Method for stimulating NK cell expansion through combination of inactivated feeder cells and cell factors

A technology of feeding cells and cytokines, which is applied in the field of cell culture, can solve the problems of incomplete inactivation, high test cost, and high requirements, and achieve the effect of targeting and killing tumor cells, high killing activity, and fast proliferation

Active Publication Date: 2019-04-05
青岛麦迪赛斯生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Radiation can completely inactivate, but it has high requirements for equipment and high test cost; the method of repeated freezing and thawing has low requirements for test equipment, but the inactivation is not complete, and repeated operations are required, and it is difficult to maintain the integrity of cell morphology. Affect NK stimulation effect

Method used

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  • Method for stimulating NK cell expansion through combination of inactivated feeder cells and cell factors
  • Method for stimulating NK cell expansion through combination of inactivated feeder cells and cell factors

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Experimental program
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Effect test

Embodiment 1

[0024] 1 Isolation of PBMCs: Collect 50ml of peripheral blood from tumor patients, anticoagulate with heparin, and centrifuge whole blood at 700g for 20min. The upper layer is the plasma layer, the middle layer is the buffy coat layer, and the lower layer is red blood cells. The plasma layer was collected, inactivated at 56°C for 30min, then allowed to stand at 4°C for 15min, 1800rpm / 800g, 4°C for 25min, and the supernatant was used as autologous plasma for later use. Collect the middle buffy coat layer, add it to 20ml PBS, mix well, then slowly add the centrifuge tube that has been added with lymphatic separation solution at a ratio of 4:3, 1800rpm / 800g, room temperature, 20min, no-break, wash PBMC; After centrifugation, suck out the white PBMC layer in the middle with a Sider pipette, add about 30ml of PBS to a new 50ml centrifuge tube to wash, 1500rpm, 8min, wash once with medium, and count.

[0025] 2 Induction and expansion of NK cells: (1) The total amount of cells in s...

Embodiment 2

[0031] 1 Isolation of PBMCs

[0032]Collect 50ml of peripheral blood from tumor patients, anticoagulate with heparin, and centrifuge whole blood at 700g for 20min. The upper layer is the plasma layer, the middle layer is the buffy coat layer, and the lower layer is red blood cells. The plasma layer was collected, inactivated at 56°C for 30min, then allowed to stand at 4°C for 15min, 1800rpm / 800g, 4°C for 25min, and the supernatant was used as autologous plasma for later use. Collect the middle buffy coat layer, add it to 20ml PBS, mix well, then slowly add the centrifuge tube that has been added with lymphatic separation solution at a ratio of 4:3, 1800rpm / 800g, room temperature, 20min, no-break, wash PBMC; After centrifugation, suck out the white PBMC layer in the middle with a Sider pipette, add about 30ml of PBS to a new 50ml centrifuge tube to wash, 1500rpm, 8min, wash once with medium, and count.

[0033] 2 Induction and expansion of NK cells

[0034] (1) The total amo...

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Abstract

The invention aims to provide a novel method for stimulating NK cell expansion through combination of inactivated reconstituted K562 cells and cell factors. The NK cells prepared by the method provided by the invention have high purity and killing activity, and tests show that the purity of the NK cells is 90% or above when the NK cells are cultured for 14 days; and the killing activity of the NKcells on the K562 cells is 80% or above when the effector-target ratio is 10 to 1. The NK cells prepared by the invention have a rapid proliferation rate, and the cell expansion multiple is up to 500or above after 14-17 days of culture. The NK cells prepared by the invention have a good targeted killing effect on tumor cells.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a method for inactivating feeder cells combined with cytokines to stimulate NK cell (Natural killer cells) expansion. Background technique [0002] Currently, cancer has become the leading cause of death worldwide. Conventional means of cancer treatment are: surgery, radiotherapy and chemotherapy three ways. Immune cell therapy is the fourth new way to treat tumors after surgery, chemotherapy and radiotherapy. It can systematically kill tumor cells, effectively inhibit the metastasis of tumor cells, have few side effects, and have good clinical effects. [0003] There are DC cells, CIK cells, DC-CIK cells and NK cells used in clinical immune cell therapy. NK cells are the third type of lymphocytes. Different from T / B lymphocytes, they can exert cytotoxic effect without antigen stimulation, can spontaneously dissolve a variety of tumor cells and cells infected b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2502/30C12N2501/515C12N2501/599C12N2501/2302
Inventor 葛淑娟徐矫健刘燕丽王玉娟
Owner 青岛麦迪赛斯生物科技有限公司
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