Method for separating and purifying CD8+T cells from liver cancer tissue

Abstract

The invention relates to a method for separating and purifying CD8+T cells from liver cancer tissue. The method for separating the CD8+T cells comprises the following specific steps: (1) separating tumor infiltration immune cells; (2) performing discontinuous density gradient centrifugation with 30% percoll and 60% percoll; and (3) sorting the CD8+T cells by using MACS (Magnetic Activated Cell Sorting) microbeads. With the combination of density gradient centrifugation and microbead sorting, the CD8+T cells are successfully purified from liver cancer tissue, the purity of the cells is identified by using a flow cytometry, the purity is up to 95% or greater, and meanwhile the cell number is up to 3*10<5> or higher.

Description

(1) Technical field [0001] The invention relates to a method for separating and purifying CD8+ T cells from liver cancer tissue. (2) Background technology [0002] CD8+ T lymphocytes are the main effector cells of anti-tumor immunity. CD8+ T cells can recognize endogenous antigenic peptides presented by MHC class I molecules, and can produce IFN-γ to mediate the killing of target cells carrying antigens. In addition, there are CD8+ T cells that can produce IL-17 in tumor tissue, and the secretion of IL-17 can promote the further development of tumor by promoting blood vessels, and at the same time, tumor-activated monocytes can secrete a series of key cytokines (IL -1β, IL-6 and IL-23) to induce the proliferation of IL-17-secreting CD8+ T cells. It can be seen that CD8 + There is a relatively complex interaction between T cells and the tumor microenvironment, which can promote the immune response to kill tumor cells, or inhibit the immune response and cannot effectively k...

AI Technical Summary

Problems solved by technology

These two methods have their own advantages and disadvantages in practical application: (1) Although the separation purity of flow separation can reach 90%, the equipment is expensive, the operation is complicated, the separation time is long, and the high-purity mode is selected for separation. There will inevitably be a loss of many cells during the selection, resulting in a final cell yield of less than 10^5

(2) Tumor tissue is complex and contains many different types of cells. At the same time, the process of digesting the tissue into single cells will produce many cell debris and dead cells. Although immunomagnetic bead sorting is simple and fast, it will produce some miscellaneous cells Non-specific binding, especially easy to bind to cell debris and dead cells, resulting in a final separation purity of less than 50%

Combining Ficoll density gradient centrifugation with immunomagnetic bead sorting removes some of the foreign cells, but this method cannot avoid non-specific binding of cell debris and dead cells, and the final sorting purity is less than 70%.

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