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Separation method for peripheral blood mononuclear cell

A separation method and peripheral blood technology, applied in the field of cell separation, can solve the problems of easy interference in the separation process, unsuitable for clinical application, and large cell damage, and achieve the effect of high purity and activity, simple operation, and small damage

Inactive Publication Date: 2017-10-17
MIAOSHUN SHANGHAI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The invention provides a method for separating peripheral blood mononuclear cells to solve the technical problems that the existing density gradient centrifugation method has great damage to cells, low cell yield, is not suitable for clinical application, and is easily disturbed during the separation process.

Method used

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  • Separation method for peripheral blood mononuclear cell
  • Separation method for peripheral blood mononuclear cell
  • Separation method for peripheral blood mononuclear cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] A preferred embodiment of the present invention discloses a method for isolating peripheral blood mononuclear cells, comprising the following steps:

[0049] (1) Before separating the cells, sterilize the biological safety cabinet and ultra-clean bench, and distribute the lymphocyte separation liquid in the centrifuge tube. The lymphocyte separation liquid includes Ficoll 400 and sodium diatrizoate meglumine. The lymphocyte separation liquid is in The density at 20°C is 1.077g / mL; wrap the centrifuge tube with foil to avoid light. Blood samples were collected on the day of cell separation, that is, fresh peripheral blood, and 10 tubes were collected with medical anticoagulant tubes, 6ml / tube, a total of 60ml. The collected blood samples were transported at room temperature and guaranteed to be sent to the laboratory within 1 hour. After receiving the blood samples, relevant personnel in the laboratory should immediately complete the registration work and immediately co...

Embodiment 2

[0058] This preferred embodiment is roughly the same as Embodiment 1, the difference is that when the diluted peripheral blood is added to the lymphocyte separation liquid, the liquid level of the lymphocyte separation liquid is at an angle of 50° to the horizontal plane; the diluted peripheral blood and the lymphocyte The volume ratio of the cell separation solution was 1:1 when mixed; during centrifugation, the speed of the centrifuge was set at 450g, and the centrifugation time was 30min.

[0059] The peripheral blood mononuclear cells obtained above were cultured for 24 hours, and analyzed by flow cytometry, and the activity of the isolated cells was 96.5%.

Embodiment 3

[0061] This preferred embodiment is roughly the same as Embodiment 1, the difference is that when the diluted peripheral blood is added to the lymphocyte separation liquid, the liquid level of the lymphocyte separation liquid is at an angle of 70° to the horizontal plane; the diluted peripheral blood and the lymphocyte The volume ratio when mixing the cell separation solution is 2:1; when centrifuging, the speed of the centrifuge is set to 600g, and the centrifugation time is 20min.

[0062] The peripheral blood mononuclear cells obtained above were cultured for 24 hours, and analyzed by flow cytometry, and the activity of the isolated cells was 97%.

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Abstract

The invention discloses a method for isolating peripheral blood mononuclear cells, which comprises the following steps: (1) mixing the peripheral blood with Duchenne's phosphate buffer to obtain diluted peripheral blood; placing the lymphocyte separation solution in a centrifuge tube and Store in shading; (2) Tilt the centrifuge tube containing the lymphocyte separation solution so that the liquid level of the lymphocyte separation solution is at an angle of 50°-70° to the horizontal plane; add the diluted peripheral blood to the lymphocyte along the side wall of the centrifuge tube. The mixed solution is obtained from the cell separation solution; the volume ratio of the diluted peripheral blood to the lymphocyte separation solution is 1-2:1; (3) centrifuge the centrifuge tube containing the mixed solution and take the buffy coat to obtain the peripheral blood mononuclear cell layer. The separation method is easy to operate and is suitable for the separation of a large number of samples; it can realize aseptic operation, and the obtained peripheral blood mononuclear cells are suitable for clinical application; the damage to the isolated peripheral blood mononuclear cells is small, and the cell yield is high. will not be disturbed.

Description

technical field [0001] The invention relates to the technical field of cell separation, in particular to a method for separating peripheral blood mononuclear cells. Background technique [0002] In clinical research, the examination of the functional and biological characteristics of specific cells often relies on cell separation techniques. Isolation of mononuclear cells from peripheral blood and various body fluids, such as pleural effusion and ascites, is an in vitro study of lymphocyte immunity. and an important prerequisite for cytology experiments. [0003] The current popular cellular immunotherapy therapy is a new technology leading the future treatment of diseases. This therapy needs to collect the human body's own immune cells, expand them through in vitro culture, and then undergo genetic engineering and special culture to make these cells have enhanced Immunity, killing pathogens and tumor cells, promoting tissue and organ regeneration and body recovery and othe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078
CPCC12N5/0634C12N2509/00
Inventor 孙俊张金保陈燕
Owner MIAOSHUN SHANGHAI BIOTECH CO LTD
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