Primary human mammary epithelial cells (HMEC) were derived from five women. Following subculture and removal of fibroblasts by differential trypsinization, cells exhibited a typical epithelial cobblestone-like morphology with some dome and ridge formation. For HMEC purity assessment, cells were stained for cytokeratins, which are usually only expressed in epithelial cells.
HMECs were derived from surgical specimens of healthy women who had undergone reduction mammoplasty. Tissues were dissected and enzymatically digested into duct-like structures termed organoids, filtered to retain only organoids, and plated or frozen in aliquots. Organoids were plated, and cells migrated off onto the plastic. There was no decrease in viability or alteration in morphology when cells were frozen before plating. Fibroblasts were removed with brief exposure to trypsin-versene (differential trypsinization). Cells were serially passed at near-confluence with trypsin-EDTA until senescence. Cells were grown in MCDB-170 medium+ SFS, a complex medium designed for growth of HMEC suplemented with estradiol, hydrocortisone, insulin, epidermal growth factor, isoproterenol and bovine pituitary extract (BPE).