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

Zubeldia-Plazaola A, Ametller E, Mancino M, Prats de Puig M, López-Plana A, Guzman F, Vinyals L, Pastor-Arroyo EM, Almendro V, Fuster G, Gascón P - Front Cell Dev Biol (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Hospital Clínic Barcelona, Spain ; Department of Medicine, University of Barcelona Barcelona, Spain.

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.

No MeSH data available.


Related in: MedlinePlus

Analysis of the expression of CD10 and CD227 by flow cytometry. The analyzed cells were obtained from two patients (Patient 1: RM108 and Patient 2: RM109), through a combination of the following techniques: (A) slow digestion and sequential filters, (B) slow digestion and differential centrifugation, (C) fast digestion and sequential filters and (D) fast digestion and differential centrifugation.
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Figure 5: Analysis of the expression of CD10 and CD227 by flow cytometry. The analyzed cells were obtained from two patients (Patient 1: RM108 and Patient 2: RM109), through a combination of the following techniques: (A) slow digestion and sequential filters, (B) slow digestion and differential centrifugation, (C) fast digestion and sequential filters and (D) fast digestion and differential centrifugation.

Mentions: Regardless of the technique investigated, once the cells started to grow they were able to form proper acinar structures when seeded on top of matrigel cultures, since myoepithelial cells (K14+) were located in the edges surrounding the epithelial cells (K19+) (Figure 4). Therefore, all the methods described here can provide viable cells (Speirs et al., 1998; Gudjonsson et al., 2002, 2004; Stingl et al., 2005; Shipitsin et al., 2007; Raouf and Sun, 2013). Moreover, the presence of both populations found in normal breast tissue, myoepithelial (CD10+) and epithelial (CD227+) cells, was analyzed by flow cytometry (Figure 5). It was found that the myoepithelial cell fraction is larger than the epithelial one. Even though the differences were not significant, a higher number of epithelial cells tended to be obtained when slow digestion was used (Figures 5A,B). This trend was found in every sample analyzed. In this context, it has been described that some markers such as CD44 are lost due to tissue dissociation protocol (Hines et al., 2014). Even if CD227 and CD10 proteins have not been reported to be disappeared, the fact that CD227+ cells are lost when fast digestion is performed, suggested that this antigen could be suffering from a similar phenomenon described for CD44 (Hines et al., 2014).


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

Zubeldia-Plazaola A, Ametller E, Mancino M, Prats de Puig M, López-Plana A, Guzman F, Vinyals L, Pastor-Arroyo EM, Almendro V, Fuster G, Gascón P - Front Cell Dev Biol (2015)

Analysis of the expression of CD10 and CD227 by flow cytometry. The analyzed cells were obtained from two patients (Patient 1: RM108 and Patient 2: RM109), through a combination of the following techniques: (A) slow digestion and sequential filters, (B) slow digestion and differential centrifugation, (C) fast digestion and sequential filters and (D) fast digestion and differential centrifugation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440402&req=5

Figure 5: Analysis of the expression of CD10 and CD227 by flow cytometry. The analyzed cells were obtained from two patients (Patient 1: RM108 and Patient 2: RM109), through a combination of the following techniques: (A) slow digestion and sequential filters, (B) slow digestion and differential centrifugation, (C) fast digestion and sequential filters and (D) fast digestion and differential centrifugation.
Mentions: Regardless of the technique investigated, once the cells started to grow they were able to form proper acinar structures when seeded on top of matrigel cultures, since myoepithelial cells (K14+) were located in the edges surrounding the epithelial cells (K19+) (Figure 4). Therefore, all the methods described here can provide viable cells (Speirs et al., 1998; Gudjonsson et al., 2002, 2004; Stingl et al., 2005; Shipitsin et al., 2007; Raouf and Sun, 2013). Moreover, the presence of both populations found in normal breast tissue, myoepithelial (CD10+) and epithelial (CD227+) cells, was analyzed by flow cytometry (Figure 5). It was found that the myoepithelial cell fraction is larger than the epithelial one. Even though the differences were not significant, a higher number of epithelial cells tended to be obtained when slow digestion was used (Figures 5A,B). This trend was found in every sample analyzed. In this context, it has been described that some markers such as CD44 are lost due to tissue dissociation protocol (Hines et al., 2014). Even if CD227 and CD10 proteins have not been reported to be disappeared, the fact that CD227+ cells are lost when fast digestion is performed, suggested that this antigen could be suffering from a similar phenomenon described for CD44 (Hines et al., 2014).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Hospital Clínic Barcelona, Spain ; Department of Medicine, University of Barcelona Barcelona, Spain.

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.

No MeSH data available.


Related in: MedlinePlus