Figure 2

Establishment of mammary epithelial cells from reduction mammoplasty/mastectomy specimens. The tissue is chopped, digested with collagenase and hyaluronidase, and plated in medium as organoids. Over a week or so, multiple types of epithelial cells and fibroblasts emerge; fibroblasts are removed by differential trypsinization (fibroblasts are loosely attached), remaining epithelial cells grow for 10–15 population doublings (PDs) followed by senescence of the majority of cells. Occasionally, an homogenous population of cells emerges that continue to proliferate for an additional 30–60 PDs, and eventually these cells also senesce (this step is referred to as agonescence).