Keratocyte-populated collagen gel as an in vitro model of excimer laser keratectomy

BACKGROUND: To develop an in vitro model to study the effects of excimer laser keratectomy on corneal stromal cells, we evaluated two types of collagen gel populated with keratocytes. METHODS: Keratocyte-populated collagen gels were prepared with type I collagen in 6-well plates or in culture plate inserts, the bottom of which consisted of a nitrocellulose membrane, contained within 6-well plates. The gels were ablated by the 193- nm excimer laser, set to ablate 50, 100, or 200 μm deep, and was observed under a phase-contrast microscope for 2 days. RESULTS: Keratocytes cultured in collagen gel developed cytoplasmic processes and formed networks of interconnected cells. Cells within the ablated area in the 6-well plates began to lose their cytoplasmic processes and became round approximately 3 hours after excimer laser ablation. These cellular changes were more prominent in the gels ablated to a depth of 200 μm. Cells outside of the ablation zones in the 6-well plates and the culture plate inserts remained intact. CONCLUSIONS: These results suggest the use of keratocyte-populated collagen gel as an in vitro model of cellular response to excimer laser keratectomy and also suggest that gel prepared in culture plate inserts is the preferred method.