Separation and purification method of goat mammary epithelial cells

A technology of mammary epithelial cells and goat mammary glands, applied in animal cells, vertebrate cells, artificial cell constructs, etc., can solve the problems of unsatisfactory separation and purification effect, and achieve neat shape, simple operation and vigorous proliferation. Effect

Inactive Publication Date: 2015-02-25
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many types of cells in breast tissue, and the breast epithelial cells obtained by the above method are a mixture of various types of cells, which must be purified and cultured before they can be used in subsequent experiments, but the separation and purification effects are not satisfactory, so it is necessary to More efficient separation and purification methods

Method used

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  • Separation and purification method of goat mammary epithelial cells
  • Separation and purification method of goat mammary epithelial cells
  • Separation and purification method of goat mammary epithelial cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Sampling and Processing of Goat Mammary Epithelial Cells

[0041] The mammary gland tissues of the purebred Laoshan dairy goats of Qingdao Aotai sheep farm in the lactation period of 30 days were directly aseptically collected by using the existing technology;

[0042] Rinse and collect mammary gland tissue blocks with PBS to remove blood and goat milk on the surface; after rinsing, immerse in DF12 medium containing 200U / ml penicillin and 200μg / ml streptomycin, put them in an ice box and bring them back to the laboratory quickly For treatment, the breast tissue was washed repeatedly with PBS containing 200 U / ml penicillin and 200 μg / ml streptomycin until the washing solution became clear, divided and trimmed on an ultra-clean bench, and the adipose tissue and connective tissue were removed and rinsed until the washing solution was clear. The obtained white granular acinar tissue can be obtained.

Embodiment 2

[0043] Example 2 Tissue Block Culture Isolation of Mammary Epithelial Cells and Fibroblasts

[0044] a. Use ophthalmic scissors and ophthalmic tweezers to divide the above-mentioned white granular mammary acinar tissue into 0.5-1mm 3 The left and right small tissue pieces were rinsed with PBS containing 200 U / ml penicillin and 200 μg / ml streptomycin at the same time;

[0045] b. Infiltrate the small tissue pieces with the medium prepared by FBS and DF12 at a volume ratio of 1:1 for 2-4 minutes, inoculate them on the bottom of the culture dish at an interval of 0.5 cm, and place them at 37°C and 5vt% CO 2 , in an incubator with saturated humidity for 4 hours;

[0046] c. Add culture medium to the above-mentioned petri dish so that it just covers the bottom of the petri dish, and place at 37°C, 5vt% concentration of CO 2 , cultured in an incubator with saturated humidity, add culture solution after 12 hours until the tissue block is completely submerged, replace half of the cu...

Embodiment 3

[0050] Fibroblasts were removed by rapid digestion combined with differential attachment method to obtain purified mammary epithelial cells. The specific steps are as follows:

[0051] a. Differential digestion method: Add digestive solution to digest the cells in the culture of mammary epithelial cells obtained in Example 2 mixed with other cell growth, observe the changes in cell morphology under an inverted microscope, when the fibroblasts break away from the bottle wall, Aspirate and discard the digestion solution to remove fibroblasts, use the remaining digestion solution to continue digestion for 1-2 minutes, add culture medium to stop digestion, and pipette the remaining cells into a single-cell suspension;

[0052] Wherein the digestion solution is an equal volume of PBS without calcium and magnesium ions and 0.25% Trypsin-EDTA solution, the digestion condition is room temperature 25°C, and the digestion time of fibroblasts is 1.5-2 minutes;

[0053] The culture soluti...

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Abstract

The invention relates to the field of tissue and cell engineering and particularly provides a separation and purification method of goat mammary epithelial cells. The method mainly comprises the following steps: 1) tissue sampling: collecting a goat mammary acinus tissue in a sterile manner; 2) cell separation: separating the mammary epithelial cells and fibroblasts by using a tissue block culture method; 3) cell purification: removing the fibroblasts by combining a differential digestion method with a differential adhesion method to obtain the purified mammary epithelial cells. The method provided by the invention is simple and convenient to operate, and the mammary epithelial cells which are fully purified can be obtained through the conventional cell culture operation. The mammary epithelial cells obtained by adopting the method provided by the invention have the advantages of tidy shapes and exuberant proliferation, and can be applied to research of breast growth and development, lactation mechanism and verification of effectiveness of a specific expression carrier of the mammary tissue.

Description

technical field [0001] The invention relates to the field of tissue and cell engineering, and in particular provides a method for separating and purifying goat mammary gland epithelial cells. Background technique [0002] The mammary gland is a compound ductular gland, which is composed of capsule, stroma and parenchyma. In general, epithelial cells make up the acinar and ductal system of the mammary parenchyma. Most of the epithelium is the acinar epithelium that lines the surface of the acinar cavity and has a secretory function. In addition, there are ductal epithelium that constitutes the duct system and myoepithelium that surrounds the outer surface of the duct and acinus. The interstitium is mainly composed of adipocytes, but fibroblasts, cells of the hematopoietic system, blood vessels, and nerves are also present. [0003] Mammary gland epithelial cells are an in vitro model for studying mammary gland growth and development, lactation mechanism, and verifying the e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071
Inventor 王建民陈存仙董飞王桂芝纪志宾秦孜娟
Owner SHANDONG AGRICULTURAL UNIVERSITY
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