Abstract
Purpose: :
Retinal pigment epithelium (RPE) plays a key role not only in physiological functions associated with neurosensory retina and choriocapillaris but also in pathogenesis of many diseases such as retinitis pigmentosa and age–related macular degeneration. However, many functions, especially on the basal aspect (the interface with Bruch's membrane), are still to be clarified. The purpose of this study is to develop a new in vitro model to provide a direct view of the basal aspect of RPE.
Methods: :
Multicellular spheroids composed of human RPE cells were made in the modified medium with F10 and methylcellulose in 96–well culture plates with no coating. Spheroids were transferred to a 75–cm2 dish after a week. At weeks 1, 2, 3, and 4, spheroids were collected and fixed with 4% paraformaldehyde. For immunohistochemistry, spheroids were reacted with the 1st antibody against occludin, elastin, or collagen type I or IV. For transmission electron microscopy (TEM), spheroids were post–fixed in 1% osmium tetroxide. Scanning electron microscopy (SEM) was also performed.
Results: :
Spheroids remodeled to establish a surface monolayer of RPE and a center of apoptotic RPE cells. RPE cells in the monolayer were hexagonal and quiescent with tight junctions. The basal surface of RPE was positioned outside of spheroids and constructed a part of Bruch's membrane involving elastic layer and collagenous elements on the surface. TEM revealed widely spaced collagen, which was identified as condensation of collagen type IV fibril by SEM. SEM revealed the deposition of membranous debris and lipid particles with approximately 120–200 nm in diameter, which have been observed in human Bruch's membrane.
Conclusions: :
Spheroids of RPE cells produced the differentiated monolayer of RPE and most elements of Bruch's membrane. This new in vitro model is fruitful to clarify unknown mechanisms of RPE on the basal aspect and helpful to elucidate the pathogenesis of RPE and Bruch's membrane–related diseases involving age–related macular degeneration.
Keywords: age-related macular degeneration • Bruch's membrane • retinal pigment epithelium