Preadipocytes Stimulate Ductal Morphogenesis and Functional Differentiation of Human Mammary Epithelial Cells on 3D Silk Scaffolds (original) (raw)
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Biomaterials, 2010
Epithelial-stromal interactions play a crucial role in normal embryonic development and carcinogenesis of the human breast while the underlying mechanisms of these events remain poorly understood. To address this issue, we constructed a physiologically relevant, three-dimensional (3D) culture surrogate of complex human breast tissue that included a tri-culture system made up of human mammary epithelial cells (MCF10A), human fibroblasts and adipocytes, i.e., the two dominant breast stromal cell types, in a Matrigel ™ /collagen mixture on porous silk protein scaffolds. The presence of stromal cells inhibited MCF10A cell proliferation and induced both alveolar and ductal morphogenesis and enhanced casein expression. In contrast to the immature polarity exhibited by co-cultures with either fibroblasts or adipocytes, the alveolar structures formed by the tri-cultures exhibited proper polarity similar to that observed in breast tissue in vivo. Only alveolar structures with reverted polarity were observed in MCF10A monocultures. Consistent with their phenotypic appearance, more functional differentiation of epithelial cells was also observed in the tri-cultures, where casein αand -β mRNA expression was significantly increased. This in vitro tri-culture breast tissue system sustained on silk scaffold effectively represents a more physiologically relevant 3D microenvironment for mammary epithelial cells and stromal cells than either co-cultures or monocultures. This experimental model provides an important first step for bioengineering an informative human breast tissue system, with which to study normal breast morphogenesis and neoplastic transformation.
PLoS ONE, 2011
Studies on the stem cell niche and the efficacy of cancer therapeutics require complex multicellular structures and interactions between different cell types and extracellular matrix (ECM) in three dimensional (3D) space. We have engineered a 3D in vitro model of mammary gland that encompasses a defined, porous collagen/hyaluronic acid (HA) scaffold forming a physiologically relevant foundation for epithelial and adipocyte co-culture. Polarized ductal and acinar structures form within this scaffold recapitulating normal tissue morphology in the absence of reconstituted basement membrane (rBM) hydrogel. Furthermore, organoid developmental outcome can be controlled by the ratio of collagen to HA, with a higher HA concentration favouring acinar morphological development. Importantly, this culture system recapitulates the stem cell niche as primary mammary stem cells form complex organoids, emphasising the utility of this approach for developmental and tumorigenic studies using genetically altered animals or human biopsy material, and for screening cancer therapeutics for personalised medicine.
A Novel 3D In Vitro Culture Model to Study Stromal–Epithelial Interactions in the Mammary Gland
Tissue Engineering Part C: Methods, 2008
Stromal-epithelial interactions mediate mammary gland development and the formation and progression of breast cancer. To study these interactions in vitro, the development of defined three-dimensional (3D) models is essential. In the present study, we have successfully developed novel 3D in vitro models that allow the formation of mammary gland structures closely resembling those found in vivo. Cocultures of a human mammary epithelial cell line MCF10A and human mammary fibroblasts obtained from reduction mammoplasties embedded in either a type I collagen or a mixed Matrigel-collagen matrix were carried out for up to 6 weeks. Histological and ultrastructural analysis confirmed the formation of ductal and alveolar structures. The importance of the stromal cells was apparent in both matrices; in the collagen gels the presence of reduction mammoplasty fibroblasts accelerated the initial formation of epithelial structures, and in the mixed Matrigel-collagen gels the presence of those fibroblasts was necessary for the formation of ductal structures. These models provide an excellent system to study tissue organization, epithelial morphogenesis, and breast carcinogenesis.
A Novel 3DIn VitroCulture Model to Study Stromal–Epithelial Interactions in the Mammary Gland
Tissue Engineering Part C-methods, 2008
Stromal-epithelial interactions mediate mammary gland development and the formation and progression of breast cancer. To study these interactions in vitro, the development of defined three-dimensional (3D) models is essential. In the present study, we have successfully developed novel 3D in vitro models that allow the formation of mammary gland structures closely resembling those found in vivo. Cocultures of a human mammary epithelial cell line MCF10A and human mammary fibroblasts obtained from reduction mammoplasties embedded in either a type I collagen or a mixed Matrigel-collagen matrix were carried out for up to 6 weeks. Histological and ultrastructural analysis confirmed the formation of ductal and alveolar structures. The importance of the stromal cells was apparent in both matrices; in the collagen gels the presence of reduction mammoplasty fibroblasts accelerated the initial formation of epithelial structures, and in the mixed Matrigel-collagen gels the presence of those fibroblasts was necessary for the formation of ductal structures. These models provide an excellent system to study tissue organization, epithelial morphogenesis, and breast carcinogenesis.
Matrix compositions and the development of breast acini and ducts in 3D cultures
In Vitro Cellular & Developmental Biology - Animal, 2010
Breast epithelial cells develop into polarized and highly organized acinar and ductal structures in response to stromal cues, including extracellular matrix composition and density, which can in part be reproduced in 3D culture conditions. Here, we present the effects of various 3D in vitro stroma compositions (termed "matrices" or "substrates") on the ability of heterotypic cultures of epithelial and mesenchymal stem cells to organize into acinar and tubular structures. Normal murine mammary gland (NMuMG) cells were cultured, either alone or in combination (30:70) with mouse mesenchymal stem cells (D1), in 3D matrices generated by agarose, collagen, and Matrigel® alone or by a combination thereof. After 3-5 d in culture, cell distribution, organization, and the presence of acinuslike and tubule-like structures were determined. The number of acinar structures was significantly higher in cultures grown in combination matrices of agarose with Matrigel® or collagen I when compared with cultures grown in Matrigel® or collagen I alone (p<0.05). No tubular structures were formed when agarose was included in the matrix, regardless of the combination. In Matrigel®, but not in collagen I/Matrigel® microenvironment, the number of tubular structures was significantly increased in NMuMG/D1 coculture when compared with culture of NMuMG cells alone (p<0.05). By immunohistochemical analysis, NMuMG cells cocultured with D1 cells were shown to form acinar structures with the NMuMG epithelial cells surrounding a lumen composed of dead cells while the D1 cells were mostly peripheral. Immunostaining for laminin indicated the presence of basement membrane when NMuMG cells were grown in Matrigel® alone or cocultured with D1 cells in a combination of Matrigel® and collagen I. These results indicate that the physical and biochemical properties of the matrix and cellular composition alter the organization of the mammary gland.
BIO-PROTOCOL, 2019
Co-culture systems utilizing reconstituted or synthetic extracellular matrix (ECM) and micropatterning techniques have enabled the reconstruction of surface epithelial tissues. This technique has been utilized in the regeneration, disease modeling and drug screening of the surface epithelia, such as the skin and esophagus. On the other hand, the reconstruction of glandular epithelia would require more intricate ECM organizations. Here we describe a protocol for a novel three-dimensional organotypic co-culture system for the reconstruction of mammary glands that utilizes the discontinuous ECM. In this technique, primary mammary fibroblasts first establish a layer of the connective tissue rich in collagen I. Then, mammary epithelial cells form acinar structures, the functional glandular units, within the laminin-rich basement membrane embedded in the connective tissue. This method allows for the regeneration of the in vivo-like architecture of mammary glands and could be utilized for monitoring the real-time response of mammary glands to drug treatment.
Frontiers in Bioengineering and Biotechnology, 2020
Breast tissue consists of an epithelial parenchyma embedded in stroma, of heterogeneous and complex composition, undergoing several morphological and functional alterations throughout females' lifespan. Improved knowledge on the crosstalk between parenchymal and stromal mammary cells should provide important insights on breast tissue dynamics, both under healthy and diseased states. Here, we describe an advanced 3D in vitro model of breast tissue, combining multiple components, namely stromal cells and their extracellular matrix (ECM), as well as parenchymal epithelial cells, in a hybrid system. To build the model, porous scaffolds were produced by extrusion 3D printing of peptide-modified alginate hydrogels, and then populated with human mammary fibroblasts. Seeded fibroblasts were able to adhere, spread and produce endogenous ECM, providing adequate coverage of the scaffold surface, without obstructing the pores. On a second stage, a peptide-modified alginate pre-gel laden with mammary gland epithelial cells was used to fill the scaffold's pores, forming a hydrogel in situ by ionic crosslinking. Throughout time, epithelial cells formed prototypical mammary acini-like structures, in close proximity with fibroblasts and their ECM. This generated a heterotypic 3D model that partially recreates both stromal and parenchymal compartments of breast tissue, promoting cell-cell and cell-matrix crosstalk. Furthermore, the hybrid system could be easily dissolved for cell recovery and subsequent analysis by standard cellular/molecular assays. In particular, we show that retrieved cell populations could be discriminated by flow cytometry using cell-type specific markers. This integrative 3D model stands out as a promising in vitro platform for studying breast stroma-parenchyma interactions, both under physiological and pathological settings.
Growth of human breast tissues from patient cells in 3D hydrogel scaffolds
Breast cancer research : BCR, 2016
Three-dimensional (3D) cultures have proven invaluable for expanding human tissues for basic research and clinical applications. In both contexts, 3D cultures are most useful when they (1) support the outgrowth of tissues from primary human cells that have not been immortalized through extensive culture or viral infection and (2) include defined, physiologically relevant components. Here we describe a 3D culture system with both of these properties that stimulates the outgrowth of morphologically complex and hormone-responsive mammary tissues from primary human breast epithelial cells. Primary human breast epithelial cells isolated from patient reduction mammoplasty tissues were seeded into 3D hydrogels. The hydrogel scaffolds were composed of extracellular proteins and carbohydrates present in human breast tissue and were cultured in serum-free medium containing only defined components. The physical properties of these hydrogels were determined using atomic force microscopy. Tissue...
Dual Regulation of Breast Tubulogenesis Using Extracellular Matrix Composition and Stromal Cells
Tissue Engineering Part A, 2012
Epithelial-mesenchymal interactions during embryogenesis are critical in defining the phenotype of tissues and organs. The initial elongation of the mammary bud represents a central morphological event requiring extensive epithelial-mesenchymal crosstalk. The precise mechanism orchestrating this outgrowth is still unknown and mostly animal models have been relied upon to explore this process. Highly tunable three-dimensional (3D) culture models are a complementary approach to address the question of phenotypic determination. Here, we used a 3D in vitro culture to study the roles of stromal cells and extracellular matrix components during mammary tubulogenesis. Fibroblasts, adipocytes, and type I collagen actively participated in this process, whereas reconstituted basement membrane inhibited tubulogenesis by affecting collagen organization. We conclude that biochemical and biomechanical signals mediate the interaction between cells and matrix components and are necessary to induce tubulogenesis in vitro.