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 microns 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 microns. 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.