Human epithelial tissue samples are obtained during routine surgical procedures under sterile condiitons or obtained commercially from a reliable, established vendor. Establishment of human epithelial cells in culture is being accomplished according to published methods or other proven techniques. Techniques to isolate and culture human epithelial cells and verify the epithelial nature of the resultant cultures are being used. Pooled tissues are used for a single isolation and early passage frozen cell stocks. An initial cytotoxicity assay is used to assess the relative toxicity of each chemopreventive test agent and to select of approximate concentration ranges for the main cytotoxicity assay(s). Human epithelial cells of sufficient quantities are plated and allowed to attach. Then the cells are exposed to test agents at five concentrations (highest= 1 mM) at log-dilutions from the highest soluble concentration in medium or highest concentration possible without solvent toxicity. At least three dishes/wells are used per treatment point. After an appropriate time period the growth or colony forming efficiency of each treatment group is assessed and compared. The cytotoxicity is determined by its relative growth or colony forming efficiency compared to (solvent) controls. An rough IC50 concentration is determined if possible from analysis of the data. Primary cultures of human epithelial cells are exposed to chemopreventive agents during the assay. The assay for cytotoxicity consists of two endpoints. The culture medium contains the chemopreventive test agent throughout t he exposure period or for limited time periods. Five concentrations at half-log dilutions of the test agent are being assayed for each agent. The positive control cultures are used using the same regimen as the chemopreventive agent. After an appropriate incubation period the cultures are stained and scored for relative growth, differentiation, or other relevant endpoint by appropriate methods. Multiple cultures per condition are being evaluated.
| Elmore, E; Luc, T T; Steele, V E et al. (2001) Comparative tissue-specific toxicities of 20 cancer preventive agents using cultured cells from 8 different normal human epithelia. In Vitr Mol Toxicol 14:191-207 |
| Elmore, E; Luc, T T; Steele, V E et al. (2000) The human epithelial cell cytotoxicity assay for determining tissue specific toxicity. Methods Cell Sci 22:17-24 |
