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Establishment of mammary gland model in vitro: culture and evaluation of a yak mammary epithelial cell line.

Fu M, Chen Y, Xiong X, Lan D, Li J - PLoS ONE (2014)

Bottom Line: The EGFP gene was successfully transferred into the YMECs, and the transfected cells could be maintained for a long duration in the culture by continuous subculturing.The cells expressed more antimicrobial peptides upon S.aureus invasion.Therefore, the established cell line could be considered a model system to understand yak mammary gland biology.

View Article: PubMed Central - PubMed

Affiliation: College of Life Science and Technology, Southwest University for Nationalities, Chengdu, China.

ABSTRACT
This study aimed to establish yak mammary epithelial cells (YMECs) for an in vitro model of yak mammary gland biology. The primary culture of YMECs was obtained from mammary gland tissues of lactating yak and then characterized using immunocytochemistry, RT-PCR, and western blot analysis. Whether foreign genes could be transfected into the YMECs were examined by transfecting the EGFP gene into the cells. Finally, the effect of Staphylococcus aureus infection on YMECs was determined. The established YMECs retained the mammary epithelial cell characteristics. A spontaneously immortalized yak mammary epithelial cell line was established and could be continuously subcultured for more than 60 passages without senescence. The EGFP gene was successfully transferred into the YMECs, and the transfected cells could be maintained for a long duration in the culture by continuous subculturing. The cells expressed more antimicrobial peptides upon S.aureus invasion. Therefore, the established cell line could be considered a model system to understand yak mammary gland biology.

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Growth curves of YMECs at the 10th, 60th, and 15th passages (resuscitated cells).
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pone-0113669-g002: Growth curves of YMECs at the 10th, 60th, and 15th passages (resuscitated cells).

Mentions: Growth curve analysis of YMECs revealed a population doubling time of approximately 36–48 h without any substantial changes among the 10th (early), 50th (late), and 15th passages (frozen thawed MECs) (Fig. 2). The cultured YMECs slowly grew within the first 4 d, and rapidly proliferated from day 5 to day 8. After day 9, the proliferation of the cells became slow. The cell growth curve exhibited an “S” shape (Fig. 2). Cell contact inhibition was observed at post-confluence stage, during which many floating dead cells appeared. Survival rate analysis showed that cell adherence and the survival rate gradually increased within 24 h, and the cell survival rate reached 85% at 24 h. These findings indicate that YMECs exhibited high viability. Bacterial and mycoplasma analyses demonstrated that the isolated cells were not contaminated by bacteria or mycoplasma. Senescence Associated β-galactosidase (SA-β- gal) assay was carried on early passage (passage 10) and late passage (passage 60) YMECs to assess the level of cell senescence. SA-β- gal staining was observed in both early and late passage YMECs cells of which positive cells stained blue, moreover, approximately 10% of YMECs cells stained for SA-β- gal (Fig. 3).


Establishment of mammary gland model in vitro: culture and evaluation of a yak mammary epithelial cell line.

Fu M, Chen Y, Xiong X, Lan D, Li J - PLoS ONE (2014)

Growth curves of YMECs at the 10th, 60th, and 15th passages (resuscitated cells).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4257549&req=5

pone-0113669-g002: Growth curves of YMECs at the 10th, 60th, and 15th passages (resuscitated cells).
Mentions: Growth curve analysis of YMECs revealed a population doubling time of approximately 36–48 h without any substantial changes among the 10th (early), 50th (late), and 15th passages (frozen thawed MECs) (Fig. 2). The cultured YMECs slowly grew within the first 4 d, and rapidly proliferated from day 5 to day 8. After day 9, the proliferation of the cells became slow. The cell growth curve exhibited an “S” shape (Fig. 2). Cell contact inhibition was observed at post-confluence stage, during which many floating dead cells appeared. Survival rate analysis showed that cell adherence and the survival rate gradually increased within 24 h, and the cell survival rate reached 85% at 24 h. These findings indicate that YMECs exhibited high viability. Bacterial and mycoplasma analyses demonstrated that the isolated cells were not contaminated by bacteria or mycoplasma. Senescence Associated β-galactosidase (SA-β- gal) assay was carried on early passage (passage 10) and late passage (passage 60) YMECs to assess the level of cell senescence. SA-β- gal staining was observed in both early and late passage YMECs cells of which positive cells stained blue, moreover, approximately 10% of YMECs cells stained for SA-β- gal (Fig. 3).

Bottom Line: The EGFP gene was successfully transferred into the YMECs, and the transfected cells could be maintained for a long duration in the culture by continuous subculturing.The cells expressed more antimicrobial peptides upon S.aureus invasion.Therefore, the established cell line could be considered a model system to understand yak mammary gland biology.

View Article: PubMed Central - PubMed

Affiliation: College of Life Science and Technology, Southwest University for Nationalities, Chengdu, China.

ABSTRACT
This study aimed to establish yak mammary epithelial cells (YMECs) for an in vitro model of yak mammary gland biology. The primary culture of YMECs was obtained from mammary gland tissues of lactating yak and then characterized using immunocytochemistry, RT-PCR, and western blot analysis. Whether foreign genes could be transfected into the YMECs were examined by transfecting the EGFP gene into the cells. Finally, the effect of Staphylococcus aureus infection on YMECs was determined. The established YMECs retained the mammary epithelial cell characteristics. A spontaneously immortalized yak mammary epithelial cell line was established and could be continuously subcultured for more than 60 passages without senescence. The EGFP gene was successfully transferred into the YMECs, and the transfected cells could be maintained for a long duration in the culture by continuous subculturing. The cells expressed more antimicrobial peptides upon S.aureus invasion. Therefore, the established cell line could be considered a model system to understand yak mammary gland biology.

Show MeSH
Related in: MedlinePlus