Isolation and Culture of Normal Rat Mammary Epithelial Cells

Abstract

Primary culture models offer researchers the opportunity to study the factors that regulate physiologically relevant development of normal mammary epithelial cells under defined conditions. This chapter provides detailed methods for the isolation and culturing of normal mammary epithelial cells. Briefly, excised rat mammary glands are mechanically and enzymatically disaggregated, and organized clusters of mammary epithelial cells are isolated as mammary epithelial organoids. The recovered organoids, free of associated stromal fibroblasts and adipocytes, are then cultured within a complex reconstituted basement membrane rich in laminin, type IV collagen, and sulfated proteoglycans. In the presence of serum-free medium supplemented with insulin, prolactin, progesterone, hydrocortisone, and epidermal growth factor, functionally immature mammary epithelial cells from pubescent virgin rats undergo extensive proliferation, branching morphogenesis, and functional differentiation in vitro. Adaptations of these protocols allow mammary gland biologists and breast cancer researchers to isolate and culture various types of normal and malignant mammary cells.