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.

Cell suspensions containing normal or tumor epithelium were readily obtained by enzymatically digesting rat mammary glands from perphenazine-treated (prolactin-hypersecreting) cycling, female virgin animals or hormone-responsive mammary tumors from animals treated with dimethylbenzanthracene. Cell suspensions were fractionated into predominantly epithelial and predominantly stromal cells by their differential rates of attachment to culture dishes. Both normal mammary and tumor epithelial cells were characterized by the presence of specific celljunctional complexes, desmosome-like structures, surface microvilli, and their ability to synthesize casein. Serum-dependent protease activity was greater in cultures derived from tumors, and cells from such cultures grew in agarose whereas those from the non-neoplastic gland did not. The addition of prolactin to the culture medium stimulated DNA synthesis in primary or secondary epithelial cultures from tumors, whereas additional insulin and hydrocortisone with prolactin were required for similar levels of DNA synthesis in cultures from nonneoplastic glands. The fraction of cells synthesizing DNA was, however, smaller than that with 10% serum measured in the same time period. Both growth hormone and epidermal growth factor stimulated DNA synthesis but to a lesser extent than did prolactin. Prolactin with hydrocortisone and insulin was relatively inactive in promoting DNA synthesis of the nonepithelial cells whereas pituitary fibroblast growth factor was more active. These mitogenic effects were obtained when the hormones were added to the medium at near physiological concentrations, and paralleled the known activities of the hormones in control of mammary gland growth and development in the rat.Growth and development of the mammary gland in mice and rats occur mainly by a process of cell multiplication rather than by hypertrophy of existing cellular units (9). The hormones which control this process in vivo have largely been identified by means of a series of endocrine gland-ablation and hormone-replacement experiments, and they include prolactin, growth hormone together with estrogens, glucocorticoids, and progesterone (18,21,34,35). In organ culture, however, Topper and others have failed to show any mitogenic effect of prolactin or growth hormone although high, nonphysiological concentrations of insulin and a corticoid were shown to stimulate thymidine incorporation into DNA (19,22,33,38,40). Since pretreatment of virgin mice with prolactin

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Increases in uterine capillary permeability after the injection of oestradiol into spayed mice, appeared to be caused by the development of vascular fenestrations and not by the separation of adjoined endothelial cells. Progesterone did not prevent the uterine weight, oedema and vascular responses to the first of two injections of oestradiol but inhibited those to the second. It was concluded that the failure of repeated oestrogen-treatment to produce uterine oedema in progesterone-treated mice resulted from the refractory state which develops after the first injection and which extends to the endometrial vasculature.

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