Natural killer cell and cultivation method thereof

A technology of natural killer cells and cells, applied in the field of natural killer cells and their culture, can solve the problems of technical difficulty in separating and purifying NK cells, NK cells cannot be widely used in clinical practice, and it is difficult to avoid T lymphocyte contamination, etc., and achieve fast cell growth. , the effect of low cost and wide application value

Active Publication Date: 2011-04-06
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, because NK cells derived from peripheral blood are difficult to expand in large quantities in vitro, and there are certain technical difficulties in isolating and purifying NK cells from peripheral blood, it is difficult to avoid contamination of T lymphocytes, and it is impossible to obtain a large number of uniform NK cells and reach clinical requirements. Therefore, NK cells cannot be widely used clinically; in addition, in some cases, NK cell-mediated anti-tumor response is weak, which may be related to NK cell killing ability, poor survival in vivo or limited migration to tumor sites. and other factors

Method used

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  • Natural killer cell and cultivation method thereof
  • Natural killer cell and cultivation method thereof
  • Natural killer cell and cultivation method thereof

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Experimental program
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Embodiment 1

[0036] The establishment of embodiment 1, NKG cell line

[0037] 1. Cell isolation

[0038] 1. Peripheral blood sample: Take 10ml of peripheral blood from a patient with non-Hodgkin's lymphoma, anticoagulate with heparin, and obtain the consent of the patient.

[0039] 2. Separation of peripheral blood mononuclear cells: After diluting the anticoagulated peripheral blood specimen with 10ml sterile PBS solution, carry out density gradient centrifugation with lymphocyte separation medium, 2200 rpm, 30 minutes, 20 degrees; For mononuclear cells, wash 3 times with PBS solution, 1500 rpm, 10 minutes, 20 degrees; use 250 μl sterile PBS solution to resuspend cells, count, and the cell concentration is 1.1×10 7 / 250μl, store at 4 degrees, and set aside.

[0040] 3. Separation and purification of NK cells: Add 20 μl of non-specific mouse IgG to the above cell solution at 4 degrees for 30 minutes to block non-specific binding. Add 100 μl CD56 MicroBeads to the cell solution, shake an...

Embodiment 2

[0047] Embodiment 2, the preparation of the culture method of NKG cell and NKG cell injection

[0048] 1. Exploration of training conditions

[0049] Inactivation of newborn bovine serum and horse serum: Place newborn bovine serum and horse serum free of bacteria, mycoplasma and virus contamination in a constant temperature water bath at 56°C for 30 minutes to inactivate complement, then store in a refrigerator at 4°C for later use.

[0050] Prepare the following media:

[0051] 1) Preparation of complete α-MEM liquid medium

[0052] Get α-MEM liquid culture medium (containing L-glutamine, sodium bicarbonate, ribonucleoside and deoxyribonucleoside) 800ml, add the following substances respectively, and the final concentration of this substance in the culture medium is in brackets: newborn cow Serum (10%, v / v), horse serum (10%, v / v), human interleukin 2 (1.0×10 5 IU / L).

[0053] 2) Preparation of complete α-MEM dry powder medium

[0054] Take 1 bag of α-MEM powder (10.2g, ...

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Abstract

The invention discloses a natural killer cell and a culturing method thereof. The natural killer cell is a human natural killer cell NKG cell of which the preservation number is CGMCC No. 2901. The NKG cell has strong lethality to tumor cells, strong survival ability in vivo, stable quality and strong tumor-resistant reaction. Cells cultured by the method have fast growing speed, large quantity, even quality, high and stable survival rate. The preparation method has easy operation, controllable quality, high yield and low cost, and realizes mass, scale, homogeneous and streamlined production for cell treating products like medicament. The method for culturing NKG cells in a large scale can ensure cell phenotype, purity, activity, cell density, culturing volume and the like to achieve clinical treatment and ensure that the NKG cells can be applied to clinical treatment for malignant tumors and injection for 1 to 5 patients at the same time. Therefore, the cells, the method for preparing cells and the preparation method for cell liquid preparation have wide application value in treating malignant tumors.

Description

technical field [0001] The invention relates to a natural killer cell and a culture method thereof. Background technique [0002] Cellular immunotherapy for tumor treatment entered the clinic earlier than other cell therapy schemes. In the schemes that have been tried, LAK cells, CIK cells, and A-NK cells are all anti-cancer systems dominated by NK cells. This is because natural Killer cells (natural killer, NK) are the main members of innate immunity, and can play an important role in the first line of defense against diseases including malignant tumors. In contrast to cells of the adaptive immune system such as T cells, NK cells can initiate cytotoxic activity without prior stimulation or sensitization, thereby providing an immediate natural response. The advantage of NK cells in killing tumors is manifested in two aspects: direct dissolution and secretion of cytokines, which can kill tumors through both perforin and FAS ligand. NK cells can produce TNF-α, IFN-γ and IL-1...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12N5/0783
Inventor 田志刚程民魏海明孙汭
Owner UNIV OF SCI & TECH OF CHINA
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