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In-vitro IAK immune cell culture method

A culture method and technology of immune cells, applied in the field of tumor prevention and treatment, can solve the problems of limited ability to kill tumor cells and low proportion of NKT cells, and achieve the effect of improving cell killing ability and good tumor killing effect.

Active Publication Date: 2017-03-08
THE FIRST AFFILIATED HOSPITAL OF ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, in the CIK cells prepared by the existing culture methods, the proportion of NKT cells (effector cells) is relatively low, generally around 10-30%, and the ability to kill tumor cells is relatively limited. Therefore, it is necessary to further optimize the culture of CIK cells Method to increase the proportion and tumor killing ability of immune effector cells

Method used

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Examples

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Effect test

Embodiment 1

[0055] Using the in vitro IAK immune cell culture method provided by the present invention, the inventor first carried out a small sample experiment, and the specific experimental process is as follows: figure 1 As shown, the details are as follows.

[0056] (1) Extract mononuclear cells, specifically:

[0057] Collect 5 mL of peripheral blood from tumor patients; centrifuge at 1500 rpm / min for 10 min, and store the supernatant at -20°C after inactivation;

[0058] The precipitated cells were resuspended in 30 mL of normal saline, placed on 15 mL of lymphocyte separation medium, subjected to density gradient centrifugation, and mononuclear cells were collected and washed three times with normal saline;

[0059] The density gradient centrifugation was performed at 2500 rpm / min for 25 min.

[0060] (2) In vitro cell culture, the culture process is as follows: figure 1 As shown, specifically:

[0061] Day 0: The peripheral blood mononuclear cells obtained in step (1) were pla...

Embodiment 2

[0080] On the basis of the small sample in Example 1, the inventor further carried out a large sample experiment. The specific process is as follows:

[0081] (1) Extract peripheral blood mononuclear cells, specifically:

[0082] Collect 60 mL of peripheral blood from tumor patients; centrifuge at 1500 rpm / min for 10 min, and store the supernatant at -20°C after inactivation;

[0083] The precipitated cells were diluted to 50 mL with normal saline, placed on four 15 mL lymphocyte separation medium respectively, subjected to density gradient centrifugation, and mononuclear cells were collected and washed three times with normal saline;

[0084] The density gradient centrifugation was performed at 2500 rpm / min for 25 min.

[0085] (2) In vitro cell culture, specifically:

[0086] The lymphocytes extracted in the above (1) were divided into two parts, one part was cultured with IAK cells, and the culture process was the same as in Example 1; the other part was cultured with tr...

Embodiment 3

[0092] The specific process of the in vitro IAK immune cell culture method provided in this example is the same as that in Example 1, except that the amount of 7DW8-5 in reagent B has been appropriately adjusted, and the specific experimental conditions are as follows.

[0093] (1) Extract mononuclear cells, as described in Example 1.

[0094] (2) In vitro cell culture, specifically:

[0095] Divide the lymphocytes extracted in the above (1) into three parts:

[0096] One of them was cultured with IAK cells, and the culture process was the same as in Example 1 (named IAK1);

[0097] When one of them was cultured with IAK cells (named IAK2), the culture procedure was the same as that in Example 1, but the amount of reagent B added was halved, that is, the amount of reagent B added was 5 μL / mL;

[0098] The last part was cultured with traditional CIK cells, and the culture process was the same as that described in Example 1.

[0099] Finally, the cells are collected, and the im...

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Abstract

The invention relates to the technical field of tumor preventing and treating, in particular to an optimized in-vitro IAK immune cell culture method. The method includes: extracting peripheral blood mononuclear cells, adding into specially designed reagent A, reagent B, reagent C and reagent D step by step and in batches according to culture time differences, culturing until the specific time is reached, and the like. The in-vitro IAK immune cell culture method has the advantages that the method can keep consistency or well improved in terms of indexes such as cell proliferation ability, effector cell phenotype and proportion and killing ability and especially effector cell proportion and killing ability are improved evidently by optimizing an IAK immune cell in-vitro culture system, and accordingly the method is well applicable to tumor treating, good in practical value and worthy of popularization and application.

Description

technical field [0001] The invention belongs to the technical field of tumor prevention and treatment, and in particular relates to an optimized in vitro IAK (Induced activated killer cell, induced activated killer cell) immune cell culture method. Background technique [0002] Autologous cell immunotherapy is to extract immature immune cells from tumor patients through blood collection, activate and cultivate them in the laboratory so that they have the ability to efficiently recognize and kill tumor cells, and then reinfuse them into tumor patients. [0003] At present, the culture method of in vitro cytokine-induced killer cell (CIK) immune cells is a kind of autologous cell immunotherapy, which is used to treat a broad spectrum of tumor patients. The technical principle of CIK therapy is: a group of heterogeneous cells obtained after co-cultivating human peripheral blood mononuclear cells with various cytokines (such as anti-CD3 monoclonal antibody, IL-2 and IFN-γ, etc.)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2501/22C12N2501/2302C12N2501/24C12N2501/515
Inventor 杨黎张毅
Owner THE FIRST AFFILIATED HOSPITAL OF ZHENGZHOU UNIV
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