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Method for separating hematopoietic stem cells from human peripheral blood

A technology for hematopoietic stem cells and human peripheral blood, applied in the field of hematopoietic stem cell separation based on nano-magnetic beads, can solve the problems of spatial direction change, change, poor monodispersity of micron magnetic beads, etc., to increase the chance of contact, improve separation efficiency, The effect of shortening the separation time

Active Publication Date: 2015-07-01
深圳市旷逸生物科技有限公司
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Problems solved by technology

However, the current separation technology based on micron-scale immunomagnetic beads has many limitations: 1) The specific surface area of ​​micron-sized magnetic beads is relatively small, which reduces the capture efficiency of magnetic beads; The combination of cells through a multiphase reaction usually takes longer to specifically capture cells in the food matrix; 3) The micron magnetic beads have poor monodispersity and are prone to self-aggregation or self-aggregation in the peripheral blood matrix Precipitation; 4) The traditional immunomagnetic separation technology often directly couples the antibody to the immunomagnetic beads. This process often leads to a greatly reduced activity of the antibody and a change in the spatial direction of the antibody, which increases the inter-antibody interaction. The steric hindrance effect reduces the capture efficiency of antibodies. 5) The blood viscosity is high and the blood cell concentration of non-hematopoietic stem cells is large. Micron magnetic beads are prone to non-specific adsorption, and it is difficult to achieve specific separation of hematopoietic stem cells in blood; 6) Excessive concentration of micron magnetic beads will cause damage to hematopoietic 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 failure of separation; 7) When the magnetic beads are coupled to antibodies, generally use Active antibodies are attached to the surface of magnetic beads by hydrophobic adsorption or chemical coupling
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 separating hematopoietic stem cells from human peripheral blood
  • Method for separating hematopoietic stem cells from human peripheral blood

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] 1. The dendritic hyperbranched polymer-antibody complex is prepared according to the following steps:

[0030] (1) Weigh 1.0 mg dendritic hyperbranched polyamidoamine aminated by dendrimer hyperbranched polymer, suspend in 4 mL phosphate buffer (PBS, 0.01 mol / L, pH 8.0), stir and add 25% of Add 545 μL of glutaraldehyde aqueous solution to make the final concentration of glutaraldehyde 3%. React at room temperature for 3.5 h at a rotating speed of 150 r / min on a shaker;

[0031] (2) Add 1 mL (ie 2.7 mg) of mouse anti-hematopoietic stem cell monoclonal antibody dropwise to the above solution to make the final concentration reach about 3 mg / mL. React at room temperature for 24 h at the speed of the shaker at 150 r / min;

[0032] (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.

[0033] 2. The long-chain biotin-d...

Embodiment 2

[0039] Example 2 Enrichment effect experiment

[0040] (1) Take 1 mL of concentration as 10 4 Cells / mL of hematopoietic stem cells were placed in a 1.5 mL sterile centrifuge tube, centrifuged at 12,000 rpm for 5 min, the supernatant was discarded, and resuspended with an equal volume of sterile PBS solution.

[0041] (2) Enrichment and capture: Set up the technical scheme group of the present invention (dendritic hyperbranched polymer group co-modified with hematopoietic stem cell antibody and long-chain biotin), nanomagnetic bead group modified with hematopoietic stem cell-specific antibody, and hematopoietic stem cell-specific Antibody-modified micron magnetic bead sets enrich target cells.

[0042] (3) After magnetic separation, pour the supernatant into a sterile centrifuge tube, and wash the isolated immunomagnetic beads with hematopoietic stem cells twice with PBST, mix well, and resuspend the immunomagnetic beads with 1 mL of sterile PBS solution. Magnetic bead compl...

Embodiment 3

[0056] Example 3 Enrichment capture experiment

[0057] Conventional magnetic stand separation time is 30min, and all the other are with embodiment 2.

[0058] The catch rate of each group is as follows:

[0059] Capture efficiency of hematopoietic stem cell-specific antibody modified micron magnetic bead set Capture efficiency of hematopoietic stem cell-specific antibody-modified magnetic nanobeads Capture rate of hematopoietic stem cell antibody and long-chain biotin co-modified dendritic hyperbranched polymer group 53.9% 42.7% 90.3%

[0060] The experimental results show that compared to the separation of 3 minutes in Example 2, when the separation time reaches 30 minutes, the capture efficiency of the three groups has been improved, especially the capture efficiency of the hematopoietic stem cell-specific antibody-modified nano magnetic bead group is the most obvious. It shows that the capture efficiency of the nano-magnetic bead group can be great...

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Abstract

The invention discloses a method for separating hematopoietic stem cells (HSC) enriched in peripheral blood, aims to better supply subsequent research on the HSC, and relates to the field of biomedicine. The method comprises the following steps of: performing covalent coupling on dendritic hyperbranched polymers and anti-rat human HSC monoclonal antibodies, wrapping the dendritic hyperbranched polymers modified by the anti-rat human HSC monoclonal antibodies by long-chain biotin molecules, capturing the HSC in a peripheral blood sample by the dendritic hyperbranched polymers co-modified by the anti-rat human HSC monoclonal antibodies and the long-chain biotins, identifying nano magnetic beads modified by streptavidin, making the nano magnetic beads be coupled with the long-chain biotin dendritic hyperbranched polymers in the peripheral blood, separating the captured HSC, performing heavy suspension on the captured HSC and the like, wherein heavy suspension liquid can be directly subjected to subsequent analysis. Compared with the conventional cell separation method, the method disclosed by the invention is more suitable for performing magnetic separation on the HSC in the complicated peripheral blood sample and the efficiency of separating the HSC from the peripheral blood sample is improved.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for separating hematopoietic stem cells based on nano magnetic beads. Background technique [0002] Hematopoietic stem cells (HSCs) refer to self-renewing abilities and can differentiate into various blood cell precursor cells, and finally generate various blood cell components, including red blood cells, white blood cells and platelets, which can also differentiate into various other cells. In the past ten years, with the continuous deepening of the understanding and research on the function of hematopoietic stem cells, hematopoietic stem cell transplantation has become an effective method for the treatment of blood diseases and malignant tumors, and is also used in the treatment of certain autoimmune diseases. disease. Peripheral blood hematopoietic stem cell transplantation is a kind of hematopoietic stem cell transplantation, which collects peripheral blood mobilized by s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0789
Inventor 许恒毅
Owner 深圳市旷逸生物科技有限公司
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