Extracellular matrix and mouse mammary cell function: Comparison of substrata in culture

Abstract

Cultured mammary cells depend on interaction with a substratum for functional differentiation, even in the presence of lactogenic hormones. Protein synthesis and secretion by mouse mammary epithelial cells on floating collagen gels and (EHS) matrix were compared. Cells were prepared by collagenase digestion of tissue from mid-pregnant mice. Protein synthesis was consistently greater in cells attached to EHS matrix, and was associated with proportionately higher rates of protein secretion into culture medium. Cells on EHS secreted protein into a luminal space formed within multicellular alveolus-like structures. Luminal secreted protein, extracted by EGTA treatment of cells in situ, constituted up to 40% of total secreted radiolabeled protein for cells on EHS matrix. The EGTA extract contained a higher proportion of casein and lactoferrin, whereas transferrin was predominantly in the medium. This indicated that cells on EHS matrix had become polarized and were secreting proteins vectorially. In contrast, EGTA treatment of cells on floating collagen gels released virtually no radiolabeled protein, showing that mammosphere formation was a property of cells on EHS. These biochemical observations were supported by ultrastructural evidence. In EHS cultures, the proportion of secreted protein in the luminal fraction, but not the distribution of secreted proteins, changed with time. This suggests that there may be leakage out of the lumen, or intraluminal degradation of protein after secretion. Nevertheless, the results suggest that cellular organization into mammospheres on EHS matrix promotes synthetic and secretory activity. This system provides a useful model for investigation of the regulation of milk secretion.

Isolation of EpH4 mammary epithelial cell subpopulations which differ in their morphogenetic properties

“…Culture of mammary epithelial ceils on Matrigel, a reconstituted laminin-rich matrix extracted from the EHS tumor (Kleinman et al, 1986), has been shown to result in the formation of well organized, alveolar-like "mammospheres" (Barcellos-Hoff et al, 1989;Petersen et al, 1993;Hurley et al, 1994). To establish whether EpH4 subpopulations would differ in their ability to form three-dimensional structures on this substrate, ceils from each clone were seeded onto a thick layer of Matrigel and incubated for 10-15 d in complete medium.…”

Mammary-tissue amino acid transport systems

“…This will lead to changes in the electrical potential of the mammary cells, which in turn will affect the activity of electrogenic transport systems. Mammary cells cultured on a reconstituted basement membrane do, however, form acinar structures enclosing a hollow lumen (Barcellos-Hoff et al 1989;Chen & Bissell, 1989;Hurley et al 1994;Blatchford et al 1995). These acini, termed mammospheres, appear to be polarized and are capable of secreting protein in a vectorial fashion (Blatchford et al 1995).…”

Unanticipated Reversal of Polarity in Primary Human Mammospheres Cultured in Ultra‐Low Attachment Plates

“…The mechanisms underlying the changes in polarity are unclear; however, previous studies of 3D cultures of primary cells and established cell lines from mouse and human suggest that culturing stem/progenitor cells on substrata such as Matrigel™ (BD Biosciences) induces acinar structures with superior cell differentiation as defined by phenotype 4,12 (and as reviewed by Elliot and Yuan 8 ).…”