Epithelial cell interaction in air-liquid interface culture

Summary A novel culture method has been developed to study the interaction of epithelial cells in the absence of a solid substratum. Starting with either a single cell suspension or aggregates, cells were floated at the interface of air and liquid culture medium. Two epithelial cell lines have been studied in this system: Madin-Darby canine kidney cells (MDCK), and a rat bladder tumor cell line (NBT-II). Starting with a single cell suspension of MDCK, the floating cells coalesced in 24 h into sheets of cells. The cells were morphologically polarized with the apical surface facing the liquid medium. Domes were observed regularly in these sheets of cells. NBT-II cells migrated actively from aggregates at the air-liquid interface. In this floating culture, NBT-II cells produced extensive cell processes similar to those seen in cells grown on a solid surface. Because cells at the air-liquid interface lack a solid substratum for adhesion, cell membrane processes such as lamellapodia, retraction fibers, pseudopods, and long, intercellular connections can only exert a tension equal to or less than the surface tension of the liquid. Dimethyl sulfoxide 2% stimulated desmosome formation in floating NBT-II cells, resulting in a cribriform pattern in the sheet of cells. This method of interface can lead to new understanding of morphogenesis of epithelial cells, and the mechanism, of cell motility and formation of cell processess. This research was supported by research grant CA14137 from the National Institutes of Health, Bethesda, MD, and in part by the W. W. Smith Charitable Trust, Rosemont, PA     

Changes in membrane-bound aminopeptidase on bone marrow-derived macrophages during their maturation in vitro

Myeloid colonies obtained by culturing mouse bone marrow cells with mouse lung conditioned medium were kept for up to 21 days in culture and the aminopeptidase content in single cells measured after staining with leucine 4-methoxy-2-naphthylamide. The enzyme was detectable only in mononuclear and not in granulocytic cells. The number of cells carrying the enzyme and the concentration of the enzyme in the mononuclear cells taken from whole dishes or single colonies increased remarkably but not uniformly from 7 days to maximal values at 13 days of culture, and then decreased again. The timing varied for individual colonies. Maximal enzyme concentrations were found in cells intermediate between the center and the fringe of a colony. However, most cells in a given colony showed concentration increases up to 13 days of culturing. During its life span in culture the mononuclear cell appears to gain aminopeptidase at the cell membrane and lose it again prior to death.     

Isolation and characterization of basement membrane and cell proteoheparan sulphates from HR9 cells

The mouse teratocarcinoma cell line HR9 was investigated for proteoheparan sulphate production. Four species of proteoheparan sulphate molecules were isolated and purified to homogeneity. The proteoheparan sulphate isolated from the tissue-culture medium contains four heparan sulphate side-chains of 25 kDa each, and its core protein has an approximate molecular mass of 50 kDa. The proteoheparan sulphates associated with the cells were separated into three individual species: cell proteoheparan sulphate I exhibits structural characteristics which are very similar to the proteoheparan sulphate isolated from the tissue culture medium; cell proteoheparan sulphates II and III contain one heparan sulphate chain of 25 kDa and 20 kDa, and core proteins of approximately 30 kDa and 25 kDa respectively. Antisera, raised against the medium form, react specifically with basement membranes in various tissues by immunofluorescence. This staining pattern was compared to the pattern observed with an antiserum which we have obtained to a proteoheparan sulphate species isolated from the plasma membrane of bovine aortic endothelial cells. The structural and immunological data suggest that basement membrane and plasma membrane proteoheparan sulphates are different biosynthetic products and are not directly related to each other.     

Effects of substrata on the polarization of bovine endometrial epithelial cells in vitro

Summary Epithelial-cell function requires cellular polarity in which apical membrane surfaces have unique characteristics and cellular organelles are stratified. Physiological investigations of endometrial, epithelial cells would be enhanced greatly by the ability of a method to polarize cells in culture. This study investigates the effects of different substrata on polarization of cultured bovine endometrial epithelial cells. Fetal bovine endometrial epithelial-cell lines were developed from explant outgrowth. Epithelial monolayers were subcultured onto amniotic membranes, Millicell-HA membranes, or Millicell-CM membranes coated with rat-tail collagen, Matrigel, laminin, Vitrogen,or fibronectin. Cultures on these substrata were maintained at the air/liquid interface. Cells grown on either collagen-coated or uncoated Milli-cell membranes also were maintained submerged in medium. Excellent polarized morphology was attained in cultures grown at the air/liquid interface on amniotic membranes and rat-tail collagen-coated membranes. Lectin-binding patterns, to apical membranes of polarized epithelial cell cultures paralleled patterns of binding to bovine endometrial surfaces in vivo. Cultures on rat-tail collagen were maintained for several weeks. These methods provide a valuable system for studying the endometrium in vitro.     

Capillary endothelial cell cultures: phenotypic modulation by matrix components

Capillary endothelial cells of rat epididymal fat pad were isolated and cultured in media conditioned by bovine aortic endothelial cells and substrata consisting of interstitial or basement membrane collagens. When these cells were grown on interstitial collagens they underwent proliferation, formed a continuous cell layer and, if cultured for long periods of time, formed occasional tubelike structures. In contrast, when these cells were grown on basement membrane collagens, they did not proliferate but did aggregate and form tubelike structures at early culture times. In addition, cells grown on basement membrane substrata expressed more basement membrane constituents as compared with cells grown on interstitial matrices when assayed by immunoperoxidase methods and quantitated by enzyme-linked immunosorbent inhibition assays. Furthermore, when cells were grown on either side of washed, acellular amnionic membranes their phenotypes were markedly different. On the basement membrane surface they adhered, spread, and formed tubelike structures but did not migrate through the basement membrane. In contrast, when seeded on the stromal surface, these cells were observed to proliferate and migrate into the stromal aspect of the amnion and ultimately formed tubelike structures at high cell densities at longer culture periods (21 d). Thus, connective tissue components play important roles in regulating the phenotypic expression of capillary endothelial cells in vitro, and similar roles of the collagenous components of the extracellular matrix may exist in vivo following injury and during angiogenesis. Furthermore, the culture systems outlined here may be of use in the further study of differentiated, organized capillary endothelial cells in culture.     

Repopulation of a human alveolar matrix by adult rat type II pneumocytes in vitro : A novel system for type II pneumocyte culture

This paper describes the preparation of lung acellular alveolar matrix fragments and culture of rat type II pneumocytes directly on the alveolar epithelial basement membrane, thereby permitting study of the effect of lung basement membrane on the morphology and function of type II cells. Collagen types I, III, IV and V, laminin and fibronectin were located by immunofluorescence in the lung matrix with the same patterns as those described for the normal human lung. Transmission electron microscopy (TEM) of the fragments revealed intact epithelial and endothelial basement membranes. The matrix maintained the normal three-dimensional alveolar architecture. Glycosaminoglycans were still present by Alcian Blue staining. Isolated adult rat type II pneumocytes cultured on 150 micron thick fragments of acellular human alveolar extracellular matrix undergo gradual cytoplasmic flattening, with loss of lamellar bodies, mitochondria, and surface microvilli. These changes are similar to the in vivo differentiation of type II pneumocytes into type I pneumocytes. The type II pneumocyte behaviour on the lung epithelial basement membrane contrasted sharply with that of the same cell type cultured on a human amnionic basement membrane. On the latter surface the cells retained their cuboidal shape, lamellar bodies and surface microvilli for up to 8 days. These observations suggest that the basement membranes from different organ systems exert differing influences on the morphology and function of type II pneumocytes and that the alveolar and amnionic basement membranes may have differing three-dimensional organizations. The technique of direct culture of type II cells on the lung basement membrane provides a useful tool for studying the modulating effect of the basement membrane on alveolar epithelial cells.     

Microporosity of the substratum regulates differentiation of MDCK cells in vitro

Summary We have analyzed the ability of the physical substratum to modulate both the ultrastructural and protein synthetic characteristics of the Madin-Darby canine kidney (MDCK) renal cell line. When MDCK cells were seeded on Millipore Millicell CM microporous membrane cell culture inserts they demonstrated a more columnar organization with an increase in cell density sixfold greater than the same cells seeded on conventional plastic substrata. After 1 wk postseeding on the microporous membrane a partial basal lamina was noted, with a contiguous basement membrane being apparent after 2 wk. One-dimensional sodium dodecyl sulfate gel electrophoresis was used to analyze detergent-solubilized proteins from MDCK cells maintained on plastic substrata vs. microporous membranes. When proteins were pulse-labeled with [³⁵S]methionine, a 55 kDa protein was evident in the cytosolic extract of cells grown on collagen, laminin, and nontreated plastic substrata; but this labeled protein was not evident in similar extracts from cells grown on collagen and laminin-coated microporous membranes. To test if the polarized, basement-membrane secreting phenotype of the MDCK cells could be generated on a microporous membrane without pretreatment with any extracellular matrix (ECM) components, cells were seeded on the Millipore Millicell HA (cellulosic) microporous membrane. This type of substrata does not need a coating of ECM components for cell attachment. A partial basement membrane was formed below cells where the basal surface of the cell was planar, but not in areas where the cell formed large cytoplasmic extensions into the filter. This led us to the conclusion that the microporous nature of the substrata can dictate both ultrastructural and protein synthetic activities of MDCK cells. Furthermore, we suggest that both the planar nature of the basal surface and the microporosity of the substrate are corequisites for the deposition of the basement membrane.     

Human keratinocytes cultured on collagen gels form an epidermis which synthesizes bullous pemphigoid antigens and alpha 2 beta 1 integrins and secretes laminin, type IV collagen, and heparan sulfate proteoglycan at the basal cell surface

Single cell suspensions of human keratinocytes when seeded onto floating three-dimensional gels constructed with type I collagen form a tissue resembling epidermis. These morphogenetic events occur in a serum-free environment in the absence of fibroblasts. Light and transmission electron microscopy show that cells form a basal layer plus suprabasilar cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasilar keratinocyte layers show morphologies which resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-fillagrin granules. The basal cell layer differs from skin in vivo in that there is no connection to a basement membrane via hemidesmosomes. Cells in the basal layers are polarized as evidenced by the secretion of type IV collagen, heparan sulfate proteoglycans, and laminin at the cell membrane interface with the collagen gel. These proteins are not organized into a cytological basement membrane. Bullous pemphigoid antigen, a protein component of hemidesmosomes, is synthesized by basal keratinocytes, but like the basement membrane proteins it is not incorporated into a definable cytological structure. Keratinocytes in the basal and suprabasilar layers also synthesize alpha 2 beta 1 integrins. The mechanisms of keratinocyte adhesion to the gel may be through the interactions of this cell surface receptor with laminin and type IV collagen synthesized by the cell and/or direct interactions between the receptor and type I collagen within the gel. This in vitro experimental system is a useful model for defining the molecular events which control the formation and turnover of basement membranes and the mechanisms by which keratinocytes adhere to type I collagen when sheets of keratinocytes are used clinically for wound coverage.     

Regulation of differentiation and polarized secretion in mammary epithelial cells maintained in culture: extracellular matrix and membrane polarity influences

Several previous studies have demonstrated that mammary epithelial cells from pregnant mice retain their differentiated characteristics and their secretory potential in culture only when maintained on stromal collagen gels floated in the culture medium. The cellular basis for these culture requirements was investigated by the monitoring of milk protein synthesis and polarized secretion from the mouse mammary epithelial cell line, COMMA-1-D. Experiments were directed towards gaining an understanding of the possible roles of cell-extracellular matrix interactions and the requirements for meeting polarity needs of the epithelium. When cells are cultured on floating collagen gels they assemble a basal lamina-like structure composed of laminin, collagen (IV), and heparan sulfate proteoglycan at the interface of the cells with the stromal collagen. To assess the role of these components, an exogenous basement membrane containing these molecules was generated using the mouse endodermal cell line, PFHR-9. This matrix was isolated as a thin sheet attached to the culture dish, and mammary cells were then plated onto it. It was found that cultures on attached PFHR-9 matrices expressed slightly higher levels of beta-casein than did cells on plastic tissue culture dishes, and also accumulated a large number of fat droplets. However, the level of beta-casein was approximately fourfold lower than that in cultures on floating collagen gels. Moreover, the beta-casein made in cells on attached matrices was not secreted but was instead rapidly degraded intracellularly. If, however, the PFHR-9 matrices with attached cells were floated in the culture medium, beta-casein expression became equivalent to that in cells cultured on floating stromal collagen gels, and the casein was also secreted into the medium. The possibility that floatation of the cultures was necessary to allow access to the basolateral surface of cells was tested by culturing cells on nitrocellulose filters in Millicell (Millipore Corp., Bedford, MA) chambers. These chambers permit the monolayers to interact with the medium and its complement of hormones and growth factors through the basal cell surface. Significantly, under these conditions alpha 1-, alpha 2-, and beta-casein synthesis was equivalent to that in cells on floating gels and matrices, and, additionally, the caseins were actively secreted. Similar results were obtained independently of whether or not the filters were coated with matrices.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of fibronectin in the early phase of avian cephalic neural crest cell migration

Immunofluorescence and immunoperoxidase labeling for fibronectin was used to study the early events of cephalic neural crest cell migration in avian embryos. Prior to crest cell appearance, fibronectin was associated with the basement membranes of all tissues. The loose mesenchymal cells were also surrounded by this glycoprotein. The crest cell individualization phase included a transient rounding up and a rapid increase in cell number in a very limited space. Whereas the neural tube basement membrane was not formed dorsally at the site of emergence of crest cells, it was partially fused laterally with the ectoderm basement membrane apparently preventing immediate crest cell emigration. Further increase in cell number occurred concomitantly with their penetration between the two developing basement membranes of the neural tube and the ectoderm. The localization of migrating crest cells is apparently greatly influenced by local interactions between the ectoderm and the neural tube, whose morphogenesis differs considerably at each axial level: at the mesencephalic-rhombencephalic levels, crest cells rapidly reached a cell-free space that was mostly devoid of fibronectin. Further migration occurred laterally in that space while pioneer crest cells became surrounded by fibronectin in their environment. Crest cells progressed as a confluent multicellular layer with an apparent velocity of 70 μm/hr. At the prosencephalic and median rhombencephalic levels, crest cells accumulated between the fibronectin-rich basement membranes of the ectoderm and the neural tube. Pioneer crest cells were arrested at the site of attachment of the ectoderm and the neural tube basement membranes (i.e., optic vesicles and otic placodes). Crest cells resumed their migration when more space became available during the constriction of the optic vesicles and the invagination of the otic placodes.     

Comparison of the ability of basement membranes produced by corneal endothelial and mouse-derived Endodermal PF-HR-9 cells to support the proliferation and differentiation of bovine kidney tubule epithelial cells in vitro

The proliferation and morphological differentiation of bovine kidney collecting-tubule epithelial cells has been examined as a function of substrata and plasma factors. Collecting kidney tubule explant maintained in vitro gave rise to two distinct cell populations; one was composed mostly of fibroblastic cells whereas the other was epithelioid (EP cells). The proliferation of fibroblastic cells when exposed to serum-supplemented medium was best expressed when cells were maintained on a basement membrane produced by bovine corneal endothelial cells. This basement membrane has a composition, which in previous studies has been shown to favor the proliferation of mesenchymal cells. In contrast, the proliferation of EP cells was best expressed when cells were maintained on a basement membrane produced by the mouse-derived endodermal cell line PF-HR-9 (HR-9-BM). This basement membrane has a biochemical composition very similar to the basement membrane underlying the kidney tubules. Although the fibroblast confluent monolayer maintained on bovine corneal endothelial cell extracellular matrix did not undergo morphogenesis, the confluent monolayer of EP cells maintained on HR-9-BM shows hemicyst formation, suggesting that they were capable of vectorial fluid transport. They also built a complex three-dimensional kidney tubulelike network. Some tubules became grossly visible and floated into the tissue culture medium, remaining tethered to the cell monolayer at either end of the tubule. On an ultrastructural level, the tubules consisted of cells held together with junctional complexes arranged so as to form a lumen. The smallest lumen were bordered by 2-3 cells, and the largest ones by 8-15 cells. The lumens of the larger tubules did contain granular fibrillar and amorphous debris. Low-density EP cell cultures maintained on HR-9-BM could be induced to proliferate at a rate approaching that of cultures exposed to serum when they were exposed to medium supplemented with high-density lipoprotein (HDL, 750 micrograms protein/ml) and transferrin (50 micrograms/ml). When exposed to HDL concentrations equal or lower than 250 micrograms protein/ml, low-density cultures proliferated at a slow rate and readily formed tubulelike structures. This observation indicates that EP cells do not need to reach confluence to undergo morphogenesis, and that HDL, which in the presence of transferrin supports the cell proliferation, can favor their differentiation into tubulelike structures once its concentration becomes limiting for mitogenesis.     

In vitro matrix assembly induced by critical assembly concentration (CAC).

L-2 cells are an immortalized cell line derived from yolk sac parietal endoderm cells, which are responsible for the production of Reichert's membrane, a thick basement membrane produced during rat gestation. Although the L-2 cells secrete all the major components of the basal lamina, they do not assemble a robust matrix in cell culture. We hypothesized that the reason L-2 cells fail to assemble a matrix in cell culture is because the concentrations of matrix components necessary for this matrix assembly do not reach a critical association concentration (CAC) under standard cell culture conditions. To limit the diffusion of secreted molecules while maintaining a nutrient-rich environment for the cells to thrive, we developed a technique that uses a dialysis membrane to limit protein diffusion in a 2-well plate format. This technique permits L-2 cells to assemble a robust matrix in as little as 24 hr that continues to be formed for at least 72 hr. This technique may address some of the physical limitations imposed by cell culture and could be readily applied to other cell types and medium conditions.     

Epithelial differentiation in the absence of extracellular matrix

Summary To investigate the regulation of epithelial differentiation, normal human epidermal keratinocytes were cultured floating on the surface of culture medium without attachment to a solid substrate. Keratinocytes spread out on the surface of the medium, proliferated and differentiated either into several flat lacy sheets 1 to 3 cells thick (on medium containing 0.15 mM calcium) or formed one single aggregate of cells from 5 to 15 cells in thickness on medium containing 1.15 mM calcium. The cell aggregates demonstrated a pattern of ordered epithelial differentiation. Levels of progressive differentiation resembling the structure of normal human epidermis were identified by light microscopy, immunohistochemistry, and electron microscopy. Differentiation proceeded from cells at the air side toward cells at the medium side with basal appearing cells on the air side and keratinocytes expressing filaggrin and involucrin on the side toward the medium. These results demonstrate that organized epithelial differentiation can occur in the absence of extracellular matrix. In contrast with other air-liquid interface cultures, epithelial differentiation in the absence of extracellular matrix progresses from air towards medium.     

Human keratinocytes cultured on collagen gels form an epidermis which synthesizes bullous pemphigoid antigens and α2β1 integrins and secretes laminin, type IV collagen, and heparan sulfate proteoglycan at the basal cell surface

Single cell suspensions of human keratinocytes when seeded onto floating three-dimensional gels constructed with type I collagen form a tissue resembling epidermis. These morphogenetic events occur in a serum-free environment in the absence of fibroblasts. Light and transmission electron microscopy show that cells form a basal layer plus suprabasilar cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasilar keratinocyte layers show morphologies which resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-fillagrin granules. The basal cell layer differs from skin in vivo in that there is no connection to a basement membrane via hemidesmosomes. Cells in the basal layers are polarized as evidenced by the secretion of type IV collagen, heparan sulfate proteoglycans, and laminin at the cell membrane interface with the collagen gel. These proteins are not organized into a cytological basement membrane. Bullous pemphigoid antigen, a protein component of hemidesmosomes, is synthesized by basal keratinocytes, but like the basement membrane proteins it is not incorporated into a definable cytological structure. Keratinocytes in the basal and suprabasilar layers also synthesize α2β1 integrins. The mechanisms of keratinocyte adhesion to the gel may be through the interactions of this cell surface receptor with laminin and type IV collagen synthesized by the cell and/or direct interactions between the receptor and type I collagen within the gel. This in vitro experimental system is a useful model for defining the molecular events which control the formation and turnover of basement membranes and the mechanisms by which keratinocytes adhere to type I collagen when sheets of keratinocytes are used clinically for wound coverage.     

Origin and deposition of basement membrane heparan sulfate proteoglycan in the developing intestine

The deposition of intestinal heparan sulfate proteoglycan (HSPG) at the epithelial-mesenchymal interface and its cellular source have been studied by immunocytochemistry at various developmental stages and in rat/chick interspecies hybrid intestines. Polyclonal heparan sulfate antibodies were produced by immunizing rabbits with HSPG purified from the Engelbreth-Holm-Swarm mouse tumor; these antibodies stained rat intestinal basement membranes. A monoclonal antibody (mAb 4C1) produced against lens capsule of 11-d-old chick embryo reacted with embryonic or adult chick basement membranes, but did not stain that of rat tissues. Immunoprecipitation experiments indicated that mAb 4C1 recognized the chicken basement membrane HSPG. Immunofluorescent staining with these antibodies allowed us to demonstrate that distribution of HSPG at the epithelial-mesenchymal interface varied with the stages of intestinal development, suggesting that remodeling of this proteoglycan is essential for regulating cell behavior during morphogenesis. The immunofluorescence pattern obtained with the two species-specific HSPG antibodies in rat/chick epithelial/mesenchymal hybrid intestines developed as grafts (into the coelomic cavity of chick embryos or under the kidney capsule of adult mice) led to the conclusion that HSPG molecules located in the basement membrane of the developing intestine were produced exclusively by the epithelial cells. These data emphasize the notion already gained from previous studies, in which type IV collagen has been shown to be produced by mesenchymal cells (Simon- Assmann, P., F. Bouziges, C. Arnold, K. Haffen, and M. Kedinger. 1988. Development (Camb.). 102:339-347), that epithelial-mesenchymal interactions play an important role in the formation of a complete basement membrane.     

Improved visualization of ultrastructural components in the avian ovarian granulosa basement membrane

By modifying and improving a fixation procedure originally designed for better conservation of tissue lipids, we were able to visualize some ultrastructural, hitherto unobserved components in the theca interna and in the neighbouring granulosa basement membrane of the wall of the largest avian ovarian follicles. The basement membrane was seen to contain a network of stacks of densely staining plasma membrane fragments, probably formed by progressive piling up of thecal membranes during the rapid expansion of the follicle wall. At the same time, uptake at the surface of the basement membrane of intercellular yolk precursor material including lipid spheres was described.     

Drosophila laminin: characterization and localization

Drosophila laminin was isolated from the medium of Drosophila Kc cell cultures. It was purified by velocity sedimentation, gel filtration, and chromatography. Drosophila laminin is a disulfide-linked molecule consisting of three chains with apparent molecular masses of 400, 215, and 185 kD. In electron micrographs, it has the cross-shaped appearance with globular domains characteristic of vertebrate laminin with closely similar dimensions. The amino acid composition and lectin-binding properties of Drosophila laminin are given. Polyclonal antibodies to Drosophila laminin were prepared and their specificity was established. In developing embryos immunofluorescence staining was detected between 6 and 8 h of development; and in sections of 8-9-h and older embryos immunostaining was seen at sites where basement membranes are present surrounding internal organs, muscles, underlying the hypodermal epithelium, and in the nervous system. Basement membrane staining was also seen in larva and adults. Cells from Drosophila embryos dissociated at the cellular blastoderm stage were grown in culture and some specific, differentiated cells synthesized laminin after several hours of culture as shown by immunofluorescence. The significance of the evolutionary conservation of the structure of this basement membrane component is discussed.     

Assembly,stability and integrity of basement membranes in vivo

Basement membranes are layered structures of the extracellular matrix which separate cells of various kinds from the surrounding stroma. One of the frequently recurring questions about basement membranes is how these structures are formed in vivo. Up to a few years ago, it was thought that basement membranes were formed spontaneously by a process of self-assembly of their components. However, it has now become clear that cell membrane receptors for basement membrane components are essential factors for the formation and stability of basement membranes in vivo. The present review highlights the modern concepts of basement membrane formation.     

Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. I. Regulation of proliferation by hormones and growth factors

Summary A serum-free primary culture system has been developed which allows for three-dimensional growth and differentiation of normal rat mammary epithelial cells (RMECs) within an extracellular matrix preparation. RMECs were isolated from mamary glands of immature 50- to 60-d-old rats and the organoids embedded within a reconstituted basement membrane matrix prepared from the Engelbreth-Holm-Swarm sarcoma. Cells grown in a serum-free media consisting of phenol red-free Dulbecco's modified Eagle's medium-F12 culture medium containing 10 μg/ml insulin, 1 μg/ml prolactin, 1 μg/ml progesterone, 1 μg/ml hydrocortisone, 10 ng/ml epidermal growth factor (EGF), 1 mg/ml fatty-acid-free bovine serum albumin (BSA), 5 μg/ml transferrin, and 5 μM ascorbic acid proliferated extensively (15- to 20-fold increase in cell number as quantitated using the MTT dye assay) over a 2- to 3-wk culture period and remained viable for months in culture. Several types of colonies were observed including the alveolarlike budding cluster which predominates at later times in culture, units with no or various degrees of ductal-like projections, stellate colonies, and two-and three-dimensional web units. Optimal proliferation required insulin, prolactin, progesterone, EGF, and bovine serum albumin. Hydrocortisone was not required for proliferation, but the colonies developing in its absence were morphologically altered, with a high frequency of colonies that formed an extensively branched network with many fine projections. Cell proliferation was also dependent on substratum, with significantly less growth and development occurring in RMECs grown within a type I collagen gel matrix compared to RMECs grown within the reconstituted basement membrane. In conjunction with other studies demonstrating extensive differentiation as well as proliferation, it is concluded that this model should prove to be an improtant tool to study the hormonal regulation of the growth and development of rat mammary cells. This work was supported by grants CA 33240 and CA 35641 and by core grant CA 24538 from the National Institutes of Health, Bethesda, MD.     

Primary culture in chemically defined medium of human fetal mammary epithelial cells within reconstituted matrix]

A serum-free primary culture system has been developed which allows for three-dimensional growth and differentiation of normal human fetal mammary epithelial cells within an extracellular matrix preparation. Human fetal mammary epithelial cells were isolated from the mammary glands of human female fetuses, 17 to 39 weeks-old. The "organoids" were embedded within a reconstituted basement membrane matrix prepared from the Engelbreth-Holm-Swarm (EHS) sarcoma according to the method of Hahm and Ip. "Organoids" were grown in either serum-free medium or in medium with fetal calf serum (FCS). The "organoid" proliferated over a 2 to 3 weeks culture period and remained viable for 1 or 2 months within the basement membrane matrix in serum free medium. Several types of colonies were observed; including alveolar-like budding clusters obtained from cultures of mammary gland from fetuses of over 20 weeks age, units with ductule-like projections and stellate-type colonies. Cell proliferation was dependent on the culture medium (with FCS no proliferation was obtained) and on the substratum (without matrix, significantly less growth and development occurred). These types of colonies are obtained when a glandular differentiation of cells budding from the malpighian epithelium is observed. Light microscopic and transmission electron microscopic studies were undertaken at the time of culture. This unique system using normal fetal mammary epithelial cells thus provides a model in which the regulation of human mammary development can be investigated.