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Lymphocyte population with memory stem T cells as main component and in-vitro efficient amplification method of lymphocyte population

A technology of lymphocytes and cells, applied in the direction of animal cells, vertebrate cells, cell culture active agents, etc., can solve the problems of loss of T cell physiological functions, etc., achieve the effect of increasing the expansion multiple, reducing dosage, and reducing toxic and side effects

Active Publication Date: 2017-12-15
THE FIRST AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV (GUANGZHOU RESPIRATORY CENT)
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional T cell expansion culture system makes some important physiological functions of T cells lost, such as long-term implantation ability and immune system reconstruction ability

Method used

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  • Lymphocyte population with memory stem T cells as main component and in-vitro efficient amplification method of lymphocyte population
  • Lymphocyte population with memory stem T cells as main component and in-vitro efficient amplification method of lymphocyte population
  • Lymphocyte population with memory stem T cells as main component and in-vitro efficient amplification method of lymphocyte population

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 A high-efficiency in vitro expansion method of a lymphocyte population mainly composed of memory stem T cells

[0043] A high-efficiency in vitro expansion method of a lymphocyte population mainly composed of memory stem T cells in this embodiment includes the following steps:

[0044] (1), with the anti-CD3mAb (PEPROTECH, USA) that final concentration is 5 μ g / ml, the anti-CD28McAb (PEPROTECH, USA) that concentration is 500ng / ml, the Novonectin (PEPROTECH, USA) of 5 μ g / ml pre-coats 75mm Culture bottle;

[0045] (2) Collect 80ml of peripheral blood from healthy people, transfer the collected blood sample to a centrifuge tube; centrifuge at 400g, 10min, absorb the upper layer of plasma for later use; reduce to 80ml with PBS, mix well; slowly add diluted blood to the peripheral blood Lymphatic separation medium, 400g, horizontal centrifugation at room temperature for 25min;

[0046] (3) Aspirate the mononuclear cells, wash the cells twice with PBS buffer, sta...

Embodiment 2

[0053] Example 2 A high-efficiency in vitro expansion method of a lymphocyte population mainly composed of memory stem T cells

[0054] In this example, a high-efficiency in vitro expansion method of a lymphocyte population mainly composed of memory stem T cells except VC, NAC, RAPA, SR1, IL-7, IL-15 and The final concentrations of IL-21 were 50mg / L, 5mM, 150nM, 0.8μM, 50μg / L, 50μg / L, and 1000μg / L respectively, and other steps were the same as in Example 1.

Embodiment 3

[0055] Example 3 A high-efficiency in vitro expansion method of a lymphocyte population mainly composed of memory stem T cells

[0056] In this example, a high-efficiency in vitro expansion method of a lymphocyte population mainly composed of memory stem T cells except VC, NAC, RAPA, SR1, IL-7, IL-15 and The final concentrations of IL-21 were 100 mg / L, 10 mM, 200 nM, 1 μM, 25 μg / L, 25 μg / L, and 700 μg / L, respectively, and other steps were the same as in Example 1.

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Abstract

The invention discloses a lymphocyte population with memory stem T cells as a main component and an in-vitro efficient amplification method of the lymphocyte population. The method comprises the steps as follows: peripheral blood mononuclear cells from healthy individuals are transferred into a culture flask precoated with anti-CD3 mAb, anti-CD28McAb and Novonectin, a serum-free medium containing IL-1alpha and IFN-gamma is added, a combination of multiple cytokines including VC, NAC, RAPA, IL-7, IL-15 and IL-21 is added, and then the serum-free medium and the combination of the multiple cytokines are supplemented; and the lymphocyte population with the memory stem T cells as the main component can be obtained after culture for 12 to 16 days. The combined application of the cytokines can have synergistic effect on amplification of lymphocytes, the amplification times are increased, and the in-vitro killing property and survival time are increased obviously; the method has the advantages of high safety, low cost and the like.

Description

technical field [0001] The invention belongs to the technical field of cell bioengineering, and more specifically, the invention relates to a lymphocyte population mainly composed of memory stem T cells and an in vitro high-efficiency expansion method thereof. Background technique [0002] Memory stem T cells are a newly discovered cell subset. This type of cells has the characteristics of stem cells, that is, multi-directional differentiation potential, and can maintain self-renewal during differentiation. It has both naive T cells (naive T) Marker, but it also shows the characteristics of memory T cells when doing cell function research. [0003] In the prior art, in order to obtain a high content of memory T cells, flow cytometry sorting is commonly used. This method has high requirements on instruments and experimental environments, and the operation is complicated and prone to pollution. The sorting process will cause certain damage and waste to the cells. [0004] In ...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0636C12N2500/32C12N2500/38C12N2501/2301C12N2501/2307C12N2501/2315C12N2501/2321C12N2501/24C12N2501/51C12N2501/515
Inventor 王雯珺何建行伍思培列璞怡
Owner THE FIRST AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV (GUANGZHOU RESPIRATORY CENT)
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