Separation and purification method, as well as preservation method for ruminant galactophore epithelial cell

A technique for mammary epithelial cells and ruminants, applied in the research field, can solve the problems of staying, incomplete morphological data, and different separation effects, so as to reduce the formation of ice crystals, improve the lactation model, and improve the protection effect.

Inactive Publication Date: 2009-04-15
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 isolatio

Method used

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  • Separation and purification method, as well as preservation method for ruminant galactophore epithelial cell
  • Separation and purification method, as well as preservation method for ruminant galactophore epithelial cell
  • Separation and purification method, as well as preservation method for ruminant galactophore epithelial cell

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Experimental program
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Embodiment 1

[0058] Embodiment 1, a method for separating, purifying and culturing ruminant mammary gland epithelial cells, the following steps are carried out in sequence:

[0059] 1) Sampling of breast tissue:

[0060] Mammary gland tissue was obtained from two lactating Holstein cows freshly slaughtered.

[0061] 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.

[0062] Since the slaughterhouse and the laboratory are not located in the same place; therefore, the above-mentioned tissue pieces were first immersed in the culture solution of 10 tim...

Embodiment 2

[0075] Embodiment 2, a method for preserving ruminant mammary gland epithelial cells, the following steps are carried out in sequence:

[0076] 1), preparation of cryoprotectant solution:

[0077] The cryoprotectant solution is composed of the following volume percentage components: 10% DMSO, 40% fetal bovine serum and 50% cell suspension; the cell suspension is a mixture formed by cells and DMEM / F12 basal medium, and the cells in the cell suspension The density in is 1×10 6 / mL, cells are primary cultured mammary epithelial cells (embodiment 1 step 3) obtained), purified mammary epithelial cells (embodiment 1 step 4) obtained) or subcultured mammary epithelial cells (embodiment 1 step 5) ) income);

[0078] 2), gradually cool down the cryoprotectant solution obtained in the above step 1) slowly:

[0079] a. From 4°C to -8°C, the cooling rate is 1°C / min; keep at -8°C for one minute;

[0080] b. From -8°C to -20°C, the cooling rate is 0.5°C / min;

[0081] c. From -20°C to -...

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Abstract

The invention discloses a method for separating, purifying and culturing a mammary epithelial cell of a ruminant animal, which includes the following steps of: 1) the sampling of a mammary tissue; 2) the preparing of a culture fluid; 3) the culturing of a tissue block for obtaining a primary cultured mammary epithelial cell; 4) the purifying of the mammary epithelial cell for obtaining a purified mammary epithelial cell; and 5) the subculturing of the mammary epithelial cell. The invention simultaneously provides a preservation method for the mammary epithelial cell of the ruminant animal, which includes the following steps of: 1) preparing a freezing protection fluid which comprises the mammary epithelial cell of the ruminant animal; and 2) gradually and slowly reducing the temperature of the freezing protection fluid. The method can be adopted for prolonging the preservation time of the mammary epithelial cell of the ruminant animal.

Description

technical field [0001] 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 [0002] 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 factors in the body system and local areas, it is very difficult for people to study the development, functional differentiation, involution, and immune defense of mammary epithelial cells. Animal cell culture models simulate the...

Claims

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Application Information

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IPC IPC(8): C12N5/06A01N1/02C12N5/071
Inventor 刘建新刘红云赵珂
Owner ZHEJIANG UNIV
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