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Cameron Postnikoff, Clémence Vigouroux, Virginie Coindre, Lena Carcreff, Maud Gorbet; Mechanistic in vitro Studies to Determine the Limitations of Monolayer Models of Corneal Staining. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4662.
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Corneal staining is a clinical phenomenon that has recently been a topic of much debate regarding its mechanism of action, its relation to biocompatibility, and ultimately its overall clinical significance. Sodium fluorescein is readily taken up by many tissues within the body, and even in monolayer corneal models, and yet a normal, healthy eye may present little or no corneal staining. The current investigation exposes monolayers of human corneal epithelial cells to benzalkonium chloride (BAK) and staurosporin (STS) to examine morphological changes, fluorescein uptake, and caspase activity by microscopy and flow cytometry.
Telomerase-immortalized human corneal epithelial cells (hTCEpi) were cultured to confluency on 24-well polystyrene cell culture plates. Monolayers were exposed to varying concentrations of STS, an initiator of apoptosis, or commercially-available wetting drops containing 0.1% BAK. BAK serves as a positive control for cytotoxicity, as it has previously been shown to be cytotoxic in vitro. As a negative control, monolayers were incubated for either two or four hours in the presence of growth medium alone, and were compared to cultures exposed to an artificial tear solution. Sodium fluorescein was diluted in phosphate buffered saline (PBS) to concentrations of 0.1% or 0.05%. For imaging, monolayers were stained for 1 minute and were subsequently washed 3 times in PBS. Fluorescence microscopy was performed following counterstaining with propidium iodide. Flow cytometry was performed post-trypsinization, staining, and washing. Apoptosis via caspase activation and necrosis via propidium iodide staining was subsequently evaluated.
All control cells took up fluorescein regardless of incubation medium. Cell death as a result of BAK or STS exposure was usually correlated with micropunctate staining inside the cell which may be due to apoptotic compartmentalization. Exposure to STS proved to induce apoptosis, however, fluorescein staining showed an overall decrease in fluorescence with increasing concentrations of STS.
In a monolayer culture, live and healthy cells readily uptake fluorescein, which does not effectively mimic the in vivo environment. Induction of apoptosis and cell death does not appear to increase sodium fluorescein uptake.
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