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High-efficiency proliferation reagent for peripheral blood NK (Natural killer) cells in vitro and operation instruction

A technology of NK cells and operating instructions, applied in the field of cell culture, can solve the problems of ineffective removal of tumor cells and small lesions, poor clinical effect of tumors, and increased proportion of tumor recurrence, and achieve good targeted killing of tumor cells and proliferation. The effect of fast speed and high killing activity

Inactive Publication Date: 2019-03-19
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

Although conventional treatment methods can effectively reduce the tumor burden in a short period of time, the clinical effect of the tumor is poor and the proportion of tumor recurrence increases because of the inability to effectively remove tumor cells and small lesions, drug resistance caused by chemotherapy, and tumor metastasis caused by radiotherapy.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 1. Isolation of PBMCs

[0028] 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; use Bath Suck out the middle white PBMC layer after centrifugation with a German pipette, add about 30ml of PBS to a new 50ml centrifuge tube to wash, 1500rpm, 8min, wash again with medium, and count.

[0029] 2. Induction and expansion of NK cells

[0030] (1) The tot...

Embodiment 2

[0038] 1. Isolation of PBMCs

[0039] Collect 45ml 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; use Bath Suck out the middle white PBMC layer after centrifugation with a German pipette, add about 30ml of PBS to a new 50ml centrifuge tube to wash, 1500rpm, 8min, wash once with medium, and count.

[0040] 3. Induction and expansion of NK cells

[0041] (1) The tota...

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Abstract

The invention relates to the technical field of cell culture, and discloses a high-efficiency proliferation reagent for peripheral blood NK (Natural killer) cells in vitro and an operation instruction. The reagent comprises the following components: an NK cell proliferation reagent: a gamma-ray inactivated recombinant K562 feeder cell, a CD52 antibody, an IL-2, a CD16 antibody, a CD3 antibody anda CD56 antibody; the gamma-ray inactivated recombinant K562 feeder cell is a K562 cell line for membrane expression of 41BBL and IL-15 which is lethally radiated by a 100G gamma-ray. According to thehigh-efficiency proliferation reagent for the peripheral blood NK cells in vitro and the operation instruction, the prepared NK cells have high purity and killing activity. Through detection, when theNK cells are cultured for 14 days, the purity of the NK cells can reach more than 90%; when the target ratio is 10:1, the killing activity of the NK cells on K562 cells can reach 80% or more; the proliferation speed of the prepared NK cells is high; when the NK cells are cultured for 14-17 days, the cell proliferation rate is more than 500; the NK cells prepared by the invention have excellent effects for targeted killing of tumor cells.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a peripheral blood NK cell high-efficiency expansion reagent in vitro and operation instructions. Background technique [0002] NK cells, natural killer cells, were discovered in 1975 and belong to non-phagocytic lymphocytes. The cytoplasm is rich in perforin and granzymes, and has the function of recognizing and lysing tumor cells. At present, cancer has become the number one cause of death in the world. Conventional means of cancer treatment are: surgery, radiotherapy and chemotherapy three ways. Although conventional treatment methods can effectively reduce the tumor burden in a short period of time, the clinical effect of the tumor is poor and the proportion of tumor recurrence increases because of the inability to effectively remove tumor cells and small lesions, drug resistance caused by chemotherapy, and tumor metastasis caused by radiotherapy. . Immune cell therapy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2501/2302C12N2501/515C12N2501/599C12N2502/30
Inventor 葛淑娟张道强刘传杰王金环徐矫健王玉娟刘燕丽
Owner 青岛麦迪赛斯生物科技有限公司
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