Separation method for separating peripheral blood mononuclear cells

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...

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 the separation medium layer and the blood layer before centrifugation will cause separation failure ;2. After the separation, the peripheral blood mononuclear cells need to be sucked with a straw. This process is easily contaminated by other cells.

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