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 2002-2003

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

Steven E. Wilson, MS, MD
University of Washington, Seattle, Washington

Many potential drug and cosmetic products must be tested for ocular toxicities prior to evaluation in humans. In many cases, living animal models are utilized for testing these substances. Since the corneal epithelium is one of the primary tissues that can be injured by drugs or cosmetics, it is our hope that an alternative in vitro testing strategy can be developed that will spare large numbers of experimental animals. Previous investigators have developed corneal epithelial cell lines from animal and humans. However, these previous cell lines have been continually (constuitively) transformed with specific genes such as SV 40 T antigen. Unfortunately, permanent continual expression of those proto-oncogenes results in loss of the normal differentiated state in these epithelial cells.

With the funding from CAAT, we have already developed methods for reversible transformation of human corneal epithelial cells. We have demonstrated that these cells can be induced to proliferate when provided with an inducer triggering expression by the vector, but that the cells assume a more differentiated cell type when the inducer is removed from the culture.

In the last year of the study, it is our intention to utilize a different transforming gene called te lomerase that is present, but closely regulated, in normal human cells and other animal cell types that undergo continuous proliferation. This normal gene is less likely to produce chromosomal abnormality in the epithelial cell lines. We will generate regulatable vectors that express this telomerase gene and insert these into cultured human corneal epithelial cells. We will study normal differentiated functions to select appropriate cell lines that will be of special importance for use in toxicity testing and other applications in which cultured human corneal epithelial cell lines are needed for research.