Preservation method of ruminant mammary gland epithelial cells

A technique for mammary epithelial cells and ruminants, applied in the research field, can solve the problems of immune identification, microstructure observation, few studies on cryopreservation, stay, and incomplete morphological data, etc., and achieve a perfect mammary epithelial cell lactation model. , Reduce the formation of ice crystals, good protection effect

Inactive Publication Date: 2010-10-20
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Scholars at home and abroad have done research on the in vitro culture of ruminant mammary gland epithelial cells, but the current research mainly stays in the aspects of culture methodology, morphology and hormone regulation of mammary gland cells, and there are no domestic and foreign reports on the isolation method. Although they are the same, the isolation effects are different, the morphological data are not comprehensive, there are not many studies involving its immunological identification, microstructure observation, and cryopreservation, and there are even fewer systematic studies on its lactation function

Method used

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  • Preservation method of ruminant mammary gland epithelial cells
  • Preservation method of ruminant mammary gland epithelial cells
  • Preservation method of ruminant mammary gland epithelial cells

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0059] Embodiment 1, a method for separating, purifying and culturing ruminant mammary gland epithelial cells, the following steps are carried out in sequence:

[0060] 1) Sampling of breast tissue:

[0061] Mammary gland tissue was obtained from two lactating Holstein cows freshly slaughtered.

[0062] Take a sample from the middle of the mammary gland, try to separate the connective tissue and fat, first soak and disinfect the mammary gland tissue with 75% absolute ethanol, and cut the tissue into 1cm 3 small pieces of tissue; wash the small pieces of tissue with 0.3% bromogeramine (that is, benzalkonium bromide aqueous solution with a mass concentration of 0.3%), and wash the small pieces of tissue repeatedly at least 3 times; % benzalkonium bromide 1ml) rinse the tissue at least 3 times.

[0063] Since the slaughterhouse and the laboratory are not located at the same place; therefore, the above-mentioned tissue pieces were first immersed in 10 times double-antibody cultu...

Embodiment 2

[0076] Embodiment 2, a method for preserving ruminant mammary gland epithelial cells, the following steps are carried out in sequence:

[0077] 1), preparation of cryoprotectant solution:

[0078] The cryoprotectant solution is composed of the following components by volume percentage: 10% DMSO, 40% fetal bovine serum and 50% cell suspension; the cell suspension is a mixture of cells and DMEM / F12 basal medium, and the cells in the cell suspension The density in is 1×10 6 / mL, the cells are primary cultured mammary epithelial cells (Example 1 step 3) obtained), purified mammary epithelial cells (Example 1 step 4) obtained) or subcultured mammary epithelial cells (Example 1 step 5) ) income);

[0079] 2), gradually cool down the cryoprotectant solution obtained in the above step 1) slowly:

[0080] a. From 4°C to -8°C, the cooling rate is 1°C / min; keep at -8°C for one minute;

[0081] b. From -8°C to -20°C, the cooling rate is 0.5°C / min;

[0082] c. From -20°C to -120°C, th...

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Abstract

The invention discloses a preservation method of ruminant mammary gland epithelial cells. The preservation method comprises the following steps of: (1) preparing a cryoprotective solution which is prepared from the following components in percentage by weight: 10 percent of DMSO (Dimethyl Sulfoxide), 40 percent of fetal calf serums and 50 percent of cell suspension; and (2) gradually and slowly lowering the temperature of the obtained cryoprotective solution until the temperature of the cryoprotective solution is lowered to -196 DEG C and cryopreserving the cryoprotective solution at -196 DEG C with liquid nitrogen. By adopting the method of the invention, the preservation time of the ruminant mammary gland epithelial cells can be prolonged.

Description

[0001] The present invention is a divisional application of "Separation, Purification and Preservation of Ruminant Mammary Epithelial Cells" with the filing date of 2008.11.24 and the application number of 200810162422.3. technical field [0002] The invention relates to the research fields of tissue and cell engineering, transgenic animals, etc., especially a method for separating, purifying, culturing and freezing mammary gland epithelial cells. Background technique [0003] The mammary gland is an important skin exocrine organ with various physiological, biochemical and immune functions, which can provide sufficient nutrition for infants or young animals and meet their developmental needs. The full play of these functions depends on a series of specific changes experienced by the mammary gland itself: mammary gland development, mammary gland differentiation, lactation, and mammary gland reconstruction and involution. In fact, due to the interaction of many variable factor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
Inventor 刘红云刘建新赵珂
Owner ZHEJIANG UNIV
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