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Preparation method of human breast milk stem cells and application of human breast milk stem cells

A stem cell and breast milk technology, applied in the field of cell biology, can solve the problems of cumbersome experimental steps, taking a lot of time, and the undifferentiated state does not have a very good effect, and achieves a large number, simple operation, and good undifferentiated state. Effect

Active Publication Date: 2017-05-24
SHENZHEN WINGOR BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, substances such as insulin and epidermal growth factor do not have a very good effect on maintaining the undifferentiated state of human breast milk stem cells. Compared with the former, culture of feeder layer cells can maintain the undifferentiated state of human breast milk stem cells slightly better, but the experimental steps more cumbersome and time-consuming

Method used

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  • Preparation method of human breast milk stem cells and application of human breast milk stem cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] A preparation method of human breast milk stem cells, characterized in that it comprises the following steps:

[0028] 1. Collection of breast milk

[0029] Collection of human breast milk: before collecting breast milk, test the donor’s ABO / Rh blood type, HLA typing and syphilis, HIV, HBV, HCV, then collect 5-200mL of the donor’s breast milk under aseptic conditions, and send it to the laboratory immediately Preparation of human breast milk stem cells.

[0030] 2. Isolation of Human Breast Milk Cells

[0031] Mix the breast milk with an equal volume of PBS buffer, centrifuge at 2000rpm for 20min at 4°C, absorb the fat layer and defatted breastmilk components, wash the remaining cell pellets with PBS buffer three times, centrifuge at 1500rpm for 10min each time, and replace with fresh culture medium. Resuspend the cell pellet and harvest the purified breast milk cell suspension.

[0032] 3. Culture of human breast milk stem cells

[0033] Transfer the purified breas...

Embodiment 2

[0037] On the basis of Example 1, the steps of cultivating human breast milk stem cells are different, as follows:

[0038] The purified breast milk cell suspension was transferred to a culture plate coated with 0.01% gelatin, which was coated for 1 h in advance, washed twice with PBS before cell culture, and then placed in CO 2 Culture in an incubator, change the medium once a day, and isolate a single cell clone on the 6th day and inoculate it into a new ultra-low adhesion culture plate to promote colony formation. When the cells are more than 80% confluent, add an appropriate amount of 0.25% trypsin to cover the cell surface, digest at 37°C for 5 minutes, stop digestion with 2 times the volume of DMEM / F12 medium containing 10% fetal bovine serum, and centrifuge at 1000rpm After 10 minutes, the cell pellet was collected, washed once with PBS buffer, centrifuged at 1000 rpm for 10 minutes, resuspended with fresh cell culture medium, and passaged at a ratio of 1:3.

[0039] The...

Embodiment 3

[0047] Carry out human breast milk stem cell proliferative activity test to embodiment 1 and comparative example, concrete process is as follows:

[0048] When the human breast milk stem cells of Example 1 and Comparative Example 1 were cultured on the 20th day, 6 fields of view (200×) were taken under an inverted microscope, and the number of undifferentiated cells was counted. The number of human breast milk stem cells prepared by the method of Example 1 was 9.17 ±0.75, the number of human breast milk stem cells prepared by the method of Comparative Example 1 was 4.17±0.75, so the number of human breast milk stem cells prepared in Example 1 was significantly higher than that of human breast milk stem cells prepared by conventional methods.

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Abstract

The invention provides a preparation method of human breast milk stem cells and application of the human breast milk stem cells. The preparation method comprises the following steps: collecting breast milk; separating human breast milk cells to obtained purified breast milk cell suspension; transferring the purified breast milk cell suspension into a culture plate of gelatin with the coating volume percentage of 0.01 to 0.05 percent, putting the culture plate into a CO2 incubator for culture, changing liquid once every day, separating single cells for cloning on the sixth day, and inoculating into a new ultralow-adherency culture plate to promote cloning formation; when the cells are fused to 80 percent or above, adding trypsin, carrying out digestion at 37 DEG C for 5 minutes, then ending the digestion, collecting cell sediments, washing and centrifugating the cells, resuspending the cells with fresh cell culture liquid, and carrying out passage according to 1 : 3. The preparation method provided by the invention is easy to operate; no co-culture of feeder cells is needed; furthermore, the undifferentiated state of the human breast milk stem cells can be better retained, and multiple human breast milk stem cells can be obtained.

Description

technical field [0001] The invention belongs to the technical field of cell biology, and in particular relates to a preparation method and application of human breast milk stem cells. Background technique [0002] Stem cells refer to those cells that have the ability of self-replication and multi-directional differentiation. They can continuously renew themselves and differentiate into one or more cells that constitute human tissues or organs under specific conditions. The use of stem cells to transplant damaged tissues can restore the tissue's ability to regenerate and restore its functions. Stem cell replacement therapy has brought great hope for the treatment of various diseases and tissue damage. Stem cells exist in embryos, mature body tissues, and blood. In recent years, studies have found that human breast milk also contains stem cell components. Breast milk stem cells show a multi-directional differentiation potential similar to human embryonic stem cells, and can ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074A61L27/38
CPCA61L27/3834C12N5/0602C12N2500/30C12N2501/11C12N2501/115C12N2501/235
Inventor 姜舒张芸纪惜銮杨顺罗朝霞
Owner SHENZHEN WINGOR BIO TECH
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