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E. Brasnu, F. Brignole-Baudouin, A. Pauly, H. Liang, L. Riancho, J.-M. Warnet, C. Baudouin; Comparative Study on the Cytotoxic Effects of Benzalkonium Chloride on Wong-Kilbourne Derivative of Chang Conjunctival and IOBA-NHC Cell Lines. Invest. Ophthalmol. Vis. Sci. 2008;49(13):6040. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The Wong-Kilbourne derivative of Chang conjunctiva-derived cell line has been widely used for toxicological and functional in vitro studies on ocular surface, but is contaminated with HeLa cells. Recently, the IOBA-NHC spontaneously immortalized conjunctival epithelial cell line has been developed and did not show other cell type contamination. In the present study, we compared in the two cell lines the toxic effects of benzalkonium chloride (BAC), a preservative which toxicity has been often reported on conjunctival in vivo and in vitro models, in order to assess whether both cell lines would be equally suitable for in vitro toxicological studies.
Microplate cytofluorometry tests (neutral red, H2DCF-DA, hydroethidine and Yopro-1), flow cytometry tests (Annexin V/7-AAD and DNA content) and standard immunofluorescence stainings were used to assess the necrotic, apoptotic and oxidative effects of BAC on Chang and IOBA-NHC cell lines. Cells were exposed to five concentrations of BAC (10-2%, 5.10-3%, 10-3%, 10-4% and 10-5%) during 15 minutes and 15 minutes followed by 24 hours of cell recovery in complete medium.
With the microplate cytofluorometry and the flow cytometry tests, the lowest cellular viability, the highest reactive oxygen species production and the highest apoptotic effects were obtained with the highest BAC concentrations and varied in a concentration-dependent manner on both IOBA-NHC and Chang cell lines. Standard immunofluorescence stainings showed increasing cell shrinkage with high concentrations of BAC on either Chang or IOBA-NHC cells.
Our findings validated the use of both IOBA-NHC and Chang cell lines for toxicological in vitro studies. However, their respective disadvantages have to be known and considered in any further study using IOBA-NHC or Chang cells.
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