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Hideaki Usui, Tsutomu Yasukawa, Aki Kato, Noriaki Takase, Masaharu Ohbayashi, Yuichiro Ogura; Structural Analysis of a Monolayer Sheet of Cultured Retinal Pigment Epithelial Cells with Bruch’s membrane. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2344.
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© ARVO (1962-2015); The Authors (2016-present)
The defect and atrophy of retinal pigment epithelium (RPE) are severe pathological findings in age-related macular degeneration and pathologic myopia. The transplantation of suspension or a sheet of cultured RPE cells has been clinically tested, partially rescuing these eyes. However, previously used RPE sheets had no native Bruch’s membrane but artificial substances, which might impair graft survival. We have previously reported a novel procedure to prepare an implantable monolayer sheet of RPE cells with Bruch’s membrane (Kato A et al, ARVO 2011 #891). The objective of this study is to reveal structural details of the monolayer RPE sheet histologically.
Isolated human RPE cells were cultured in F-10 with 10% fetal calf serum. RPE cells at passages 6-10 were tripsinized, collected, and re-seeded onto a flat bottom plate with high density (106 cells/cm2), allowed to form a RPE monolayer sheet. Light microscopy was performed to observe morphological characteristics of RPE cells cultured. The RPE sheet was extracted and processed for Western blotting to evaluate the expression of components of tight junction and Bruch’s membrane. The sheet was fixed, carefully detached from the bottom of the plate, and forwarded to scanning electron microscopy (SEM), transmission Electron Microscope (TEM), and confocal immunofluorescence microscopy.
Light microscopy revealed the hexagonal shape of RPE cells with an overlying fibrous membrane, suggesting upside-down polarity of RPE and the sheet was easily detached from the bottom of the plate by the gentle pipetting technique. The RPE sheet expressed occludin and ZO-1 as tight junction markers as well as collagen type IV and elastin as a Bruch’s membrane component in Western blotting. In SEM images, the upper side showed microvilli and lamellipodia of RPE cells and, thereafter, Bruch’s membrane-like structures involving elastic fibers and collagen. TEM disclosed the tight junctions of the RPE cells. Confocal immunofluorescence microscopy showed the expression of ZO-1 and occludin among RPE cells.
The RPE sheet manufactured by our original method was well-structured as an implantable sheet with Bruch’s membrane and tight junction. This RPE sheet may have a potential as a better option for RPE transplantation.
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