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 apoptosis. We investigated whether reattachment of RPE to a substrate will prevent apoptosis.
Methods: Second-passage human RPE cells were plated onto tissue culture plastic precoated with extracellular matrix, fibronectin or laminin, uncoated tissue culture plastic, untreated plastic and untreated plastic coated with 4% agarose. Reattachment rates were determined for each substrate 24 h after plating. The TUNEL technique was used to determine apoptosis rates in attached cells, unattached cells and the entire cell population.
Results: Attachment rates were as follows: ECM-coated tissue culture plastic-->fibronectin-coated tissue culture plastic-->laminin-coated tissue culture plastic-->uncoated tissue culture plastic-->untreated plastic-->agarose-coated untreated plastic. Apoptosis rates for the entire cell population increased as the RPE cell attachment rate decreased. The proportion of apoptotic cells in the entire population was inversely related to the percent 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.