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An immunomagnetic bead for sorting human cells, its preparation and excision method

A technology of magnetic beads and cells, applied in the field of medical biology experiments, can solve the problems of cell waste, time-consuming, unfavorable culture, etc., and achieve the effect of high excision efficiency

Active Publication Date: 2018-11-23
SHANGHAI BAIZE MEDICAL APP & INSTR CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because this method takes a long time, it is not conducive to subsequent cell culture, and the purity of the collected PBMC cells is 80%, and the recovery rate is about 80%, which causes a waste of cells to a certain extent.

Method used

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  • An immunomagnetic bead for sorting human cells, its preparation and excision method
  • An immunomagnetic bead for sorting human cells, its preparation and excision method
  • An immunomagnetic bead for sorting human cells, its preparation and excision method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Example 1: Preparation of magnetic bead-nucleic acid-antibody complexes

[0115] 〔1〕 Take the magnetic beads (0.5mg) with amino groups on the surface into a 1.5ml EP tube, magnetically separate, and discard the supernatant.

[0116] 〔2〕Resuspend the magnetic bead pellet in 1ml of PBS buffer, then magnetically separate, and discard the supernatant.

[0117] [3] Repeat step 2 two to three times.

[0118] 〔4〕 Add 600-800ul of glutaraldehyde as a coupling agent, place in a mixing rotator and incubate at room temperature for 1.5h.

[0119] [5] Take the nucleic acid fragment (SEQ ID NO: 1) (magnetic beads: 1mg of nucleic acid: 50ug) into a 1.5ml centrifuge tube, add 600-800ul of glutaraldehyde, place it in a mixing rotator and incubate at room temperature for 1.5h.

[0120] 〔6〕Remove the incubated magnetic beads from the rotator, separate them magnetically, and remove the supernatant.

[0121] [7] Add the nucleic acid fragments after incubation in step 4, resuspend the mag...

Embodiment 2

[0129] Example 2: Magnetic bead-nucleic acid-antibody complex cell sorting method

[0130] [1] Take 1ml of human peripheral blood mononuclear cells (PBMC), and centrifuge at 1500rpm for 5min. figure 1 The proportion of positive CD3-PECy7 detected by flow cytometry of peripheral blood mononuclear cells (initial cell number 1×10 8 , the proportion of CD3 positive cells was 34.1%, which contained 3.41×10 7 CD3 positive cells).

[0131] 〔2〕Discard the supernatant, add PBS to resuspend the cells, and centrifuge at 1500rpm for 5min.

[0132] [3] Repeat step 2 twice.

[0133] [4] Resuspend the cells with a small amount of PBS, add the magnetic beads prepared in Example 1, mix well, place in a mixing rotator and incubate at room temperature for 1 hour.

[0134] 〔5〕Magnetically separate the cells after incubation, and aspirate the supernatant, which is negative cells. figure 2 It shows the positive ratio (2.5%) of CD3-PECy7 detected by flow cytometry in the negative cells after t...

Embodiment 3

[0138] Example 3: Magnetic Bead Excision Method

[0139] [1] The positive cells collected in Example 2 were centrifuged at low speed.

[0140] 〔2〕Discard the supernatant and add 90ul of double digestion buffer to resuspend the cells.

[0141] 〔3〕Add the enzyme and bathe in water at 37°C for 10 minutes.

[0142] 〔4〕Magnetic separation, aspiration of the supernatant into a new centrifuge tube.

[0143] 〔5〕Centrifuge, discard the supernatant, and resuspend in a small amount of PBS, that is, the positive cells without magnetic beads after sorting.

[0144] Table 1 below shows the excision time, excision efficiency and cell viability obtained by using different enzyme digestion methods for magnetic bead excision experiments.

[0145] Table 1

[0146]

[0147] 1: The time it takes to completely remove the magnetic beads, this experiment is the recovery of 3.1×10 7 cells shall prevail.

[0148] 2: Live cells and dead cells are counted by trypan blue staining, and then counte...

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Abstract

The invention provides an immunomagnetic bead for sorting human cells and a preparation and cutting-off method of the immunomagnetic bead and particularly relates to a magnetic bead-nucleic acid-antibody compound or a kit containing the magnetic bead-nucleic acid-antibody compound. The compound comprises a magnetic bead, nucleic acid covalently coupled to the magnetic bead and an antibody covalently coupled with the nucleic acid, wherein the nucleic acid comprises at least one enzyme digestion site, and the antibody can specifically bind with antigen on the surfaces of to-be-screened cells. The invention further comprises the purpose and preparation method of the magnetic bead-nucleic acid-antibody compound and a method for using the magnetic bead-nucleic acid-antibody compound to perform cell sorting.

Description

technical field [0001] The invention belongs to the technical field of medical biology experiments, and relates to an immunomagnetic bead for sorting human cells, its preparation and excision method. Background technique [0002] Tumor is one of the diseases that seriously endanger human health. Traditional treatment methods such as surgery, radiotherapy, and chemotherapy have been difficult to kill the remaining tumor cells in the body. In the 21st century, with the scientific and technological progress of tumor treatment, the technical system of biotherapy has been developed. Among them, biological therapy is currently the most mature and widely used tumor biological treatment method. Biological therapy is to collect mononuclear cells (PBMC) from the patient's peripheral blood, and then send them to the GMP studio for culture, amplification, induction, and tumor antigen stimulation, so as to obtain DC cells that can recognize cancer cells and have high tumoricidal propert...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/53C12Q1/02
CPCC12Q1/6804C12Q2521/301
Inventor 钱其军陆元修杨华宋启平李林芳师传胤
Owner SHANGHAI BAIZE MEDICAL APP & INSTR CO LTD
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