Abstract
P 072
Multicellular spheroid culture of retinal pigment epithelial cells enhancing differentiation as an epithelium and formation of Bruch's membrane
Tsutomu Yasukawa1, Peter Wiedemann2, Wolfram Eichler2, Yousef Yafai2, Akiko Nishiwaki1, Andreas Bringmann2, Yuichiro Ogura1
1Ophthalmology and Visual Science, Department of Ophthalmology, Nagoya City University Hospital, Nagoya, Japan; 2Klinik und Poliklinik für Augenheilkunde, Universitätsklinikum Leipzig, Leipzig
Objective
Retinal pigment epithelium (RPE) plays an important role in retinal physiology. Its disorder results in a variety of retinal diseases involving age-related macular degeneration (AMD). RPE cells tend to dedifferentiate and transdifferentiate into myofibroblasts in the conventional culture, which is a critical demerit in investigating physiological functions. The objective of this study is to develop a three-dimensional culture system to enhance differentiation of RPE cells as an epithelium.
Methods
Isolated human RPE cells were cultured with viscotic medium on a 96-well round bottom culture plate, allowed to form a spheroid. Light and electron microscopies were performed. Western blotting was carried out to detect elastin, a component of Bruch's membrane and apoB-100 in RPE-derived lipoproteins.
Results
RPE cells per well were aggregated into a sphere. This spheroid is composed of inner material derived from apoptotic cells and a monolayer of RPE cells on the outer surface. A monolayer or RPE cells was well-differentiated as an epithelium, acccompanying Bruch’s membrane on the most-outer surface. Bruch’s membrane was composed of type I and IV collagens and elastic fibers. Clustered lipoproteins expressing apoB-100 were observed deposited across Bruch’s membrane.
Conclusions
A spheroid reproduced well-differentiated epithelium of RPE cells with the basal side out. This 3D-culture system may have a great poteintial to clarify unknown basal functions of RPE and pathogenesis of AMD and other RPE-related diseases.
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