A kind of NK cell with enhanced killing activity and preparation method thereof

A technology of NK cells and killing activity, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve the problems of NK cells unable to maintain proliferation, low purity of NK cells, complicated procedures, etc., to achieve proliferation effect and cost Inexpensive, enhanced lethality effects

Active Publication Date: 2021-03-26
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In general, however, NK cells are unable to sustain proliferation, so their proliferative responses to cytokines are modest and transient, whether they are co-cultured with other cells or not
[0006] Expansion-based methods ar...

Method used

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  • A kind of NK cell with enhanced killing activity and preparation method thereof
  • A kind of NK cell with enhanced killing activity and preparation method thereof
  • A kind of NK cell with enhanced killing activity and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Preparation of NK cells

[0048] 1. Collection of peripheral blood specimens: 120ml of peripheral blood was collected in a sodium heparin anticoagulant blood bag or an anticoagulant tube, and the patient’s consent was obtained.

[0049] 2. Preparation of autologous plasma: Transfer the peripheral blood in the anticoagulant blood bag to a 50ml centrifuge tube, centrifuge at 800g for 10 minutes, absorb the light yellow liquid in the upper layer, which is the autologous plasma, transfer it to a new centrifuge tube, and place it at 56 Water bath in a water bath for 30 minutes to inactivate, after inactivation, 1200G, 10min, put in a refrigerator at 4 degrees for standby, add 10% plasma each time as a medium additive.

[0050] 3. Separation of mononuclear cells: take physiological saline for use, add human lymphocyte separation medium Ficoll (density 1.077) in advance to two 50mL sterile centrifuge tubes, 15mL per tube. Use normal saline to reduce the sample after...

Embodiment 2

[0058] Characterization of embodiment 2 NK cells

[0059] The obtained cells were collected, the number of obtained cells and the viability were calculated, and the purity of NK cells (CD3-CD56+) was analyzed by flow cytometry as shown in the figure and the expression of activating receptors.

[0060] Antibodies conjugated to cells with appropriate concentrations of fluorescent dyes and CD3 (clone No. UCHT1), CD56 (clone No. B159), NKG2D (clone No. 149810), NKp30 (clone No. ) and NKp46 (clone number 9E2), all antibodies were purchased from BD (Franklin Lakes, NJ, USA) and the expression of NK-activating receptors was determined by flow cytometry on the combination of the above antibodies.

[0061] The experimental results are shown in Figures 2 to 4 middle.

Embodiment 3

[0062] Example 3 Killing rate of NK cells on human chronic myeloid leukemia cells K562 cells as target cells

[0063] NK cells (effector cells: E) and K562 (target cells: T) were mixed according to different ratios of E to T (E:T). K562 target cells were labeled with 1 ng / ml fluorescent dye CFSE (invitrogen) in PBS at 37°C for 15 min, calf serum was added to the cells within 15 min, and then the cells were washed and resuspended in RPMI medium. 100ul of K562 cells according to 5X10 3 Cells / well were placed in a 96-well round-bottom well plate. 100uL of unstained activated NK cells (experimental group) and unactivated NK cells (control group) were mixed according to the E:T ratio of 5:1, 2.5:1, 1:1 (respectively 2.5X10 4 , 1.25X10 4 , 5X10 3 cells / well) were added to K562 cells. After 2 to 28 h, the killing effect of target cells K562 in the cell line was determined by FACS (Fluorescent Flow Cytometry) as the percentage of propidium iodide (PI)-positive (dead) CFSE-labeled...

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Abstract

The invention relates to NK cells with enhanced killing activity and a preparation method thereof. The method includes the steps that a, feeder cells are prepared from peripheral blood mononuclear cells PBMCs; b, NK cell proliferation is conducted, wherein in the step a, the PBMCs are primarily stimulated with monoclonal antibodies CD3 and continue to be activated in a first culture medium containing IL-2, IFN-r, IL-15 and IL-18, primarily activated PBMCs with antigen presentation are obtained and irradiated with gamma rays to be used as the feeder cells, and part of the feeder cells are subjected to freeze preservation with liquid nitrogen; in the step b, CD3+T lymphocytes are removed from the original PBMCs not activated through an immunomagnetic bead sorting method, NK cells after CD56+are gathered again selectively, the NK cells and the feeder cells not subjected to freeze preservation with liquid nitrogen are subjected mixed hatching, NK cell proliferation culturing is conductedin a second culture medium containing IL-2, IL-15, OK432 and nicotinamide, then the NK cells are transferred into a third culture medium containing IL-2 and nicotinamide to be subjected to proliferation culturing, and finally the feeder cells which are thawed from liquid nitrogen freeze preservation are added to continue proliferation culturing.

Description

technical field [0001] The invention relates to an NK cell with enhanced killing activity and a preparation method thereof, belonging to the field of biotechnology. Background technique [0002] Human natural killer (natural killer, NK) cells rely on the interaction between their own surface activating receptors and tumor cell surface ligands, and can recognize MHC-I without being restricted by the major histocompatibility complex (MHC) Tumor cells that do not express or down-regulate the expression of similar molecules and effectively remove and kill them. The unique anti-tumor immune function of NK cells has received extensive attention, and has been increasingly used in adoptive immunotherapy of tumors. Since NK cells account for a very small proportion (5%-10%) in PBMC, and NK cells isolated by conventional methods are in an inactivated state, an effective method for in vitro activation and expansion is to develop NK cells. The foundation and key of immunotherapy. [...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12N5/078C12N13/00A61K35/17A61P35/00
CPCA61K35/17A61P35/00C12N5/0634C12N5/0646C12N13/00C12N2500/38C12N2501/06C12N2501/2302C12N2501/2315C12N2501/2318C12N2501/24C12N2501/515C12N2502/11
Inventor 方维佳彭富强许震宇郑怡涂晓璇谢信飞
Owner ZHEJIANG UNIV
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