viability of ARPE-19 and R28 cells has not been reported to date. 

The mitochondrial dehydrogenase assay measures the metabolic activity of cells and a decrease in the activity of this enzyme would suggest a temporary decline in cell function. Recently, Gale et al. ³⁰ reported that trypan blue with light did not affect the mitochondrial dehydrogenase activity in ARPE-19 cells, and our study is in agreement with their results. In addition, we performed the mitochondrial dehydrogenase assay on neurosensory retinal precursor cells also, and to our knowledge, the effect of trypan blue on mitochondrial dehydrogenase activity of R28 cells has not been reported. Trypan blue 0.1% with or without light caused a significant reduction in mitochondrial dehydrogenase activity in R28 cells. However, trypan blue at concentrations of 0.05% or less did not significantly reduce the activity of mitochondrial dehydrogenase even with simultaneous exposure of light for 10 minutes. 

Finally, we also found that trypan blue, with or without light, does not significantly alter DNA synthesis in both ARPE-19 and R28 cells. The tritiated [³H] thymidine incorporation assay was used to quantify the rate of DNA synthesis. An increase in this assay could mean that the toxic insult due to trypan blue is not severe enough to cause the arrest of cell proliferation, but instead, the cells try to compensate for the cellular injury by increased replication. This is the first study to report the effects of trypan blue on DNA synthesis in ARPE-19 and R28 cells. 

In this study, using three different assays, we have shown that trypan blue, with or without light, is not toxic to ARPE-19 cells. The response of neurosensory R28 cells was more complex. For the cell viability assay and the tritiated thymidine assay of DNA synthesis, toxicity was not observed for any of the trypan concentrations and light exposure times. However, R28 cells showed evidence of toxicity using the mitochondrial dehydrogenase assay at the higher doses and light exposure times. Therefore, clinical studies may be warranted to determine the safety and efficacy of ILM staining by trypan blue. 

 Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, April 2004.

 Supported by The Discovery Fund for Eye Research, the Henry L. Guenther Foundation, and the Skirball Molecular Ophthalmology Program.

 Submitted for publication June 15, 2004; revised September 9 and October 7, 2004; accepted October 17, 2004.

 Disclosure: R. Narayanan, None; M.C. Kenney, None; S. Kamjoo, None; T.-H.T. Trinh, None; G.M. Seigel, None; G.P. Resende, None; B.D. Kuppermann, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Dye-exclusion assay after treatment with trypan blue and light. (A) R28 cells and (B) ARPE-19 cells did not show any significant decrease in cell viability.

Figure 2.

 

WST-1 mitochondrial dehydrogenase assay. Optical density (OD) at 490 nm after exposure to trypan blue and light. (A) R28 cells and (B) ARPE-19 cells. ARPE-19 cells did not have a significant reduction in enzyme activity at any concentration of trypan blue. *P < 0.05, **P < 0.01 when compared with serum-free DMEM control samples exposed to light only, for 10 minutes.

Table 1.

 

WST-1 Mitochondrial Dehydrogenase Assay of R28 Cells Exposed to Trypan Blue and Light Compared with Control

% Concentration	Light Exposure (min)	P
0.1	10	<0.01
0.1	5	<0.05
0.1	0	<0.05
0.05	10	>0.05
0.05	5	>0.05
0.05	0	>0.05
0.025	10	>0.05
0.025	5	>0.05
0.025	0	>0.05
0.0125	10	>0.05
0.0125	5	>0.05
0.0125	0	>0.05
0.0	10	>0.05

 Trypan blue 0.05% with light exposure for 5 minutes did not significantly reduce mitochondrial dehydrogenase activity. Values in bold show significantly decreased mitochondrial dehydrogenase activities compared with serum-free DMEM control samples exposed to light only, for 10 minutes.

Table 2.

 

WST-1 Mitochondrial Dehydrogenase Assay of ARPE-19 Cells Exposed to Trypan Blue and Light compared with the Control

% Concentration	Light Exposure (min)	P
0.1	10	>0.05
0.1	5	>0.05
0.1	0	>0.05
0.05	10	>0.05
0.05	5	>0.05
0.05	0	>0.05
0.025	10	>0.05
0.025	5	>0.05
0.025	0	>0.05
0.0125	10	>0.05
0.0125	5	>0.05
0.0125	0	>0.05
0.0	10	>0.05

 There was no significant reduction in the mitochondrial dehydrogenase activity in ARPE-19 cells.

Figure 3.

 

[³H] thymidine assay in counts per minute of β-ray emission after exposure to trypan blue and light. (A) R28 cells and (B) ARPE-19 cells. There was no significant difference in counts between control cells and those exposed to trypan blue and light.