Development and validation of immortalized bovine mammary epithelial cell line as an in vitro model for the study of mammary gland functions
- 225 Downloads
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.
- Booth C, O’Shea JA (2002) Isolation and culture of intestinal epithelial cells. In: Freshney RI, Freshney MG (eds) Culture of epithelial cells, 2nd edn. Copyright 2002 Wiley-Liss Inc (2002) ISBNs: 0-471-40121-8 (Hardback); 0-471-22120-1 (Electronic)Google Scholar
- Geraghty RJ, Capes-Davis A, Davis JM, Downward J, Freshney RI, Knezevic I, Lovell-Badge R, Masters JR, Meredith J, Stacey GN, Thraves P, Vias M; Cancer Research UK (2014) Guidelines for the use of cell lines in biomedical research. Br J Cancer 111:1021–1046. doi: 10.1038/bjc.2014.166 CrossRefGoogle Scholar
- Gibson CA, Vega JR, Baumrucker CR, Oakley CS, Welsch CW (1991) Establishment and characterization of bovine mammary epithelial cell lines. In Vitro Cell Dev Biol 1993 Jan;29A:86.Google Scholar
- Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell Biol Int 100:57–70Google Scholar
- Hong HX, Zhang YM, Xu H, Su ZY, Sun P (2007) Immortalization of swine umbilical vein endothelial cells with human telomerase reverse transcriptase. Mol Cells 24:358–363Google Scholar
- Hou M, Xu D, Bjorkholm M, Gruber A (2001) Real-time quantitative telomeric repeat amplification protocol assay for the detection of telomerase activity. Clin Chem 47:519–524Google Scholar
- Jedrzejczak MA (2010) Bovine mammary epithelial cell culture an alternative model for study of modification and mammary gland functions. PhD dissertation, West Pomeranian University of Technology, SzczecinGoogle Scholar
- Kozłowski M, Motyl T (2007) Use of three-dimensional cultures (3D) in the investigation of bovine mammary gland biology. Med Weter 63:1417–1420Google Scholar
- Moore CM, Janish C, Eddy CA, Hubbard GB, Leland MM, Rogers J (1999) Cytogenetic and fertility studies of a rheboon, rhesus macaque (Macaca mulatta) x baboon (Papio hamadryas) cross: further support for a single karyotype nomenclature. Am J Phys Anthropol 110:119–127. doi: 10.1002/(SICI)1096-8644(199910)110:2<119:AID-AJPA1>3.0.CO;2-S CrossRefGoogle Scholar
- Povey RC, Osborne AD (1969) Mammary gland neoplasia in the cow. A review of the literature and report of a fibrosarcoma. Pathol Vet 6:502–512Google Scholar
- Riley LG, Gardiner-Garden M, Thomson PC, Wynn PC, Williamson P, Raadsma HW, Sheehy PA (2010) The influence of extracellular matrix and prolactin on global gene expression profiles of primary bovine mammary epithelial cells in vitro. Anim Genet 41:55–63. doi: 10.1111/j.1365-2052.2009.01964.x CrossRefGoogle Scholar
- Serakinci N, Christensen R, Graakjaer J, Cairney CJ, Keith WN, Alsner J, Saretzki G, Kolvraa S (2007) Ectopically hTERT expressing adult human mesenchymal stem cells are less radiosensitive than their telomerase negative counterpart. Exp Cell Res 313:1056–1067. doi: 10.1016/j.yexcr.2007.01.002 CrossRefGoogle Scholar
- Tait L, Soule HD, Russo J (1990) Ultrastructural and immunocytochemical characterization of an immortalized human breast epithelial cell line, MCF-10. Cancer Res 50:6087–6094Google Scholar
- Taylor-Papadimitriou J, Stampfer M, Bartek J, Lewis A, Boshell M, Lane EB, Leigh IM (1989) Keratin expression in human mammary epithelial cells cultured from normal and malignant tissue: relation to in vivo phenotypes and influence of medium. J Cell Sci 94:403–413Google Scholar
- Wang W, Said A, Wang Y, Fu Q, Xiao Y, Lv S, Shen Z (2015) Establishment and characterization of duck embryo epithelial (DEE) cell line and its use as a new approach toward DHAV-1 propagation and vaccine development. Virus Res 29:260–268Google Scholar