Method for enriching and separating human peripheral blood CD34+ and CD91+ lymphocytes

A technology of human peripheral blood and lymphocytes, applied in the field of biomedicine, can solve the problems of separation failure, antibody spatial direction change, small specific surface area, etc., and achieve the effect of increasing the chance of contact, stabilizing the reaction solution, and improving the capture efficiency

Inactive Publication Date: 2015-05-20
北京佑仁生物科技集团有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current separation technology based on micron-scale immunomagnetic beads has many limitations: 1) Compared with nano-magnetic beads, the specific surface area of ​​micro-magnetic beads is relatively small, which reduces the capture efficiency of magnetic beads; The particle properties of micron magnetic beads, which combine with cells through a multiphase reaction (multiphase reaction), usually take longer to specifically capture cells in the food matrix; 4) The traditional immunomagnetic separation technology often directly couples the antibody to the immunomagnetic beads, which often causes a significant decrease in antibody activity and changes in the spatial direction of the antibody. And increase the steric hindrance effect between antibodies, thereby reducing the capture efficiency of antibodies 5) Blood viscosity is high and non-CD34 + and CD90 + Stem cells have a high concentration of blood cells, micron magnetic beads are prone to non-specific adsorption, and it is difficult to achieve CD34 in blood + and CD90 + Specific separation of stem cells; 6) Excessive concentration of micron magnetic beads will cause CD34 + and CD90 + The damage of stem cells (the magnetic field causes the magnetic beads on the cell surface to attract each other, causing the cells to be squeezed or even ruptured), resulting in the failure of separation; 7) When the magnetic beads are coupled to antibodies, hydrophobic adsorption or chemical coupling is generally used to combine active antibodies. Attached to the surface of magnetic beads
The distance between the antibody and the surface of the magnetic bead is too close, the nature of the magnetic bead itself and the residual hydrophobic or strong hydrophilic groups on the surface are likely to cause changes in the spatial conformation of the antibody, resulting in a decrease in the biological activity of the antibody

Method used

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  • Method for enriching and separating human peripheral blood CD34+ and CD91+ lymphocytes
  • Method for enriching and separating human peripheral blood CD34+ and CD91+ lymphocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] 1. Multi-Armed Well Star Polymer-Mouse Anti-Human CD34 + Antibody complexes were prepared according to the following steps:

[0036] (1) Dissolve 1.0 mg Dobby Star Polymer Dobby Star Polyamide-amine in 2 mL, 0.02 M, pH 6.5 PBS, add 0.6 mg NHSS, 0.4 mg EDC, place on a mixer at room temperature Stir and activate for 15 min;

[0037] (2) Take 2.14 mg mouse anti-human CD34 + Antibody was added to the above reaction solution, placed on a mixer at room temperature and stirred for 30 min;

[0038] (3) The above solution was spin-dried under reduced pressure, dissolved in deionized water, and dialyzed in PBS and deionized water for 1 day; after the dialysis, the obtained solution was freeze-dried.

[0039] 2. Multi-armed well star polymer-mouse anti-human CD90 + Antibody complexes were prepared according to the following steps:

[0040] (1) Dissolve 1.0 mg of aminated multi-arm well star polymer in 2 mL, 0.02 M, pH 6.5 PBS, add 0.6 mg NHSS, 0.4 mg EDC, stir on a mixer at r...

Embodiment 2

[0049] Example 2 Enrichment effect experiment

[0050] (1) Take 1 mL of concentration as 10 4 CD34 cells / mL + or CD90 + Centrifuge the cells at 12000 rpm for 5 min in a 1.5 mL sterile centrifuge tube, discard the supernatant, and resuspend with an equal volume of sterile PBS solution.

[0051] (2) Enrichment and capture: respectively set the technical solution group of the present invention (CD34 + or CD90 + Cell antibody and long-chain biotin co-modified multi-armed well star polymer group), CD34 + or CD90 + Cell-specific antibody-modified nanomagnetic bead set, CD34 + or CD90 + Cell-specific antibody-modified micron magnetic bead sets enrich target cells.

[0052] (3) After magnetic separation, pour the supernatant into a sterile centrifuge tube, and the separated CD34 + or CD90 + The immunomagnetic beads of the cells were washed twice with PBST, mixed evenly, and the immunomagnetic bead complex was resuspended with 1 mL sterile PBS solution.

[0053] (4) Capture...

Embodiment 3

[0065] Example 3 Enrichment capture experiment

[0066] Conventional magnetic stand separation time is 30min, and all the other are with embodiment 2.

[0067] CD34 + Capture efficiency of cell-specific antibody-modified micron magnetic bead sets CD34 + Capture efficiency of cell-specific antibody-modified nanomagnetic bead sets CD34 + Capture efficiency of multi-armed well star polymer sets co-modified with cellular antibodies and long-chain biotin 57.2% 44.7% 86.3% CD90 + Capture efficiency of cell-specific antibody-modified micron magnetic bead sets CD90 + Capture efficiency of cell-specific antibody-modified nanomagnetic bead sets CD90 + Capture efficiency of multi-armed well star polymer sets co-modified with cellular antibodies and long-chain biotin 55.9% 43.9% 88.2%

[0068] The experimental results show that compared with the separation of 3min in Example 2, when the separation time reaches 30min, the capture efficiency of th...

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Abstract

The invention discloses a method for enriching and separating human peripheral blood CD34<+> and CD91<+> stem cells, which provides basis for the subsequent researches on CD34<+> and CD91<+> stem cells better, and relates to the field of biomedicine. The method comprises the following steps of: covalently coupling a multi-arm well star-shaped polymer with a mouse anti-human CD34<+> or CD91<+> monoclonal antibody; coating long-chain biotin molecules on the multi-arm well star-shaped polymer modified by the mouse anti-human CD34<+> or CD91<+> monoclonal antibody; capturing the CD34<+> and CD91<+> stem cells in a peripheral blood sample by the multi-arm well star-shaped polymer modified by the mouse anti-human CD34<+> or CD91<+> monoclonal antibody; identifying and coupling the long-chain biotinylated well star-shaped polymer in the peripheral blood by nanometre magnetic beads modified by streptavidin; separating and re-suspending the captured CD34<+> and CD91<+> stem cells, and the like, wherein the re-suspension can be directly used for subsequent analysis. Compared with the traditional cell separation method, the method is more suitable for performing magnetic separation on the CD34<+> and CD91<+> stem cells in the complex peripheral blood sample, and capable of increasing the separation efficiency for the CD34<+> and CD91<+> stem cells in the peripheral blood sample.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to CD34 based on nano magnetic beads + and CD90 + Stem Cell Isolation Methods. Background technique [0002] Isolation and purification of stem cells are the basic means to study various biological characteristics of stem cells, and have potential clinical therapeutic value. CD34 + cells are earlier stem cells, but CD34 + Cells are heterogeneous, among which pluripotent stem cells only account for a small part, and the more primitive part should be CD34 + / CD90 + Wait. Only by purifying these more primitive parts for in vitro culture can we truly understand the characteristics, proliferation and differentiation characteristics of primitive hematopoietic stem cells. However, CD34 in the peripheral blood of normal people + and CD90 + The content of stem cells is very low, and the existing methods cannot detect the trace amount of CD34 in peripheral blood. + and CD90 + Stem cells ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0783
Inventor 许恒毅傅芬黄小林刘雯婷范丽娟熊勇华
Owner 北京佑仁生物科技集团有限公司
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