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Induced culture composition capable of promoting proliferation of spleen-derived CD8 positive T cell and application of induced culture composition

A technology of cell proliferation and composition, applied in the direction of cell culture active agent, culture process, tissue culture, etc., can solve the problems of unfavorable acquisition of CD8-positive T cells and low amount of CD8-positive T cells, etc.

Inactive Publication Date: 2018-11-23
深圳灵赋拓普生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the proportion of lymphocytes in peripheral blood is small, which is not conducive to obtaining a large number of CD8-positive T cells
Some studies promote the expansion of CD8-positive T cells by adding stimulating factors such as interleukin-2 in the process of immune cell proliferation, but the amount of CD8-positive T cells obtained is still low and cannot meet actual needs

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Extract total cells from the spleen:

[0075] (1) SD rats were taken, anesthetized with anesthesia, transferred to an ultra-clean workbench, the abdominal cavity was opened, the spleen tissue was cut out, and placed in HBSS buffer precooled to 4°C.

[0076] (2) Transfer the spleen tissue placed in the HBSS buffer to the sterilized cell operation room. Place a clean 60mm cell culture dish on ice, add 3 mL of pre-cooled HBSS buffer solution to the cell culture dish, and transfer the spleen tissue to the cell culture dish containing HBSS buffer.

[0077] (3) Cut up the spleen tissue with scissors, grind the chopped spleen tissue, put the ground spleen tissue on a 200-mesh sieve, wash the sieve with 1 mL of HBSS buffer, and collect the filtrate, which is Cell fluid containing total cells of the spleen.

Embodiment 2

[0079] Extract mononuclear cells:

[0080] (1) Get a clean 15mL centrifuge tube, fill it with ficoll lymphocyte separation solution, add the cell solution obtained in Example 1 along the tube wall of the centrifuge tube, so that a clear boundary is formed between the ficoll lymphocyte separation solution and the cell solution . Among them, the total number of cells in the cell solution is 5×10 6 cells / mL, the volume ratio of ficoll lymphocyte separation medium to cell fluid was 1:2.

[0081] (2) Carefully put the centrifuge tube into a horizontal centrifuge (TD5A-WS from Xiangyi Company), and centrifuge at 25° C. and 1500 rpm for 15 minutes. After centrifugation, take out the centrifuge tube carefully, and a buffy coat layer is formed between the ficoll lymphocyte separation liquid layer and the cell liquid layer, which is the mononuclear cell layer.

[0082] (3) Take out the buffy coat cells with a pipette and transfer to another clean 15mL centrifuge tube. Add 3mL of PBS...

Embodiment 3

[0084] Isolation of CD8-positive T cells from mononuclear cells:

[0085] (1) The precipitate obtained in Example 2 is resuspended with RPMI1640 medium to obtain a resuspension, and the number of cells in the resuspension is 5×10 6 individual / mL.

[0086] (2) Transfer the resuspension to a culture bottle containing RPMI1640 medium, and place it at 37°C, 5% CO 2 Cultivate in an incubator for 1 day to obtain a culture solution, and the volume ratio of the resuspension solution to the RPMI1640 medium is 10. The culture solution was centrifuged at 4°C and 1600 rpm for 10 minutes to obtain a precipitate.

[0087] (3) Resuspend the precipitate with PBS buffer, add double anti-magnetic beads and incubate at 4°C in the dark for 10 min. Among them, the amount of PBS buffer added is: 4 × 10 7 Mononuclear cells were added to 160 μL of PBS buffer. The amount of double anti-magnetic beads added is: 4×10 7 Add 40 μL of double-antimagnetic beads to each mononuclear cell.

[0088] (4) ...

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PUM

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Abstract

The invention relates to an induced culture composition capable of promoting proliferation of a spleen-derived CD8 positive T cell and application of the induced culture composition. The induced culture composition comprises a culture medium and inducing factors added in the culture medium, the inducing factors comprises phytohemagglutinin, a CD3 antibody and a CD28 antibody, the final concentration of the phytohemagglutinin is 1 mu g / mL to 10 mu g / mL, the final concentration of the CD3 antibody is 1 mu g / mL to 10 mu g / mL, the final concentration of the CD28 antibody is 1 mu g / mL to 10 mu g / mL. The induced culture composition provided by the invention can promote proliferation of the spleen-derived CD8 positive T cell.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an induction culture composition for promoting the proliferation of spleen-derived CD8 positive T cells and an application thereof. Background technique [0002] T lymphocytes are derived from pluripotent stem cells of the bone marrow. During the embryonic and nascent stages, some pluripotent stem cells or pre-T cells in the bone marrow migrate into the thymus, differentiate and mature under the induction of thymus hormones, and become immune-competent T lymphocytes. T lymphocytes can be divided into helper T cells (Th), suppressor T cells (TS), effector T cells (Te) and cytotoxic T cells (Tc) according to their different functions in the immune response. Among them, cytotoxic T cells, also known as CD8-positive T cells, express CD8 molecules on the surface, and are a type of immune cells that can kill target cells. CD8-positive T cells play a very important role in fighting agains...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0636C12N2500/12C12N2500/14C12N2500/16C12N2500/32C12N2500/34C12N2500/38C12N2501/51C12N2501/515C12N2501/59
Inventor 饶秀茸王康易艳琼李鹏李泓彦
Owner 深圳灵赋拓普生物科技有限公司
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