Establishment And Culture Of Goat Mammary Epithelial Cell Line

We used both methods of explant culture and enzymatic digestion in this study to separate goat mammary epithelial cells. Purified cells were established for single-cell clone and identified. MTT colorimetric assay was developed to determine the optimum medium of goat mammary epithelial cells with the design of orthogonal regression rotation. The normal physiological function of mammary epithelial cells was identified by enzyme linked immunosorbent assay and polyacrylamide gel electrophoresis. By this, we successfully established the Goat mammary epithelial cell line.Experiment 1 Both methods of explant culture and enzymatic digestion were used to separate goat mammary epithelial cells. The result showed that the viable epithelial cells which have high capacity of proliferation can be observed by explant culture. Proliferative epithelial cells were achieved by using collagenaseⅠ/ hyaluronidase digestion technique. The proliferation capacity of epithelial cells declined after several passages. After 2-3 passages, Purified cells were obtained by scraping, difference digestion and difference adherence.Experiment 2 Single-cell clone lines were carried out from goat mammary epithelial cells. Three cell cloning were obtained in this study, there was no one cloning by Mouth sucker method, 5.9% by cloning cylinder method and 3.5% by cell limiting dilution method. It was characterized as goat mammary alveolus epithelial cells by the cellular morphology and cytokeratin 18. We concluded that cell cloning efficiency was highest by the cloning cylinder method, which was difficult to practice and could result in cell impurity. Cell limiting dilution method is the easiest practice, least damage and higher cloning efficiency. Experiment 3 MTT colorimetric assay was developed to allow a reproducible and quantitative measurement to determine the effects of fetal bovine serum (FBS), hydrocortisone, insulin, transferring and epidermal growth factor (EGF) on the growth of goat mammary epithelial cells with the design of orthogonal regression rotation. The results indicated that the optimum medium was DMEM/F12 with 12% FBS, 4μg/ml hydrocortisone, 10μg/ml insulin, 8μg/ml transferring and 6ng/ml EGF.Experiment 4 Goat mammary epithelial cells planted in the coated culture plate were induced by prolactin.β-casein in the cell supernatant was tested by ELISA. The protein level were 0.5mg/ml after 48h culture, 0.75mg/ml after 72h. The secretion ofβ-casein was further identified by the polyacrylamide gel electrophoresis. It meant the mammary epithelial cells had the normal physiological function.