title: Three-dimensional spheroidal culture visualization of membranogenesis of Bruch's membrane and basolateral functions of the retinal pigment epithelium.
abstract: OBJECTIVE
  Aging changes in the RPE involve lipid accumulation and membranous basal deposits onto the underlying Bruch's 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 Bruch's 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 Bruch's membrane. Western blotting showed expression of RPE differentiation markers and components of Bruch's 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.
authors.affiliation: Department of Ophthalmology and Visual Science, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
authors.forename: Rina
authors.initials: R
authors.lastname: Sato
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Yasukawa
authors.forename: Johannes
authors.initials: J
authors.lastname: Kacza
authors.forename: Wolfram
authors.initials: W
authors.lastname: Eichler
authors.forename: Akiko
authors.initials: A
authors.lastname: Nishiwaki
authors.forename: Ianors
authors.initials: I
authors.lastname: Iandiev
authors.forename: Masaharu
authors.initials: M
authors.lastname: Ohbayashi
authors.forename: Aki
authors.initials: A
authors.lastname: Kato
authors.forename: Yousef
authors.initials: Y
authors.lastname: Yafai
authors.forename: Andreas
authors.initials: A
authors.lastname: Bringmann
authors.forename: Ayae
authors.initials: A
authors.lastname: Takase
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Ogura
authors.forename: Johannes
authors.initials: J
authors.lastname: Seeger
authors.forename: Peter
authors.initials: P
authors.lastname: Wiedemann
mesh_terms: D053299:Apolipoprotein B-100 / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D001896:Boron Compounds; D016570:Bruch Membrane / Q000254:growth & development*; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002470:Cell Survival; D005109:Extracellular Matrix / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D021621:Imaging, Three-Dimensional; D020287:In Situ Nick-End Labeling; D046529:Microscopy, Electron, Transmission; D009024:Morphogenesis / Q000502:physiology*; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000378:metabolism; D018874:Spheroids, Cellular / Q000166:cytology* / Q000378:metabolism
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D053299:Apolipoprotein B-100; D015415:Biomarkers; D001896:Boron Compounds