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Culture medium and cultural method for endometrial stem cells

A culture method and culture medium technology, applied in biochemical equipment and methods, culture process, tissue culture and other directions, can solve the problems of poor cell quality, low cell purity, differentiation of endometrial stem cells during culture time, etc., to overcome easy differentiation , to avoid virus contamination, the effect of high cell purity

Active Publication Date: 2019-04-09
GUANGDONG CHINAHEALTH LIFE SCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method of natural cell attachment is adopted, and the purity of the cells obtained by this method is low, which is not conducive to the subsequent purification of endometrial stem cells
In addition, the existing endometrial stem cells are directly inoculated after isolation, which will affect the adhesion of endometrial stem cells; and the culture system is a common culture system, resulting in slow proliferation of endometrial stem cells, and Excessive culture time makes differentiation of endometrial stem cells
[0005] In summary, the traditional stem cell culture medium is not suitable for the culture of endometrial stem cells, and the traditional stem cell culture medium will lead to low proliferation efficiency and poor cell quality of endometrial stem cells; after limited generations of proliferation of endometrial stem cells, Differentiation occurs; it cannot meet the clinical cell dosage requirements

Method used

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  • Culture medium and cultural method for endometrial stem cells
  • Culture medium and cultural method for endometrial stem cells
  • Culture medium and cultural method for endometrial stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The embodiment of the present invention provides some reagent preparation methods:

[0038] 1. The preparation method of the human mesenchymal stem cell adhesion-promoting reagent: mix the human mesenchymal stem cell adhesion-promoting reagent and 1640 basal medium according to the volume ratio (v:v) of 1:99, and then obtain the human mesenchymal stem cell promoting agent. Adherence reagents are stored at 4°C for later use.

[0039] 2. The preparation method of platelet lysate: take 100mL of human peripheral blood at 4°C, centrifuge at 2000rpm for 15min, transfer the upper plasma to a new centrifuge tube; centrifuge the plasma at 3000rpm for 20min, the remaining 20mL of plasma and Platelets, the upper layer of plasma was aspirated and discarded. After blowing evenly with a Pasteur pipette, it was ultrasonically lysed in an ultrasonic instrument for 10 minutes. After the lysate was completed, the platelet lysate was obtained and transferred to a 4°C environment for late...

Embodiment 2

[0045] The embodiment of the present invention provides a method for culturing endometrial stem cells:

[0046] 1. Add the human mesenchymal stem cell adhesion-promoting reagent prepared in Example 1 to the culture dish, make the human mesenchymal stem cell adhesion-promoting reagent infiltrate the bottom of the dish, and incubate at 37° C. After 1 hour, discard the human mesenchymal stem cell. Adhesion-promoting reagent for mesenchymal stem cells.

[0047] 2. Collect menstrual blood from the second day to the third day.

[0048] 3. Reference literature (Zhou Yunfan, Yang Bo, Hu Xiang, etc.; Isolation, Culture and Identification of Endometrial Mesenchymal Stem Cells from Menstrual Blood [J], Chinese Tissue Engineering Research and Clinical Rehabilitation; 2010, 14(32): 5952 ~5956.), the menstrual blood in step 2 was separated from the buffy coat by Ficoll density gradient centrifugation, and the red blood cells in the menstrual blood were lysed with red blood cell lysate (Red...

Embodiment 3

[0053] The embodiment of the present invention provides surface antigen detection of endometrial stem cells, and the specific steps are as follows:

[0054] Take endometrial stem cells and make 1×10 6 Cells / mL cell suspension, take 5 μL of monoclonal antibodies against human CD45, CD14, CD29, CD90, CD105, and HLA-DR respectively, add 500 μL of cell suspension cultured in group 4, and incubate at room temperature in the dark for 20 minutes, At the same time, a blank isotype control was set up, centrifuged at 1500r / min for 5min, the supernatant was discarded, washed twice with PBS containing 10% FBS, resuspended with 500 μL of PBS, and tested on the machine, according to the domestic patent CN105586308A (stem cell culture medium and cultured endometrium) The method of stem cells, denoted as patent 1) and CN106801034A (a large-scale preparation method of endometrial stem cells and its application, denoted as patent 2) respectively obtain two patented endometrial stem cells, and u...

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Abstract

The invention belongs to the field of biotechnology, in particular to a culture medium and a cultural method for endometrial stem cells. The culture medium provided by the invention comprises a serum-free medium, a blood platelet lysate, N-acetylcysteine, galacturonic acid and an antibiotic. The culture medium and the cultural method for endometrial stem cells provided by the invention are used for solving the technical defects of low value increase efficiency of the endometrial stem cells, low cell quality and easy differentiation caused by existing culture medium culture.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a culture medium and a method for culturing endometrial stem cells. Background technique [0002] Stem cells refer to a class of early undifferentiated cells with high proliferation potential, self-renewal ability and high differentiation ability. Mesenchymal stem cells are currently the most concerned member of stem cells, with high self-renewal ability and multi-directional differentiation potential. The human uterus is a tissue with a high ability to regenerate and differentiate. The endometrium contains stem cells with a high ability to proliferate. Scientists can separate these stem cells from the menstrual blood of healthy women and name them endometrial stem cells, also known as menstrual blood sources. stem cell. [0003] Endometrial stem cells (endometrial derived stem cells, EDSCs) refer to undifferentiated endometrial cells with self-renewal, multidirectional...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0682C12N2500/30C12N2500/32
Inventor 李文东宋云庆卢瑞珊李捷
Owner GUANGDONG CHINAHEALTH LIFE SCI CO LTD
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