Cellular photoablation to control postoperative fibrosis in filtration surgery: in vitro studies

The purpose of this study was to evaluate the feasibility of cellular photoablation using fluorescence-generated photoreaction products as a method to control postoperative fibrosis in filtration surgery. The fluorescent probe, 2', 7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM) is a cell membrane permeable compound rendered membrane-impermeable and fluorescent upon cleavage by intracellular esterases. Human scleral and Tenon's capsule fibroblasts were cultured and used as the target cells. Uptake and retention of the probe were determined with a fluorescence multi-well plate reader. Fibroblasts with or without intracellular probe were irradiated under conditions of fluorescence microscopy with diffuse blue light (450-490 nm, 1.68x10(2)mW m(2-1)). The viability of cells was examined by trypan blue exclusion and crystal violet test. To better mimic a wound healing process the effect of cellular photoablation was verified in artificial lesions produced in cultured monolayers loaded with different concentrations of the probe. Uptake and retention of BCECF-AM is dependent on ambient concentration. When incorporated the probe is lethal to those cells exposed to the appropriate photo-irradiation. Cells exposed to BCECF-AM (for 45 min) at a concentration of approximately 10 microm and irradiated for 1 min resulted in 100% cell death. Cellular photoablation in contrast to chemotherapeutic agents acts only on the targeted cells. This method shall be pursued as an alternative therapy to control postoperative fibrosis in filtration surgery.