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Culture method for improving peripheral blood CTL (cytotoxic lymphocyte) cells

A culture method and peripheral blood technology, applied in the field of cell culture, can solve the problems of poor cell activity and low amplification CTL magnification, and achieve good amplification effect

Inactive Publication Date: 2019-02-22
见多视光(北京)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 existing CTL culture methods in vitro generally have problems such as low amplification CTL magnification and poor cell activity.

Method used

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  • Culture method for improving peripheral blood CTL (cytotoxic lymphocyte) cells
  • Culture method for improving peripheral blood CTL (cytotoxic lymphocyte) cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] This embodiment relates to an improved culture method of peripheral blood CTL.

[0055] 1. Peripheral blood CTL cell culture medium

[0056] 1) Coating solution: D-PBS, CD3 monoclonal antibody concentration 400ng / mL, CD28 antibody concentration 600ng / mL.

[0057]2) CTL initial medium: VIVO medium (containing 4% autologous plasma), IFN-γ900U / mL.

[0058] 3) CTL medium: AIM-V medium (containing 4% autologous plasma), IL-2 concentration 1100U / mL.

[0059] 2. Peripheral blood CTL cell culture method

[0060] 1) On the 0th day of culture, take a T25 culture bottle, add 2.5 mL of coating solution, place it in an incubator for coating for 2.5 hours, and set aside. Take out the coated T25 culture bottle, pour off the coating solution, and take the prepared PBMC 0.8×10 7 , inoculate into a T25 culture flask, add 13ml of CTL initial medium, place at 37°C, 5% CO 2 in the incubator.

[0061] 2) On the first day of culture, take out the culture bottle, add IL-1α and IL-2, the ...

Embodiment 2

[0067] This embodiment relates to an improved culture method of peripheral blood CTL.

[0068] 1. Peripheral blood CTL cell culture medium

[0069] 1) Coating solution: D-PBS, CD3 monoclonal antibody concentration 600ng / mL, CD28 antibody concentration 400ng / mL.

[0070] 2) CTL initial medium: VIVO medium (containing 6% autologous plasma), IFN-γ 1100 U / mL.

[0071] 3) CTL medium: AIM-V medium (containing 6% autologous plasma), IL-2 concentration 900 U / mL.

[0072] 2. Peripheral blood CTL cell culture method

[0073] 1) On the 0th day of culture, take a T25 culture bottle, add 3.5mL of coating solution, place it in an incubator for coating for 1.5h, and set aside. Take out the coated T25 culture bottle, pour off the coating solution, and take the prepared PBMC 1.2×10 7 , inoculate into a T25 culture flask, add 17ml CTL initial medium, place at 37°C, 5% CO 2 in the incubator.

[0074] 2) On the first day of culture, take out the culture bottle, add IL-1α and IL-2, the final...

Embodiment 3

[0080] This embodiment relates to an improved culture method of peripheral blood CTL.

[0081] 1. Peripheral blood CTL cell culture medium

[0082] 1) Coating solution: D-PBS, CD3 monoclonal antibody concentration 450ng / mL, CD28 antibody concentration 550ng / mL.

[0083] 2) CTL initial medium: VIVO medium (containing 5% autologous plasma), IFN-γ950U / mL.

[0084] 3) CTL medium: AIM-V medium (containing 5% autologous plasma), IL-2 concentration 1050U / mL.

[0085] 2. Peripheral blood CTL cell culture method

[0086] 1) On the 0th day of culture, take a T25 culture bottle, add 2.8 mL of coating solution, place it in an incubator for coating for 2.2 hours, and set aside. Take out the coated T25 culture bottle, pour off the coating solution, and take the prepared PBMC 1.1×10 7 , inoculate into a T25 culture flask, add 14ml CTL initial medium, place at 37°C, 5% CO 2 in the incubator.

[0087] 2) On the first day of culture, take out the culture bottle, add IL-1α and IL-2, the fi...

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Abstract

The invention relates to the technical field of cell culture, in particular to a culture method for improving peripheral blood CTL (cytotoxic lymphocyte) cells. The method includes the steps: 1) inoculating PBMC separated by peripheral blood into a culture vessel coated by a CD3 antibody and a CD28 antibody to cultural the PBMC; 2) adding IL-1 alpha and IL-2 with the final concentration of 900U / mL-1100U / mL, IL-7 with the concentration of 40ng / mL-60ng / mL and IL-12 with the concentration of 15ng / mL-25ng / mL into a culture system; 3) exchanging liquid. A cell factor in the culture system is IFN-gamma with the concentration of 900U / mL-1100U / mL, and a cell factor in a new culture system is IL-2 with the concentration of 900U / mL-1100U / mL. According to the method, CTL amplification effects are good, activated CTL proportion is higher, and the proportion of T-reg cells is lower.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to an improved culture method for peripheral blood CTL cells. Background technique [0002] Cytotoxic T lymphocytes (cytotoxic lymphocyte, CTL), is a specific, T cells with killing function, specialized in secreting various cytokines to participate in immune function. It has a killing effect on antigenic substances such as certain viruses and tumor cells, and forms an important line of defense for the body's anti-virus and anti-tumor immunity with natural killer cells. [0003] Activated CTL can produce specific tumor killing activity, this cytotoxicity mainly includes (1) perforin-granzyme pathway (2) activated CTL highly expresses FaSL, which can induce Target cell apoptosis (3) secrete TNF and other cytokines to directly kill target cells (4) produce a variety of chemokines, attract natural immune cells, and kill target cells (5) release a variety of serine acetylases, thro...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
Inventor 张权
Owner 见多视光(北京)科技有限公司
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