Impact of photodynamic inactivation (PDI) using the photosensitizer chlorin e6 on viability, apoptosis, and proliferation of human keratocytes in vitro

Photodynamic inactivation (PDI) may be a potential alternative in cases of therapy-resistant infectious keratitis. The purpose of our study was to determine the impact of PDI using the photosensitizer chlorin e6 (Ce6) on viability, apoptosis, and proliferation of human keratocytes, in vitro. Primary human keratocytes were isolated by digestion in collagenase (1 mg/ml) from human corneal buttons, and cultured in DMEM/Ham's F12 medium supplemented with 10 % FCS. Keratocyte cell cultures underwent illumination using red (670 nm) light for 13 min following exposure to 50 nM to 64 mu M concentrations of Ce6 in the culture medium. Twenty-four hours after PDI, cell viability was evaluated by the Alamar blue assay, total DNA content of the cells and apoptosis using the APO-DIRECT (TM) Kit, and cell proliferation by the BrdU Cell Proliferation Assay Kit. Using Ce6 or illumination only, we did not detect significant changes of cell viability, apoptosis, and proliferation. Using illumination, viability of keratocytes decreased significantly above 100 nM (P < 0.01), and proliferation at 250 nM Ce6 concentration (P = 0.01) and the percentage of apoptotic keratocytes increased significantly at 500 nM (P < 0.01) concentration. In the short term, photodynamic inactivation using Ce6 decreases viability and proliferation, and also triggers apoptosis of human keratocytes, in vitro.