Early Attachment of Uncultured Retinal Pigment Epithelium from Aged Donors onto Bruch's Membrane Explants

Abstract

Retinal pigment epithelium (RPE) transplantation might replace cells lost as a consequence of choroidal neovascular membrane excision in patients with age-related macular degeneration (AMD). Autologous transplantation of RPE cells harvested from a peripheral biopsy may overcome problems of immune rejection. To study the feasibility of autologous RPE cell transplantation, the authors examined the attachment of freshly harvested RPE cells from aged donors onto Bruch's membrane explants, debrided to (1) remove or (2) preserve the RPE basement membrane. Human retinal pigment epithelial sheets were harvested from adult donor eyes (N=12, mean age 79.00±9.40 years) and, following incubation in collagenase, were mechanically fragmented into microaggregates. Microaggregates (approximately 120000 cells) were seeded onto the paired explants (7mm diameter) and incubated for 20min, 1, 4, or 24hr at 37°C. The percent coverage of the debrided surface by microaggregates was determined by sampling the center of the explants with scanning electron microscopy. RPE microaggregate attachment to Bruch's membrane was significantly greater at all time points analysed in samples with intact basement membrane versus those with an exposed inner collagenous layer. Coverage of debridements retaining intact RPE basement membrane was 1.83±1.10% at 20min, 3.54±2.14% at 1hr, and 8.68±2.63% at 4hr. Coverage of debridements lacking basement membrane was 0.10±0.04% at 20min, 0.39±0.25% at 1hr, and 0.63±0.42% at 4hr. Based on their morphologic appearance, many cells were dying as early as 1hr following seeding. To increase surface coverage, the authors seeded four times the above number of cells and incubated the specimens for 1hr. Coverage on explants lacking RPE basement membrane showed no increase in the number of cells attached to the inner collagenous layer. There was a significant approximately three-fold increase in the number of cells attached in the presence of basement membrane. These results indicate that if RPE cells from aged human donors are used for transplantation, some modification of the Bruch's membrane surface or the cells must be considered for cell attachment and eventual cell survival.