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Aki Kato, Tsutomu Yasukawa, Ayae Takase, Rina Sato, Shinya Ugawa, Yuichiro Ogura; Novel Procedure To Prepare An Implantable Sheet Of Well-differentiated Monolayer Of Cultured Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):891.
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© ARVO (1962-2015); The Authors (2016-present)
The sheets of cultured retinal pigment epithelial (RPE) cells on an amniotic membrane or other artificial films have been developed for the transplantation in the treatment of age-related macular degeneration and other diseases. However, such membranes alternative to Bruch’s membrane may disturb the achievement of physiological functions of implanted RPE. The objective of this study is to develop a new simple method to prepare an implantable, well-differentiated RPE cell sheet without such alternative materials.
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 (2×106 cells/cm2), allowed to form a RPE monolayer sheet. Light microscopy was performed to observe morphological characteristics of RPE cells. The sheet was fixed, carefully detached from the bottom of the plate, and forwarded to scanning electron microscopy (SEM). The RPE sheet was extracted and processed for Western blotting to evaluate the expression of differentiation markers and components of Bruch’s membrane.
Light microscopy revealed the hexagonal shape of RPE cells and overlying membrane-like structure, suggesting upside-down polarity of RPE as compared with conventional culture. The sheet was easily detached from the bottom of the plate by the gentle pipetting technique. SEM disclosed Bruch’s membrane-like structures on the upper side of the RPE sheet. The RPE sheet expressed cytokeratin, occludin, and ZO-1 as a differentiation marker as well as collagen type IV and elastin as a Bruch’s membrane component.
Implantable, well-differentiated monolayer sheet was constructed without any supporting materials by the culture of RPE cells with cell density equal to or higher than that in the physiological condition. The sheet produced Bruch’s membrane-like structures with upside-down polarity. This novel culture system may be useful to elucidate Bruch’s membranogenesis and provide the sheet for RPE transplantation.
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