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Treatment method for de-proliferation ability of feeder cells for NK cell culture

A feeder cell and NK cell technology, which is applied in the field of feeder cell deproliferation treatment, can solve the problems of affecting NK cell expansion effect, incomplete cell shape, and high test cost, so as to achieve convenient promotion and use, low cost, and high lethality active effect

Inactive Publication Date: 2020-07-17
WEIHAI CENT HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, radiation can completely inactivate, but it requires high equipment and high test costs. At the same time, the shape after radiation inactivation is different from that before treatment; while the method of repeated freezing and thawing has low requirements for test equipment, but the inactivation is not complete and needs to be repeated. Multiple operations may result in incomplete cell morphology, which will affect the expansion effect of stimulating NK cells

Method used

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  • Treatment method for de-proliferation ability of feeder cells for NK cell culture
  • Treatment method for de-proliferation ability of feeder cells for NK cell culture
  • Treatment method for de-proliferation ability of feeder cells for NK cell culture

Examples

Experimental program
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Effect test

Embodiment 1

[0021] Example 1 Mitomycin C treatment method for feeder cells

[0022] First add appropriate amount of DMSO to dissolve mitomycin C so that the concentration is 33 mg / ml, that is, prepare mitomycin C stock solution, aliquot and store at -20°C for later use. 1200rpm, 5min centrifugation to collect 41BBL-mbIL-21-K562 feeder cells, and use RPMI1640 medium to dilute the feeder cells to a concentration of 1x10 7 cells / ml of cell suspension. Add mitomycin C to the cell suspension to a final concentration of 33 μg / ml at 37 °C in 5% CO 2 After being treated in the dark for 90 minutes, the cells were collected at 1200 rpm for 5 minutes, and washed 4 times with DPBS to remove residual mitomycin C as much as possible to avoid toxicity to the cultured cells. The treated feeder cells can be frozen in cell freezing medium for future use, or directly used for NK cell expansion or other experiments.

[0023] In order to verify the proliferative ability of the feeder cells treated by the m...

Embodiment 2

[0028] Embodiment 2 NK cells are cultured after 14 days feeder cell retention situation

[0029] The feeder cells used are K562 cells, and the ratio of feeder cells can be detected by flow cytometry with Anti-41BBL PE antibody (R&D product number FAB2295P).

[0030] 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, 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; Suck out the white PBMC ...

Embodiment 3

[0034] Example 3 Use of Treated Cells to Cultivate NK Cells

[0035] 1. Isolation of PBMCs

[0036] 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; Suck out the white PBMC layer in the middle after centrifugation with a Sider pipette, add about 30ml of PBS to a new 50ml centrifuge tube to wash, 1500rpm, 8min, wash with culture medium again, and count.

...

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Abstract

The invention provides a treatment method for the de-proliferation ability of feeder cells for NK cell culture, and uses mitomycin C to process the feeder cells. The de-proliferation operation methodof the feeder cells provided by the invention has low costs, and is simple to operate and convenient to promote and use; the cell morphology of the feeder cells processed by the method remains intact,the expansion of NK cells can be better stimulated, so that the prepared NK cells have very high purity and killing activity, through detection, when the cells are cultured for 14 days, the purity ofthe NK cells reaches 94% or more, and the amplification multiple reaches 720 or more; when the effector-target ratio is 5:1, the killing activity of the NK cells on K562 cells is 60% or more; and thefeeder cells treated by the method are almost all killed by the NK cells on the fourth day of culture without post-residue residues, and the method can be applied and promoted in clinical research.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a method for treating the deproliferation ability of feeder cells used for NK cell culture. Background technique [0002] NK cells are non-specific immune cells. They can directly kill certain tumors and virus-infected target cells without antigen pre-sensitization, and can also secrete a variety of cytokines and chemokines to participate in immune regulation. Therefore, it plays an important role in the immune process of the body's anti-tumor and early anti-virus or intracellular parasitic infection. NK immune cell therapy has broad application prospects in antiviral and antitumor treatments. In recent years, the clinical use of NK cells for immunotherapy has been increasing. [0003] At present, feeder cells using gene recombination, such as K562 cells stably expressing 41BBBL and membrane-expressing IL-21 factors (41BBL-mbIL-21-K562 cells), are widely used f...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2502/30
Inventor 张道强刘传杰宫琪葛淑娟
Owner WEIHAI CENT HOSPITAL
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