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Method for efficiently separating and expanding mesenchymal stem cells in human umbilical cord blood

A technology for stem cells and human umbilical cord blood, which is applied in the biological field to achieve the effect of improving purity and efficiency and realizing large-scale expansion

Active Publication Date: 2013-01-16
夏亮
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, the application of umbilical cord blood in the treatment of diabetes, nerve injury and vascular necrosis has also been frequently reported.

Method used

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  • Method for efficiently separating and expanding mesenchymal stem cells in human umbilical cord blood

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Coating cell culture plates

[0023] Laminin (Laminin; BD Bioscience, CA) and gelatin protein (Gelatin; BD Bioscience, CA) were diluted 2 mg / ml according to the instructions. After mixing according to the volume ratio of 1:2, transfer to the cell culture plate and place at room temperature for 1 Hour. Under sterile conditions, it can be stored at 4°C for 3 months.

Embodiment 2

[0024] Example 2: Isolation and Expansion of Umbilical Cord Blood Mesenchymal Stem Cells

[0025] The umbilical cord blood collected under sterile conditions was mixed and diluted with cells in α-MEM at a volume ratio of 1:1, mixed with 5g / L methylcellulose at a ratio of 4:1, and allowed to stand for 30 minutes to settle red blood cells. Aspirate the supernatant, after centrifugation, use PBS to make a single cell suspension, superimpose on the lymphocyte separation medium Ficoll-Hypaque with a relative density of 1.077, centrifuge at 2500rpm for 20min, take the interface layer, add PBS to make a single cell suspension, and wash by centrifugation .

[0026] Since Ficoll-Hypaque has a specific gravity of 1.077 g / ml, which is heavier than monocytes but lighter than erythrocytes, monocytes can be separated from residual erythrocytes. Pure mononuclear cells can be collected at the interface level.

[0027] Dilute the obtained mononuclear cells with PBS, centrifuge at 2000rpm for...

Embodiment 3

[0031] Example 3: Cell surface antigen characterization of cultured mesenchymal stems

[0032] In order to confirm that the above-mentioned cells have the characteristics of surface antigens of mesenchymal stem cells, the cell surface was analyzed using FACs. The results are shown in Table 1.

[0033] Table 1

[0034] indicator

[0035]Table 1 shows that for the stem cells isolated and cultured in the present invention, CD34, CD45 and CD3 are negative, while CD73, CD105 and CD90 are positive. This result indicates that the cells isolated and expanded in the present invention are mesenchymal stem cells.

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Abstract

The invention relates to an optimal method for efficiently separating and expanding mesenchymal stem cells in umbilical cord blood in cell therapy. The fact that the mesenchymal stem cells with higher differentiative capacity and smaller immunological rejection are contained in the umbilical cord blood has been proved. The establishment of an umbilical cord blood bank provides a basis for achieving transplantation of autologous cells. However, since the content of the mesenchymal stem cells in the umbilical cord blood is very low (only 0.5 to 30 mesenchymal stem cells are contained in 1*108 mononuclear cells), how to efficiently separate and expand the mesenchymal stem cells becomes a problem hindering the transformation of the mesenchymal stem cells in the umbilical cord blood to the clinical application. At present, in most situations, the mesenchymal stem cells in the umbilical cord blood are separated in virtue of adsorption capacity of the mesenchymal stem cells in a culture vessel, but the success ratio is low, and the characteristics of stem cells are difficult to maintain in an expanding process. The method comprises the steps of coating the culture vessel with protein components, adding multiple growth factors in the culture vessel in a coordination manner, and building the expansion environment of the mesenchymal stem cells in the umbilical cord blood, so as to achieve the efficient separation and expansion of the mesenchymal stem cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for efficiently separating and culturing human umbilical cord blood mesenchymal stem cells. Background technique [0002] Stem cells refer to a group of cells in an organism that can perform self-renewal and maintain the metabolism of various tissue cells in the body. At the same time, when various tissues in the body are damaged by endogenous or exogenous sources, stem cells can be activated to perform differentiation functions, so as to achieve the purpose of repairing the tissues. Under appropriate conditions, mesenchymal stem cells have strong proliferation and differentiation capabilities, and can repair a variety of tissue damage, including bone, muscle, nerve and other tissues. Therefore, mesenchymal stem cells have attracted more and more attention in the field of regenerative medicine. more attention. [0003] Bone marrow is rich in mesenchymal stem cells, so it h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/0789
Inventor 孙博肖忠党
Owner 夏亮
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