Abstract A150: A methodology for the isolation, culture, and immortalization of human breast epithelial and myoepithelial cells.

Abstract

Abstract Introduction: Breast tissue is composed by different cell types including epithelial and myoepithelial cells, macrophages, fibroblasts, adipocytes, and cells from the immune system. Two main lineages, epithelial and myoepithelial cells, are the most essential cell populations in the normal mammary gland and also in breast cancer. Traditionally, cancer research has been performed using commercial cell lines, but primary cell cultures suppose a powerful tool to study with a higher reliability new aspects of mammary gland biology including normal and diseased tissue. However, normal primary human epithelial cells in culture achieve senescence at 10 to 40 population doubling, hampering their long-term culture. In addition, some studies have described lineage-specific marker limitations to be used in order to isolate these two cell types, since some of the markers tend to change during cell culture. Experimental Procedures: In this protocol we compared two different methodologies in an effort to improve isolation, culture and immortalization of different cell populations of human normal mammary epithelium. We based our approaches in previously described methods incorporating additional steps to improve cell survival, to preserve cellular antigens used for isolation and validation of cell subpopulations and to enhance cellular transformation. For these purposes, we determined cell growth and viability, and also cell-lineage specific markers of cytospinned cells in both methodologies used. Results: One of the goals of the present work to yield on cell survival and antigens preservation is in fact overnight digestion at low enzymatic concentration. Other crucial steps are cell immortalization and also addition of critical compounds to the cell culture media. Conclusions: In the present work, we propose some guidelines to establish more efficiently mammary epithelial cell lines allowing long-term cell culture system and providing us a strong instrument to better understand the role of the different epithelial cell types and the origins of breast cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A150. Citation Format: Gemma Fuster, Arantzazu Zubeldia-Plazaola, Mario Mancino, Patricia Fernández-Nogueira, Elisabet Ametller, Pedro Gascón, Vanessa Almendro. A methodology for the isolation, culture, and immortalization of human breast epithelial and myoepithelial cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A150.