Can widely used cell type markers predict the suitability of immortalized or primary mammary epithelial cell models?

BACKGROUND: Mammary 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 (bMEC US) or Swiss Holstein-Friesian (bMEC CH) 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. RESULTS: The 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. CONCLUSIONS: The 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.

Estudio lectinhistoquímico y morfométrico de la glándula mamaria de llama lama glama durante el período de lactancia / Lectinhistochemical and morphological study of llama lama glama mammary gland during lactation

Se evaluó la expresión de oligosacáridos en la glándula mamaria de llamas (Lama glama) durante la lactogénesis utilizando lectinas. Mediante análisis morfométrico se evaluó el diámetro nuclear de los lactocitos, diámetros promedio de los alvéolos y el porcentaje del área ocupada por la luz alveolar. Se realizaron biopsias a 15 hembras, a los 5,15,30,60 y 120 días postparto. Se utilizaron siete lectinas biotiniladas (Con-A, WGA, DBA, SBA, PNA; RCA-1, UEA-1), siguiendo protocolos preestablecidos (Método ABC). El citoplasma del dominio apical de los lactocitos reaccionó intensamente con las lectinas WGA y RCA; y en forma moderada a Con-A, durante toda la lactogénesis. El glucocálix de los lactocitos reaccionó intensamente con SBA y PNA, en todos los períodos estudiados; desde el día 5 hasta el día 15 la reacción fue marcada para la RCA, y desde el 15 hasta los 120 días de lactogénesis con la DBA. No hubo marcación del parénquima con UEA- en ningunos de los períodos estudiados. Los resultados mostraron que los hidratos de carbono se expresan de diferentes maneras a medida que progresa la producción láctea, lo que indica la ocurrencia de cambios biomoleculares debidos a una intensa actividad funcional. El análisis morfométrico reveló que no se produce variaciones en el área relativa ocupada por los alvéolos, pero sí un aumento significativo (p<0.05) en el tamaño de los alvéolos a partir del día 15 acompañado por una disminución en el tamaño de los núcleos de lactocitos, a partir de los 60 días