Abstract
Purpose :
To test whether the survivability and surface coverage of human RPE on aged BM can be restored by repairing the age-related defects with patch grafting BM harvested from young donors.
Methods :
BM explants were harvested from freshly enucleated (<24 hours) three aged (>65) and three young (<50) adult human eyes. Defects in age-related macular degeneration were mimicked by flattening the old BM on a HTTP membrane and stripping away the RPE basal lamina mechanically. Explants were then stabilized on 4% agarose in 30 mm petri dishes exposing the aged inner collagenous layer onto which two 8-mm patch grafts of young donor BM was attached using a biocompatible photopolymerizable gel composed of 100 mg/mL poly(ethylene glycol) diacrylate (PEG-DA) with 10 mg/mL hyaluronic acid (HA, viscosity 5200 mP.sec). After sterilization of the construct, 500,000 viable and synchronized ARPE-19 cells (700 cell/mm2/22% surface coverage) were seeded. RPE cells were fed with Fluoresbrite microsphere beads and surface coverage was calculated by image analyses of the fluorescence microphotographs taken at regular intervals for up to 2 weeks. RPE plated onto treated culture plates served as control. Results were also confirmed with light microscopy.
Results :
ARPE-19 cells did not attach to agarose. Cells attached initially onto aged BM but remained round, eventually detach from the surface and failed to cover the aged BM. RPE cells that fell onto grafted young donor BM revealed the high attachment rates and form confluent sheets over the young BM patch graft on day 12. Surface coverage on aged BM, young BM patch grafts and tissue culture plate on days 4 (4.8±0.5%, 11.3±0.2%, 14.9±0.3%), 6 (0.1±0.01%, 57.8±0.2%, 99.9±3.6%) and 12 (0.0±0.0, 98.7±1.5%, 100±4.3%) indicated that cells plated on young BM patches attach and cover the surface comparable to tissue culture plastic. However, after confluence RPE cells do not cross the boundaries between the aged and young grafts to cover the aged BM. Grafted BM patches remain flat and attached to aged-BM throughout RPE proliferation and surface coverage.
Conclusions :
RPE transplantation strategies should involve methods to repair aged BM for graft survival. Age-related defects of BM can be repaired by young BM patch grafts. These patch grafts can increase survival and surface coverage of transplanted RPE.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.