Dissociated normal mammary epithelial cells from prelactating mice were plated on different substrates in various medium-serum-hormone combinations to find conditions that would permit maintenance of morphological differentiation. Cells cultured on floating collagen membranes in medium containing insulin, hydrocortisone and prolactin maintain differentiation through 1 month in culture. The surface cells form a continous epithelial pavement. Some epithelial cells below the surface layer rearrange themselves to form alveolus-like structures. Cells at both sites display surface polarization; microvilli and tight junctions are present at their medium-facing of luminal surface and a basal lamina separates the epithelial components from the gel and stromal cells. Occasional myoepithelial cells, characterized by myofilaments and plasmalemmmal vesicles, are identified at the basal surface of the secretory epithelium. In contrast, cells cultured on plastic, glass or collagen gels attached to Petri dishes form a confluent epithelial sheet showing surface polarization, but lose secretory and myoepithelial specializations. If these dedifferentiated cells are subsequently maintained on floating collagen membranes, they redifferentiate. There is little DNA synthesis in cells on collagen gels, in contrast to Petri-dish controls. Protein synthesis in cells on floating collagen membranes increases over TO values and remains constant through 7 days in culture whereas it decreases on attached gels; however, if the gels are freed to float, protein synthesis increases sharply and parallels that seen on floating membranes.

In cell culture a kidney epithelial cell line, MDCK, forms a continuous sheet of identically oriented asymmetrical cells joined by circumferential occluding junctions. The reconstructed epithelial membrane has transport and permeability qualities of in vivo transporting epithelia.The cell layer can e readily manipulated when cultured on a freely permeable membrane filter and, when placed in an Ussing chamber, electrophysiological measurements can be taken. In the absence of a chemical gradient, the cell layer generates an electrical potential of 1.42 mV, the apical surface negative. It is an effective permeability barrier and lacks significant shunting at the clamped edge, as indicated by a resistance of 84 ohms.cm2, which increased when bulk flow from basolateral to apical was induced by an osmotic gradient or electroosmosis. The MDCK cell layer is cation selective with a relative permeability ratio, PNa/PCI, of 1.7. Net water flux, apical to basolateral, was 7.3 Ml cm2 hr'1 in the absence of a chemical gradient. The morphological and functional qualities of a transporting epithelium are stable in cell culture, and the potentia use of a homogeneous cell nopulation in cell culture would enhance studies of epithelial transport at the cellular and subcellular levels. In 1969 Leighton et al. (1) and Auersperg (2) described the occurrence of domes, turgid, fluid-filled, blister-like hemicysts, in cell cultures of renal and uterine cervix epithelia, respectively. Subsequently, domes have been reported in cell cultures of normal (3) and. neoplastic (4) mouse mammary epithelia, mouse liver (5), human breast adenocarcinoma (6), pig kidney (7), and frog urinary bladder epithelium (unpublished observation). In each instance, the cells cultured were from transporting epithelia. That domes represent a transport phenomenon is suggested by the presence of morpho--logical polarity unique to transporting epithelia (8), apical microvilli extending upward into the medium and occluding junctions joining adjacent cells at the apical-basolateral membrane border. As well, time-lapse photography revealed domes to be localized regions of the cell layer that lift off the culture dish substratum, gradually expand to a maximum, and then rapidly collapse (9). The establishment and characterization of epithelial transport function in cell culture has both experimental and biological significance. The cells were cultured in Waymouth's 752/1 medium (GIBCO) supplemented with penicillin (100 units/ml), streptomycin (100,gg/ml), insulin (26 IU/ml) (11), and 10% (vol/vol) fetal calf serum. Experiments were performed on cultures 4-21 days after plating. Membrane filters, 25 mm (Millipore HAMK 02512), were boiled for 5-10 min to remove the wetting agent and sterilize. The wet filters were affixed to plastic culture dishes by droplets of Millipore Cement Formulation no. 1 applied around the edge to hold the filter flat. The cultured cells were plated directly into the culture dish over the filter. When the cell layer and underlying filter w...

The nature and distribution of cell contacts have been examined in thin sections and freeze-fracture replicas of mammary gland samples from female C3H/Crgl mice at stages from birth through pregnancy, lactation, and postweaning involution. Epithelial cells of major mammary ducts at all stages examined are linked at their luminal borders by junctional complexes consisting of tight junctions, variable intermediate junctions, occasional small gap junctions, and one or more series of desmosomes. Scattered desmosomes and gap junctions link ductal epithelial and myoepithelial cells in all combinations; hemidesmosomes attach myoepithelial cells to the basal lamina. Freeze-fracture replicas confirm the erratic distribution of gap junctions and reveal a loose, irregular network of ridges comprising the continuous tight-junctional belts. Alveoli develop early in gestation and initially resemble ducts. Later, as alveoli and small ducts become actively secretory, they lose all desmosomes and most intermediate junctions, whereas tight and gap junctions persist, The tight-junctional network becomes compact and orderly, its undulating ridges oriented predominantly parallel to the luminal surface. It is suggested that these changes in junctional morphology, occurring in secretory cells around parturition, may be related to the greatly enhanced rate of movement of milk precursors and products through the lactating epithelium, or to the profound and recurrent changes in shape of secretory cells that occur in relation to myoepithelial cell contraction, or to both.

We have develo ed a method for embedding cells within a collagen matrix which allows sustained growth of mouse mammary tumor epithelial cells in primary culture. A characteristic and reproducible pattern of organization and growth occurs: the cells rearrange themselves and produce duct-like structures extending into the matrix, resulting in a three-dimensional outgrowth. Autoradiography showed continuous [3H] The limited in vitro growth capacity of mammary epithelial cells in conventional primary culture in tissue culture dishes is a well-known phenomenon. The cells generally undergo a few rounds of division, but proliferation cannot be sustained nor can these cells be passaged. Mouse mammary epithelial cells cultured at low density become flattened and multinucleated, and rarely do they attain confluence (1, 2). Cells plated at high density are maintained well but little growth is ever achieved.Most studies to date dealing with proliferation of mammary epithelial cells in vitro have used established cell lines adapted to grow in conventional monolayer culture (3-6). Considerable effort is being devoted in several laboratories to analysis of hormone and drug sensitivity in these selected cell lines. An inherent limitation of this approach is that such lines may not be representative of the original cell population. Therefore, it is of considerable importance to improve the conditions for culture of primary cells. Our demonstration of differentiated function in mouse mammary cells cultured on floating collagen gels, as indicated by levels of casein (7), mammary tumor virus (8), and prolactin receptor (9), has prompted us to examine collagen as an appropriate substrate for growth. The most encouraging results were obtained when mammary cells were embedded within the collagen gel. The present study describes (10), and normal mammary gland from C3H/Crgl and BALB/cNIV/Crgl mice were dissociated by a modification of a previously described method (11,12). Briefly, finely minced mammary tissues were placed in 125-or 250-ml erlenmeyer flasks containing 0.1% collagenase (CLS III; 120-150 units/mg; Worthington) in Hanks' balanced salt solution (10 ml/g of tissue) and swirled on a gyratory water bath shaker (model G76, New Brunswick) at 120-150 rpm at 370C for approximately 90 min or until the suspensions were uniform without macroscopic lumps. The suspension was passed through Nitex cloth (mesh size, 150 ,um); the cells were collected by centrifugation at 80 X g for 5 min, and washed twice with Hanks' solution. The resulting preparation consisted mainly of small clumps of cells. Cell number was estimated by mixing 1 vol of cell suspension with 9 vol of 0.02% crystal violet in 0.1 M citric acid and counting stained nuclei in a hemocytometer. Dissociated human mammary epithelial cells were obtained from excised tissue (reduction mammoplasties and mastectomies) by modification of the above procedure and subsequent gradient centrifugation (details will be reported elsewhere).Culture Procedure. Collagen solution and ge...

Cultured on floating collagen membranes in the presence of lactogenic hormones, dissociated normal mammary epithelial cells from prelactating mice acquire the ultrastructural and biochemical characteristics of differentiated mammary secretory cells in vivo. The cells on floating collagen membranes in medium containing insulin alone have sparse secretory organelles, and a small amount of casein can be detected in these cells with a sensitive radioimmunoassay. These cells resemble counterpart cells in early-pregnant mice. When the cells are exposed to insulin, cortisol, and prolactin, the secretory apparatus is elaborated and significant increases in intracellular and extracellular casein are observed. In this environment, the intracellular casein content is generally four to eight times greater than in freshly dissociated cells or cells cultured in insulin alone. The amount of casein secreted into the medium by floating-collagen-membrane cultures in the three hormones is from 25 to 200 times greater than that secreted by cultures in insulin alone. Cells cultured on plastic substrates in either hormone combination fail to show any increase in intracellular or extracellular casein. On floating collagen membranes, the cells differentiate in response to hormones as they do in vivo and in organ culture. This cell-culture system provides an opportunity to study direct effects of environmental factors on mammary differentiation at the cellular level. Preparation of Floating Collagen Membranes. The collagen solution and collagen gels were prepared according to the method of Michalopoulos and Pitot (11) as modified by us (7).Eighteen-twenty hours after the cells were seeded onto collagen-gel-coated dishes, the gels were removed from the plastic substrate by rimming the gels with a scalpel blade and gently

Ca 2+ dependence of occluding junction structure and permeability, well documented in explanted or cultured epithelial sheets, presumably reflects inherent control mechanisms. As an approach to identification of these mechanisms, we induced disassembly of zonulae occludentes in confluent monolayers of mouse mammary epithelial cells by exposure to low concentrations of the chelators, EGTA or sodium citrate. Stages in disassembly were monitored during treatment by phase-contrast microscopy and prepared for transmission and scanning electron microscopy. Cellular response included several events affecting occluding junctions: (a) Centripetal cytoplasmic contraction created tension on junction membranes and displaced intramembrane strands along lines determined by the axis of tension. (b) Destabilization of junction position, probably through increased membrane fluidity, augmented tensioninduced movement of strands, resulting in fragmentation of the junction belt. (c) Active ruffling and retraction of freed peripheral membranes remodeled cell borders to produce many filopodia, distally attached by occluding-junction fragments to neighboring cell membranes. Filopodia generally persisted until mechanically ruptured, when endocytosis of the junction and adhering cytoplasmic bleb ensued. Junction disassembly thus resulted from mechanical tensions generated by initial centripetal contraction and subsequent peripheral cytoskeletal activity, combined with destabilization of the junction's intramembrane strand pattern.

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Application of fragmentation and thin‐sectioning techniques to Tetrakymena pyriformis, Colpidium compiles and Glaucoma chattoni has permitted an analysis of the ultra‐structure of their silverline and fibrillar systems. The classical silverline system consists of a mosaic of flat, membrane‐bound blisters whose rims represent the sites of selective silver deposition. Cilia and protrichocysts emerge between adjacent blisters. I The pellicle consists of the membranes outlining the blisters, overlain by a continuous outer membrane that covers the whole cell and cilia. Fibrillar structures, which are not argentophilic, include: (1) tapering, striated kinetodesmal fibers arising singly from the kinetosomes, passing to the right and anteriad, and overlapping to form a loose bundle accompanying each kinety; (2) a longitudinal fibril band immediately beneath the pellicle at the right of each kinety, consisting of overlapping individual fibrils; (3) a transverse band of fibrils arising at the left side of each kinetosome and passing to the left under the pellicle; and (4) a set of postciliary fibrils arising at the right posterior edge of each kinetosome and passing posteriad under the pellicle. The fibrils of sets (2), (3), and (4) all are about 20 Mμ in diameter and appear tubular in cross‐section; they are very unlike the heavier, solid kinetodesmal fibers. None of the fibril sets directly interconnect, although transverse and postciliary fibrils end in the vicinity of the longitudinal fibril band.