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Breast cancer targeted Car-NK cell preparation method

A breast cancer cell technology, applied in the field of biomedicine, can solve problems such as easy shedding, loss of breast cancer cells, and loose cell connections

Inactive Publication Date: 2017-10-20
ANHUI HUIEN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The mammary gland is not an important organ to maintain human life activities, breast cancer in situ is not fatal; but because breast cancer cells lose the characteristics of normal cells, the connections between cells are loose and easy to fall off

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] S1. Collect the peripheral enriched blood of the patient, filter out the white blood cells, slowly transfer it into a centrifuge tube filled with lymphocyte separation fluid, and centrifuge it by density gradient centrifugation. , to obtain peripheral blood mononuclear cells;

[0024] S2. Wash the peripheral blood mononuclear cells obtained in S1 with PBS, discard the supernatant, take the cell pellet and add GT551 to resuspend, and adjust the cell concentration to 2x10 6 ;

[0025] S3. Take 90 mL of the resuspension in S2, add 10 mL of inactivated autologous serum and IL-210 ng / mL, inoculate into six-well culture plate, add 0.9% sodium chloride injection and Lymactin- NK, wherein the volume ratio of sodium chloride injection and Lymactin-NK is 4:1, the final volume of liquid in each well is 3mL, placed at 37°C, 5% CO 2 Cultivate in a saturated humidity incubator;

[0026] After S4 was cultured for 36-48 hours, add final concentrations of 800IU / ml IL-2, 100IU / ml IL-1...

Embodiment 2

[0029] S1. Collect the peripheral enriched blood of the patient, filter out the white blood cells, slowly transfer it into a centrifuge tube filled with lymphocyte separation fluid, and centrifuge it by density gradient centrifugation. , to obtain peripheral blood mononuclear cells;

[0030] S2. Wash the peripheral blood mononuclear cells obtained in S1 with PBS, discard the supernatant, take the cell pellet and add GT551 to resuspend, adjust the cell concentration to 2-2.5x10 6 ;

[0031] S3. Take 90 mL of the resuspension in S2, add 10 mL of inactivated autologous serum and IL-210 ng / mL, inoculate into six-well culture plate, add 0.9% sodium chloride injection and Lymactin- NK, wherein the volume ratio of sodium chloride injection and Lymactin-NK is 4:1, the final volume of liquid in each well is 3mL, placed at 37°C, 5% CO 2 Cultivate in a saturated humidity incubator;

[0032] After S4 was cultured for 36-48 hours, add final concentrations of 1200IU / ml IL-2, 200IU / ml IL-...

Embodiment 3

[0035] S1. Collect the peripheral enriched blood of the patient, filter out the white blood cells, slowly transfer it into a centrifuge tube filled with lymphocyte separation medium, and centrifuge with density gradient centrifugation. The centrifugation parameters are 2100r / min, 23°C, 19min, and absorb the buffy coat , to obtain peripheral blood mononuclear cells;

[0036] S2. Wash the peripheral blood mononuclear cells obtained in S1 with PBS, discard the supernatant, take the cell pellet and add GT551 to resuspend, and adjust the cell concentration to 2.2x10 6 ;

[0037] S3. Take 90 mL of the resuspension in S2, add 10 mL of inactivated autologous serum and IL-210 ng / mL, inoculate into six-well culture plate, add 0.9% sodium chloride injection and Lymactin- NK, wherein the volume ratio of sodium chloride injection and Lymactin-NK is 4:1, the final volume of liquid in each well is 3mL, placed at 37°C, 5% CO 2 Cultivate in a saturated humidity incubator;

[0038] After S4 ...

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Abstract

The invention discloses a method for preparing Car-NK cells for breast cancer. The method comprises the following steps: collecting peripheral enriched blood from a patient, filtering out leukocytes, slowly moving it into a centrifuge tube containing lymphocyte separation liquid, and using a density gradient Centrifuge, absorb the buffy coat, and obtain peripheral blood mononuclear cells; take 90 mL of the resuspension, add inactivated autologous serum 10 mL and IL-2 10 ng / mL, inoculate into six-well culture plates, and in six-well culture plates Add 0.9% sodium chloride injection and Lymactin-NK; after culturing for 36-48h, add IL-2, IL-15 and IL-21 to the culture plate at a final concentration; transfect and amplify NK cells with lentivirus cultured to obtain CAR-NK cells. The invention discloses a preparation method of Car-NK cells, which comprises separating nuclear cells from peripheral blood of breast cancer patients by Ficoll method, and amplifying NK cells in vitro through IL-2+IL-15+IL-21 combined culture scheme, The preparation method is convenient and easy to operate, and the obtained Car-NK cells can be injected into the patient's body through the method of preparing cell preparations, which is of great help to the treatment of breast cancer.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to a method for preparing Car-NK cells for breast cancer. Background technique [0002] The female mammary gland is composed of skin, fibrous tissue, mammary glands and fat. Breast cancer is a malignant tumor that occurs in the mammary gland epithelial tissue. 99% of breast cancers occur in women and only 1% in men. [0003] The mammary gland is not an important organ to maintain human life activities, and breast cancer in situ is not fatal; but because breast cancer cells lose the characteristics of normal cells, the connections between cells are loose and easy to fall off. Once the cancer cells fall off, the free cancer cells can spread throughout the body with the blood or lymph fluid, forming metastasis, which is life-threatening. At present, breast cancer has become a common tumor that threatens women's physical and mental health. [0004] The global incidence of breast cancer has...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N5/0783
CPCC12N5/0646C07K14/7051C12N2501/2302C12N2501/2315C12N2501/2321C12N2510/00
Inventor 张正亮
Owner ANHUI HUIEN BIOTECH
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