[CANCER RESEARCH 32, 2407-24-12, November 1972] Growth of Normal Human Mammary Gland Epithelium in Vitro1 B. Allen Flaxman and Eugene J. Van Scott The Skin and Cancer Hospital of Philadelphia, Department of Dermatology, Temple University Health Sciences Center, Philadelphia, Pennsvlvania 19140 SUMMARY gland and used as a specific source of epithelial cells, is described. The cells proliferated readily in vitro and retained A method is described whereby ducts of the nonlactating features of differentiation present in vivo. This system should human mammary gland can be identified in vivo and isolated make it possible to conduct baseline studies of normal so as to serve as a specific source of epithelial cells for studies function and behavior that may be of value in understanding in vitro. The method overcomes the inherent difficulty in factors leading to development of human breast cancer. obtaining epithelial cells from random expiants of nonlactating mammary gland where the duct system is poorly developed. MATERIALS AND METHODS Ducts from the nipple and subjacent mammary tissue were first dissected from the surrounding connective tissue, The clinically normal nipple and subjacent mammary tissue cut into small fragments and explanted on glass coverslips. was obtained from autopsy specimens of 2 postmenopausal Epithelial cells grew out onto the coverslip as a stratified (ages 59 and 79) females. The tissue was removed aseptically, squamous epithelium in which mitoses were numerous. placed in Gey's balanced salt solution, and then bisected. With Ultrastructurally, cells remaining on the expiant and those in the aid of a dissecting microscope, ducts were seen in the outgrowth were characterized by an abundance of longitudinal or transverse section. The connective tissue was cytoplasmic filaments indicating their origin from filament- gently dissected, thus freeing the ducts which were white, firm containing cells normally found in breast epithelium. There tubes that stood out clearly if the dissection was carefully was no convincing morphological evidence of milk synthesis or performed. Some large ducts were 2 mm in diameter and could secretion. Further exploitation of this system may allow be seen without the aid of a microscope. The epithelium had a correlative studies of function and carcinogenesis in the human distinct yellow-orange tint that also aided in identification. mammary gland in vivo and in vitro, studies heretofore not Ducts were excised, transferred to a new dish of balanced salt possible. solution, and excess connective tissue was removed (Fig. 1). Ducts were opened by a longitudinal slit, placed with the INTRODUCTION epithelial surface up, and cut into pieces approximately 3x3 mm. These expiants were then transferred to glass coverslips Experimental analysis of development, function, and on the bottom of plastic Petri dishes and held in place by a carcinogenesis of the mammary gland has been limited mainly clot comprised of 1 drop each of chick plasma and embryo extract (9). Cultures were immersed in Eagle's minimal to nonhuman species such as the mouse, the rabbit, and the cow. Although breast cancer is a major cause of human essential medium containing 10% fetal calf serum and 100 units each of penicillin, streptomycin, and mycostatin per ml. mortality, there is little experimental information on the Cultures were grown in a high-humidity incubator at 37°in an biology of normal human mammary tissue. While numerous attempts have been made to culture cells from a variety of atmosphere of 5% CO2 in air. Culture fluid was changed every benign and malignant human mammary gland lesions (5, 10, 3 to 4 days. 14, 26), few attempts have been made to grow normal For light microscopy, cultures attached to their coverslips epithelium (18, 19). Both explantation and enzymatic were fixed in 10% buffered formalin. The expiants were digestion methods have been used to obtain tumor cells when removed, embedded in paraffin, sectioned, and stained with large numbers of such cells are present. The lactating hematoxylin and eosin. Epithelial cells remaining on the glass mammary gland would provide an abundant supply of were stained with hematoxylin. epithelial cells, but such material is not readily obtainable for For electron microscopy, cultures were fixed in 2.5% obvious reasons. In the normal, nonlactating mammary gland, glutaraldehyde in 0.1 M cacodylate buffer at room the duct system is not well developed; hence, the number of temperature for 1 hr. They were washed in buffer, postfixed in epithelial cells is minimal and explantation or enzymatic a 1:2 mixture of Os04 in collidine (2), dehydrated through digestion is most likely to yield fibroblasts rather than alcohol and propylene oxide, removed from the glass coverslip, epithelial cells. A method that circumvents this problem, in and embedded in Araldite. which individual ducts were dissected from the mammary 'Supported by Grant l PO l CA 11536 from the National Cancer RESULTS Institute, and Grant l PO l AM 1551-01 from the National Institute of Arthritis and Metabolic Diseases. Duct Epithelium in Dissected Specimens. In sections cut Received June 16, 1972; accepted July 28, 1972. perpendicular to the surface, the epithelium was comprised NOVEMBER 1972 2407 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1972 American Association for Cancer Research. B. Allen Flaxman and Eugene J. Van Scott predominantly of a double layer of cuboid cells (Fig. 2). In growth of which is oriented toward connective tissue (expiant) sections that seemed to have been cut more tangentially, the and toward a nonliving surface (glass coverslip). This should epithelium appeared thicker. make possible experiments designed to elucidate those aspects Epithelial Outgrowths. Twenty-seven expiants were cultured of normal mammary gland epithelial cell behavior that are for 10 days. In all cases ductal epithelial cells grew onto the connective-tissue dependent. Epithelial cell outgrowths from surface of the glass coverslip. Sections of cultures showed fragments of normal human mammary gland have been direct continuity of the outgrowing cells with epithelial cells reported before (18, 19), but the exact origin of the cells, on the expiant (Fig. 3). Outgrowing cells formed a ring of whether from terminal ductules or larger ducts, was not epithelium surrounding the expiant (Fig. 4). Within the known. Information regarding the region of the ductal tree epithelium, cells were mainly polygonal and closely adherent from which the epithelium was obtained may be important to one another (Fig. 5). The amount of overlap was variable with respect to an understanding of epithelial carcinogenesis. for, while the epithelium seemed to be monolayer at the Histological and ultrastructural characteristics of the human periphery, centrally it appeared stratified. Mitoses were mammary gland during development and in response to numerous (Fig. 5), and the mitotic index was 32 ±7/1000 in hormonal stimulation have not been adequately studied. As a 10-day-old cultures. The sheet of cells enlarged radially during consequence, there is a lack of baseline information with the 10-day period of growth. In 7 cultures fibroblasts became regard to the "normal" ultrastructural appearance. The ductal numerous but grew mainly outside the ring of epithelium, the system starts at the skin level as large ducts in the nipple, growth of which did not seem to be affected. In the remaining branches downward into smaller ductules and, during cultures, few or no fibroblasts emerged. pregnancy, presumably, develops terminal alveoli. In ultra- Ultrathin sections for electron microscopy cut vertical to structural studies of terminal ductules, different authors have the surface of the outgrowth showed that the cells formed a not been able to agree on the number of cell types comprising stratified squamous epithelium (Fig. 6). The flattened cells the epithelium (22-24). Whether differences are real is not were piled 2 to 5 layers thick. In some regions cell contact was clear, since the hormonal status of the women from whom quite intimate; elsewhere large spaces were present. The material was obtained was not known. In at least one of the plasma membrane often showed short villous projections. Cells published studies (22), however, a cell has been described were connected by fairly numerous desmosomes with attached which contains bundles of cytoplasmic filaments resembling tonofilaments. Similar filaments were also present in the epidermal tonofilaments. These cells were connected by cytoplasm. Mitochondria, smooth and rough endoplasmic desmosomes. The majority of cells observed in the present reticulum, and free ribosomes were present. Round, dense experiments had similar characteristics, and it seems possible bodies, probably representing lipid, were found in some cells. that they were derived from the filament-containing cells of Many vesicles were also present. Evidence of protein secretion the duct. was not found. There was no convincing morphological evidence for Expiants. The behavior of the epithelium was variable. In 2 secretion by cells either on the expiant or in the outgrowth. cases the cells retained a cuboid or columnar shape and a Such evidence of secretion would include the presence of large certain amount of upward proliferation appeared to have amounts of rough and smooth endoplasmic reticulum, vesicles occurred (Fig. 7). Mitoses were present. In the remaining containing milk protein, and lipid (8, 17). While