Extracellular matrix promotes mammary epithelial growth and differentiation in vitro.

Abstract

The study of growth and differentiation of mammary epithelium has been hampered by the difficulty of maintaining these functions in vitro. We describe a system for the primary culture of rat mammary epithelium on an acellular matrix derived from whole rat mammary glands that maintains growth and differentiation for months. Cultures plated on this complex substratum produce 50 times the ei-lactalbumin of those on tissue culture dishes and 5 times the a-lactalbumin of those on floating collagen gels as determined by radioimmunoassay. Unlike cultures grown on floating collagen gels, which rapidly lose the ability to secrete the milk sugar lactose, mammary cells on this matrix retain this ability for over 30 days in culture. The organ specificity ofthis mammary extracellular material is shown by the failure of extracellular matrix prepared from rat liver to support mammary differentiation. Within a given culture dish, cells on the surface of mammary extracellular matrix are more differentiated than those on the adjacent plastic. This is demonstrated by their increased a-lactalbumin content as shown by indirect immunofluorescence, and by their increased ability to bind fluorescein-conjugated peanut lectin. Cells on the surface ofthe matrix continue to synthesize DNA as determined by [3H]thymidine incorporation and autoradiography. Even when mammary epithelial cells are plated at low density, cell division continues until the matrix is covered with a confluent layer. We propose that the limited growth, differentiation, and survival of mammary cells in previously described in vitro systems may have been due to substrata that were inadequate to support these functions.The study of mammary growth and differentiation has been hampered by the lack of a suitable system that is capable of maintaining these functions in vitro. When normal mammary epithelial cells from rodents or humans are cultured on tissue culture plastic surfaces they undergo only a few rounds of cell division and rapidly lose differentiated function (1-3). Sometimes, continuous cell lines that are easy to manipulate in vitro can be established from these cultures (4, 5). However, because these cells are highly selected to proliferate under artificial conditions, their control mechanisms may have little relevance to those ofmammary cells in vivo. Organ culture has the advantage of maintaining more normal tissue orientations. However, these systems have limited viability and the presence of stromal cells makes quantitation of epithelial growth difficult (6, 7).It has been appreciated for some time that cell behavior in vitro may be influenced by placing cells on matrices of stromal collagen (8,9). More recently, Emerman and Pitelka described a system for the culture ofmouse mammary cells on floating gels of stromal collagen. Mammary epithelial cells isolated from midpregnant mice produced considerably more ofthe milk protein casein when plated on these floating collagen gels than when plated on attached collagen gels or tissue culture plastic dishe...

Primary culture, cellular stress and differentiated function

“…Epithelial cells are found to be rounded and dispersed singly or in small clumps [24,26]. Maintenance of normal in vivo levels of constitutively synthesized tissue specific proteins is significantly enhanced by culturing these cells on or within specific biomatrices such as collagen gels [53,54], or matrices from other natural tissue glycoproteins such as fibronectin [SO]. Complex biomatrices have also been employed, as for example, that secreted by bovine corneal epithelial cells [41], or isolated from rat liver [55].…”

Topographical arrangement of basement membrane proteins in lactating rat mammary gland: comparison of the distribution of type IV collagen, laminin, fibronectin, and Thy-1 at the ultrastructural level.

“…However, the mesenchyme in mice is partially responsible for supporting the growth of normal mammary epithelium (30) and, in early development phases, for either the destruction of the glands in males (31) or organization of the branching pattern in females (32). Similarly, the extracellular matrix is required for the maintenance of the fully differentiated lactational state in vitro (33). Such epithelial-mesenchymal interactions would have to occur across the basement membrane.…”

Tissue Stiffness Dictates Development, Homeostasis, and Disease Progression

“…For example, ECM mechanics and geometry have been shown to be critical for epithelial cell differentiation, 33 and proper ECM composition and elasticity are required for maintenance of the differentiated state of mammary epithelial cells, as ECM stiffening and loss of laminin signaling were found to reduce mammary epithelial cell-specific gene expression. 34 Further, a complete loss or disruption of ECM structure leads to mammary gland regression 35 and the involution of capillary blood vessels, 36 demonstrating the importance of ECM structure for cellular differentiation and tissue development. Some tissues have unique mechanisms for altering their mechanics throughout morphogenesis.…”