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High cytotoxic CIK (cytokine induced killer) cell preparation and culture method thereof

A technology of cytotoxicity and culture method, which is applied in the field of tumor treatment, and can solve problems such as impaired immune function, weakened immune cell function, and low ability of CIK cells to kill tumors

Inactive Publication Date: 2018-08-17
龙口南山养生谷肿瘤医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the impaired immune function of tumor patients and the weakened immune cell function, the CIK cells prepared by the existing technology have low ability to kill tumors, which affects the efficacy of CIK

Method used

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  • High cytotoxic CIK (cytokine induced killer) cell preparation and culture method thereof
  • High cytotoxic CIK (cytokine induced killer) cell preparation and culture method thereof

Examples

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preparation example Construction

[0021] Further, the preparation method of autologous plasma comprises: placing the plasma layer at 50-60° C. for 25-35 minutes, then standing at 0-10° C. for 10-20 minutes, and centrifuging to obtain the supernatant. More preferably, the plasma layer is placed at 54-58° C. for 28-32 minutes, then at 2-6° C. for 18-22 minutes, and centrifuged to obtain the supernatant.

[0022] Step S2: adjusting the cell concentration of peripheral blood mononuclear cells with lymphocyte culture medium, adding IL-2, IFN-γ and autologous plasma, and placing them in a culture bottle for culturing.

[0023] Further, the cell concentration of peripheral blood mononuclear cells is 0.5×10 5 ~0.5×10 7 individual / mL.

[0024] Further, in the culture flask, the final concentration of IL-2 is 800-1200 IU / mL, and the final concentration of IFN-γ is 800-1200 IU / mL.

[0025] Further, in the culture bottle, the final concentration of autologous plasma is 0.5-1.5%.

[0026] More preferably, the culture s...

Embodiment 1

[0034] This embodiment provides a highly cytotoxic CIK cell preparation, the culture method comprising:

[0035] (1). Blood collection: take 100mL of peripheral blood through the transfer window (EDTA anticoagulant is not allowed), spray alcohol and put it into a safety cabinet, mix the collected peripheral blood and add it to a 50mL sterile centrifuge tube, at room temperature, under 700g Centrifuge for 20 minutes, the upper layer A is the plasma layer (accounting for about 50%), and the lower layer B is the Buffy coat+erythrocyte layer.

[0036] (2). Preparation of autologous plasma: put the plasma layer in a new 50ml tube, place it at 56°C for 30min, and let it stand at 4°C for 15min; then, centrifuge at 800g, 4°C for 25min, take out the supernatant and put it in 50mL centrifuge tube as autologous plasma.

[0037] (3). Peripheral blood mononuclear cell isolation:

[0038] a. Mix the lower layer B (i.e. Buffy coat + erythrocyte layer) with D-PBS evenly, and the total volum...

Embodiment 2

[0046] This example provides a highly cytotoxic CIK cell preparation, the culture method of which is basically the same as that of Example 1, the difference lies in the in vitro expansion culture process:

[0047] (1). Transfer bag:

[0048] a. On the 4th day of culture, observe the cell morphology, viability and contamination under a microscope;

[0049]b. Subsequently, collect the cell suspension in the culture bottle into a centrifuge tube, dilute it 4 times with PBS and count. Maintain 1×10 by cell concentration 6 Units / mL to calculate the amount of fluid replacement, IL-2 and autologous plasma.

[0050] c. Add a fixed amount of GT-T551H3 medium into a 50mL centrifuge tube, add 500 units / ml of IL-2 (V / 2μl) and 0.5% autologous plasma (10 times the amount of IL-2) according to the total volume of the culture system ), mix well.

[0051] d. Take the GT-T610A culture bag, unscrew the long tube, wipe the mouth with an alcohol cotton ball, connect it to a 60ml syringe, pour ...

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Abstract

The invention provides a high cytotoxic CIK (cytokine induced killer) cell preparation and a culture method thereof, and relates to the field of tumor treatment. The culture method comprises: centrifuging anticoagulated peripheral blood, removing a plasma layer, mixing with a lymphocyte separation solution, and separating peripheral blood mononuclear cells; adjusting the cell concentration of theperipheral blood mononuclear cells with a lymphocyte culture medium, adding IL-2, IFN-gamma and autologous plasma, and culturing in a culture flask; performing expansion in vitro for culture, and centrifugally collecting the obtained CIK cells. Through the culture method, the cell culture time can be shortened, the cell activity of the obtained CIK cell preparation is more than 95 percent, the ownimmunity of a patient can be enhanced after reinfusion, and the anti-tumor effect is increased.

Description

technical field [0001] The invention relates to the field of tumor treatment, in particular to a highly cytotoxic CIK cell preparation and a culture method thereof. Background technique [0002] As one of the largest public health problems in the world, cancer has greatly endangered human health and has become the number one killer of human beings. Biological immunotherapy is currently an emerging treatment method. It mainly expands and cultures immune cells in vitro, and then infuses them back into the body to enhance the immunity of patients. This kind of biological immunotherapy has strong cancer-killing power, wide cancer-killing spectrum, and kills the remaining cancer cells. It does not damage normal cells, and can restore the damage caused by surgery, improve the sensitivity of chemotherapy, reduce side effects, and restore physical strength. Relieve symptoms and prolong the survival of cancer patients. [0003] In recent years, adoptive immune cell therapy, such as...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0638C12N2500/84C12N2501/2302C12N2501/24
Inventor 王立平王炜玮曲迅李家敏王方聚张明徽刘君周武松田明一吕春明隋丽战美娜韩佳佳宁应梅
Owner 龙口南山养生谷肿瘤医院
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