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In-vitro culture method for T lymphocytes

A lymphocyte, in vitro culture technology, applied in animal cells, vertebrate cells, blood/immune system cells, etc., can solve the problems of inconvenient use, high cost, unfavorable medical research and clinical application, etc. Reduced requirements for transport conditions and the effect of reducing accidental infections

Active Publication Date: 2012-05-02
LIAONING MEDI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This culture method also uses two mitogens, phytohemagglutinin and anti-CD3 monoclonal antibody, to jointly activate the cells. Although it can enhance the stimulation intensity of the cells, this method needs to be completed with the help of a bioreactor, which is not only costly, but also inconvenient to use. And it is not conducive to medical research and clinical application

Method used

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  • In-vitro culture method for T lymphocytes
  • In-vitro culture method for T lymphocytes
  • In-vitro culture method for T lymphocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Human peripheral blood is now used as a processed sample, and the contents of the kit of the present invention are used to sort and induce CIK cells, and apply it to clinical biological treatment. The specific operation steps are as follows:

[0039] step one. blood processing

[0040] 1. Pour 50ml of anticoagulated blood into a 50ml centrifuge tube and centrifuge at 1600~2000×g for 10min at 4°C. If it cannot be centrifuged immediately, it can be stored at 4°C, but not more than 4 hours.

[0041] 2. Use a pipette to transfer the upper layer of plasma into a new 50ml centrifuge tube, and mark the patient information. Inactivate at 56°C for 30min.

[0042] 3. Dilute the blood cells with normal saline to 50ml, and mix with a pipette. The above is the process of diluting blood cells.

[0043] 4. Slowly pour the diluted blood cells into each tube of 7ml cell sorting medium at a ratio of 1:1. Be careful not to break the interface.

[0044] 5. Centrifuge at 2200~2500×g...

Embodiment 2

[0102] The invention can also be used for the in vitro induction and expansion of T lymphocytes in human umbilical cord blood. The specific operation steps are as follows:

[0103] step one. blood processing

[0104] 1. Inject 80ml of anticoagulated umbilical cord blood into four 50ml centrifuge tubes, and centrifuge at 1600~2000×g for 10min at 4°C. If it cannot be centrifuged immediately, it can be stored at 4°C, but not more than 4 hours.

[0105] 2. Discard the upper plasma layer.

[0106] 3. Dilute 4 tubes of blood cells with normal saline to 50ml, and mix with a pipette. The above is the process of diluting blood cells.

[0107] 4. Slowly pour the diluted blood cells into each tube of 7ml cell sorting medium at a ratio of 1:1. Be careful not to break the interface.

[0108] 5. Centrifuge at 2200~2500×g for 20min at 20°C. Pipette the second layer of cells into a new 50ml centrifuge tube.

[0109] 6. Add physiological saline to the newly collected cells to 50ml, an...

Embodiment 3

[0128] The invention can also be used for the in vitro induction and expansion of T lymphocytes in human pleural fluid or ascites. The specific operation steps are as follows:

[0129] step one. Take 1000ml of pleural fluid or ascites, and centrifuge at 1600-2000×g for 4-5min at 4°C.

[0130] Step two. Discard the supernatant, flick the bottom of the centrifuge tube to break up the precipitate.

[0131] Step three. Rinse the cells 3 times with saline.

[0132] Step four. Take the coated bottle, rinse it once with normal saline or culture medium and discard it.

[0133] Step five. Take 25ml of culture medium to resuspend the cells, mix thoroughly with a pipette and transfer them into the coating bottle.

[0134] Step six. Rinse the centrifuge tube with 25ml of medium, and transfer the rinsing solution into the coating bottle again.

[0135] Step seven. Place the flask in 5% CO 2 , cultured in a 37°C incubator.

[0136] Subsequent steps are the same as in Example 1 ...

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Abstract

The invention discloses an in-vitro culture method for T lymphocytes, and radically solves the problems of high cost, inconvenience of use and unfavorable medical research and clinical application existing in the conventional in-vitro culture method for the T lymphocytes. The method comprises the following steps of: directionally sorting the T lymphocytes, inducing, differentiating, culturing, amplifying and the like. A special cell factor combination is used for performing induction differentiation on the T lymphocytes; three factors are a CD3 monoclonal antibody, gamma-interferon and interleukin-1 alpha respectively; the optimal concentration ratio of the three factors is 10:1:1; the total final concentration of the three factors in a culture medium is 1,200ng / ml; and the experiment shows that the combination is an extremely effective way of inducing the T lymphocytes to differentiate.

Description

technical field [0001] The present invention relates to a method for treating mononuclear cells in human peripheral blood, umbilical cord blood, pleural effusion, ascites and other samples to become cytokine-induced killer cells (Cytokine-Induced Killer, referred to as CIK), especially a method for The method for culturing T lymphocytes in vitro can be applied to clinical cell biotherapy, basic medical research, clinical application research and biotechnology research, especially immune system research and tumor killing effect research. Background technique [0002] As tumors pose an increasingly serious threat to human survival and health, the treatment models for tumors are also changing rapidly. Various new drugs, new technologies, and new methods emerge in an endless stream. Among them, cell biology therapy is developed after surgery, radiotherapy, and chemotherapy. The fourth type of tumor treatment method has become an important development direction in tumor biologica...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
Inventor 李文欣
Owner LIAONING MEDI BIOTECH CO LTD
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