title: In vitro transplantation of fetal human retinal pigment epithelial cells onto human cadaver Bruch's membrane.
abstract: Retinal pigment epithelium transplantation has been proposed as adjunctive treatment for age-related macular degeneration following surgical excision of choroidal neovascular membranes. The goal of this study was to develop a model to evaluate retinal pigment epithelium transplantation onto human Bruch's membrane in vitro. We investigated the ability of cultured fetal human retinal pigment epithelium to colonize human cadaver Bruch's membrane, determined the incubation time needed to form a monolayer and to exhibit apical microvilli and tight junctions, and assessed the production of basement membrane. Freshly enucleated (less than 48 hours old) human eyes were cut through the pars plana, and the anterior segment, vitreous, and retina were removed. The native retinal pigment epithelium was debrided with a surgical sponge. Bruch's membrane and choroid at the macula were trephined with a 7.0 mm diameter trephine and then incubated with 1/2 ml of Dulbecco's modified Eagle's medium +15% fetal calf serum+basic fibroblast growth factor (1 ng ml-1), and fetal human retinal pigment epithelium at a concentration of 242,000 cells ml-1. Specimens were incubated for 1, 4, 6, 8, 12, or 24 hours. The specimens were fixed in half strength Karnovsky's fixative, processed, and analysed with scanning and transmission electron microscopy. The retinal pigment epithelium covered the debrided macular specimens to different degrees at different incubation times. After 1 hour, the cells started to attach and flatten (median percent coverage: 78%). The extent of Bruch's membrane coverage by fetal retinal pigment epithelium varied greatly between specimens. After 4-6 hours, the cells covered the entire debrided surface in a monolayer (median percent coverage: 97.2% at 4 hours, 99.8% at 6 hours). Tight junctions were observed, and the cells had few apical microvilli. The lateral cell borders were obliquely oriented with respect to Bruch's membrane, and the nuclei were elongated, exhibited prominent nucleoli, and were oriented parallel to Bruch's membrane. After 6-8 hours, cells started to become hexagonal (median percent coverage at 8 hours: 99.97%). Cells attached to the inner collagenous layer tended to be flatter than cells attached to residual native basement membrane. At 12 and 24 hours, expression of hexagonal shape, tight junctions, and apical microvilli were observed more frequently (median percent coverage: 99.87% at 12 and 100% at 24 hours). No newly formed basement membrane was observed at these time points. In separate experiments comparing attachment in the presence and absence of native RPE basement membrane, the presence of native retinal pigment epithelial basement membrane promoted the early attachment of the cells and more rapid expression of normal morphology. This in vitro system provides a reproducible way to study the adherence of retinal pigment epithelium to normal and diseased human Bruch's membrane.
authors.affiliation: University of Medicine and Dentistry, New Jersey Medical School, Department of Ophthalmology, 90 Bergen Street, DOC 6th floor, Newark, New Jersey, 07103-2499, USA.
authors.forename: A A
authors.initials: AA
authors.lastname: Castellarin
authors.forename: I K
authors.initials: IK
authors.lastname: Sugino
authors.forename: J A
authors.initials: JA
authors.lastname: Vargas
authors.forename: B
authors.initials: B
authors.lastname: Parolini
authors.forename: G M
authors.initials: GM
authors.lastname: Lui
authors.forename: M A
authors.initials: MA
authors.lastname: Zarbin
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D016570:Bruch Membrane / Q000166:cytology / Q000601:surgery* / Q000648:ultrastructure; D002102:Cadaver; D018929:Cell Culture Techniques; D002455:Cell Division; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D008297:Male; D008854:Microscopy, Electron; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000196:embryology / Q000637:transplantation*