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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

Flowchart of methodological approaches used to obtain cellular fractions from human reduction mammoplasty tissue. Human tissue was minced into small fragments and sequentially digested using two digestion protocols. (A) Slow digestion (overnight at low enzymatic concentration) and fast digestion (4–6 h at high enzymatic concentration). Afterwards, the digested tissues obtained from each method were processed using two cell separation techniques: (B) sequential filtering and differential centrifugation.
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Figure 1: Flowchart of methodological approaches used to obtain cellular fractions from human reduction mammoplasty tissue. Human tissue was minced into small fragments and sequentially digested using two digestion protocols. (A) Slow digestion (overnight at low enzymatic concentration) and fast digestion (4–6 h at high enzymatic concentration). Afterwards, the digested tissues obtained from each method were processed using two cell separation techniques: (B) sequential filtering and differential centrifugation.

Mentions: For our purpose, the tissue digestion was performed following the two methods described below (Figure 1), and each sample was distributed as shown in Table S1. When two methods were performed, the samples were equally divided (40–75 g in each method).


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)

Flowchart of methodological approaches used to obtain cellular fractions from human reduction mammoplasty tissue. Human tissue was minced into small fragments and sequentially digested using two digestion protocols. (A) Slow digestion (overnight at low enzymatic concentration) and fast digestion (4–6 h at high enzymatic concentration). Afterwards, the digested tissues obtained from each method were processed using two cell separation techniques: (B) sequential filtering and differential centrifugation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Flowchart of methodological approaches used to obtain cellular fractions from human reduction mammoplasty tissue. Human tissue was minced into small fragments and sequentially digested using two digestion protocols. (A) Slow digestion (overnight at low enzymatic concentration) and fast digestion (4–6 h at high enzymatic concentration). Afterwards, the digested tissues obtained from each method were processed using two cell separation techniques: (B) sequential filtering and differential centrifugation.
Mentions: For our purpose, the tissue digestion was performed following the two methods described below (Figure 1), and each sample was distributed as shown in Table S1. When two methods were performed, the samples were equally divided (40–75 g in each method).

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