title: Amniotic membrane maintains the phenotype of rabbit retinal pigment epithelial cells in culture.
abstract: The success of surgical removal of choroidal neovascularisation followed by transplantation of autologous retinal pigment epithelial cells (RPE) for age-related macular degeneration (ARMD) may be limited by damage in Bruch's membrane. We investigated whether amniotic membrane (AM) might be used as an alternative basement membrane-containing matrix to support RPE growth and differentiation. Primary RPE plastic cultures were established from freshly enucleated Dutch belted rabbit eyes in DMEM/F12 containing 0.1 mM Ca(++) and 10% dialysed FBS. Upon subconfluence, cells were subcultured at 5000-9000 cells cm(-2) in the above-mentioned culture medium on intact AM (iAM), epithelially denuded AM (dAM) or plastic. After confluence, the Ca(++) concentration in the medium was increased to 1.8 mm for 4 weeks. Growth and morphology were monitored by phase contrast microscopy, and the phenotype by immunostaining with antibodies against cytokeratin 18, tight junction protein ZO-1, and RPE65 protein, and by transepithelial resistance (TER) measurement. Immunostaining to cytokeratin 18 confirmed the epithelial origin of isolated cells in both primary culture and subcultures. Compared to plastic cultures, RPE increased pigmentation within 24 hr after seeding on AM, with iAM being more pronounced than dAM. RPE adopted a hexagonal epithelial phenotype with more organised pigmentation, strong expression of ZO-1 and RPE65, and a significantly higher TER 4 weeks after Ca(++) switch on dAM. Our results indicate that AM may be used as a basement membrane-containing matrix to maintain RPE phenotype in vitro, and may facilitate subsequent transplantation to treat ARMD.
authors.affiliation: L. Boltzmann Institute for Retinology and Biomicroscopic Lasersurgery, Juchgasse 25, 1030 Vienna, Austria.
authors.forename: Boris V
authors.initials: BV
authors.lastname: Stanzel
authors.forename: Edgar M
authors.initials: EM
authors.lastname: Espana
authors.forename: Martin
authors.initials: M
authors.lastname: Grueterich
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Kawakita
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Parel
authors.forename: Scheffer C G
authors.initials: SC
authors.lastname: Tseng
authors.forename: Susanne
authors.initials: S
authors.lastname: Binder
mesh_terms: D000650:Amnion; D000818:Animals; D001485:Basement Membrane / Q000166:cytology / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects; D005136:Eye Proteins / Q000032:analysis; D007633:Keratins / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis; D008858:Microscopy, Phase-Contrast / Q000379:methods; D010641:Phenotype; D010750:Phosphoproteins / Q000032:analysis; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000187:drug effects; D011506:Proteins / Q000032:analysis; D011817:Rabbits; D062826:Zonula Occludens-1 Protein
chemical_list: D003470:Culture Media; D005136:Eye Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D011506:Proteins; D062826:Zonula Occludens-1 Protein; D007633:Keratins; D002118:Calcium