A. Takase

Three-Dimensional Spheroidal Culture Visualization of Membranogenesis of Bruch's Membrane and Basolateral Functions of the Retinal Pigment Epithelium

PURPOSE Aging changes in the RPE involve lipid accumulation and membranous basal deposits onto the underlying Bruchs membrane, which may be related to AMD. Conventional in vitro cell culture is limited in its ability to observe the epithelial functions on the basal side. The purpose of this study was to develop a three-dimensional culture system to observe basolateral functions of the RPE. METHODS Isolated human RPE cells were cultured in a viscous medium on a rounded-bottom culture dish, resulting in spheroid formation. The appearance and size of the spheroids were assessed by light microscopy. Spheroids were fixed in 4% paraformaldehyde for immunohistochemistry or sampled for Western blotting. For transmission electron microscopy (TEM) and scanning electron microscopy (SEM), spheroids were postfixed in 1% osmium tetroxide. RESULTS The spheroids had a differentiated RPE monolayer with a thin elastic layer, a main layer of Bruchs membrane, on their surface and showed outward deposition of lipoproteins with apoB-100. TEM revealed widely spaced collagen, which was identified as condensation of collagen fibrils by SEM. SEM showed deposition of membranous debris and lipid particles, which have been observed in human Bruchs membrane. Western blotting showed expression of RPE differentiation markers and components of Bruchs membrane and RPE lipoproteins. CONCLUSIONS This model provides direct views of epithelialization processes involving elastogenesis and functions at the basolateral side such as lipoprotein deposition and may elucidate not only unknown epithelial behaviors but also the pathogenesis of RPE-related diseases.