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Biological Research

, 49:1

First Online: 06 January 2016Received: 01 September 2015Accepted: 21 December 2015DOI: 10.1186-s40659-015-0063-2

Cite this article as: Ontsouka, E.C., Bertschi, J.S., Huang, X. et al. Biol Res 2016 49: 1. doi:10.1186-s40659-015-0063-2

Abstract

BackgroundMammary cell cultures are convenient tools for in vitro studies of mammary gland biology. However, the heterogeneity of mammary cell types, e.g., glandular milk secretory epithelial or myoepithelial cells, often complicates the interpretation of cell-based data. The present study was undertaken to determine the relevance of bovine primary mammary epithelial cells isolated from American Holstein bMECUS or Swiss Holstein–Friesian bMECCH cows, and of primary bovine mammary alveolar epithelial cells stably transfected with simian virus-40 SV-40 large T-antigen MAC-T for in vitro analyses. This was evaluated by testing their expression pattern of cytokeratin CK 7, 18, 19, vimentin, and α-smooth muscle actin α-SMA.

ResultsThe expression of the listed markers was assessed using real-time quantitative PCR, flow cytometry and immunofluorescence microscopy. Characteristic markers of the mesenchymal vimentin, myoepithelial α-SMA and glandular secretory cells CKs showed differential expression among the studied cell cultures, partly depending on the analytical method used. The relative mRNA expression of vimentin, CK7 and CK19, respectively, was lower P < 0.05 in immortalized than in primary mammary cell cultures. The stain index based on flow cytometry of CK7 and CK19 protein was lower P < 0.05 in MAC-T than in bMECs, while the expression of α-SMA and CK18 showed an inverse pattern. Immunofluorescence microscopy analysis mostly confirmed the mRNA data, while partly disagreed with flow cytometry data e.g., vimentin level in MAC-T. The differential expression of CK7 and CK19 allowed discriminating between immortal and primary mammary cultures.

ConclusionsThe expression of the selected widely used cell type markers in primary and immortalized MEC cells did not allow a clear preference between these two cell models for in vitro analyses studying aspects of milk composition. All tested cell models exhibited to a variable degree epithelial and mesenchymal features. Thus, based on their characterization with widely used cell markers, none of these cultures represent an unequivocal alveolar mammary epithelial cell model. For choosing the appropriate in vitro model additional properties such as the expression profile of specific proteins of interest e.g., transporter proteins should equally be taken into account.

KeywordsCell characterization Flow cytometry Gene expression Lactation marker Mammalian Mammary epithelial cell AbbreviationsCKcytokeratin

MECmammary epithelial cells

MGmammary gland

SIstain index

SMAsmooth muscle actin

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Autor: Edgar Corneille Ontsouka - Janique Sabina Bertschi - Xiao Huang - Michael Lüthi - Stefan Müller - Christiane Albrecht

Fuente: https://link.springer.com/







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