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Method for culturing peripheral blood containing CIK cells

A culture method and peripheral blood technology, applied in the biological field, can solve the problems of low cytotoxicity and poor reproductive ability of CIK cells, and achieve the effects of increasing the multiplier, solving the poor reproductive ability of CIK cells, and improving cytotoxic activity

Pending Publication Date: 2021-10-15
上海南滨江细胞生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The object of the present invention is to provide a kind of culture method that peripheral blood contains CIK cell, in order to solve the technical problem that the CIK cell cultured out of the current CIK cell culture method has poor reproductive ability and low cytotoxic activity

Method used

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  • Method for culturing peripheral blood containing CIK cells
  • Method for culturing peripheral blood containing CIK cells
  • Method for culturing peripheral blood containing CIK cells

Examples

Experimental program
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Effect test

Embodiment 1

[0020] A method for culturing peripheral blood containing CIK cells, comprising the following steps:

[0021] Step 1. Extraction of peripheral blood and culture of mononuclear cells: Collect peripheral blood from patients or volunteers in a sterile environment, add heparin sodium, and centrifuge with lymphatic separation solution at a temperature of 15°C and a centrifugal force of 260xg Mononuclear cells were isolated in 18 minutes; the mononuclear cell layer was aspirated with capillaries, washed with normal saline, and then centrifuged at 20°C and 300xg for 23 minutes, repeated twice; then suspended and adjusted cells with medium A Density is 4×10 6 / L, and inoculate this medium A in 30cm 2 culture flask, and then place the culture flask at 37 °C, 5% CO 2 Collect the cells after culturing in an incubator at 97% humidity for 24 hours, centrifuge for 17 minutes at a temperature of 17°C and a centrifugal force of 250×g, discard the supernatant, and obtain cultured mononuclear...

Embodiment 2

[0028] A method for culturing peripheral blood containing CIK cells, comprising the following steps:

[0029] Step 1. Extraction of peripheral blood and culture of mononuclear cells: Collect peripheral blood from patients or volunteers in a sterile environment, add heparin sodium, and centrifuge with lymphatic separation solution at a temperature of 16°C and a centrifugal force of 260xg Mononuclear cells were isolated in 18 minutes; the mononuclear cell layer was aspirated with capillaries, washed with normal saline, and then centrifuged at 20°C and 300xg for 23 minutes, repeated twice; then suspended and adjusted cells with medium A Density is 4×10 6 / L, and inoculate this medium A in 30cm 2 culture flask, and then place the culture flask at 37 °C, 5% CO 2 Collect the cells after culturing in an incubator at 98% humidity for 24 hours, centrifuge for 17 minutes at a temperature of 19°C and a centrifugal force of 250×g, discard the supernatant, and obtain cultured mononuclear...

Embodiment 3

[0035] A method for culturing peripheral blood containing CIK cells, comprising the following steps:

[0036] Step 1. Extraction of peripheral blood and culture of mononuclear cells: Collect peripheral blood from patients or volunteers in a sterile environment, add heparin sodium, and centrifuge with lymphatic separation solution at a temperature of 18°C ​​and a centrifugal force of 260xg Isolate the mononuclear cells in 18 minutes; absorb the mononuclear cell layer with capillaries, then wash with normal saline, then centrifuge at 23°C and 300xg for 23 minutes, repeat twice; then use medium A to suspend and adjust the cells Density is 4×10 6 / L, and inoculate this medium A in 30cm 2 culture flask, and then place the culture flask at 37 °C, 5% CO 2 Collect the cells after culturing in an incubator at 100% humidity for 24 hours, centrifuge for 17 minutes at a temperature of 22°C and a centrifugal force of 250×g, discard the supernatant, and obtain cultured mononuclear cells; ...

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Abstract

The invention discloses a method for culturing peripheral blood containing CIK cells, and belongs to the technical field of biology, and the culture method for peripheral blood containing CIK cells comprises the following steps: step 1, extracting peripheral blood and culturing mononuclear cells; and step 2, culturing the CI K cells. The CD28 monoclonal antibody is added in the culture process of the CI K cells, and the CD28 can be used for increasing the activation of the T cells, promoting the generation of the T cells IL-2 and the proliferation of the antigen-specific polyclonal T cells, obviously increasing the proliferation multiple of the CI K cells and improving the reproductive capacity of the CI K cells; IL-1alpha, IL-15 and IL-21 stimulating factors are also added to induce the expression of perforin, granzyme B and CD107a in the cultured CIK cells, so that the cytotoxic activity of the CIK cells is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a culture method for peripheral blood containing CIK cells. Background technique [0002] CIK (Cytokine-Induced Killer Cells) in Chinese is called cytokine-induced killer cells. It is a group of heterogeneous cells obtained after human peripheral blood mononuclear cells are co-cultured with various cytokines for a period of time in vitro. It has not only the strong anti-tumor activity of T lymphocytes, but also the non-MHC (major histocompatibility antigen) restricted tumor killing ability of NK cells (natural killer cells). CIK cells have the characteristics of high anti-tumor activity, broad anti-tumor spectrum, low toxicity to normal tissues, and high expansion in vitro. They are currently widely used adoptive immunotherapy cells in clinical practice. [0003] At present, the commonly used activation method of CIK cells is to use CD28 monoclonal antibody, interleukin-...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12N5/078
CPCC12N5/0638C12N5/0634C12N2501/51C12N2501/2315C12N2501/2321C12N2509/00C12N2500/32C12N2501/115C12N2500/38C12N2501/727C12N2501/33C12N2501/998C12N2500/05C12N2501/2302C12N2501/2301C12N2501/24
Inventor 占震锋李庆静
Owner 上海南滨江细胞生物科技有限公司
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