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Isolated culture method for mammary epithelial cells

A technology for separating and culturing mammary gland epithelial cells, which is applied in the direction of cell culture active agents, epidermal cells/skin cells, tissue culture, etc., can solve the problems of immature mammary gland epithelial cells cultured in vitro, shorten the separation time, reduce pollution, and facilitate The effect of the operation

Inactive Publication Date: 2021-04-02
BEIJING YULONG SHENGSHI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the in vitro culture of mammary epithelial cells is still immature

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] In order to achieve the above object, the technical scheme adopted in the present invention is:

[0035] Experimental supplies; curved tweezers, curved forceps, surgical scissors, surgical scalpels; culture medium and additives: F12 basal culture medium, 10% heat-inactivated fetal bovine serum, hydrocortisone, insulin, penicillin, streptomycin, human Recombinant epidermal growth factor; culture plate, culture bottle; collagenase solution: collagenase A, 2.6mg / mlHepes, 1.2mg / mlNaHCO 3 , trypsin, 9.8mg / ml HamsF10 and 10% fetal bovine serum; trypsin / EDTA mixed digestion solution, PBS, stop solution (10% fetal bovine serum was added to the F12 basal medium).

[0036] The invention provides a method for isolating and culturing human breast epithelial cells, comprising the steps of:

[0037] Step (1): select pregnant mammary gland tissue, put the breast tissue into PBS buffer solution with double antibody and wash repeatedly for several times, and remove fat tissue with surg...

Embodiment 2

[0046] A method for separating and culturing mammary gland epithelial cells, similar to Example 1, the difference is that in step (6), hydrocortisone 1ug / ml, insulin 8ug / ml, penicillin 80ug / ml, streptocin were added to the F12 basal medium Mycin 80ug / ml, human recombinant epidermal growth factor 8ug / ml.

Embodiment 3

[0048] A method for separating and culturing mammary gland epithelial cells, similar to Example 1, the difference is that in step (6), hydrocortisone 3ug / ml, insulin 12ug / ml, penicillin 120ug / ml, streptocin were added to the F12 basal medium Mycin 120ug / ml, human recombinant epidermal growth factor 12ug / ml.

[0049] Cell Viability Detection

[0050] The cell viability was determined by the traditional trypan blue method in the laboratory. Use the addition of complete medium in step (8) to terminate the digested cell solution, centrifuge and resuspend, draw a small amount of cell suspension and trypan blue to mix at 9:1, and use a cell counting plate to calculate the staining and concentration within 3 minutes. Number of unstained cells.

[0051] Cell viability = number of unstained cells / total number of cells × 100%

[0052] Experimental results, the average value was taken four times successively, according to the formula, the average values ​​of Examples 1-3 were 97.23%, ...

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PUM

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Abstract

The invention discloses an isolated culture method for mammary epithelial cells. The method comprises the following steps: selecting and placing pregnant mammary tissue into a double-antibody-added PBS buffer solution, and carrying out repetitive scrubbing to remove fatty tissue; shearing the mammary tissue into tissue pieces; placing the sheared mammary tissue into a culture dish, and adding a collagenase solution for digestion; carrying out centrifugation to collect precipitated cells, carrying out centrifugation with a culture solution, carrying out washing, and suspending cell mass again;suspending the cells again with an inoculation culture medium, and culturing the cells in an incubator; and replacing a growth culture medium, carrying out culture until a transferred generation is overgrown, dumping supernatant, carrying out washing, adding trypsin for digestion until cells completely fall off, and adding terminating liquid to terminate digestion . According to the isolated culture method, the defects and shortages in the prior art that the cell vitality is low, the isolated quantity is small, and the storability is poor are overcome to the maximum; and compared with cells obtained by the traditional laboratory methods, the mammary epithelial cells cultured by the method have the advantages that the vitality is higher, the quantity is larger, and adherence is easier.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a method for separating and culturing 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. Mammary gland epithelial cells are derived from mammary gland lobules, and together with glandular ducts and adipose tissue, they form a complex network structure in the mammary gland. Mammary gland epithelial cells are regulated by hormones during birth, development and pregnancy for a series of growth, migration and differentiation. Imbalanced hormone levels, changes in the extracellular matrix, and other genetic factors can lead to malignant growth of mammary epithelial cells and eventually cause breast cancer. Culture and understanding of the characteristics of ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0631C12N2501/30C12N2501/33C12N2501/11
Inventor 张晓南吴芳春谷涌泉侍晓云张斌
Owner BEIJING YULONG SHENGSHI BIOTECH CO LTD