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Isolation medium and isolation method of bone marrow endothelial progenitor cells

A technology of endothelial progenitor cells and separation method, which is applied in the field of separation medium and separation of bone marrow endothelial progenitor cells, can solve the problems of unfavorable economic benefits, inconsistent phenotypic expression rate, cumbersome culture methods, etc., and achieve high viability and cell mass. big effect

Active Publication Date: 2021-02-05
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The traditional culture method is relatively cumbersome. In the process of sorting MNCs with CD34+ antibody-coated immunomagnetic beads, the loss of MNC CD34+ and the damage to the cells by the magnetic beads themselves will inevitably reduce the cell acquisition rate. The method of immunomagnetic bead separation is expensive, which is not conducive to economic benefits. Especially after the concept of CD34- was proposed by George et al., it was proved that the phenotypic expression rates on EPCs were inconsistent, so this method has gradually not been adopted.

Method used

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  • Isolation medium and isolation method of bone marrow endothelial progenitor cells
  • Isolation medium and isolation method of bone marrow endothelial progenitor cells
  • Isolation medium and isolation method of bone marrow endothelial progenitor cells

Examples

Experimental program
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Effect test

Embodiment 1

[0052] 1. Extract 5mL of human bone marrow;

[0053] 2. Add an equal volume of high-sugar DMEM basal medium and mix well;

[0054] 3. Slowly add to the centrifuge tube filled with Ficoll solution, centrifuge at 700g for 30min;

[0055] 4. Add 10ng / ml FN to T25, put it in a carbon dioxide incubator at 37°C for 60min;

[0056] 5. Absorb the middle mononuclear cell layer;

[0057] 6. Add 10ml of normal saline containing 0.2% human serum, centrifuge at 500g for 10min, repeat once;

[0058] 7. Discard the supernatant, resuspend in medium 1, take 50 μL of the cell suspension, stain with trypan blue, and count the cells;

[0059] 8. According to the counting result, press 1×10 5 / cm 2 Cells were inoculated in T25 culture flasks coated with human fibronectin at a density of ;

[0060] 9. After 24 hours, take the supernatant, centrifuge at 500g for 10 minutes, and re-inoculate the cell pellet into a new T25 culture flask that has been coated with human fibronectin;

[0061] 10. ...

Embodiment 2

[0064] 1. Extract 10mL of human bone marrow;

[0065] 2. Add an equal volume of high-sugar DMEM basal medium and mix well;

[0066] 3. Slowly add to the centrifuge tube filled with Ficoll solution, centrifuge at 700g for 30min;

[0067] 4. Add 10ng / ml FN to T25, put it in a carbon dioxide incubator and cover it for 30-60min;

[0068] 5. Absorb the middle mononuclear cell layer;

[0069] 6. Add 10ml of normal saline containing 0.2% human serum, centrifuge at 500g for 10min, repeat once;

[0070] 7. Discard the supernatant, resuspend in medium 1, take 50 μL of the cell suspension, stain with trypan blue, and count the cells;

[0071] 8. According to the counting result, press 2×10 6 cell / cm 2 Cells were inoculated in T25 culture flasks coated with human fibronectin at a density of ;

[0072] 9. After 24 hours, take the supernatant, centrifuge at 500g for 10 minutes, and re-inoculate the cell pellet into a new T25 culture flask that has been coated with human fibronectin; ...

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Abstract

The invention relates to the technical field of cell culture, in particular to a medium and a method for separating EPCs (endothelial progenitor cells) of bone marrow. When cultured with the medium and the method, the EPCs can grow and proliferate rapidly, and 5*10<7> cells can be obtained in about 20 days for 5-10 ml of the bone marrow.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a culture medium and a method for isolating bone marrow endothelial progenitor cells. Background technique [0002] EPCs are the precursor cells of vascular endothelial cells, that is, the progenitor cells that can differentiate into mature vascular endothelial cells, also known as angioblasts or vascular endothelial stem cells, derived from bone marrow primitive cells, and human embryonic hemangioblasts) and HUVEC Similarly, differentiation into mature ECs can be induced under certain conditions. In 1997, Asahara et al. first isolated and confirmed the existence of endothelial progenitor cells that can differentiate into vascular endothelial cells in adult peripheral blood, and confirmed their ability to generate blood vessels in vivo. People began to study various aspects of endothelial progenitor cells. At present, endothelial progenitor cells have been isolated from umb...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071
CPCC12N5/0692C12N2501/105C12N2501/11C12N2501/115C12N2501/165C12N2501/39C12N2509/00
Inventor 陈海佳葛啸虎王一飞张梦晨王小燕
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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