Method for efficiently separating and purifying mammary epithelial cells

A technique for separation and purification of mammary epithelial cells is applied in the field of high-efficiency separation and purification of animal mammary epithelial cells in vitro to achieve the effects of less cell damage, simple and easy experimental operation, and improved culture efficiency.

Inactive Publication Date: 2016-02-10
北京大北农科技集团股份有限公司动物医学研究中心 +3
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to overcome the deficiencies of the existing methods for separating and purifying mammary gland epithelial cells, and provide a method for efficiently separating and purifying mammary gland epithelial cells that can obtain uniformly purified mammary gland epithelial cells in a short period of time without affecting cell-specific functions. method

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  • Method for efficiently separating and purifying mammary epithelial cells
  • Method for efficiently separating and purifying mammary epithelial cells
  • Method for efficiently separating and purifying mammary epithelial cells

Examples

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Effect test

Embodiment 1

[0023] Example 1 Separation and Purification of Cow Mammary Epithelial Cells

[0024] 1. Sampling of breast tissue

[0025] The sample was taken from an adult lactating black-and-white dairy cow in a local slaughterhouse. The mammary gland was cut open by aseptic surgery. In the parenchyma, avoid blood vessels, fat and connective tissue, and collect 8mm 3 Small and large tissue pieces were soaked in 75% alcohol for 2-3 seconds, washed in Hank’s solution containing 200 U / mL penicillin and 200 μg / mL streptomycin for about 30 seconds, incubated in the above-mentioned Hank’s solution with ice packs and brought back to the laboratory.

[0026] Trim off the visible fat and connective tissue of the tissue block in an ultra-clean workbench, wash twice with Hank’s solution containing 150 U / mL penicillin and 150 μg / mL streptomycin, and chop to 1 mm 3 Add 30mL of this Hank’s solution, shake at room temperature for 5min, discard the cleaning solution, and repeat this until the supernatan...

Embodiment 2

[0043] Example 2 Separation and purification of sheep mammary gland epithelial cells

[0044] The sample was taken from an adult late-gestation sheep, the mammary gland was aseptically cut open, and in the parenchyma, avoiding fat and connective tissue, the sample was collected 8mm 3Small and large tissue pieces were soaked in 75% alcohol for 2-3 seconds, washed in Hank’s solution containing 200 U / mL penicillin and 200 μg / mL streptomycin for about 30 seconds, incubated in the above-mentioned Hank’s solution with ice packs and brought back to the laboratory. Trim off the visible fat and connective tissue of the tissue block in an ultra-clean workbench, wash twice with Hank’s solution containing 150 U / mL penicillin and 150 Uμg / mL streptomycin, and chop to 1 mm 3 Add 30mL of this Hank’s solution to the following size, shake at room temperature for 5min, and collect about 10g of small tissue pieces for later use.

[0045] Put spare tissue pieces into 15ml of collagenase digestion...

Embodiment 3

[0046] Example 3 Separation and Purification of Yak Mammary Epithelial Cells

[0047] The sample was taken from an adult lactating yak, the mammary gland was aseptically cut open, and a 10mm sample was collected in the parenchyma, avoiding fat and connective tissue 3 Small and large tissue pieces were soaked in 75% alcohol for 2-3 seconds, washed in Hank’s solution containing 200 U / mL penicillin and 200 μg / mL streptomycin for about 30 seconds, incubated in the above-mentioned Hank’s solution with ice packs and brought back to the laboratory. Trim off the visible fat and connective tissue of the tissue block in an ultra-clean workbench, wash twice with Hank’s solution containing 300 U / mL penicillin and 300 Uμg / mL streptomycin, and chop to 1 mm 3 Add 30mL of this Hank’s solution to the following size, shake at room temperature for 5min, discard the cleaning solution, repeat this process until the supernatant is clear, and collect about 10g of small tissue pieces for later use. ...

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Abstract

The invention discloses a method for efficiently separating and purifying mammary epithelial cells. The method is suitable for a plurality of mammals. The mammary tissues of adult mammals, which are in the middle and later periods of gestation or in the lactation period, are collected through aseptic operation. According to the provided method, the separation and purification can be achieved in one step, the whole experiment is simple and efficient; the purification, which comprises repeated differential digestion and differential attachment, in the conventional method is eliminated, the purity of mammary epithelial cells obtained by the provided method can reach 98% or more, the obtained mammary epithelial cells can be continuously cultured in-vitro and passed down for 40 generations, and the lactating function of mammary epithelial cells is not affected. The obtained mammary epithelial cells can be applied to the researches on growth of mammary gland, lactating mechanism, mammary bioreactor, transgenic animals, mammary diseases, and the like.

Description

technical field [0001] The invention relates to a method for separating and purifying animal cells in vitro, in particular to a method for efficiently separating and purifying animal mammary gland epithelial cells in vitro. Background technique [0002] The main function of the mammary gland is lactation. Mammary epithelial cells are the only cells with secretory function in the mammary gland, and they are the starting point for studying the mechanism of mammary gland lactation. The proliferation and differentiation of mammary epithelial cells run through the process of mammary gland development and lactation. Therefore, it is very meaningful to study the regulation of mammary epithelial cell proliferation and differentiation at the cellular level. The in vitro isolation and culture of mammary epithelial cells has been widely used in the research of mammary gland development mechanism, milk secretion mechanism, mammary gland bioreactor construction and breast cancer mechanis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 郝明超刘长辉王敏王贵华赵亚荣
Owner 北京大北农科技集团股份有限公司动物医学研究中心
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