The Center for Alternatives to Animal Testing is an academic center affiliated with the Division of Toxicological Sciences in the Department of Environmental Health Sciences of the Johns Hopkins University Bloomberg School of Public Health.

 

Johns Hopkins School of Public Health

Research Grants 1999-2000

Development of Reversibly Transformed Human Corneal Epithelial Cells as an Optimal In Vitro Model

Steven E. Wilson, M.S., MD
University of Washington, Seattle, Washington

Appropriate in vitro models could markedly reduce animal usage in testing for irritation or injury during safety evaluation of consumer products. A promising new in vitro test has recently been developed, the transepithelial permeability to fluorescein (TEP) test. In this model, the barrier function to fluorescein of a multi-layered culture of transformed human corneal epithelial cells is analyzed to model corneal epithelial injury in the living eye. The available transformed human corneal epithelial cells, however, continuously express the transforming genes that extend the life span of the cells so that sufficient cells are produced for study. The genes used (such as SV40 large T antigen) extend the life span of cells by blocking important regulators of cell proliferation, apoptosis, and differentiation. It is unlikely that these cell lines could provide an optimal model for a critical differentiated function of the corneal epithelium such as barrier function since differentiation is affected. The aim of this study is to develop methods to genetically engineer human corneal epithelial with DNA sequences that allow the transforming genes such as SV40 large T antigen to be turned on to grow the cells and off to allow the cells to normally differentiate for testing. Optimal cells will be studied by evaluating normal functions of differentiated corneal epithelial cells such as differentiation markers and normal responses to growth factors that stimulate proliferation. Finally, we will test the utility of the engineered cell lines in the TEP test to ascertain whether they provide a good model for pharmacological testing.