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In-vitro culture method of mammary gland epithelial cells

A mammary epithelial cell, in vitro culture technology, applied in the field of biological culture, can solve the problems of difficult removal of fibroblasts, decreased mammary epithelial cell viability, decreased biological function, etc., to achieve uniform size, larger size and high activity Effect

Inactive Publication Date: 2014-12-10
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

This makes it difficult to completely remove fibroblasts during digestion and passage, and the viability and biological function of breast epithelial cells decrease after multiple digestion passages

Method used

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  • In-vitro culture method of mammary gland epithelial cells
  • In-vitro culture method of mammary gland epithelial cells
  • In-vitro culture method of mammary gland epithelial cells

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Embodiment 1

[0021] 1. Culture medium preparation

[0022] Mix 50 milliliters of FBS, 2.5 milliliters of penicillin-streptomycin mixed solution [penicillin-streptomycin mixed solution (100X), the content of penicillin is 10000U / ml, the content of streptomycin is 10mg / ml], 0.15 gram of taurine and 0.4 ml of epidermal growth factor (10 μg / ml) was dissolved in 450 ml of DMEM / F12 culture medium, filtered through a 0.22-micron membrane filter, and distributed into sterilized 50-ml centrifuge tubes, 40 ml per tube, and stored at 4°C. Half an hour before cell culture, put two tubes of a total of 80 ml of culture solution in a 37°C water bath to preheat.

[0023] 2. Treatment of culture bottles

[0024] Take out the aseptically packaged T25 culture bottle in the ultra-clean bench, place the bottom wall of the culture bottle upwards, use a scale to draw a horizontal line every 0.4 cm from the bottom wall to the bottom of the bottle until it is close to the mouth of the bottle, Then start from one s...

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Abstract

The invention discloses an in-vitro culture method of mammary gland epithelial cells. The method comprises the following steps: treating a mammary tissue into chylific tissue blocks with a tissue homogenizer, sequentially inoculating the tissue blocks on labeled culture bottle bottom walls, and culturing, wherein after 2-3 days, fibroblasts around each tissue block are free and perform adherent culture growth; and after 4-5 days, epithelial cells start to be free and perform adherent culture growth, and the fibroblasts are driven to the periphery of the epithelial cell and grow around the epithelial cells; and at this time, adding trypsinase to digest the adherent-culture fibroblasts, and continuing culturing for 5-7 days after finishing digestion until about 80-90% of cells are adherent to the wall, thereby obtaining the higher-purity mammary gland epithelial cells. Compared with the existing culture method, the method disclosed by the invention performs digestion treatment on the fibroblasts in the early culture stage, and at this time, only small amounts of epithelial cells are free and grow (can be completely removed in the digestion treatment); and afterwards, the free epithelial cells can not be damaged by the digestion treatment, and the cell activity and purity are very high.

Description

technical field [0001] The invention relates to a biological culture technology, in particular to a method for in vitro culture of breast epithelial cells. Background technique [0002] The mammary gland is an organ unique to mammals. It has the function of synthesizing and secreting milk, and is a very active place for protein synthesis. Studies have shown that the expression of milk protein-encoding genes has obvious tissue-specific and stage-specific, that is, the synthesis of milk protein is only carried out in mammary epithelial cells, and occurs for a long time before and after the lactation mother gives birth During lactation, mammary epithelial cells are recognized as target cells for making mammary gland bioreactors. That is to use breast epithelial cells cultured in vitro to produce medicinal proteins or other proteins with great medical value by constructing specific expression vectors. In addition, mammary gland epithelial cells are also a useful tool for study...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 游伟谭秀文赵红波刘晓牧刘桂芬成海建刘倚帆万发春宋恩亮
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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