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R. Yeilding, R. T. Olmstead, R. W. Frey, R. Shah, C. B. Orizu, I. L. Thornton; Lens Culture System for Long Term Study of Porcine Lenses Pre and Post Laser Photodisruption Treatment. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3838.
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To study the effects of photodisruption in a lens over time, a culture system is needed to maintain lens health. We have developed a lens culturing system that is able to maintain health of porcine lenses for periods exceeding a month post mortem. Additionally, our results show that we are able to maintain the health of photodisrupted lenses.
Lenses were excised from room temperature porcine eyes less than 4 hours post mortem and then placed in well plates. The lenses were inspected for damage, placed in a wash solution, and then placed in culture medium. The wash solution and culture media consisted of a Modified Liquid medium M199 that was adjusted to a pH of 7.6. The cultured lenses were kept in an incubator at 37C and in a 4% CO2- 96% air atmosphere for the duration of the experiments with the media solution being replaced every 48 hours. Lens health was determined by using the AlamarBlue assay and qualitative clarity measurements. The AlamarBlue assay is a commercial preparation of dye used to quantify cell proliferation, cytotoxicity and viability. It incorporates resazurin and resorufin as a fluorometric-colorimetric oxidation-reduction indicator that fluoresces in response to cell metabolism reduction. After 1 week of pre-incubation, the fluorescence levels of the lenses were measured with a fluorescence multi-well plate reader. The readings obtained at week one were taken as baseline and were compared to subsequent readings taken weekly along with photography documenting the clarity of the lenses.
The fluorescence multi-well plate reader reported that the control lenses were viable for periods over a month, on average only losing 17% of there initial metabolic rate. We found that when normalized, the average lens metabolic activity decayed at an exponential decay rate of approximately .05 per week. During the test period, the clarity in the lenses dropped minimally until the fifth week when the experiment was terminated. When lenses were treated with the laser, the average metabolic rate did not decrease.
We have been able to maintain viable lens specimens for a period of one month post mortem for use in photodisruption experiments. The lenses showed both clarity and metabolic activity. In addition we have been able to maintain lenses that have been photodisrupted for periods in excess of a week with active metabolic rates. This ability to extend the life of lenses has aided in the further study of lens photodisruption.
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