Breast cancer is primarily derived from mammary epithelial cells, the main cell type in human mammary glands. The majority of knowledge gained thus far around breast cancer has come from research using immortalized epithelial cell lines. The use of primary cells derived from breast tissue can be used in research to provide more biological relevance representative of the heterogeneous nature of breast cancer development and metastasis in its natural microenvironment. However, the successful isolation and propagation of human primary mammary gland cells can be costly and difficult due to their complex in vivo microenvironment and sensitivity when isolated. Here, we present a gentle isolation method for viable human mammary epithelial cells (hMECs) and donor-matched human mammary fibroblasts (hMFbs) from human mammary gland tissue. We isolated, expanded and passaged the hMECs and hMFbs in vitro and characterized cultures using cell-specific markers. A total of four primary cell lines were isolated and established from normal breast tissue and characterized through various markers, including pan cytokeratin (panCK), CK14, CD44, CD31, fibronectin and vimentin by immunofluorescence. To determine functional potential for subsequent studies, epithelial cells were examined via Matrigel® assays to assess spheroid development. Both cell type cultures expressed lineage specific markers with hMECs but not hMFbs forming spheroid structures in 3D Matrigel® assays. Our analyses confirm the successful isolation of two different cell phenotypes from normal breast tissues. This robust technique provides an inexpensive and accessible approach for mammary cell isolation.
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