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Method for hypoxia culture of endometrial stem cell from menstrual blood

An endometrial and stem cell technology, applied in the field of stem cells, can solve the problems of high oxygen concentration and differences in tissue microenvironment, and achieve the effects of strong proliferation and differentiation ability, strong survival ability and good stability

Pending Publication Date: 2019-08-02
左凤琼
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the general conditions for in vitro expansion and culture of endometrial stem cells are O 2 The concentration is 21%, the oxygen concentration is high, and there is a big difference between the microenvironment of endometrial stem cells and the tissue microenvironment of their source during in vitro culture

Method used

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  • Method for hypoxia culture of endometrial stem cell from menstrual blood
  • Method for hypoxia culture of endometrial stem cell from menstrual blood
  • Method for hypoxia culture of endometrial stem cell from menstrual blood

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Collection of menstrual blood samples from adult women

[0034] (1) Preparation of specimen preservation solution: PBS buffer solution, respectively add gentamicin, 0.2 mg / ml amphotericin, and 1.0 mg / ml EDTA-NA2 at a final concentration of 200 U / ml. Mix well.

[0035] (2) Eight healthy adult female volunteers were recruited 3 days before the menstrual cycle, and about 10ml of their menstrual blood samples were collected, and then the menstrual blood samples were transferred to a 50ml centrifuge tube containing 10ml of specimen preservation solution, and mixed gently. The menstrual blood sample preservation solution was stored at 4°C, and the separation and extraction of endometrial stem cells were completed within 24 hours.

Embodiment 2

[0036] Example 2 Preparation of hypoxic endometrial stem cells

[0037] Take 4 parts of the menstrual blood sample preservation solution in Example 1 and centrifuge at 300 g for 5 min respectively. Collect part of the supernatant and reserve samples for microbial detection, and discard the rest, leaving the precipitate for further operation in a biological safety cabinet.

[0038] For further precipitation, add 20ml of normal saline and mix well, slowly add to the upper layer of 10ml of Ficoll lymphocyte separation medium, add liquid carefully to keep a clear interface between the blood sediment and Ficoll lymphocyte separation medium. Centrifuge at 400g for 20 minutes, carefully suck off the uppermost liquid, and retain the buffy coat cells and endometrial fragments. Wash twice with PBS buffer, then add 5ml of complete medium (low sugar DMEM and F12 are mixed in proportion, then add plasma replacement with a final concentration of 10%, gentamicin 100U / ml, amphotericin 0.1mg / ...

Embodiment 3

[0040] Example 3 Growth curve of endometrial stem cells derived from menstrual blood

[0041] Detection of growth curve of endometrial stem cells in menstrual blood: culture endometrial stem cells to the third passage in hypoxic carbon dioxide incubator and normal oxygen carbon dioxide incubator, digest cells with 0.25% trypsin, and resuspend with appropriate volume of medium cells, take 100 μl single cell suspension (1×10 3) were seeded in 96-well plates. The cells were placed in the hypoxic incubator and the normoxic incubator for 8 days, and each day was used as a time point and three replicate holes were set up; 10 μl of CCK-8 solution was added to the 96-well plate 4 hours before the measurement; The standard instrument detects the OD value of each well at a wavelength of 490nm, and records the results. Draw the growth curve, see the results figure 1 .

[0042] from figure 1 It can be seen that MenESCs cultured in normoxia and hypoxia both showed a good proliferation...

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Abstract

The invention discloses a method for hypoxia culture of an endometrial stem cell from menstrual blood. The endometrial stem cell is derived from the menstrual blood of an adult woman, and the culturecondition is 1 to 10% of oxygen concentration(preferably 5%). The method has the characteristics that the preparation is simple and convenient, the scale production is easy, the stability is good, andthe like; compared with the endometrial stem cell which is cultured under a normoxic culture condition, the hypoxia cultured endometrial stem cell in the oxygen-deficient environment has stronger survival ability.

Description

technical field [0001] The invention relates to the technical field of stem cells, in particular to a method for hypoxic culture of menstrual blood-derived endometrial stem cells. Background technique [0002] Mesenchymal Stem Cell (MSC) is a kind of pluripotent stem cell, which is derived from the mesoderm in the early stage of development. It is named for its ability to differentiate into mesenchymal tissue. It has the ability of self-renewal and multi-directional differentiation, and can be used for hematopoiesis Support and promote various functions such as stem cell implantation and immune regulation. [0003] Endometrial stem cells have high differentiation potential, proliferation ability and low immunogenicity, and can reduce inflammation as an immunomodulator. Animal experiments have shown that they have a unique role in protecting the colon from colitis. In remodeling and tissue engineering, it can promote vascular regeneration and angiogenesis, which is superior ...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0665
Inventor 左凤琼杨嘉洁
Owner 左凤琼
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