Impact Of Photodynamic Therapy (PDT) On Viability, Apoptosis And Proliferation Of Human Keratocytes In Vitro

Abstract

Purpose: : Photodynamic therapy (PDT) may be a potential alternative in case of therapy resistant infectious keratitis. The purpose of our study was to determine the impact of PDT on viability, apoptosis and proliferation of human keratocytes in vitro.

Methods: : 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 different concentrations of the photosensitizer chlorin e6 (Ce6) in the culture medium. Twenty-four hours after PDT, cell morphology was evaluated with light microscopy, cell viability by the Alamar blue assay, viability and apoptosis using the APO-DIRECT^TM Kit and cell proliferation by the BrdU Cell Proliferation Assay Kit.

Results: : Using Ce6 or illumination only, we did not detect significant changes of cell morphology, viability, apoptosis and proliferation. Following PDT, keratocytes lost cytoplasm and cell nuclei became condensed. Viability of keratocytes decreased significantly above 50 nM Ce6 concentrations using Alamar blue assay, and above 250 nM Ce6 concentrations using APO-DIRECT^TM Kit (p< 0.05). Proliferation of keratocytes decreased significantly (p< 0.05) using 100 nM and 250 nM concentrations of Ce6 and the percentage of apoptotic keratocytes increased significantly with increasing concentrations of Ce6 from 250 nM to 64 μM (p< 0.05).

Conclusions: : Photodynamic therapy decreases viability and proliferation. In addition, PDT triggers apoptosis of human keratocytes in vitro.

Keywords: photodynamic therapy • cornea: stroma and keratocytes • apoptosis/cell death