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Culture method of peripheral blood CTL cells

A culture method and peripheral blood technology, applied in the direction of cell culture active agent, cell culture support/coating, blood/immune system cells, etc., can solve the problems of poor cell activity and low expansion CTL magnification, and achieve expansion good effect

Inactive Publication Date: 2019-03-19
汇麟生物科技(北京)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 of peripheral blood CTL cells
  • Culture method of peripheral blood CTL cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] This embodiment relates to a method for culturing peripheral blood CTLs.

[0052] 1. Peripheral blood CTL cell culture medium

[0053] 1) Coating solution: D-PBS, CD3 monoclonal antibody concentration 400ng / mL, CD28 antibody concentration 600ng / mL.

[0054] 2) CTL initial medium: VIVO medium (containing 4% autologous plasma), IFN-γ900U / mL.

[0055]3) CTL medium: VIVO medium (containing 4% autologous plasma), IL-2 concentration 1100 U / mL, IL-7 2ng / mL.

[0056] 2. Peripheral blood CTL cell culture method

[0057] 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 10 mL of CTL initial medium, and place at 37°C, 5% CO 2 in the incubator.

[0058] 2) On the first day of culture, take out the culture bottle, add IL-1α an...

Embodiment 2

[0064] This embodiment relates to a method for culturing peripheral blood CTLs.

[0065] 1. Peripheral blood CTL cell culture medium

[0066] 1) Coating solution: D-PBS, CD3 monoclonal antibody concentration 600ng / mL, CD28 antibody concentration 400ng / mL.

[0067] 2) CTL initial medium: VIVO medium (containing 6% autologous plasma), IFN-γ 1100 U / mL.

[0068] 3) CTL medium: VIVO medium (containing 6% autologous plasma), IL-2 concentration 900U / mL, IL-7 3ng / mL.

[0069] 2. Peripheral blood CTL cell culture method

[0070] 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 10 mL of CTL initial medium, and place at 37°C, 5% CO 2 in the incubator.

[0071] 2) On the first day of culture, take out the culture bottle, add IL-1α and IL...

Embodiment 3

[0077] This embodiment relates to a method for culturing peripheral blood CTLs.

[0078] 1. Peripheral blood CTL cell culture medium

[0079] 1) Coating solution: D-PBS, CD3 monoclonal antibody concentration 450ng / mL, CD28 antibody concentration 550ng / mL.

[0080] 2) CTL initial medium: VIVO medium (containing 5% autologous plasma), IFN-γ950U / mL.

[0081] 3) CTL medium: VIVO medium (containing 5% autologous plasma), IL-2 concentration 1050U / mL, IL-7 2.2ng / mL.

[0082] 2. Peripheral blood CTL cell culture method

[0083] 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 10 mL of CTL initial medium, and place at 37°C, 5% CO 2 in the incubator.

[0084] 2) On the first day of culture, take out the culture bottle, add IL-1α ...

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Abstract

The invention relates to a culture method of peripheral blood CTL cells. The method comprises the following steps: 1) inoculating PBMC obtained by separating from placental blood to a culture vessel coated with a CD3 antibody and a CD28 antibody for culture, wherein the cell factor in the culture system is IFN-gamma 900U / mL-1100U / mL; 2) adding IL-1alpha and IL-2 into the culture system, with the final concentrations being both 900U / mL-1100U / mL, and also adding IL-7 into the culture system, with the final concentration being 4-6ng / mL; and 3) changing the solution, wherein cell factors in the new culture system are IL-2 900U / mL-1100U / mL, IL-7, 2-3ng / mL, and the basal culture medium is a VIVO culture medium containing 4-6% autologous plasma. The method has the advantages that the CTL amplification effect is good, the activated CTL proportion is relatively high, and the T-reg cell proportion is relatively low.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for culturing 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, through the ...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0638C12N2501/2301C12N2501/2302C12N2501/2307C12N2501/24C12N2501/51C12N2501/515C12N2533/50
Inventor 施琳
Owner 汇麟生物科技(北京)有限公司
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