Damage Thresholds for Exposure to NIR and Blue Lasers in an In Vitro RPE Cell System

Until reliable nonanimal systems of analysis are available, animal models will be necessary for ocular laser hazard analysis and for evaluating clinical applications. The purpose of this work was to demonstrate the utility of an in vitro system for laser bioeffects by identifying photothermal and photochemical cytotoxicity thresholds for continuous-wave (cw) and mode-locked (ml) laser exposures.

Exogenous melanosomes were added to hTERT-RPE1 cells in exposure wells 1 day before laser exposure. Thermal or photochemical laser exposures were delivered to artificially pigmented retinal pigment epithelial (RPE) cultures, with subsequent assay for viability 1 hour after exposure. Beam delivery for the 1-hour photochemical exposures was via a modified culture incubator. The cytoprotective effect of pretreatment with two antioxidants was investigated.

Phagocytosis of melanosomes by the RPE cells was efficient, yielding cultures of uniform pigmentation. The damage threshold for the thermal exposure was consistent with published in vivo results. Thresholds for both blue exposures (cw and ml) were identical. Overnight treatment of cells with ascorbic acid (AA) minimized cell death from both cw and ml blue laser exposure, whereas similar treatment with N-acetyl-L-cysteine (NAC) was less effective.

The in vitro system described is suitable for measuring meaningful thermal and photochemical laser damage thresholds. The system is also useful in comparative laser bioeffects studies, such as comparisons between cw and ml laser exposures, cells with various degrees of pigmentation, and studies determining the efficacy and mechanisms of treatments altering the response of cells to lasers.