Functional and morphological differentiation of nonpigmented ciliary body epithelial cells grown on collagen rafts

Summary We have examined the effect of alteration in cell shape on promoting differentiated morphology and physiology in cultured nonpigmented epithelial cells from the ciliary body. We have grown pure populations of nonpigmented cells on collagen gels released from the culture dish to create collagen rafts. Shortly after the gels were detached, the cells shrank in diameter and increased in height while they contracted the gel. Concurrently, the actin cytoskeleton reorganized to the cell cortex as found in vivo. After this differentiated morphology developed, large changes in intracellular Ca²⁺ could be elicited by simultaneous activation of acetylcholine and epinephrine or acetylcholine and somatostatin receptors as seen in intact tissue. Explant cultures of isolated nonpigmented cell layers maintained their actin distribution and also showed synergistic Ca²⁺ increases. Spread cells, grown on rigid substrates, had a disorganized cytoskeleton and rarely showed synergism. These data suggest that the mechanism underlying synergistic Ca²⁺ responses in the ciliary body is functional in nonpigmented cells grown on collagen rafts. In addition, this pathway appears to be sensitive to the disposition of the cell’s cytoarchitecture.     

The differentiation behaviour of MDCK cells grown on matrix components and in collagen gels

MDCK cells are grown on various substrates (Thermanox pure, extracellular matrix (ECM), dried or wet collagen type I or type III), on floating collagen and enclosed in collagen gels, and their differentiation behaviour is investigated electron microscopically. The cells grown on ECM or dried collagen (type I and type III) do not show any changes as compared with the controls (Thermanox). Differentiation processes can only be observed when the cells are grown on wet collagen (type I and type III), especially on floating collagen and enclosed in collagen gels. These differentiation processes comprise changes in the cell shape, an increase in the number of microvilli, an increase in the length of the lateral contact zone with the formation of gap junctions and desmosomes, and an increase in the number and size of the cell organelles. A basement membrane only develops in the form of short segments. Moreover, on floating collagen and in collagen gels three-dimensional, organoid structures develop: cell aggregates with central lumina and tubuli. They are formed by cuboid cells that also exhibit indications of differentiation. Basement membrane fragments occur more often and are longer. It can be concluded from these findings that the chemical structure of the substrate does not play the primary role in the described process. It is rather the physical properties, probably the plasticity, that are of significance. Due to this property the cells change their shape and the contact areas increase in size. The establishment of contacts might be the triggering factor for differentiation. Organoid structures with lumina develop when the apical surface comes into contact with other cells or collagen gels. The pronounced tendency towards polarization necessitates a re-arrangement of three-dimensionally growing cells to structures with lumina. The formation of the basement membrane is the result and not the cause of differentiation.     

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).     

Regulation of cytoskeletal structure in human mammary carcinoma cells MCF-7 by culture substrata

Three-dimensional cellular structures formed by MCF-7 human mammary carcinoma cells within collagen gels were isolated with collagenase and cultivated on plastic substratum to examine whether the cytoskeleton specific for cells forming cellular structures (S-type) changes to that specific for cells grown as monolayers (M-type). The cytoskeleton isolated as 0.05% Triton-insoluble fraction from the cellular structures after culture for 1 day on plastic was exclusively S-type. However, both types of cytoskeletons were observed in the cellular structures cultivated for 7 days on plastic as well as in the cells grown as monolayers for 2 days after dissociation of the cellular structures with trypsin. By use of an antibody raised against a 65-kD polypeptide that was specific for the M-type cytoskeleton, the presence of the polypeptide was found to be restricted to the cells grown out as monolayers from the edge of the cellular structures. In the cells grown for 2 days as monolayers, a mixture of cells both having and lacking the polypeptide was observed. After a 7-day culture of the dissociated cells as monolayers on plastic, however, most of the cells had M-type cytoskeletons. The present results show that the apparent change in the cytoskeleton of MCF-7 cells from S-type to M-type does not occur in cells involved in the three-dimensional cellular structures even in the absence of collagen gels, but that it occurs in cells which are grown as monolayers for at least 7 days on plastic substratum.     

Induction of cAMP receptors by disaggregation of the multicellular complexes of Dictyostelium discoideum

Subcultivated rat lingual epithelial cells when grown on collagen gels at a liquid-gas interface achieve a highly ordered state that closely resembles the parent tissue. Three distinct cell layers are present; basal, spinous, and keratinized. Basal cells are cuboidal in shape and form a complex interface with the underlying collagen fibrils. Spinous cells form a layer 5–10 cells thick and, with the exception of keratohyalin granules, possess an organellar complement identical with native cells, including membrane-coating granules. The keratinized cell layer increases in thickness as a function of time spent in culture. Forty or more plies of terminally differentiated cells are observed following a 30-day culture period. Terminally differentiated cells while retaining pycnotic nuclei and some other organellar debris are principally envelope-enclosed squames filled with tonofilaments. Keratinization is a continuing process which occurs simultaneously across the full expanse of the culture surface. The high degree of tissue organization observed appears to be the result of feeding the cultures from the undersurface.     

Control of type IV collagen production in rat mammary epithelial and myoepithelial-like cells

A rat mammary myoepithelial-like cell line (Rama 401) produces 3.5 times more type IV collagen than a mammary epithelial cell line (Rama 25), as measured by the formation of protein hydroxyproline. However, using quantitative "dot" hybridization techniques, the level of poly (A)-containing mRNA hybridizing to a type IV collagen cDNA probe is only 50% higher in Rama 401 cells than in Rama 25 cells. The total amount of hydroxyproline synthesized per cell by the two cell lines is similar. However, in the Rama 25 cells approximately 70% of the hydroxyproline is found as free hydroxyproline against 13% for Rama 401 cells. When Rama 25 cells are grown on collagen gels, they accumulate 2.5-fold more type IV collagen. However, type IV collagen mRNA levels are only 30% higher in Rama 25 cells grown on collagen. The total amount of hydroxyproline synthesized is the same as cells grown on plastic, whereas the extent of collagen degradation is reduced from 71% to 30% in cells grown on collagen gels. No degradation of type IV collagen can be detected in the culture medium of Rama 25 cells. These results indicate that the increased accumulation of type IV collagen in Rama 401 cells is not due to increased synthesis but to a decreased rate of intracellular degradation, and that for Rama 25 cells, the extracellular matrix modulates type IV collagen production by regulating the rate of intracellular collagen degradation.     

Reorganization of polymerized actin: a possible trigger for induction of procollagenase in fibroblasts cultured in and on collagen gels

Changes in cell shape are postulated to modulate gene expression during differentiation of a number of cell types, including rabbit synovial fibroblasts, which are inducible for expression of the zymogen form of the metalloendopeptidase, collagenase. In the work presented here, fibroblasts cultured on and within hydrated collagen gels were allowed to contract by release of the gels from the sides of the culture dish. Within 24 h of cell release, synthesis and secretion of procollagenase was initiated in the absence of any chemical manipulation. Fibroblasts grown in and on collagen also responded to 12-O-tetradecanoylphorbol-13-acetate and cytochalasin B with morphologic change and induced procollagenase. However, colchicine, which altered morphology to varying degrees in cells on plastic, on collagen, and within collagen gels, did not induce procollagenase expression. In all cases, the enzyme was induced only after reorganization of polymerized actin, rather than after a change in cellular morphology per se. As a first approach to identifying other aspects of the stimulated phenotype that could affect collagen turnover, the expression of collagen and endogenous metalloproteinase inhibitors in relation to procollagenase secretion was investigated. Collagen secretion by fibroblasts decreased when procollagenase secretion was induced by the pharmacologic agents, but not when cells were stimulated by contraction on or within collagen gels. The expression of two endogenous inhibitors was not coordinately regulated with induction of procollagenase. Therefore, the extracellular matrix and the cellular actin cytoskeleton may transduce signals that modulate the tissue remodeling phenotype of fibroblasts.     

Morphological and functional differentiation of cryopreserved lactating bovine mammary cells cultured on floating collagen gels

Cryopreserved bovine mammary epithelial cells prepared from lactating mammary tissue synthesize and secrete the milk proteins alpha_s1-casein, lactoferrin (Lf), and alpha-lactalhumin during in vitro culture on collagen gels in serum-free medium. Each milk protein is differently regulated by detachment and thickness of the collagen substratum, fetal calf scrum, and prolactin in the medium. Collagen detachment did not modulate lactoferrin secretion but strongly induced casein secretion, with detachment on day 6 (after formation of cell sheets) inducing casein secretion to 3 μg/ml medium, which was 2–3-fold higher than for cells on collagen detached on day 2 (prior to cell spreading to form sheets), and ten-fold higher than for cells grown on collagen not detached. Alpha-lactalbumin secretion was also induced, but only to low levels, in cells grown on detached but not on attached collagen. Cells grown on thin collagen gels secreted lower levels of lactoferrin and casein compared to cells on thick collagen. Lactoferrin but not casein secretion was increased in cells grown in the presence of fetal calf serum. Casein but not lactoferrin secretion was completely dependent on prolactin. Cells grown serum-free on collagen gels detached on day 6 of culture showed a polarized epithelial cell layer with high differentiation evidenced by the apical microvilli, tight junctions, and fat droplets surrounded by casein-containing secretory vesicles. An underlying layer of myoepithelial-like cells was also evident. These studies show for eryopreserved primary bovine mammary cells prepared from lactating mammary tissue the induction of highly differentiated and polarized cell morphology and ultrastructure with concomitant induction of the secretion of casein, lactoferrin. and alpha-lactalbumin in vitro, and that the non-coordinate regulation of milk protein secretion by substratum, prolactin, and serum likely involves alternate routing and control of secretion pathways for casein and lactoferrin.     

Prolonged expression of differentiated phenotype by chondrocytes cultured at low density on a composite substrate of collagen and agarose that restricts cell spreading

The dedifferentiation of chondrocytes in culture is frequently associated with transition from a rounded to a spread morphology. A number of culture methods which prevent cell spreading have been described; however, all have disadvantages that limit their widespread use. In this paper we describe a new technique which allows prolonged cultivation of attached chondrocytes at low density while inhibiting spreading: the cells are grown on a composite substrate of agarose and collagen. By varying the ratio of agarose to collagen in the gel, the degree of spreading can be varied. The cultures are suitable for ultrastructural and immunofluorescence analysis and for studies of the synthesis and secretion of macromolecules. In order to determine whether the differentiated phenotype was maintained on composite gels, we compared the levels of messenger RNAs for cartilage-specific proteoglycan, link protein, alpha 1 (II) and alpha 1 (I) collagens in chondrocytes grown at low density on composite gels or at high or low density on tissue culture plastic for up to 21 days. The rate of decline in the level of mRNAs encoding the cartilage-specific products and the rate of increase in the level of alpha 1 (I) collagen mRNA were slower in the composite cultures than in the cultures on plastic. This culture technique may, therefore, prolong expression of the differentiated phenotype of chondrocytes relative to cultivation on plastic and will be useful for further studies on the role of cell shape in regulating differentiated gene expression.     

Specific association of iduronic acid-rich dermatan sulphate with the extracellular matrix of human skin fibroblasts cultured on collagen gels.

Human skin fibroblasts cultured on collagen gels produced two dermatan sulphate species, one, enriched in iduronic acid residues, that bound specifically to the collagenous fibres of the gel, the other, enriched in glucuronic acid, that accumulated in the culture medium. Collagen-binding and collagen-non-binding dermatan sulphates were also produced by cells grown on plastic surfaces, but in these cultures each constituent was released into the growth medium. Net synthesis of dermatan sulphate was 3-fold higher in cells maintained on collagen gels. In contrast, heparan sulphate synthesis was not influenced by the nature of the culture surface. The concentration of heparan sulphate in surface-membrane extracts was similar for cells grown on plastic and on collagen gels, but cells cultured on collagen showed a notable increase in the content of surface-membrane dermatan sulphate. The patterns of synthesis and distribution of sulphated glycosaminoglycans observed in skin fibroblasts maintained on collagen gels may reflect differentiated cellular functions.     

Collagen modulates cell shape and cytoskeleton of embryonic corneal and fibroma fibroblasts: Distribution of actin, α-actinin,and myosin

In the embryo, fibroblasts migrating through extracellular matrices (ECM) are generally elongate in shape, exhibiting a leading pseudopodium with filopodial extensions, and a trailing cell process. Little is known about the mechanism of movement of embryonic cells in ECM, for studies of fibroblast locomotion in the past have been largely confined to observations of flattened cells grown on planar substrata. We confirm here that embryonic avian corneal fibroblasts migrating within hydrated collagen gels in vitro have the bipolar morphology of fibroblasts in vivo, and we show for the first time that highly flattened gerbil fibroma fibroblasts, grown as cell lines on planar substrata, can also respond to hydrated collagen gels by becoming elongate in shape. We demonstrate that the collagen-mediated change in cell shape is accompanied by dramatic rearrangement of the actin, α-actinin, and myosin components of the cytoskeleton. By immunofluorescence, the stress fibers of the flattened corneal fibroblasts grown on glass are seen to stain with antiactin, anti-α-actinin, and antimyosin, as has been reported for fibroma and other fibroblasts grown on glass. Stress fibers, adhesion plaques, and ruffles do not develop when the corneal or fibroma fibroblast is grown in ECM; these features seem to be a response to strong attachment of the cell underside to a planar substratum. When the fibroblasts are grown in ECM, antimyosin staining is distributed diffusely through the cytoplasm. Antiactin and anti-α-actinin stain the microfilamentous cell cortex strongly. We suggest that locomotion of the fibroblast in ECM is accompanied by adhesion of the cell to the collagen fibrils and may involve an interaction of the myosin-rich cytosol with the actin-rich filamentous cell cortex. Interestingly, the numerous filopodia that characterize the tips of motile pseudopodia of cells in ECM are very rich in actin and α-actinin, but seem to lack myosin; if filopodia use myosin to move, the interaction must be at a distance. Soluble collagen does not convert flattened fibroblasts on planar substrata to bipolar cells. Thus, the effect of collagen on the fibroblast cytoskeleton seems to depend on the presence of collagen fibrils in a gel surrounding the cell.     

Alteration of Cellular Behavior and Response to PI3K Pathway Inhibition by Culture in 3D Collagen Gels

Most investigations into cancer cell drug response are performed with cells cultured on flat (2D) tissue culture plastic. Emerging research has shown that the presence of a three-dimensional (3D) extracellular matrix (ECM) is critical for normal cell behavior including migration, adhesion, signaling, proliferation and apoptosis. In this study we investigate differences between cancer cell signaling in 2D culture and a 3D ECM, employing real-time, live cell tracking to directly observe U2OS human osteosarcoma and MCF7 human breast cancer cells embedded in type 1 collagen gels. The activation of the important PI3K signaling pathway under these different growth conditions is studied, and the response to inhibition of both PI3K and mTOR with PI103 investigated. Cells grown in 3D gels show reduced proliferation and migration as well as reduced PI3K pathway activation when compared to cells grown in 2D. Our results quantitatively demonstrate that a collagen ECM can protect U2OS cells from PI103. Overall, our data suggests that 3D gels may provide a better medium for investigation of anti-cancer drugs than 2D monolayers, therefore allowing better understanding of cellular response and behavior in native like environments.     

A comparison of the effects of different substrata on chondrocyte morphology and the synthesis of collagen types IX and X

Summary Embryonic chick sternal chondrocytes were cultured either within three dimensional gels of type I collagen, type II collagen or agar, or as monolayers on plastic dishes coated with air-dried films of these matrix macromolecules. It was observed that cell shape and cell growth varied markedly between the different culture conditions. Flattened monolayers of cells on plastic or films of type I or type II collagen, proliferated more rapidly and reached a higher final cell density per culture than the more rounded cells found in the cultures on agar films or within three-dimensional gels. Biosynthetic studies demonstrated that in addition to the synthesis of type II collagen, all the cultures were producing collagen types IX and X. Chondrocytes cultured on plastic or films of the different matrix macromolecules all showed a similar expression of types IX and X collagen, independent of whether they displayed a flattened or round cell morphology. In contrast, marked variations in the proportions of the minor collagens, particularly type X collagen, were observed when the cells were cultured within three-dimensional gels. The data suggest that direct interaction of the cell surface with matrix constituents displaying a particular spatial array could be an important aspect in the control of type IX and X collagen expression by chondrocytes. The financial support of the Arthritis & Rheumatism Council and the Medical Research Council is gratefully acknowledged.     

Cell-matrix interactions of in vitro human skin fibroblasts upon addition of hyaluronan

Normal human skin fibroblasts were grown in a three-dimensional collagen gel or in monolayer in the presence or absence of high molecular weight hyaluronan (HA) to assess the influence of extracellular HA on cell-matrix interactions. HA incorporated into the collagen gel or added to the culture medium did not modify lattice retraction with time. The effect was independent from HA molecular weight (from 7.5 x 10(5) to 2.7 x 10(6) Da) and concentration (from 0.1 up to 1 mg/ml). HA did not affect shape and distribution of fibroblasts within the gel, whereas it induced the actin filaments to organise into thicker cables running underneath the plasma membrane. The same phenomenon was observed in fibroblasts grown in monolayer. By contrast, vimentin cytoskeleton and cell-substrate focal adhesions were not modified by exogenous HA. The number of fibroblasts attached to HA-coated dishes was always significantly lower compared to plastic and to collagen type I-coated plates. By contrast, adhesion was not affected by soluble HA added to the medium nor by anti-CD44 and anti-RHAMM-IHABP polyclonals. After 24-h seeding on collagen type I or on plastic, cells were large and spread. Conversely, cells adherent to HA-coated surfaces were long, thin and aligned into rows; alcian blue showed that cells were attached to the plastic in between HA bundles. Therefore, normal human skin fibroblasts exhibit very scarce, if any, adhesion to matrix HA, either soluble or immobilised. Moreover, even at high concentration, HA molecules do not exert any visco-mechanical effect on lattice retraction and do not interfere with fibroblast-collagen interactions nor with focal adhesion contacts of fibroblasts with the substrate. This is probably relevant in organogenesis and wound repair. By contrast, HA greatly modifies the organisation of the actin cytoskeleton, suggesting that CD44-mediated signal transduction by HA may affect cell locomotion and orientation, as indicated by the fusiform shape of fibroblasts grown in the presence of immobilised HA. A role of HA in cell orientation could be relevant for the deposition of collagen fibrils in regeneration and tissue remodelling.     

INTEGRIN EXPRESSION AND COLLAGEN TYPE II IMPLICATED IN MAINTENANCE OF CHONDROCYTE SHAPE IN MONOLAYER CULTURE: AN IMMUNOMORPHOLOGICAL STUDY

Chondrocytes grown in monolayer culture at low density, with serum added, either dedifferentiate after several days whereby their cell shape changes or they are overgrown by fibroblast-like cells. The aim of this study was to optimize the cultivation of chondrocytes in monolayer culture and to slow down their transformation or their overgrowth by fibroblast-like cells. For this purpose freshly isolated chondrocytes of cartilage anlagen from 17-day-old mouse embryos were grown on plastic or collagen type II-coated substrates. With this model: (a) chondrocytes grown on plastic substrates had almost completely changed to fibroblast-like cells after 5 days in culture. (b) When grown on collagen type II, the chondrocytes maintained their round phenotype for more than 2 weeks in culture. (c) Immunomorphological investigations showed that chondrocytes produce collagen type II and fibronectin and express specific surface receptors (integrins of the β1-group) on the membrane from day 1 until the end of the culture period when grown on collagen type II. (d) Treatment with β1-integrin antibodies clearly reduces chondrocyte adhesion on collagen type II by about 70%. Hence, these data indicate that the most probable influence of collagen type II on cellular behaviour depends on the integrins participating in a chondrocyte—collagen type II interaction, and this model represents a pure chondrocyte culture which allows cell growth for an extended period.     

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.     

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.     

The different effects of type I and IV collagens on the survival and proliferation of cultured BSC-1 cells

The effects of type I and IV collagens on the survival and proliferation of cells were investigated to clarify a possible involvement of the substratum in the regulation of cell function. BSC-1 cells attached, spread and sustained their viability in the absence of calf serum on culture dishes coated with type IV collagen, but were unable to spread and survive on untreated culture dishes. The effects of adding type IV collagen in solution were similar to those of type IV coating. The fraction of the solution of type IV collagen with molecular mass of more than 100 kDa enhanced spreading and survival of cells, but the fraction of less than 100 kDa did not. Type I collagen did not support cell viability in the absence of calf serum. Moreover, coating of culture dishes with type I collagen, but not with type IV collagen, inhibited DNA synthesis and cell proliferation in the presence of calf serum. The cells grown on type I collagen were long, thin and spindle-shaped, and their stress fibers were not well developed, but the cells grown on type IV collagen, as well as those grown on untreated culture dishes, were polygonal in shape with well-developed stress fibers. These results indicate that the interactions of BSC-1 cells with the substratum, when it is derived from type I and IV collagens, differentially modulate the survival and proliferation of BSC-1 cells.     

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.     

Extracellular matrix organization modulates fibroblast growth and growth factor responsiveness

To learn more about the relationship between extracellular matrix organization, cell shape, and cell growth control, we studied DNA synthesis by fibroblasts in collagen gels that were either attached to culture dishes or floating in culture medium during gel contraction. After 4 days of contraction, the collagen density (initially 1.5 mg/ml) reached 22 mg/ml in attached gels and 55 mg/ml in floating gels. After contraction, attached collagen gels were well organized; collagen fibrils were aligned in the plane of cell spreading; and fibroblasts had an elongated, bipolar morphology. Floating collagen gels, however, were unorganized; collagen fibrils were arranged randomly; and fibroblasts had a stellate morphology. DNA synthesis by fibroblasts in contracted collagen gels was suppressed if the gels were floating in medium but not if the gels were attached, and inhibition was independent of the extent of gel contraction. Therefore, growth of fibroblasts in contracted collagen gels could be regulated by differences in extracellular matrix organization and cell shape independently of extracellular matrix density. We also compared the responses of fibroblasts in contracted collagen gels and monolayer culture to peptide growth factors including fibroblast growth factor, platelet-derived growth factor, transforming growth factor-beta, and interleukin 1. Cells in floating collagen gels were generally unresponsive to any of the growth factors. Cells in attached collagen gels and monolayer culture were affected similarly by fibroblast growth factor but not by the others. Our results indicate that extracellular matrix organization influenced not only cell growth, but also fibroblast responsiveness to peptide growth factors.