title: Comparison of methods for the isolation of human breast epithelial and myoepithelial cells.
abstract: 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.
authors.affiliation: Department of Medical Oncology, Hospital Clínic Barcelona, Spain ; Department of Medicine, University of Barcelona Barcelona, Spain.
authors.forename: Arantzazu
authors.initials: A
authors.lastname: Zubeldia-Plazaola
authors.affiliation: Department of Medical Oncology, Hospital Clínic Barcelona, Spain ; Institut d'Investigacions Biomediques August Pi i Sunyer Barcelona, Spain.
authors.forename: Elisabet
authors.initials: E
authors.lastname: Ametller
authors.affiliation: Department of Medical Oncology, Hospital Clínic Barcelona, Spain ; Institut d'Investigacions Biomediques August Pi i Sunyer Barcelona, Spain.
authors.forename: Mario
authors.initials: M
authors.lastname: Mancino
authors.affiliation: Department of Senology, Clínica Planas Barcelona, Spain.
authors.forename: Miquel
authors.initials: M
authors.lastname: Prats de Puig
authors.affiliation: Department of Medical Oncology, Hospital Clínic Barcelona, Spain ; Institut d'Investigacions Biomediques August Pi i Sunyer Barcelona, Spain.
authors.forename: Anna
authors.initials: A
authors.lastname: López-Plana
authors.affiliation: Histopathology-Citology, Anatomical Pathology Service, Centro Medico Teknon Barcelona, Spain.
authors.forename: Flavia
authors.initials: F
authors.lastname: Guzman
authors.affiliation: Department of Medical Oncology, Hospital Clínic Barcelona, Spain ; Institut d'Investigacions Biomediques August Pi i Sunyer Barcelona, Spain.
authors.forename: Laia
authors.initials: L
authors.lastname: Vinyals
authors.affiliation: Kidney and Acid-base Physiology Research Group, Institute of Physiology, University of Zurich Zurich, Switzerland.
authors.forename: Eva M
authors.initials: EM
authors.lastname: Pastor-Arroyo
authors.affiliation: Department of Medical Oncology, Hospital Clínic Barcelona, Spain ; Institut d'Investigacions Biomediques August Pi i Sunyer Barcelona, Spain ; Division of Medical Oncology, Department of Medicine, Harvard Medical School, Dana-Farber Cancer Institute, Brigham and Women's Hospital Boston, MA, USA.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Almendro
authors.affiliation: Department of Medical Oncology, Hospital Clínic Barcelona, Spain ; Institut d'Investigacions Biomediques August Pi i Sunyer Barcelona, Spain.
authors.forename: Gemma
authors.initials: G
authors.lastname: Fuster
authors.affiliation: Department of Medical Oncology, Hospital Clínic Barcelona, Spain ; Department of Medicine, University of Barcelona Barcelona, Spain ; Institut d'Investigacions Biomediques August Pi i Sunyer Barcelona, Spain.
authors.forename: Pedro
authors.initials: P
authors.lastname: Gascón
keywords: epithelial cells; isolation; myoepithelial cells; primary cell culture