Primary Bovine Mammary Epithelial Cells Were Isolated, Cultured And Passaged Technology

Use of animal mammary gland to produce pharmaceutical proteins are important biological high-tech content, exploration of mammary epithelial cells never stop. Starting from transgenic mice, an animal cell technology platform has been gradually built up. Mammary epithelial secretory cells, is a function of the cell, is relying on the breast cells to lactation. As a basis for the lactating mammary gland, mammary epithelial cells to physiological lactation sta rt the inquiry process, to maintain the function and degradation, it is an essential material, this material does not, and can not signal transduction studies of gene expression problems.Currently, there are some problems cultured bovine mammary epithelia l cells, complicated process and limited the number of passages leading to the success rate is not high. Under normal circumstances, even if successful culture, mammary epithelial cells will lose some biological functions, which makes these cells are generally only used for scientific research, using its practical application in industrial production, etc., more difficult. Domestically, the example successfully cultured bovine mammary epithelial cells much. Even in successful cases, for how isolated, cultured and passaged mammary epithelial cells, nor a more definite conclusion. As can be seen from the above background, the current problems to be solved is the creation of a new method, or the improvement of existing methods to effectively isolated and cultur ed and passaged bovine mammary epithelial cells, and the other is to establish what the technical indicators to detect purified mammary epithelial cells. Therefore, the study of bovine mammary epithelial cells were isolated, cultured and passaged technology is of great significance.Holstein cow mammary tissue studied experimental material selected from healthy tissue using inoculation in vitro and has been bovine mammary epithelial cells. And after a successful Cow mammary epithelial cells using cytokeratin immunofluorescence(IF) staining and protein immunoblotting(WB) on mammary epithelial cells were identified. Through cell isolation, culture, observed under an optical microscope passages and research on cryopreservation and recovery methods, the establishment of a clear primary bovine mammary epithelial cell culture methods. At the same time, with different methods of cryopreservation of bovine mammary epithelial cells to recover, and then measured the rate of adherent cells. In addition, also examined the bovine mammary epithelial cells, triglycerides, lactose and casein these nutrients and proliferation of cells law. These test results, from all aspects reflect the difference in different generation of cows lactating mammary epithelial cells, which in turn provides the basis and reference for the bovine mammary epithelial cells behind the experiment.The experimental results show that, by tissue explants adherent cultures were obtained separation into mammary epithelial cells and primary cultures of fib roblasts. After keratin for immunofluorescence(IF) staining and protein immunoblotting(WB) identified in mammary epithelial cells subculture. Different ways frozen bovine mammary epithelial cells to recover, group B method(placing the vials made of foam box) after cryopreservation best adherence was 91.2%. This method is recommended cryopreserved cells. Through lactose, milk fat, milk protein and cell proliferation of different generation of mammary epithelial cells were detected, the results show that the top ten generations mammary epithelial cells which lactose, milk fat, milk protein secretion change is not obvious, wherein the lactose, milk fat, milk protein when the fifteenth generation reduced secretion is obvious, slow the 12 th. Make sure 1-10 on behalf of the bovine mammary epithelial cells can be used in in vitro studies.