Dahanayaka, Sudath Anilaka (2016-07). Isolation and Characterization of Porcine Mammary Epithelial Cells from Non-Lactating and Non-Pregnant Gilt. Master's Thesis.
Thesis
Mammary epithelial cells (MEC) are the functional units of the mammary gland during lactation. There are well-established MEC cell lines, such as bovine MAC-T, as well as mouse and human cell lines. Primary MEC lines are useful models to understand metabolism, growth, differentiation and other functions of the mammary gland epithelium. It was recently reported that dietary supplementation with either branched-chain amino acids or glutamate enhanced milk production by sows. The availability of a stable porcine MEC (PMEC) cell line is important to elucidate the underlying mechanisms responsible for effects of amino acids on enhancing milk production by lactating sows. In the present study, PMEC were isolated from mammary glands of a 9-month-old non-pregnant and non-lactating female pig. Cells were then cultured at 37^oC and 5% CO2 in 10 ml Dulbecco's Eagle Medium-F12 medium containing 5 ug/ml insulin, 1 ug/ml hydrocortisone, 5 ng/ml epidermal growth factor, 50 ug/ml Gentamycin, 5% fetal calf serum, and antifungal and antibiotics from Gibco. The PMEC plated on culture plates displayed a monolayer with a cobblestone epithelial-like morphology and formed island monolayer aggregates with typical characteristics of MEC. PMEC cells expressed cytokeratin-18, but not Vimentin. These cells also expressed the mammary gland-specific gene for beta-casein and synthesized and released beta-casein protein into the culture medium, based on results of reverse transcription polymerase chain reaction and western blot analyses.
