Lack of toxicity of methylene blue chloride to supravitally stained human mammary tissues

Methylene blue chloride (MBC) has been used previously as a supravital stain to facilitate the excision and subsequent transplantation of mammary epithelial structures by delineating them from surrounding connective tissue and fat, which stain less intensely. This study was undertaken to determine why MBC selectively stains epithelium and if it has any long-term toxicity to epithelial cells. Light microscopy indicated that MBC was taken up into cell cytoplasm, but neither thin-section nor scanning electron microscopy indicated the mechanism of uptake or cytoplasmic localization. Spectrophotometry indicated that relative MBC uptake was greatest by macrophage and mammary epithelial cell lines and least by endothelial and fibroblast cell lines, offering an explanation for the more intense staining of epithelium. Toxicity of MBC to the cell lines was dose dependent and corresponded roughly to the MBC uptake capacity of the cell lines. Low concentrations were stimulatory to growth of some cell lines in serum-free medium. Comparisons of primary epithelial cultures from 28 pairs of MBC-treated and untreated human breast tissues indicated no significant effect of MBC on the growth of cells of any classification (normal, nonmalignant atypical, and malignant). These results confirm that supravital MBC staining of mammary tissues may be used with confidence that it will not bias the outcome of subsequent experiments requiring healthy, proliferating mammary epithelium.