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MA Gamulescu, C Framme, S Warzecha, J Schröder, J Roider; A New Method for the Preparation of Coherent RPE-Sheets Suitable for Transplantation . Invest. Ophthalmol. Vis. Sci. 2002;43(13):708.
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Purpose:Surgical therapies in age-related macular degeneration (AMD), e.g. extraction of a neovascular membrane, are mostly associated with loss of the surrounding retinal pigment epithelium (RPE). In this study we investigated the potential of a new technique for the preparation of RPE-sheets suitable for transplantation. Methods:In freshly enucleated porcine eyes (2-4 h after enucleation) the anterior segment, the vitreous body and the retina were carefully removed and the eyes were divided into four quarters. Following a superficial incision of 3 x 3 mm, the RPE as a sheet was slowly detached by the flush of saline from a thin and soft subretinal cannula. We compared the preparation of RPE-sheets without and with additional application of calcium-magnesium-free solution (CMFS, up to 30 minutes), in order to loosen the adhesion of the RPE to the Bruch membrane. Finally, the RPE-sheets obtained with this technique were stained and examined by light and electron microscopy. Results:The detachment of the RPE cell layer as a coherent sheet was possible, the morphological analysis with light and electron microscopy revealed intact tight junctions and intracellular mikrofilaments and showed only little cell damage. No differences in cell morphology were seen when comparing the preparation without and with additional use of CMFS. With increasing incubation time of CMFS, we observed an easier preparation of the RPE, however with the disadvantage of increasing cell loss and discontinuation of the cell layer. Thus, the optimal incubation time seemed to be 1-5 min. Despite the fragileness of the prepared RPE-sheets, they could easily be transferred to another site by means of a soft instrument. Due to a different reflex and a characteristic rolling-in to one side, the choroidal and retinal side of the sheet could be distinguished. We also noted that the preparation of the RPE, when it was started in the periphery of the eye, resulted in greater difficulties in separation of the RPE and in obtaining a coherent sheet. Conclusion:It is possible to obtain a coherent RPE-sheet by detaching the cells from the underlying tissue with the flush of saline from a subretinal cannula. Additional application of CMFS may help to prepare a functional RPE-sheet suitable for dislocation within the eye. However, there seems to be a time dependency and only a short time window in which cellular and structural damage of the thin cell layer can be avoided with this procedure. These results may help to further develop a more efficient and satisfactory subretinal surgery technique in AMD.
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