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Alternatives to Laboratory Animals - ATLA

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Evaluation of a Porcine Lens and Fluorescence Assay Approach for In Vitro Ocular Toxicological Investigations

Olanrewaju M. Oriowo, Anthony P. Cullen, Kristin Schirmer, B. Ralph Chou, Niels C. Bols and Jacob G. Sivak

Cell biology, as monitored with the fluorescent indicator dyes Alamar BlueTM and 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), and lens optical quality, as measured with an in vitro scanning laser system, have been used to evaluate in vitro the condition of porcine lenses after being placed in a culture medium. The measurements, beginning from week one of culture, were compared statistically. Optical quality and cellular viability, as measured with either dye, were unchanged in lenses that had been maintained for 6 weeks in modified M199 medium. Some lenses were treated with 0.152J/cm2 UVB radiation, and a decline was observed after 48 hours in both optical and metabolic capabilities, as indicated by a decreased capacity of the lenses to reduce Alamar Blue. The measurements with CFDA-AM did not show complete concordance with the other indicators of lens health after UV treatment, making this dye less reliable as applied currently to lens cultures. Overall, the findings suggest that porcine lenses can be maintained for weeks in culture, and that their condition can be evaluated quantitatively by assays that probe cellular functions and optical properties. Such a system should prove valuable for in vitro ocular pharmacotoxicological research.