Separation method for separating peripheral blood mononuclear cells

A separation method and nuclear cell technology are applied in the field of separation of peripheral blood mononuclear cells, which can solve the problems of cumbersome operation, failure of separation, easy to be polluted by other cells, etc., and achieve the effect of simple preparation

Inactive Publication Date: 2020-05-08
JIANGSU TAIZHOU PEOPLES HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has two obvious disadvantages: 1. The operation is cumbersome: it is necessary to carefully add Ficoll and anticoagulated whole blood layer by layer to form a well-defined separation medium layer and blood layer, and the mixing of ...

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  • Separation method for separating peripheral blood mononuclear cells
  • Separation method for separating peripheral blood mononuclear cells
  • Separation method for separating peripheral blood mononuclear cells

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Embodiment Construction

[0023] Method embodiment of the present application:

[0024] Use the same source of human peripheral anticoagulated whole blood to repeat three times, as follows:

[0025] First prepare the separation tube: first add 4ml of Percoll cell separation solution with a density of 1.075-1.0796g / ml to the 15ml centrifuge tube, draw 1ml of separation gel with a density of 1.06-1.07g / ml and add it to the mouth of the centrifuge tube, 1000g at room temperature Centrifuge for 2 minutes, and the separation gel forms an isolation layer on the surface of the Percoll cell separation medium;

[0026] Then carry out the separation of peripheral blood mononuclear cells: draw 4ml of anticoagulated whole blood and add it to the prepared 15ml separation tube. After horizontal centrifugation for 10 minutes, a layer of peripheral blood mononuclear cells visible to the naked eye is formed above the adjacent separation gel layer, while red blood cells are completely deposited at the bottom of the tub...

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Abstract

The invention provides a separation method for separating peripheral blood mononuclear cells, and particularly relates to the field of biological medicines. The separation method comprises the following steps: S1, preparation of a separation tube: firstly, adding 2-6 ml of Percoll or polysucrose or meglumine diatrizoate cell separation liquid with the density of 1.075-1.0796 g/ml into a centrifugal tube, sucking 0.5-1.5 ml of separation gel with the density of 1.06-1.07 g/ml, adding the separation gel into a tube opening of the centrifugal tube, and carrying out horizontal centrifuging for 1-3minutes at room temperature under the centrifugal force of 800-1200 g, so that the separation gel forms an isolation layer on the liquid surface of the Percoll or polysucrose or meglumine diatrizoatecell separation liquid, and preparation of the separation tube is finished; and S2, separation of the peripheral blood mononuclear cells: adding 2-6 ml of anticoagulant whole blood into the preparedseparation tube, and carrying out horizontal centrifuging for 8-12 minutes at room temperature under the centrifugal force of 800-1200 g; sucking and discarding the uppermost liquid to remove cell fragments and platelets, directly pouring the liquid above the isolation layer into a collection tube, carrying out centrifuging for 4-6 minutes at room temperature under the centrifugal force of 600-1000 g, and resuspending the cells by using PBS to obtain the peripheral blood mononuclear cells. According to the separation method, it can be ensured that the anticoagulant whole blood can be absolutely not mixed with a cell separation medium after being added, and other cells are not polluted when the peripheral blood mononuclear cells are harvested.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a separation method for separating peripheral blood mononuclear cells. Background technique [0002] Peripheral blood mononuclear cells (PBMCs) refer to cells with a single nucleus in peripheral blood, mainly including lymphocytes (T cells, B cells and NK cells), monocytes, phagocytes and A small number of other cell types. Peripheral blood mononuclear cells are important cells involved in the body's immune response function, and the frequency of its cell subsets varies among individuals. 70%-90% are lymphocytes, monocytes account for 10%-30%, and only 1%-2% are dendritic cells. Among lymphocyte subsets, 70%-85% are CD3+T cells, 5%-20% are B cells, and 5%-20% are NK cells. Peripheral blood mononuclear cells are often required research materials in the fields of infectious diseases, blood system diseases, antibody drug and vaccine development, tumor immunotherapy, and tra...

Claims

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Application Information

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IPC IPC(8): C12N5/078
CPCC12N5/0634C12N2509/00
Inventor 周成林刘勇王春香彭海林
Owner JIANGSU TAIZHOU PEOPLES HOSPITAL
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