Cultivation and characterization of a bovine in vitro model of the cornea.

ABSTRACT

The aim of this study was to develop an in vitro model of the cornea of bovine cells, to characterise the model by histochemical methods and to investigate permeation of ophthalmic drugs through the model. As in the in vivo situation, an in vitro model of the cornea should consist of all three different types of cells. In the current study, the construction of the in vitro cornea was performed using cells prepared from primary cultures. To investigate the state of the cells in the cultures, growth curves were established. Immunocytochemical determination of keratin and vimentin was performed for all three isolated and sub-cultivated cell types of the bovine cornea. To further simulate the in vivo conditions, corneal epithelial cells were seeded onto the collagen-gel base containing the stromal cells with an underlying sheet of endothelium. Permeation experiments were performed with pilocarpine hydrochloride and timolol hydrogen maleate as model drugs and excised bovine cornea and the in vitro cornea as permeation barriers. The immunohistochemical investigations show that excised bovine cornea and the in vitro model of the cornea are comparable with respect to the expression of keratin K3, indicating that the primarily isolated cells correspond to the different cell types of the cornea. Culturing of the epithelial cells on the complex basis has led to the formation of a corneal epithelium with several layers, closely resembling the morphology of the in vivo epithelium. Although the permeation rates of the drug through the in vitro cornea were always higher, the sequence in which the drugs permeate through the two types of barriers was the same. The drug permeation through the in vitro cornea may therefore be a useful predictive tool to estimate the permeability coefficients of drugs through excised cornea.