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Erythrocyte de-nucleation culture medium and kit

A technology for red blood cells and culture medium, which is applied in the field of red blood cell denucleation and can solve the problems of unclear denucleation mechanism and other problems.

Active Publication Date: 2019-08-06
ACADEMY OF MILITARY MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although it has been recognized how red blood cells undergo denucleation under some physiological conditions, the research on the denucleation mechanism is not clear at present, and the preparation technology of in vitro mature red blood cells still needs to be improved, especially for the induction of various types of stem cells. Increased denucleation efficiency in red blood cells

Method used

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  • Erythrocyte de-nucleation culture medium and kit
  • Erythrocyte de-nucleation culture medium and kit
  • Erythrocyte de-nucleation culture medium and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] 1. Isolation of cord blood mononuclear cells

[0056] 1. Sedimentary red blood cells

[0057] (1) Transfer the remaining umbilical cord blood from the first centrifugation into a 250ml glass bottle, and add normal saline to the original volume of umbilical cord blood.

[0058] (2) Add 6% hydroxyethyl starch to 1 / 3 of the total volume of cord blood, mix thoroughly, and settle at room temperature for 20-30 minutes.

[0059] (3) Carefully aspirate the supernatant, collect it into 2-3 50ml centrifuge tubes, centrifuge at 1800rpm for 5min, and room temperature 20-25°C.

[0060] 2. Isolation of cord blood mononuclear cells (mononuclear cell, MNC)

[0061] (1) Take the above-mentioned umbilical cord blood centrifuged cell pellet without red blood cells, discard the supernatant, add 10mL PBS to each tube to resuspend the cells, centrifuge at 1800rpm / min at room temperature for 5min, after washing, merge and resuspend all the cells with 10mL PBS;

[0062] (2) Add human lympho...

Embodiment 2

[0081] The difference between Example 2 and Example 1 is that the Bcl-2 small molecule inhibitor used in Example 2 is ABT-199 (also known as Venetoclax, Venetoclax, Venetoclax, Venetoclax , GDC-0199):

[0082] According to the formula in Table 1, the EM20-25 in the erythrocyte denucleation medium was replaced with ABT-199 (the molecular formula is as follows, and the concentration used was 20 μM), and the denucleation detection results were as follows image 3 shown.

[0083]

[0084] image 3 The results showed that the denucleation rate was 14% in the control group, and 37% in the treatment with the Bcl-2 small molecule inhibitor ABT-199. The results showed that the denucleation rate was at least doubled by using the Bcl-2 small molecule inhibitor ABT-199 compared with the control group without ABT-199. The Bcl-2 small molecule inhibitor ABT-199 has the same denucleation-promoting effect as EM20-25.

[0085] In addition, for the Bcl-2 small molecule inhibitor ABT-199,...

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Abstract

The invention relates to the field of erythrocyte de-nucleation, and specifically relates to an erythrocyte de-nucleation culture medium and a kit. The culture medium includes a basic culture medium,transferrin, recombinant human insulin, heparin and a Bcl-2 small molecule inhibitor; and optionally, the culture medium also includes at least one of blood plasma, serum, glutamine or erythropoietin.The kit is used for the de-nucleation cultivation of erythrocyte and includes the Bcl-2 small molecule inhibitor. The de-nucleation rates of the erythrocyte can be significantly enhanced by utilizingthe provided culture medium or kit to prepare the mature erythrocyte.

Description

technical field [0001] The invention relates to the field of erythrocyte denucleation, in particular to a erythrocyte denucleation medium and a kit. Background technique [0002] In the treatment of hematopoietic dysfunction, routine anemia and shock, blood transfusion is a key link. Although there is a relatively sufficient blood supply in developed countries, there is a problem that blood-borne viruses cannot be completely eliminated. In developing countries, blood supply has been in short supply for a long time. In recent years, many cities in our country have even appeared "blood shortage". To solve the problem of lack of blood sources, the development of new blood sources has become an urgent need in current medical treatment. One of the ways is to produce red blood cells by autologous or related means of stem cell research, including umbilical cord, adult bone marrow, hematopoietic stem / progenitor cells derived from peripheral blood, embryonic stem cells (human embry...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078
CPCC12N5/0641C12N2500/24C12N2500/32C12N2501/14C12N2501/33C12N2501/91
Inventor 裴雪涛谢小燕房芳陈琳曲洺逸
Owner ACADEMY OF MILITARY MEDICAL SCI
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