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In vitro culture kit for T-lymphocyte cells

A lymphocyte, in vitro culture technology, applied in the field of in vitro culture kits for T lymphocytes, can solve the problems of unfavorable medical research and clinical application, high cost, inconvenient use, etc. The effect of easy quality control

Active Publication Date: 2012-06-27
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 kit for T-lymphocyte cells
  • In vitro culture kit for T-lymphocyte cells
  • In vitro culture kit for T-lymphocyte cells

Examples

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preparation example Construction

[0044] The preparation and source of kit content of the present invention are as follows:

[0045] 1. Preparation of coated bottles

[0046] Take CD3 monoclonal antibody, γ-interferon, and interleukin-1α and add them to 40ml of phosphate buffer. The optimal concentration ratio of CD3 monoclonal antibody, γ-interferon, and interleukin-1α is 10 :1:1, make a mixed solution with a total concentration of 1200ng / ml, and mix well; add the mixed solution into a 600ml culture bottle, and let it stand at 4°C for at least 24 hours to make an initial coating bottle; Freeze the bottle at -40°C for 24 hours; start the compressor of the freeze dryer to reduce the temperature in the machine chamber to -40°C, unscrew the mouth of the coated bottle and put it in the drying room; turn on the vacuum pump to make Pressure reached 2 Intake valve, close the vacuum pump; close the CO after the pressure reaches 110K 2 Inlet valve, take out the coated bottle, tighten the cap, seal the mouth, store at...

Embodiment 1

[0054] 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:

[0055] step one. blood processing

[0056] 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.

[0057] 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.

[0058] 3. Dilute the blood cells with normal saline to 50ml, and mix with a pipette. The above is the process of diluting blood cells.

[0059] 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.

[0060] 5. Centrifuge at 2200~2500×g...

Embodiment 2

[0118] 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:

[0119] step one. blood processing

[0120] 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.

[0121] 2. Discard the upper plasma layer.

[0122] 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.

[0123] 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.

[0124] 5. Centrifuge at 2200~2500×g for 20min at 20°C. Pipette the second layer of cells into a new 50ml centrifuge tube.

[0125] 6. Add physiological saline to the newly collected cells to 50ml, an...

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Abstract

The invention relates to an in vitro culture kit for T-lymphocyte cells, which fundamentally solves the problems of existing T-lymphocyte cell in vitro culture methods that the used materials and culture time are inconsistent, and the culture periods, quality and quantity of effective cells are different. The technical key points are as follows: the content comprises a coating bottle for inducting the differentiation of the T-lymphocyte cells; a CD3 monoclonal antibody, gamma- interferon and interleukin-1 alpha are pre-coated in the coating bottle; the component of a cell factor A, a cell factor B and a cell factor A for stimulating the division and proliferation of the T-lymphocyte cells is 200000 units of interleukin-2, and the component of the cell factor B is 2 million units of interleukin-2. According to the in vitro culture kit for the T-lymphocyte cells, the composite reagent and materials required for the in vitro culture of the T-lymphocyte cells are combined together in a standard way, all the operation is standardized and modularized, so that not only is the working efficiency improved, but also the cell quality and yield difference caused by the operation of different people is reduced.

Description

technical field [0001] The present invention relates to a kit for treating mononuclear cells in human peripheral blood, umbilical cord blood, pleural effusion, ascites and other samples into cytokine-induced killer cells (Cytokine-Induced Killer, referred to as CIK), especially a In vitro culture kit for T lymphocytes, the kit can be used in clinical cell biotherapy, basic medical research, clinical application research and biotechnology research, especially in immune system research and tumor killing 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 biological treatment. Cancer is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M3/00C12N5/0783
Inventor 李文欣
Owner LIAONING MEDI BIOTECH CO LTD
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