Assessment of the toxic effect of benzalkonium chloride on human limbal stem cells.

Benzalkonium chloride (BAC) is the most commonly used preservative in eye drops. Unfortunately, it is potentially toxic and considered a leading cause of iatrogenic dry eye disease (DED) associated with local damage to the corneal epithelium. Corneal epithelium can be reconstituted thanks to the ability of limbal epithelial stem cells (LESCs) to self-renew, migrate, and differentiate, and can potentially be damaged by BAC. The aim of this study was to characterize the phenotype of human limbal stem cells (LSCs) isolated from the whole corneoscleral rims, and treated with BAC in vitro. The BAC dose was determined based on LSC viability assessment (MTT assay). The 48-h incubation period of LSCs with BAC was chosen to simulate long-term exposure of cells to preservative-containing eye drops. The cells were characterized by specific marker immunofluorescence staining; expression of genes related to proliferation, apoptosis, and inflammation (RT-qPCR); colony-forming ability and wound healing (scratch assay). Cell cycle stages were identified by flow cytometry. A BAC concentration of 0.0002% in the culture medium was chosen as an effective dose to inhibit LSC proliferation and migration and stimulate the expression of genes related to cell cycle, apoptosis, and inflammation. LSCs lose their clonogenic potential under the influence of BAC. It was concluded that benzalkonium chloride can develop toxic activity against limbal stem cells, limiting their regenerative potential.