Comparison of methods for the isolation of human breast epithelial and myoepithelial cells

Frontiers in Cell and Developmental Biology

Flavia eGuzmán¹  Miquel ePrats de Puig²  Vanessa eAlmendro³  Laia eVinyals⁴  Gemma eFuster⁴  Elisabet eAmetller⁴  Mario eMancino⁴  Arantzazu eZubeldia-Plazaola⁴  Anna eLópez-Plana⁴  Pedro eGascon⁴  Eva Maria ePastor-Arroyo⁵ 

[1]Anatomical Pathology Service, Centro Medico Teknon

[2]Clínica Planas

[3]Dana-Farber Cancer Institute, Brigham and Women’s Hospital and Harvard Medical School

[4]IDIBAPS

[5]Institute of Physiology, University of Zurich

关键词: Primary Cell Culture;    breast cancer;    Breast epithelial cells;    breast myoepithelial cells;    isolation protocol;   

DOI  :  10.3389/fcell.2015.00032

来源: DOAJ

【 摘 要 】

Two lineages, epithelial and myoepithelial cells, are the main cell populations in the normal mammary gland and in breast cancer. Traditionally, cancer research has been performed using commercial cell lines, but primary cell cultures obtained from fresh breast tissue are a powerful tool to study more reliably new aspects of mammary gland biology, including normal and pathological conditions. Nevertheless, the methods described to date have some technical problems in terms of cell viability and yield, which hamper work with primary mammary cells. Therefore, there is a need to optimize technology for the proper isolation of epithelial and myoepithelial cells. For this reason, we compared four methods in an effort to improve the isolation and primary cell culture of different cell populations of human mammary epithelium. The samples were obtained from healthy tissue of patients who had undergone mammoplasty or mastectomy surgery. We based our approaches on previously described methods, and incorporated additional steps to ameliorate technical efficiency and increase cell survival. We determined cell growth and viability by phase-contrast images, growth curve analysis and cell yield, and identified cell-lineage specific markers by flow cytometry and immunofluorescence in 3D cell cultures. These techniques allowed us to better evaluate the functional capabilities of these two main mammary lineages, using CD227/K19 (epithelial cells) and CD10/K14 (myoepithelial cells) antigens. Our results show that slow digestion at low enzymatic concentration combined with the differential centrifugation technique is the method that best fits the main goal of the present study: protocol efficiency and cell survival yield. In summary, we propose some guidelines to establish primary mammary epithelial cell lines more efficiently and to provide us with a strong research instrument to better understand the role of different epithelial cell types in the origin of breast cancer.

【 授权许可】

Unknown