Isolation and Propagation of Mammary Epithelial Stem and Progenitor Cells

Several methods of mammary gland dissociation have been described that utilize a combined strategy of mechanical and enzymatic dissociation to isolate mammary epithelial cells (MECs) from intact tissue (Smalley et al., J Mammary Gland Biol Neoplasia 17:91-97, 2012). Here we detail a robust method that enables the isolation of all major stem and progenitor MEC populations, which has been successfully used to study stem cell behavior when coupled with transplantation and in vitro assays (Shackleton et al., Nature 439:84-88, 2006; Bouras et al., Cell Stem Cell 3:429-441, 2008; Sheridan et al., BMC Cancer 15:221, 2015; Jamieson et al., Development 144:1065-1071, 2017). Furthermore, we outline two prominent methods for culturing MECs for the purposes of ex vivo manipulation or study: 2D feeder layer cultures and 3D Matrigel colony assays. Importantly, all outlined methods retain stem and progenitor cell behaviors and can be used in combination with downstream in vivo, in vitro, or in silico analyses.