Method for efficient in-vitro amplification and culture of natural killer T cells with high killing capacity

An in vitro expansion and killing technology, which is applied in the direction of cell culture active agent, animal cells, tissue culture, etc., can solve the problems of difficult expansion, low NKT cell content, and limit the clinical application of NKT cells, so as to achieve easy raw material acquisition , The preparation method is simple and easy, and the effect of killing tumor cells is strong

Pending Publication Date: 2019-09-27
INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the content of NKT cells in vivo is extremely low, and it is not e...

Method used

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  • Method for efficient in-vitro amplification and culture of natural killer T cells with high killing capacity
  • Method for efficient in-vitro amplification and culture of natural killer T cells with high killing capacity
  • Method for efficient in-vitro amplification and culture of natural killer T cells with high killing capacity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Obtaining human peripheral blood leukocytes

[0041] Take 5ml of fresh human peripheral blood, add hydroxyethyl starch (peripheral blood: hydroxyethyl starch = 5:1), and let stand at room temperature for 1 hour. Take the supernatant (white blood cell coat) and remove the lower layer of red blood cells.

[0042] Add the same volume of lymphocyte separation medium as the supernatant to the centrifuge tube, and carefully transfer the supernatant to the separation medium (operate gently to avoid breaking the interface liquid); centrifuge at 1800rpm, 30°C, 15 minutes; after centrifugation, the solution separates , the supernatant is serum, aspirated and transferred to a new centrifuge tube (for spare); the middle is the buffy coat layer (lymphocyte layer), aspirated and transferred to a new centrifuge tube.

Embodiment 2

[0043] Example 2: Pre-activation of lymphocytes

[0044]Configure NK / NKT cell culture medium: cell culture medium (Daktronics, product number DKW37-NKP001, main components: amino acids, glucose, folic acid, phenol red, etc.), add cell activator (Daktronics, product number DKW37-NKP001) in proportion , medium 1L: cell activator 1ml), and autologous serum (Example 1) was added at the same time to configure a medium containing 5% autologous serum, which was filtered before use.

[0045] Add PBS to the lymphocytes (Example 1) and mix well, centrifuge at 1800 rpm for 10 minutes, collect the precipitate, resuspend the buffy coat cells with the prepared NK / NKT cell culture medium, and count them. About 5ml of fresh peripheral blood can obtain a total of 6.2 ×10 6 leukocyte.

[0046] The above white blood cells 6.2×10 6 Divided into two parts, one part added 2ml 5% autologous serum medium, the other part added 2ml 5% autologous serum medium containing 4-1BB-L factor, the cell c...

Embodiment 3

[0047] Embodiment 3: measure the concentration of NK cells and NKT cells in peripheral blood

[0048] Take the above 1×10 5 White blood cells, add 50ul PBS to mix, add 1ul Antibody-human CD45 (APC-Cy7), Antibody-CD56 (PE-CY7), Antibody-CD16 (PE) (purchased from BD Company) and mix, and incubate at room temperature for 30 minutes, Wash once with PBS, resuspend in 300ul PBS, and detect the proportion of various cells by flow cytometry.

[0049] Take the above 1×10 5 White blood cells, add 50ul PBS to mix, add 1ul Antibody-human CD45 (APC-Cy7), Antibody-CD33 (Percp-cy5.5), Antibody-CD3 (APC), Antibody-CD19 (FITC) (purchased from BD Company) Mix well, incubate at room temperature for 30 minutes, wash once with PBS, resuspend in 300ul PBS, and detect the proportion of various cells by flow cytometry.

[0050] Table 1 Various types of cells in 5ml peripheral blood

[0051]

[0052] Remarks: CD45 is a marker on the surface of white blood cells, CD56 / CD16 is a marker on the s...

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Abstract

The invention discloses a method for efficient in-vitro amplification and culture of natural killer T cells with high killing capacity. The method comprises the following steps: a, taking fresh human peripheral blood and obtaining mononuclear cells with a gradient centrifugation method; b, pre-stimulating mononuclear cells in an in vitro medium; c, expanding culture of peripheral blood derived mononuclear cells, detecting the proportion of various cells, and judging amplification efficiency and activity of the target NK/NKT cells; d, detecting the proportion of NK/NKT cells to the cell population after culture for 3-4 weeks, and detecting cell killing efficiency; e, further purifying the NK/NKT cells. The method is simple to operate, the NK cell population rich in NKT cells with high killing activity is obtained and contains no impurity cells, immunogenicity caused by the impurity cells is avoided, and side effects in clinical application are lower. Besides, the method has the advantages that materials are easily available, the cells are suitable for large-scale culture, damage to the body is small and the like, and is more suitable for adjuvant treatment of tumor patients.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for efficiently expanding and culturing natural killer T cells with high lethality in vitro. Background technique [0002] Tumor immunotherapy is a new type of tumor treatment mode. It uses cytokine induction, antigen induction, genetic modification and other methods to activate immune cells, and through the activation of these cells, the purpose of tumor treatment is achieved. Tumor immunotherapy has achieved great results in the treatment of malignant tumors, and has become the fourth tumor treatment method after surgery, radiotherapy and chemotherapy. Since the 1990s, with the in-depth understanding of the body's immune system, tumor immunotherapy has made rapid progress, such as immune detection antibodies, tumor vaccines and chimeric antigen receptors (chimeric antigen receptor, CAR), etc. It has a good application prospect. The core of tumor immunotherapy is...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/17A61P35/00A61P35/02
CPCC12N5/0646A61K35/17A61P35/00A61P35/02C12N2501/599
Inventor 汪晓敏袁胜男袁卫平
Owner INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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