A method for isolating and culturing endometrial stem cells and its special medium

A technique for separating and culturing the endometrium, which is applied in the field of separating and culturing endometrial stem cells and its special medium, can solve the problems of restricting the research of endometrial stem cells, the composition of the culture medium is complex, and the number of stem cells is small, and achieves good results. Proliferation and differentiation ability, increased cell expansion rate, highly reproducible effect

Active Publication Date: 2021-04-30
CENTURY BIOSTRENGTH BEIJING PTY LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the complexity and scarcity of endometrial stem cells, the research on endometrial stem cells is seriously restricted. Using conventional stem cell separation and culture methods to prepare endometrial stem cells in vitro has the advantages of low purity of the obtained cells and small number of stem cells. The number of stem cells with high differentiation potential is less, and the number of passages is small, the yield is low, and the cells are severely differentiated, and there are also problems such as complex culture medium components and low repeatability

Method used

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  • A method for isolating and culturing endometrial stem cells and its special medium
  • A method for isolating and culturing endometrial stem cells and its special medium
  • A method for isolating and culturing endometrial stem cells and its special medium

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Effect test

Embodiment 1

[0028] A method for isolating and culturing endometrial stem cells, said method comprising the steps of:

[0029] 1) Washing: the isolated endometrium is obtained under sterile conditions, and rinsed with sodium chloride injection;

[0030] 2) digestion: the endometrium obtained in step 1) was cut into 1mm 3 To the pieces, add sodium chloride injection containing 0.05mg / mL collagenase I and 0.2mg / mL DispaseⅡ, digest in a shaker at 37°C for 30min, and then replace the digestion solution with sodium chloride containing 1mg / mL hyaluronase The injection was digested at 37°C for 1 hour. After the digestion was terminated, it was rinsed in sodium chloride injection, centrifuged, and the obtained precipitate was washed with sodium chloride injection and blown evenly, passed through a 40 μm cell sieve, and the filtrate was collected. The supernatant was discarded by centrifugation, and the cell pellet was obtained, stained with trypan blue, and the living cells were counted;

[0031...

Embodiment 2

[0034] A method for isolating and culturing endometrial stem cells, said method comprising the steps of:

[0035] 1) Washing: the isolated endometrium is obtained under sterile conditions, and rinsed with sodium chloride injection;

[0036] 2) digestion: the endometrium obtained in step 1) was cut into 1mm 3The fragments were digested at 37°C for 1 hour by adding digestive solution. After the digestion was terminated, they were rinsed in sodium chloride injection, centrifuged, and the obtained precipitate was washed with sodium chloride injection and blown evenly. The filtrate was centrifuged and the supernatant was discarded to obtain a cell pellet, which was stained with trypan blue and counted viable cells;

[0037] 3) Primary culture: Add culture medium to the cell pellet obtained in step 2) to obtain a single cell suspension, according to 5×10 4 cells / mL, inoculated in a culture dish, cultured for 3 hours, transferred the non-adhered cells in the culture dish to a new c...

Embodiment 3

[0040] A method for isolating and culturing endometrial stem cells, said method comprising the steps of:

[0041] 1) Washing: the isolated endometrium is obtained under sterile conditions, and rinsed with sodium chloride injection;

[0042] 2) digestion: the endometrium obtained in step 1) was cut into 2mm 3 The fragments were digested at 37°C for 2 hours by adding digestive solution. After the digestion was terminated, they were rinsed in sodium chloride injection, centrifuged, and the obtained precipitate was washed with sodium chloride injection and blown evenly. The filtrate was centrifuged and the supernatant was discarded to obtain a cell pellet;

[0043] 3) Primary culture: Add culture medium to the cell pellet obtained in step 2) to obtain a single cell suspension, according to 4.5×10 4 cells / mL, inoculated in a culture dish, cultured for 8 hours, transferred the non-adherent cells in the culture dish to a new culture dish, after replacing the culture medium, added 6...

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Abstract

The invention relates to a method for isolating and culturing endometrial stem cells, which comprises the steps of washing, digestion, primary culture and subculture, wherein in the process of primary culture, culture for 3-8 hours, and move unattached cells in the culture dish into new Petri dish, continue to culture for 3‑4 days after changing the medium, then collect the adherent cells and mark P 01 For generation of cells, move the non-adherent cells in the medium into a new culture dish, continue to culture for 4-6 days after changing the medium, collect the adherent cells, and mark them as P 02 Generation cells; in the process of subculture, the first generation adopts P 01 Generation cells and P 02 The first generation of cells is cultured independently, and after the second generation, mixed culture is carried out. By adopting the above method, the obtained endometrial stem cells have a high yield and high purity, and the obtained endometrial stem cells can be passaged for multiple times, and the stem cells obtained after multiple passages have high differentiation potential.

Description

technical field [0001] The invention belongs to the field of cell culture, and in particular relates to a method for separating and culturing endometrial stem cells and a special culture medium thereof. Background technique [0002] In recent years, more and more studies have shown that there is a small group of stem cells with strong proliferative ability and multi-directional differentiation potential in the endometrium, which enables the endometrium to maintain continuous self-renewal and regeneration capabilities. In view of the close relationship between endometrial stem cells and endometrial growth, endometrial stem cells can be isolated and cultured in vitro, and then transplanted into the body to treat endometrial injury or disease, endometriosis, infertility, and intrauterine Diagnosis and treatment of diseases such as membrane cancer open up new avenues. [0003] At present, there are relatively few studies on endometrial stem cells at home and abroad. As adult st...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
Inventor 董明珠李霞云潘新刘世红卢家堃
Owner CENTURY BIOSTRENGTH BEIJING PTY LTD
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