REATTACHMENT TO A SUBSTRATE PREVENTS APOPTOSIS OF HUMAN RETINAL-PIGMENT EPITHELIUM

Background: Epithelial cells generally fail to survive in suspension. Harvesting human retinal pigment epithelium (RPE) for transplantation may separate the cells from their extracellular matrix and induce apop tosis. We investigated whether reattachment of RPE to a substrate will prevent apoptosis. Methods: Second-passage human RPE cells were plate d onto tissue culture plastic precoated with extracellular matrix, fib ronectin or laminin, uncoated tissue culture plastic, untreated plasti c and untreated plastic coated with 4% agarose. Reattachment rates wer e determined for each substrate 24 h after plating. The TUNEL techniqu e was used to determine apoptosis rates in attached cells, unattached cells and the entire cell population. Results: Attachment rates were a s follows: ECM-coated tissue culture plastic --> fibronectin-coated ti ssue culture plastic --> laminin-coated tissue culture plastic --> unc oated tissue culture plastic --> untreated plastic --> agarose-coated untreated plastic. Apoptosis rates for the entire cell population incr eased as the RPE cell attachment rate decreased. The proportion of apo ptotic cells in the entire population was inversely related to the per cent attached cells (r = -0.95). Conclusion: Reattachment of harvested RPE to a substrate decreased the rate of RPE apoptosis in vitro. RPE cells which are removed from their substrate prior to transplantation must reattach rapidly to a substrate to prevent apoptosis.