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.

Research Grants 2001-2002

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

For molecular and cellular investigations, as well as toxicity testing, it is frequently useful to have available large quantities of normal human corneal epithelial cells. Unfortunately, sufficient tissue to grow large quantities of these cells are not available. In addition, the expense involved in generating large numbers of primary cells, even if tissue were available, is prohibitive. In the past, several epithelial cell lines have been produced, both in our laboratory and the labs of others, by expressing oncogenes such as SV40 large T antigen, or E6/E7. Unfortunately, all of these cell strains formed, to date, have constitutively (constantly) expressed these oncogenes. Since these oncogenes influence the function of several proteins involved in terminal differentiation and performance of normal functions, for many applications these cells have not been optimal. Our goal in this study is to use a retroviral vector system to transduce E6/E7 into normal human corneal epithelial cells using a vector that can be regulated through the presence or absence of a non-toxic chemical called doxycycline.

We have been successful in generating several strains of these human corneal epithelial cells. We have demonstrated the expression of E6/E7 occurs in the presence of doxycycline, but not in its absence.

We are in the process of evaluating additional epithelial cell functions in these cell lines, including transepithelial permeability to fluorescein and transepithelial electrical resistance in the presence or absence of doxycycline. Epithelial barrier function is an important property of normal corneal epithelium. This is critical in control of corneal hydration, as well as preventing penetration of harmful substances into the cornea. Retention of normal barrier functions by these transduced epithelial cell strains will indicate that these cells are useful for toxicity investigations, as well as cell and molecular studies of epithelial cell function.