Novel method for preparing menstrual blood mesenchymal stem cells

A technology of stem cells and menstrual blood, applied in the field of biomedicine, to achieve the effect of high passage times, avoiding damage and high cell viability

Active Publication Date: 2015-06-17
ZHEJIANG SHENGCHUANG PRECISION MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But as mentioned above, the younger the girl, the more hesitant about invasive in vivo harvesting, so the source of viable menstrual blood stem cells becomes a problem

Method used

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  • Novel method for preparing menstrual blood mesenchymal stem cells
  • Novel method for preparing menstrual blood mesenchymal stem cells
  • Novel method for preparing menstrual blood mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1 In vitro collection of blood

[0076] The urine cup is used to collect menstrual blood outside the body. The main process is as follows:

[0077] Before that, the menstrual blood was collected in the body. This method not only has certain requirements for the volunteers, such as married women, but also has certain damage to the volunteers’ bodies. Our method is to collect them outside the body, that is, There are no strict requirements for volunteers. This method requires the volunteers to collect when the menstrual blood flow is relatively large. figure 1 shown) at room temperature for collection, pour the collected menstrual blood into a screw-cap collection tube with a scale on the tube wall and containing 25ml of menstrual blood collection fluid (capacity is 50ml, figure 2 ), about 20ml of menstrual blood needs to be collected each time, and the cap should be tightened immediately after pouring, and the tube should be shaken to mix. The purpose is to pr...

Embodiment 2

[0079] Example 2 Isolation of mesenchymal stem cells from menstrual blood (centrifugation)

[0080] The mixture of menstrual blood sample and collection fluid was filtered with a 100 μm cell strainer (brand: BD; product number: 352360) to remove most mucus and clots, and then mononuclear cells were collected by density gradient centrifugation. Mononuclear cells were counted with a hemocytometer.

[0081] Mononuclear cells are collected and subjected to cell expansion, purification and cryopreservation.

[0082]1. The filtered menstrual blood sample is obtained by density gradient centrifugation to obtain mononuclear cells: slowly add the sample to the human lymphocyte separation medium (TBD), and the volume ratio of the sample to the TBD separation medium is 2:1. Centrifuge at 800g for 15 minutes in a tabletop centrifuge.

[0083] 2. Absorb the central buffy coat cells, that is, mononuclear cells, into another clean centrifuge tube, add PBS and repeat centrifugal washing for...

Embodiment 4

[0110] Example 4 The tumorigenicity of menstrual blood mesenchymal stem cells separated by the present invention

[0111] 64 nude mice were divided into the positive control group and the mesenchymal stem cell group obtained in Example 2, 32 in each group, half male and half male. The mesenchymal stem cells obtained in Example 2 at the fifth passage were inoculated into nude mice subcutaneously, and 5×10 6 Menstrual blood stem cells and gastric cancer BGC823 cell line. The experimental observation time ranged from 2 weeks to 2 months, and none of the test groups inoculated with menstrual blood stem cells of the present invention had tumorigenicity. Exemplary results such as Figure 5 Shown (observation results after four weeks of inoculation), where the upper picture is menstrual blood stem cells (5 × 10 6 cells); the figure below is gastric cancer cell line BGC823 (5×10 6 cell).

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Abstract

The invention relates to a novel method for preparing menstrual blood mesenchymal stem cells. The method comprises the following steps: a, collecting menstrual blood in vitro, and storing in menstrual blood collecting liquid to obtain menstrual blood mixed liquid; b, separating single karyocyte from the menstrual blood mixed liquid; C, culturing the single karyocyte in an adherent manner, proliferating cells, and then through adherent purification, obtaining purified cells, namely the menstrual blood mesenchymal stem cells. The novel method has the advantages of being nondestructive in the body, strong in uniformity and high in cell activity which is reflected in the characteristics of high cell proliferation speed, high passage times and the like.

Description

technical field [0001] The invention relates to a novel method for preparing menstrual blood mesenchymal stem cells. Specifically, the present invention relates to a novel method for collecting, separating and storing menstrual blood mesenchymal stem cells, which belongs to the field of biomedicine. Background technique [0002] Stem cells are a kind of pluripotent cells with self-replication ability, which can differentiate into various functional cells under certain conditions. Stem cell research has received more and more attention. At present, stem cell sources can be divided into embryonic stem cells and adult stem cells. However, due to the ethical and moral controversies involved in embryonic stem cells, its clinical application is severely limited. Adult stem cells are divided into neural stem cells, blood stem cells, mesenchymal stem cells, epidermal stem cells, etc. Among them, mesenchymal stem cells (mesenchymalstem cells, MSCs) are currently one of the most co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 余艳春
Owner ZHEJIANG SHENGCHUANG PRECISION MEDICAL TECH CO LTD
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