ABSTRACT
A routine, reproducible procedure was developed for the preparation and characterization of stromal cells from normal human breast tissue obtained by reduction mammaplasty. Isolates (n = 15) all exhibited enhanced rates of proliferation, even in the presence of 20% fetal calf serum, when exposed to epidermal growth factor or transforming growth factor a (both 10(-8) M). Cellular responsiveness to these growth factors was consistent with expression of specific surface receptors for epidermal growth factor (approximately 10(4)/cell). In cultures, stromal cells elaborated an extensive, cross-linked, insoluble extracellular matrix which remained firmly associated with the plastic surface of tissue culture ware upon lysis of cells. The insoluble matrix material was analyzed using enzymatic digestion procedures following incorporation of radiolabelled precursors into macromolecular material prior to lysis and preparation. The relative proportion of glycoconjugate (glycopeptides and proteoglycans) and collagenous material present in matrix material was approximately 45% and approximately 55%, respectively, and this was modulated by inclusion of epidermal growth factor into culture medium to approximately 60% and approximately 40%, respectively. Under similar culture conditions stromal cells synthesized twice as much hyaluronate as was produced by control cultures. By use of specific antibody preparations we identified at least four species of glycopeptide present in stromal matrices (namely, fibronectin, laminin, tenascin, and thrombospondin) as well as three types of collagen (types I, III, and IV). The rapid and reproducible procedure for the preparation of radiolabelled insoluble matrix material from normal human breast tissue allows for the study of cellular interaction involving extracellular matrix turnover and degradation.
