Isolation and differentation of cloned epithelial cell lines from normal rat mammary glands

Summary Single-cell-cloned cell lines have been established from primary cultures of neonatal rat mammary glands. A representative cuboidal cell line, Rama 704, shows the presence of intermediate filamental proteins keratin and vimentin, and occasional cells express milk fat globule membrane antigens on their apical surfaces. Rama 704 cells grow as a cuboidal pavement in culture and produce hemispherical blisters or domes when confluent. Noteworthy ultrastructural features are the presence of junctional complexes, desmosomes, and apical microvilli typical of epithelia. Cells seeded within floating collagen gels with form a variety of multicellular outgrowths, some of which are ductlike in morphology and are composed of polarized cells surrounding a central lumen. The cuboidal cells produce elongated cells under conditions of high cell density and also when cells float off collagen gels and reattach to the plastic substrate. The former elongated cells have been cloned and three cel lines established: Rama 710, 711, and 712; the latter uncloned elongated cells are termed Rama 704E. The cloned elongated cells show an increase in the amounts of basement membrane proteins deposited, a lack of junctional complexes and microvilli, and an increase in the amount of rough endoplasmic reticulum compared with their parental cells. Rama 704E cells show an enhanced deposition of basement membrane proteins and increased amounts of actin in the cytoplasm over the elongated cell lines and contain microfilaments and pincocytotic vesicles similar to those seen in myoepithelial cells. All the elongated cells and lines fail to form ductlike structures within collagen gels. None of the cell lines form tumors in syngeneic rats although they all produce some tumors in nude mice, which are composed of cords of epithelioid cells and spindle cells in varying proportions. In addition, some of the Rama 704 tumors contain rhabdomyoblastic elements that penetrate the host fat pad. This is the first report of the isolation and characterization of a stable cuboidal cell line from a neonatal rat mammary gland. The Rama 704 cell line shows morphological and biochemical features of mammary epithelial cells and converts at high cell density to elongated cells that have also been cloned.     

Isolation and properties of rat cell lines morphologically intermediate between cultured mammary epithelial and myoepithelial-like cells

The cloned cuboidal epithelial cell line Rat Mammary (Rama) 25 converts at low frequency in culture to elongated cells that possess some of the properties of myoepithelial cells; one such clonal cell line is termed Rama 29. Three morphologically intermediate clonal cell lines have been isolated from Rama 25 which form a morphological series in the order: Rama 25 cuboidal cells, Rama 25-Intermediate 2(I2), Rama 25-I1, Rama 25-I4, and Rama 29 elongated cells. This same order is largely maintained for increasing percentages of elongated cells, decreasing percentages of cuboidal cells, decreasing tubular structures on collagen gels, and increasing times of appearance of tumors in nude mice. The fully elongated cells fail to revert to cuboidal cells and to form tumors. Binding of antisera to epithelial-specific milk fat globule membranes and human keratin declines whereas binding of antisera to myoepithelial-associated laminin, vimentin, and Thy-1 increases in the cell lines in the same order. Similarly 7 polypeptides characteristic of elongated cells increase and 4 polypeptides characteristic of cuboidal cells decrease in the cell lines in the same way. Anti-actin serum binds equally to all cell lines grown on plastic, except for Rama 25-I4, where its binding is increased. Rama 25-I1 and Rama 25-I4 cells also give rise to anti-actin, anti-myoglobin, and phosphotungstic acid hematoxylin-staining giant, striated cells on collagen gels and in tumors that also have ultrastructural characteristics of skeletal muscle. Fresh elongated converts of Rama 25 bind appreciably more anti-actin serum than many of the clonal elongated cell lines such as Rama 29. Ultrastructural analysis confirms the gradual loss of epithelial characteristics and the acquisition of immature myoepithelial characteristics in the same sequence of cell lines. It is suggested that such a linear sequence of intermediate morphological states occurs between the Rama 25 cuboidal cells and the elongated myoepithelial-like cells in vitro, and that a similar morphological sequence may exist in terminal ductal structures in vivo.     

Morphogenetic behavior of simian virus 40-transformed human mammary epithelial stem cell lines on collagen gels

Summary Transformation of primary cultures of human breast cells with simian virus 40 and clonal selection has yielded single-cell-cloned, epithelial cell lines, as well as myoepithelial-related cell lines. When grown on floating collagen gels, the epithelial cell lines give rise to branching rays of cells, thick fingerlike protrusions, saclike structures, and degenerating areas. The myoepithelial-related cell lines give rise only to the branching rays. Epidermal growth factor stimulates the production of the thick protrusions, whereas cholera toxin stimulates the production of the degenerating areas. Immunocytochemical staining of these cultures using reagents directed against the cell surface-extracellular matrix or the cellular cytoskeleton confirms the epithelial and myoepithelial nature of the cells, and demonstrates that the degenerating areas are undergoing squamous metaplasia. The fingerlike protrusions consist of cords of cells composed of inner, epithelial and outer, myoepithelial-related cells sometimes surrounding a central lumen reminiscent of ducts. The saclike structures resemble alveoli. Ultrastructural analysis confirms the identification of the basic cell types and also identifies indeterminate cells possessing features of both epithelial and myoepithelial cells. It is suggested that the epithelial cell lines represent human mammary stem cells that can undergo processes of morphogenesis and differentiation in vitro to form many of the three-dimensional structures found within the breast. This work was supported by the North West Cancer Research Fund and the Cancer and Polio Research Fund.     

Effect of cell shape change on the function and differentiation of rabbit mammary cells in culture

We examined the role of cell shape, cytodifferentiation, and tissue topography on the induction and maintenance of functional differentiation in rabbit mammary cells grown as primary cultures on two-dimensional collagen surfaces or in three-dimensional collagen matrices. Mammary glands from mid-pregnant rabbits were dissociated into single cells, and epithelial cells were enriched by isopycnic centrifugation. Small spheroids of epithelial cells (approximately 50 cells) that formed on a rotary shaker were plated on or embedded in collagen gels. The cells were cultured for 1 d in serum-containing medium and then for up to 25 d in chemically defined medium. In some experiments, epithelial monolayers on gels were mechanically freed from the dishes on day 2 or 5. These gels retracted and formed floating collagen gels. On attached collagen gels, flat monolayers of a single cell type developed within a few days. The cells synthesized DNA until the achievement of confluence but did not accumulate milk proteins. No morphological changes were induced by prolactin (PRL). On floating gels, two cell types appeared in the absence of cell proliferation. The cells in direct contact with the medium became cuboidal and developed intracellular organelles typical of secretory cells. PRL-induced lipogenesis, resulting in large fat droplets filling the apical cytoplasm and accumulation of casein and α-lactalbumin in vesicles surrounding the fat droplets. We detected tranferrin in the presence or absence of PRL intracellularly in small vesicles but also in the collagen matrix in contact with the cell layer. The second cell type, rich in microfilaments and reminiscent of the myoepithelial cells, was situated between the secretory cell layer and the collagen matrix. In embedding gels, the cells formed hollow ductlike structures, which grew continuously in size. Secretory cells formed typical lumina distended by secretory products. We found few microfilament-rich cells in contact with the collagen gels. Storage and secretion of fat, caseins and alpha-lactalbumin required the presence of PRL, whereas the accumulation and vectorial discharge of transferrin was prolactin independent. There was no differentiation gradient between the tip and the cent of the outgrowth, since DNA synthesis and milk protein storage were random along the tubular structures. These results indicate that establishment of functional polarity and induction of cytodifferentiation are influenced by the nature of the interaction of the cells with the collagen structure. The morphological differentiation in turn plays an important role in the synthesis, storage, and secretion of fat and milk proteins.     

Different substrates influence the expression of intermediate filaments and the deposition of basement membrane proteins

Summary A primary culture of serous cystadenocarcinoma of the ovary was used to study the expression of intermediate filament proteins and the deposition of basal lamina proteins. It was found that cells grown on type I and IV collagens or in collagen gels failed to express vimentin, which was readily demonstrable in cultures of the same cells grown on plastic or glass. Furthermore cells grown in collagen gels formed colonies demonstrating a cystic architecture Unlike what is commonly observed on glass or plastic where laminin and fibronectin are deposited as disorganized fibrils in the extracellular space, in or on collagen these proteins appear solely at the interface between the epithelial cells and matrix. The results suggest that the extracellular matrix influences the cytoskeletal organization of the intermediate filaments and determines cell polarity. They confirm that collagen substrates permit epithelial cell cultures to progress toward a more differentiated state. Supported by grants from the Italian Assciation for Cancer Research (AIRC).     

Extracellular matrix control of collagen production by rat mammary epithelial and myoepithelial-like cells in vitro

A rat mammary myoepithelial cell line (Rama 401) grown on plastic produces 5 times more collagen (largely type IV) than a mammary epithelial cell line (Rama 704) grown on the same surface. When the cells are grown on collagen gels, the amount of collagen produced by Rama 704 cells increases 3.3 times, whereas there is no increase in collagen production by Rama 401 cells. Increased production of collagen by Rama 704 cells is due to both an increased rate of synthesis and a decreased rate of degradation. These results indicate that for mammary epithelial cells, unlike myoepithelial cells, the rate of production of collagen can be regulated by the extracellular matrix.     

Morphology and lactose synthesis in tissue culture of mammary alveoli isolated from lactating mice

Summary Mammary epithelial cells from lactating mice synthesize and secrete lactose in culture and retain many features of their in vivo morphology if mammary glands are only partially dissociated to alveoli, rather than completely dissociated to single cells. After 5 d in culture lactose synthesis by alveoli cultured on floating collagen gels is 10 to 20 times higher than in cultures of single cells on floating collagen gels. Moreover, mammary alveoli in culture retain sensitivity to lactogenic hormones; the synthesis of lactose by alveoli depends on the continued presence of insulin and either hydrocortisone or prolactin. In addition, within alveoli the original juxtaposition of constituent epithelial cells is retained, and cells are cuboidal and have many microvilli and fat droplets. In contrast, alveoli on attached gels flatten and lose their secretory morphology. These results indicate that the shape of the cells, presence of lactogenic hormones, and maintenance of epithelial:epithelial cell contacts are required for maintenance of mammary epithelial cell differentiation in culture. This research was supported by Grants CA-16392 and AG-02909 from the National Institutes of Health and Institutional Grant IN 119 from the American Cancer Society.     

Expression of epithelial antigens in primary cultures of normal human breast analysed with monoclonal antibodies

Abstract. Primary cultures of normal human breast were stained with monoclonal antibodies to see if antigens characteristic of luminal epithelial cells are retained in culture. Three monoclonal antibodies were used, LICR-LON-M8, LICR-LON-M18, and LICR-LON-M24, all specific for the cell surface of luminal epithelial as opposed to myoepithelial or stromal cells in the breast, and each staining a different subset of the epithelial cells in the intact tissue. Cultures were prepared from reduction mammoplasty samples by digestion with collagenase. The surface layer of cells was stained by immunofluorescence without fixation. (Cells underneath the surface layer were not accessible to this mode of staining). The antibodies stained patches of cells resembling flattened epithelium. These patches of cells cannot be distinguished by phase contrast microscopy without reference to the staining, in fact the boundaries of the cells are not usually resolved by phase contrast microscopy. Electron microscopy of sections through these cells show they are very flattened. They lie on top of the polygonal and elongated cells that dominate the phase contrast image. Two of the antibodies, M8 and M24, stain subsets of these epithelial-like cells at all stages of culture. The third antibody, Ml8, stains such cells initially, but after the first few days staining is predominantly found on the polygonal and elongated cells, then this also gradually disappears. It is possible that the cells stained by antibody Ml8 are converting from the epithelial-like morphology to the cuboidal and elongated morphology. Many cells are not stained by any of the antibodies, so appear either to by myoepithelial in origin or to have lost their luminal epithelial surface antigens at an early stage. This analysis draws attention to the variety of cell types in these cultures and the limitations of phase contrast microscopy as a means of analysing them.     

Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes

Summary 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 continuous 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 or luminal surface and a basal lamina separates the epithelial components from the gel and stromal cells. Occasinal myoepithelial cells, characterized by myofilaments and plasmalemmal 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 T₀ 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. The work was supported by USPHS Grants CA-05388 and CA-05045 from the National Cancer Institute, DHEW.     

Expression of epithelial antigens in primary cultures of normal human breast analysed with monoclonal antibodies

Primary cultures of normal human breast were stained with monoclonal antibodies to see if antigens characteristic of luminal epithelial cells are retained in culture. Three monoclonal antibodies were used, LICR-LON-M8, LICR-LON-M18, and LICR-LON-M24, all specific for the cell surface of luminal epithelial as opposed to myoepithelial or stromal cells in the breast, and each staining a different subset of the epithelial cells in the intact tissue. Cultures were prepared from reduction mammoplasty samples by digestion with collagenase. The surface layer of cells was stained by immunofluorescence without fixation. (Cells underneath the surface layer were not accessible to this mode of staining). The antibodies stained patches of cells resembling flattened epithelium. These patches of cells cannot be distinguished by phase contrast microscopy without reference to the staining, in fact the boundaries of the cells are not usually resolved by phase contrast microscopy. Electron microscopy of sections through these cells show they are very flattened. They lie on top of the polygonal and elongated cells that dominate the phase contrast image. Two of the antibodies, M8 and M24, stain subsets of these epithelial-like cells at all stages of culture. The third antibody, M18, stains such cells initially, but after the first few days staining is predominantly found on the polygonal and elongated cells, then this also gradually disappears. It is possible that the cells stained by antibody M18 are converting from the epithelial-like morphology to the cuboidal and elongated morphology. Many cells are not stained by any of the antibodies, so appear either to by myoepithelial in origin or to have lost their luminal epithelial surface antigens at an early stage. This analysis draws attention to the variety of cell types in these cultures and the limitations of phase contrast microscopy as a means of analysing them.     

The use of colloidal gold complexes in an ultrastructural study of lectin-binding sites and matrix deposition of normal human primary breast epithelium on collagen gels

Summary Colloidal gold probes were used to study the distribution of peanut agglutinin binding sites and the deposition of extracellular fibronectin and type IV collagen in cultured human breast cells grown on type I collagen gels. Qualitative analysis was performed at the ultrastructural level and appraised in relation to the possible role of peanut agglutinin, fibronectin and type IV collagen as functional markers for distinguishing cell types using this methodology.Peanut agglutinin bound to the surface of cuboidal epithelial cells but not on basal, putative myoepithelial cells in the cell islands, suggesting that it may be a useful functional marker. The binding on the epithelial cells was markedly increased by pre-treatment of the cells with neuraminidase. No correlation was seen between the amount of binding and either the surface topography or cellular ultrastructure.Fibronectin and type IV collagen were demonstrated on the fibrillar network left on the collagen gels after removal of the cell sheet. Any cells still adhering to the gel surface showed no evidence of gold probe binding on their upper surfaces. Examination of the under surfaces of the cell sheet showed gold probe binding equivalent to that found on the gels under the cells. However, it was not proven conclusively which cells produce the fibronectin and type IV collagen.     

Distinction between vascular smooth muscle cells and myoepithelial cells in primary monolayer cultures of human breast tissue

Summary We report on the discrimination of vascular smooth muscle cells and myoepithelial cells in primary cultures of human breast tissue. Breast tissue was disaggregated enzymatically and the resulting organoids seeded in monolayer culture on collagen-coated plastic in serum-free medium CDM3a. Two main types of organoids were present after enzymatic digestion. One resembled small blood vessels and the other interlobular ducts or acini of the breast gland epithelium. Within 3 to 8 d after plating the organoids migrated into typical monolayer islets. These monolayer islets were evaluated using phase contrast microscopy and further tagged with monoclonal antibodies for immunocytochemical demonstration of Factor VIII-related antigen, muscle iso-forms of actin, type IV collagen, vimentin, desmin, and keratins. It is concluded that vascular smooth muscle cells resembled myoepithelial cells by expressing vimentin filaments, depositing type IV collagen, and showing immunoreactivity to muscle iso-forms of actin. However, whereas vascular smooth muscle cells were associated with endothelial cells and sometimes expressed desmin, myoepithelial cells appeared together with luminal epithelial cells and expressed cytokeratins. This work was supported by the Danish Medical Research Council, the Danish Cancer Society, the NOVO Foundation, and the Thaysen Foundation.     

Immunocytochemical identification of myoepithelial cells in normal and neoplastic rat mammary glands with the lectins Griffonia simplicifolia-1 and pokeweed mitogen

Peroxidase-conjugated Griffonia simplicifolia-1 (GS-1) and pokeweed mitogen (PWM) histochemically stain only the myoepithelial cells and not the epithelial or fibroblastic cells of rat mammary glands preserved in methacarn or glutaraldehyde and embedded in paraffin. This pattern of staining occurs in other rat exocrine glands except the pancreas, but is the reverse of that seen in most lining epithelium. The histochemical binding of GS-1 and PWM to myoepithelial cells is inhibited specifically by D-galactose and by polymers of N-acetylglucosamine, respectively. GS-1 and its subcomponent, GS-1-B4, also bind to extracellular structures similar to those stained by anti-laminin serum. At the ultrastructural level, both conjugated GS-1 and PWM bind to the plasma membrane of the myoepithelial cells, as well as to the adjacent basement membrane. Non-metastasizing rat mammary tumors produced by dimethylbenz[a]anthracene, by derivative epithelial stem-cell lines, and by a transplantable tumor all contain more elongated myoepithelium-like cells as well as cuboidal epithelium-like cells; both cell types are neoplastic. The more elongated myoepithelium-like cells are stained by GS-1 and PWM, whereas the cuboidal epithelium-like cells are unstained. Moderately and strongly metastatic rat mammary tumors produced by epithelial cell lines and by transplantable tumors, respectively, contain no such neoplastic cells that bind either lectin. We suggest that the carbohydrate receptors for GS-1 and PWM are consistent markers for the presence of the myoepithelial cell in normal and tumorous rat mammary glands.     

Production of albumin and α-fetoprotein in primary culture of fetal human liver cells on collagenous substrata in the presence of hydrocortisone

Summary When primary cultures of fetal human liver cells established on type I collagen gels were compared to sister cultures developed on tissue culture plastic, the cells in contact with type I collagen secreted albumin at a higher rate than those without contact. The albumin secretion was dependent on the presence of hydrocortisone (HC) in the medium. Also, α-fetoprotein (AFP), of which the level decreased gradually and became undetectable after 6 d regardless of the presence or absence of HC in the cells cultured on plastic, was maintained for longer periods of time by plating the cells on type I collagen gels in the presence of HC. Different secretion rates of albumin and AFP were observed after Day 13 and Day 16, respectively, between cells maintained on type I collagen gels and those on film plastic. The cells secreted larger amounts of both albumin and AFP in plates coated with type IV or I collagens than with fibronectin after Day 10. The cells cultured on type I collagen gels were cuboidal in shape, whereas those on plastic were flattened in cultures with HC. These data indicate that the secretion of human albumin and AFP is facilitated by synergies between HC and collagenous substrata.     

Secretory function of lactating mouse mammary epithelial cells cultured on collagen gels

Mammary epithelial cells dissociated from lactating mouse mammary glands form confluent monolayer cultures on collagen gel substrates. For these cultures, the substrate is more significant than the presence of lactogenic hormones in the maintenance of cell differentiation, as indicated by both morphological and biochemical criteria. Only cells cultured on floating collagen gels are able to maintain their lactose pool over several days in culture, although their ability to synthesize and secrete lactose becomes impaired. These cells are cuboidal in shape. In contrast, cells cultured on attached gels, which are constrained from changing shape and whose basolateral surfaces are inaccessible, lose their differentiation with time in culture. These flattened, dedifferentiated cells respond to the same hormonal environment by showing a mild proliferative response. Therefore, the response of cells to their hormonal milieu may be correlated with their shape: the squamous cells dedifferentiate and proliferate; the cuboidal cells maintain their differentiation and do not proliferate.     

Synthesis of basement membrane proteins by rat mammary epithelial cells

A mammary epithelial cell line, Rama 25, growing on plastic, deposits fibronectin, type IV collagen, and laminin in punctate structures located beneath the basal surface of the cells. When grown on the surface of collagen gels, Rama 25 cells deposit these basement membrane proteins in a continuous layer between the basal surface of the cells and the surface of the collagen matrix. Rama 25 cells also penetrate the collagen matrix forming rudimentary duct-like structures. These structures are surrounded by a discontinuous layer of basement membrane proteins. The ducts of fetal and neonatal rat mammary glands contain few mature myoepithelial cells and our results suggest that some mammary epithelial cells, in contact with a collagenous stroma, are capable of synthesizing a basal lamina-like structure.     

Epithelial organoids and mononuclear phagocytes from DMBA-induced mammary tumors of the rat secrete collagenase in vitro

The production of collagenase has been examined in primary cultures of multicellular epithelial organoids and of stromal cells isolated from DMBA-induced mammary tumors of the rat. Plastic culture dishes and dishes coated with collagen fibrils were used to study the effect of such a substrate on collagenase release. Cultures of 51-μm epithelial organoids consisted of cuboidal cells and a myoepithelial-like cell type which formed a continuous layer under the cuboidal cells. A transient low production of collagenase with an apparent molecular weight (MW) of 72 kD was detected on both substrates. Upon separation by trypsin only cuboidal cells released collagenase. Cultures of 27-μm organoids contained only few myoepithelial-like cells. On plastic, they formed dense monolayers of cuboidal cells and released more collagenase than the greater aggregates. On collagen fibrils, these organoids formed cords and ridges and collagenase production was about 4- to 6-fold higher. These results indicate that collagenase release is influenced by the nature of the interaction of cuboidal cells with the substrate on which they grow. Similar organoids prepared from virgin mammary glands failed to secrete collagenase on either substrate. Primary cultures of stromal cells derived from tumor tissues comprised one basic cell type that expressed a series of properties characteristic for monocytes/macrophages. These cultures were capable of producing collagenase with an apparent MW of 56 kD. Collagenase with a similar size was detected in the extracts of 51 from 65 mammary tumors.     

Ultrastructural features of bovine mammary epithelial cells grown on collagen gels

Bovine mammary epithelial cells cultured on floating gels of rat tail collagen showed two principal cell types, columnar and squamous, with ultrastructural features resembling secretory and myoepithelial cells respectively. Cultures of freshly prepared cells released alpha-lactalbumin into the culture medium and in some cases contained fat droplets, although these did not appear to be released. No ultrastructural evidence of casein synthesis was observed. A notable feature was the failure to secrete a continuous basement membrane. Intermediate filaments were present in abundance in squamous epithelial cells.     

Collagenous substrata regulate the nature and distribution of glycosaminoglycans produced by differentiated cultures of mouse mammary epithelial cells

We have investigated the influence of culture substrata upon glycosaminoglycans produced in primary cultures of mouse mammary epithelial cells isolated from the glands of late pregnant mice. Three substrata have been used for experiments: tissue culture plastic, collagen (type I) gels attached to culture dishes, and collagen (type I) gels that have been floated in the culture medium after cell attachment. These latter gels contract significantly. Cells cultured on all three substrata produce hyaluronic acid, heparan sulfate, chondroitin sulfates and dermatan sulfate but the relative quantities accumulated and their distribution among cellular and extracellular compartments differ according to the nature of the culture substratum. Notably most of the glycosaminoglycans accumulated by cells on plastic are secreted into the culture medium, while cells on floating gels incorporate almost all their glycosaminoglycans into an extracellular matrix fraction. Cells on attached collagen gels secrete approx. 30% of their glycosaminoglycans and assemble most of the remainder into an extracellular matrix. Hyaluronic acid is produced in significant quantities by cells on plastic and attached gels but in relatively reduced quantity by cells on floating gels. In contrast, iduronyl-rich dermatan sulfate is accumulated by cells on floating gels, where it is primarily associated with the extracellular matrix fraction, but is proportionally reduced in cells on plastic and attached gels. The results are discussed in terms of polarized assembly of a morphologically distinct basal lamina, a process that occurs primarily when cells are on floating gels. In addition, as these cultures secrete certain milk proteins only when cultured on floating gels, we discuss the possibility that cell synthesized glycosaminoglycans and proteoglycans may play a role in the maintenance of a differentiated phenotype.