Reduction mammoplasty tissue was used to obtain short-term cultures of human epithelial cell populations. Digestion of tissue with collagenase and hyaluronidase resulted in cell clusters (organoids) resembling ductal and alveolar structures; these could be separated by filtration from the stromal components. Epithelial outgrowth from these organoids was greatly enhanced by the addition of conditioned medium from other human epithelial and myoepithelial cell lines. Additionally, the mammary epithelial growth was stimulated by insulin, hydrocortisone, epidermal growth factor, and steroid hormones. With this enriched nutritional environment, active cell division could be maintained for 1 to 3 months and cells could be serially subcultured 1 to 4 times.

We characterized two human cell lines (Hs578T and Hs578Bst), which provide several unique features that should be useful in the study of breast disease. Hs578T, derived from a carcinosarcoma, is epithelial in origin. Hs578Bst, established from normal tissue peripheral to the tumor, is myoepithelial in origin. This is the first report of companion cell lines, one malignant and one normal, established from the same organ.

Nine epithelial cell strains were established from normal mouse liver, mammary gland, ovary, and ear skin. After overnight digestion by collagenase in growth medium, intact glandular fragments produced many islands of epithelial outgrowth when plated in an enriched medium. Contaminating fibroblasts were removed by selective trypsinization. These strains continue to show morphologic and ultrastructural features typical of epithelial cells after 10-50 subcultures. In addition, several strains continue to form secretory vesicles, or hemicysts, on confluent cell sheets. All but 1 strain have maintained a majority population of diploid cells, but all strains show increasing numbers of near-triploid cells. Five strains produced tumors (benign cystadenomas, adenocarcinomas, and sarcomas) when inoculated into isogeneic mice. Type-C oncornaviruses were seen in all but 2

We have qualitatively evaluated the retention of the fluorescent dye rhodamine 123 by malignant or non-malignant breast epithelial cells in passively-infused fresh surgical specimens. Our findings demonstrate a microscopically-visible increase in the ability of primary and metastatic tumor cells to retain the dye, as compared to non-malignant epithelium. Some variability in fluorescence intensity was seen within and between tumor specimens. The optimal length of incubation in the presence of the dye was critical in achieving differential fluorescence intensity between normal and malignant cells. This method of examining rhodamine 123 uptake and retention in tissue explants provides a reliable means for direct, comparative visualization in situ of any tissue and its associated disorders. The results of this study also demonstrate the validity of extending the use of lipophilic, cationic compounds such as rhodamine 123 as antitumor agents, from model systems to the treatment of malignant disease.

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