Method for establishing uterine membrane stem cell bank

A technology of uterine stem cells and construction method, which can be applied to non-embryonic pluripotent stem cells, animal cells, vertebrate cells, etc., can solve the method of sterilization and the effect of sterilization. Reports are unfavorable and cannot guarantee the success rate of uterine stem cells. And other issues

Inactive Publication Date: 2014-06-11
上海坤爱生物科技股份有限公司
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  • Claims
  • Application Information

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Problems solved by technology

[0003] However, after searching the prior art literature, it was found that among the reported methods for obtaining uterine stem cells, no one reported on the method of sterilization during the separation process and the effect of sterilization, and the success rate of uterine stem cell isolation cannot be guaranteed. , which is not conducive to the subsequent process

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  • Method for establishing uterine membrane stem cell bank
  • Method for establishing uterine membrane stem cell bank
  • Method for establishing uterine membrane stem cell bank

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, the collection of menstrual blood

[0037] After the donor signs the informed consent, store the 5ml-15ml menstrual blood collected by the menstrual cup in an equal volume of menstrual blood preservation solution (PBS, and add 100U / ml penicillin and 100U / ml streptomycin) at 4°C Delivered to the nationally certified GMP workshop within 48 hours under certain conditions.

Embodiment 2

[0038] Example 2, the separation of uterine stem cells

[0039] Put 5ml of menstrual blood mixture directly on a 100mm plate, add 5ml of α-MEM (containing 20% ​​serum), at 37°C, 5%CO 2 cultured under static conditions. After 48 hours, replace with 10ml of fresh culture medium. Perform a sterility test on the discarded supernatant (see Table 1).

[0040] Table 1 Sterility detection of uterine stem cells

[0041]

[0042] (Note: positive means the culture medium in the test tube is clarified, negative means the culture medium in the test tube is turbid)

Embodiment 3

[0043] Example 3, Sterilization and Separation of Uterine Stem Cells

[0044] Slowly add 5ml of menstrual blood mixture into a 15ml centrifuge tube containing 5ml of Ficoll separation solution, use the method of density gradient centrifugation, set the acceleration and descent speed to 1, and the rotation speed to 300-500×g, centrifuge for 20-40 minutes and collect Buffy coat, or mononuclear cells (PBMC). Rinse the PBMC with 10ml of PBS, centrifuge at 1000rpm-1500rpm for 5-10 minutes, and discard the waste liquid. This process is repeated 3 times to remove residual bacteria and other substances. Resuspend the isolated target cells with 3ml α-MEM (containing 10%-20% serum), place in a six-well plate at 37°C, 5% CO 2After 48 hours, 3 ml of fresh culture medium was replaced, and adherent cells were observed under the microscope, and the cells were spindle-shaped. Perform a sterility test on the discarded supernatant (see Table 1).

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Abstract

The invention provides a method for establishing a uterine membrane stem cell bank. The method comprises the steps of menstrual blood collection, uterine membrane stem cell separation and degerming, amplification, analysis, detection, long-term storage and regular detection. The invention provides a degerming method which is simple and easy to operate. The method provided by the invention can obtain a large quantity of higher-purity uterine membrane stem cells by carrying out amplification culture on the uterine membrane stem cells with substances (such as bacteria) removed and can be used for storing the uterine membrane stem cells for a long time, thereby providing a large quantity of uterine membrane stem cell resources for future clinical and scientific research application study.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the construction of a stem cell bank, and more specifically to the construction of a uterine stem cell bank. Background technique [0002] In 1999, "Science" rated the research results of human embryonic stem cells as the top ten scientific and technological progress in the world that year. In 2000, Time magazine listed it as the top ten scientific and technological achievements in the world at the end of the 20th century. Genome will simultaneously become the most promising field of development and application in the new century. So far, stem cell research has become one of the most striking hot spots in the fields of biology and medicine in recent years. Stem cells (SC) are a kind of pluripotent cells with self-renewing ability, which can differentiate into various functional cells under certain conditions. Stem cells are not yet fully differentiated and immature cells, which have ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12N5/074C12N5/0789
Inventor 刘召青陈彦田齐瀚实
Owner 上海坤爱生物科技股份有限公司
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