Development and validation of immortalized bovine mammary epithelial cell line as an in vitro model for the study of mammary gland functions
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This study aimed to develop a bovine mammary epithelial (BME) cell line model, which provides a possibility to determine functional properties of the bovine mammary gland. The primary cell culture was derived from bovine mammary gland tissues and processed enzymatically to obtain cell colonies with epithelial-like morphology. The cultures of BME cells were purified and optimally cultured at 37 °C in DMEM/F12 medium supplemented with 10% fetal bovine serum. The BME cells were identified as epithelial cell line by the evaluating the expression of keratin-18 using immunofluorescence staining. A novel gene expression system strongly enhances the expression of telomerase, has been used to immortalize BME cell line termed hTBME cell line. Interestingly, telomerase remained active even after over 60 passages of hTBME cell line, required for immortalization of BME cells. In addition, the hTBME cell line was continuously subcultured with a spontaneous epithelial-like morphology, with a great proliferation activity, and without evidence of apoptotic and necrotic effects. Further characterization showed that hTBME cell line can be continuously propagated in culture with constant chromosomal features and without tumorigenic properties. Finally, established hTBME cell line was evaluated for mammary gland specific functions. Our results demonstrated that the hTBME cell line was able to retain functional-morphological structure, and functional differentiation by expression of beta (β)-casein as in the bovine mammary gland in vivo. Taken together, our findings suggest that the established hTBME cell line can serve as a valuable tool for the study of bovine mammary gland functions.
KeywordsBovine mammary epithelial cell Immortalization Milk protein expression
The work was supported by the Key Program of the National Science Foundation of China [Grant No: 2008AA101006, China], and supported by grants from the National Natural Science Foundation of China (Grant Nos. 31572492, 31072109), the Natural Science Foundation of Tianjin, China (Grant No. 12JCZDJC22100), the Veterinary Biotechnology Scientific Research Innovation Team of Tianjin, China (Grant No. TD12-5019), and the Veterinary “Leading Talent Culture Project” of Tianjian, China.
JL and AS contributed equally to this work by designing, carrying out experiments, analyzing and interpreting the data. XG and WW contributed to analyze and interpret the data of this work. AS, JL, YZ and TJ contributed to the drafting of the manuscript, revising it critically, and giving final approval of the version to be published. All authors read and approved the final manuscript. All contributors who do not meet the criteria for authorship should be listed in an acknowledgements section.
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
Bovine mammary gland tissue was obtained from a local slaughterhouse in Shannxi, Xian, China. Animals were not killed for this scientific research, therefore, no ethical approval was needed for tissue collection.
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