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Method for in-vitro culture and enrichment of CD8+ T cells

A technology of cells and culture medium, applied in the field of cell culture

Active Publication Date: 2017-04-19
英威福赛生物技术(天津)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CD8+ T cells are the main cell subpopulation that exerts specific killing function, but there is no mature method for high-efficiency directed expansion of CD8+ T cells in vitro. Therefore, the development of high-efficiency CD8+ T cell directed proliferation culture technology and reagents in vitro It is of great significance to improve CTL-based tumor therapy, so it is in clinical demand, especially the culture method that contains a higher content of central memory T cells and enriches CD8+ T cells

Method used

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  • Method for in-vitro culture and enrichment of CD8+ T cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029]Embodiment 1. Separation of peripheral blood lymphocytes (PBMCs) of volunteers:

[0030] The lymphocytes used in the present invention are obtained from the venous peripheral blood of an individual. After the selected individuals pass the physical examination of the clinician, the experimenter will inform the specific project process and the amount of blood required. After the volunteers agree and sign the informed consent, the clinical medical staff will collect blood from the volunteers. In the end, this project screened 2 cases of healthy volunteers D1 and D2, and used EDTA-K 2 Anticoagulant 9ml disposable vacuum blood collection tube (VACUETTE, Greiner Company, Austria), about 20-25ml blood was collected from each volunteer, and the blood sample was immediately inverted to prevent coagulation after blood collection.

[0031] 1) Dilute the freshly collected peripheral blood by one time with cooled phosphate buffered saline (0.01M PBS, pH7.4, autoclaved at 121°C), and...

Embodiment 2

[0037] Embodiment 2, T cell sorting

[0038] The T lymphocytes in the isolated PBMCs were sorted and enriched by immunomagnetic bead method to obtain high-purity T cells for subsequent culture.

[0039] 1) Firstly, the isolated PBMCs obtained above were divided into 1 × 10 7 The cells were mixed with 20 μl of CD4 antibody-coupled magnetic beads and 20 μl of CD8 antibody-coupled magnetic beads (purchased from Miltenyi Biotechnology Co., Ltd., Germany), and then incubated at 4°C for 15 minutes. (FBS, American Thermo Fisher company brand, Australian source) and 2mM EDTA-Na PBS buffer (PBS-F buffer) were washed twice, and centrifuged at 500g for 5 minutes at 25°C.

[0040] 2) Resuspend the above cells with 500 μl of PBS-F buffer, and filter through a 200-mesh disposable sterile filter (purchased from Miltenyi Biotechnology Co., Ltd., Germany) to remove impurities in the cell suspension, so that the cells can Pass through the separation column (MS separation column, purchased fro...

Embodiment 3

[0045] Example 3, T cell stimulation expansion in vitro

[0046] 1) The high-purity T cells mainly composed of CD4+ and CD8+ T cells obtained in step 5) of Example 2 were subjected to the following three treatment methods of A, B, and C respectively:

[0047] A. Add CD3 and CD28 antibody-coupled magnetic beads;

[0048] B. Placed in a cell culture plate pre-coated with CD3 antibody and CD28 antibody;

[0049] C. Add CD3 and CD28 antibodies in solution state;

[0050] In order to compare the proliferation level of T cells and the expanded T cell subsets and differentiation levels under different stimulation combinations and culture conditions. The specific addition or cultivation conditions are as follows:

[0051] A. CD3 and CD28 antibody-coupled magnetic beads culture: the magnetic beads coated with CD3 and CD28 antibodies (purchased from LifeTechnology company Human T-Activator CD3 / CD28) was taken out according to the ratio of magnetic beads: T cells = 1:1 and added to ...

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Abstract

The invention belongs to the field of cell culture, particularly relates to a method for in-vitro culture and enrichment of CD8+ T cells, and more particularly relates to a method for directional amplification of a large amount of CD8+ T cells in peripheral blood mononuclear cells of a human body by using an irritant. The T cells obtained by the technical scheme are large in proliferation number and high in activity; the ratio of the CD8+ T cells to CD4+ T cells can be increased to 2-5 : 1 from about 1 : 2 in a normal physiological status; and in a target culture, the content of central memory T cells exceeds 30% based on the total cell number.

Description

technical field [0001] The invention belongs to the field of cell culture, and in particular relates to a method for culturing and enriching CD8+ T cells in vitro, more specifically, the invention relates to a method of using stimulators to amplify CD8+ T cells in a large amount from human peripheral blood mononuclear cells Methods. Background technique [0002] In 2011, cancer surpassed heart disease as the leading cause of death worldwide. The WHO announced in December 2013 that the number of new cancer patients worldwide has exceeded 14 million each year, which is a substantial increase compared with the 2008 statistics of 12.7 million. During the same period, the number of deaths of cancer patients also increased, from 7.6 million in the past to 8.2 million. New cancer cases will increase by 50% to 21.6 million a year by 2030, the report said. In 2013, immune anti-cancer therapy was rated as the top 10 scientific and technological breakthroughs of the year by Science ...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0636C12N2501/2302C12N2501/51C12N2501/515
Inventor 张卫红李燕谭曙光
Owner 英威福赛生物技术(天津)有限公司
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