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In-vitro amplification kit and amplification method for regulatory T cells in human peripheral blood

A technology of human peripheral blood and kits, which is applied in the direction of cell culture active agents, blood/immune system cells, animal cells, etc., and can solve the problems of in vitro function experiments that cannot be carried out smoothly, biological safety that needs to be studied, and effective amplification, etc. problems, to achieve the effect of retaining biological functions, stable phenotype and function, and low cost

Active Publication Date: 2020-05-08
北京纽莱福生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The number of cells obtained by the above separation method is small and the purity is low, and the regulatory T cells that actually play an inhibitory role in the in vitro expansion process have not been effectively expanded, resulting in a very small number of Tregs in the finally obtained cells, which cannot be successfully followed up. in vitro function test
In addition, the cost of the above method is high, and the biological safety needs to be studied

Method used

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  • In-vitro amplification kit and amplification method for regulatory T cells in human peripheral blood
  • In-vitro amplification kit and amplification method for regulatory T cells in human peripheral blood
  • In-vitro amplification kit and amplification method for regulatory T cells in human peripheral blood

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Embodiment approach

[0034] According to a preferred embodiment of the present invention, the coating antibody is one selected from CD4 monoclonal antibody, CD25 monoclonal antibody and FoxP3 monoclonal antibody, preferably CD4 monoclonal antibody.

[0035] The present inventors have found that the CD4 monoclonal antibody has a wide range of sources, low cost, and is easy to obtain. It is selected as the first coating antibody, which can efficiently and massively expand CD4-positive T cells in peripheral blood mononuclear cells, and can expand the proliferation of Treg cells. The number of basic cells cultured is convenient for further screening and culture operations in the later stage.

[0036] In a further preferred embodiment, the first activating factor is interferon-γ (IFN-γ).

[0037] In the present invention, gamma interferon (IFN-γ) is selected as the activation factor because it can promote the development and differentiation of Treg and negatively regulate the occurrence and development...

Embodiment 1

[0157] Such as figure 1 As shown, the in vitro expansion of the human peripheral blood regulatory T cells is carried out according to the following steps:

[0158] Step 1: Take 50-100ml of anticoagulated peripheral blood, centrifuge at 2000rpm for 20min, separate and collect plasma, inactivate at 56°C for 30min, and centrifuge at 1500rpm for 10min to obtain autologous plasma;

[0159] Add 1-1.5 times the plasma volume of sterile PBS buffer to the blood from which the plasma has been removed to dilute the blood, and then use the lymphocyte separation medium Ficoll-Paque (purchased from Beijing Biolab Technology Co., Ltd., product number BTN131136) to separate Peripheral blood mononuclear cells were obtained.

[0160] Step 2: Resuspend the mononuclear cells obtained in step 1 with a selection medium, wherein the selection medium consists of: RPMI 1640 medium + inactivated autologous plasma + β-mercaptoethanol + IL-2 + Hepes, wherein , the final concentration of inactivated aut...

Embodiment 2

[0169] The method used in this example is similar to Example 1, except that in step 2, the final concentration of the CD4 monoclonal antibody is 22 μg / ml, and the final concentration of IFN-γ is 1500 U.

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Abstract

The invention discloses an in-vitro amplification kit for regulatory T cells in human peripheral blood. The kit comprises an activating agent I, an activating agent II, a screening culture medium andan amplification culture medium. The invention further discloses an in-vitro amplification method for the regulatory T cells in human peripheral blood. According to the method, mononuclear cells of peripheral blood are directly obtained and are paved in culture containers coated with different antibodies for culture, and activators such as interleukin and AICAR are added to promote cell proliferation. The method has the advantages of low cost, simple operation, and capability of obtaining of a large number of regulatory T cells with high cell viability in a short time; and the obtained cells are stable in phenotypes and functions and high in biological safety.

Description

technical field [0001] The invention belongs to the technical field of cell separation and expansion, and in particular relates to an in vitro expansion kit and expansion method of human peripheral blood regulatory T cells. Background technique [0002] Regulatory T cells (Treg) are a kind of T cell subsets that control the body's autoimmune reactivity, and can suppress immune responses. important role. Although the proportion of peripheral blood cells is low, accounting for only 5% to 10% of CD4+ T cells in peripheral blood and spleen tissue of normal people, it is closely related to tumors, autoimmune diseases, and graft acceptance. [0003] In recent years, the clinical application of cancer immunotherapy has made significant progress, and has become another effective treatment for cancer after surgery, radiotherapy, chemotherapy, and targeted therapy. Among various immunotherapy methods, immune checkpoint therapy has achieved outstanding efficacy in recent years. It re...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0637C12N2500/44C12N2500/60C12N2501/505C12N2501/599C12N2501/51C12N2501/515C12N2501/24C12N2501/2302C12N2501/2307C12N2501/2312C12N2501/2315C12N2501/2321C12N2500/38C12N2500/40C12N2501/15C12N2533/50Y02A50/30
Inventor 段海峰薛冰华于婷婷解晶肖秀孝庞如梦弓景波张群伟鲁飞
Owner 北京纽莱福生物科技有限公司
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