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

Arantzazu Zubeldia-Plazaola,Vanessa Almendro,Mario Mancino,Elisabet Ametller,Flavia Guzman,Pedro Gascã³N,Gemma Fuster,Miquel Prats De Puig,Laia Vinyals,Eva M Pastor-Arroyo,Anna Lã³Pez-Plana

doi:10.3389/fcell.2015.00032

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.

Highlights

The mammary gland is a tubuloalveolar structure consisting of a branching network of ducts ending in terminal ductal lobular units (TDLUs) that constitute the functional domains of the pre-menopausal breast
20 and 60 years old was obtained from 13 patients who underwent reduction mammoplasty and 2 patients who had a mastectomy after breast cancer in the contralateral breast
In order to understand breast cancer etiology, it is crucial to comprehend the behavior of the cells in the normal human mammary breast gland

Summary

Introduction

The mammary gland is a tubuloalveolar structure consisting of a branching network of ducts ending in terminal ductal lobular units (TDLUs) that constitute the functional domains of the pre-menopausal breast. Approaches vary in the mechanical manipulation (discarding adipose tissue or not, the size of pieces), digestion (time of digestion, type/concentration of enzymes added such as collagenase, hyaluronidase or a combination of both), cell fraction separation (by sequential filtering or differential centrifugation), and final cell isolation (by immunomagnetic beads or by sorting) used to isolate the mammary epithelial cells (Speirs et al, 1998; Gudjonsson et al, 2004; Stingl et al, 2005; Shipitsin et al, 2007; Labarge et al, 2013; Raouf and Sun, 2013) Each of these procedures differs in the cell yield and viability due to the digestion, the fractionation steps, and the cell culture.

Methods

Results and Discussion

Conclusions