Method for efficiently separating and culturing porcine mammary epithelial cells

Abstract

The invention relates to a method for efficiently separating and culturing porcine mammary epithelial cells. The method comprises the steps of mammary tissue collection, pretreatment, mammary tissue block digestion, inoculation, cell purification, cell digestion passage and cell cryopreservation. According to the separation method, the porcine mammary epithelial cells are separated by using an enzyme digestion tissue block method, a porcine mammary epithelial cell line with stable growth is established, the time for separating the cells is short, the obtained cells are high in purity and do not need to be repeatedly purified, and the high-purity cells can be obtained only by purifying for 1-2 times; damage to cells is small, and the obtained cells are high in activity; and the cell growth is stable, the cell proliferation rate is not obviously changed when the cell is cultured to the tenth generation, the long-term experimental research is facilitated, and a stable experimental material is provided for the research of the physiological metabolism mechanism of the pig breast tissue.

Description

technical field [0001] The invention relates to a method for efficiently separating and culturing porcine mammary gland epithelial cells, which belongs to the field of biology. Background technique [0002] Sow milk is synthesized by mammary epithelial cells under the stimulation of prolactin, secreted into mammary acinar cavity, and finally excreted through mammary ducts and nipples, so mammary epithelial cells are the core of lactation. A large number of studies have focused on the regulation of nutrition on the lactation performance of sows, and porcine mammary epithelial cells can be used as an effective biological model for the study of the mechanism of nutrition regulation of sows' lactation performance. At present, the separation and culture technology of human, mouse and bovine mammary gland epithelial cells is becoming more and more mature, and high-quality cell lines can also be purchased in China. However, the separation technology of porcine mammary gland epithel...

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Problems solved by technology

These two methods have certain disadvantages. The "collagenase method" mainly uses collagenase to fully lyse breast tissue into individual cells and then culture them. The lysed breast tissue mainly contains mammary epithelial cells and fibroblasts, which need to be purified several times before Breast cells with high purity can be obtained, and the collagenase digestion process is likely to cause cell damage and affect cell activity; the "tissue block method" mainly separates and cultures breast tissue blocks until new breast epithelial cells grow. Better avoid the problems of fibroblast contamination and cell damage, but the cycle time required to obtain a large number of mammary epithelial cells is longer

[0004] Therefore, it is difficult to buy porcine mammary epithelial cells with high purity and strong activity in the current market, and the existing porcine mammary epithelial cell isolation methods have long separation cell cycle, many miscellaneous cells, cumbersome purification, and large cell damage. problem, further research is needed on the isolation method of porcine mammary epithelial cells

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