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A high-purity allogeneic nk cell culture medium and in vitro expansion method

A technology for NK cells and in vitro expansion, applied in cell dissociation methods, cell culture active agents, and culture processes, etc., can solve the problems of poor killing activity and low purity of NK cells, and achieve low cost, high purity, and simple steps Effect

Active Publication Date: 2022-07-29
圣至润合(北京)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

It can solve the problems of low purity and poor killing activity of NK cells in the existing NK culture system and technology

Method used

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  • A high-purity allogeneic nk cell culture medium and in vitro expansion method
  • A high-purity allogeneic nk cell culture medium and in vitro expansion method
  • A high-purity allogeneic nk cell culture medium and in vitro expansion method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0046] The main implementation steps of this embodiment are as follows:

[0047] 1. Isolation of peripheral blood mononuclear cells (PBMC) from placental blood and preparation of plasma

[0048] 1.1. Transfer the collected 30mL placental blood sample to a T75 bottle. The sample was donated voluntarily by volunteers who signed the corresponding informed consent form, and the whole process complied with ethical requirements.

[0049] 1.2. Take another 50mL centrifuge tube and add 15mL of human lymphocyte separation solution to the bottom of the centrifuge tube with a pipette.

[0050]1.3. On the 15mL human lymphocyte separation medium, slowly add 15~30mL blood sample with a pipette.

[0051] 1.4. Centrifuge in a horizontal rotor centrifuge, 4°C, 800g, 20min.

[0052] 1.5. After centrifugation, without disturbing the buffy coat layer, aspirate the plasma layer and transfer it to a 50mL centrifuge tube.

[0053] 1.6. Aspirate the buffy coat layer (PBMC layer) and transfer it t...

experiment example 1

[0073] Experimental example 1. Detection of NK cell purity

[0074] 1. Cell staining: Add 2 μL of CD56 and CD3 antibodies to 100 μL of cell suspension, respectively, shake and mix, and store in the dark for 20 minutes.

[0075] 2. On-machine detection: add 400 μL of PBS and mix well, and then on-board detection, the color scheme is CD56 (APC-Cy7-A), CD3 (FITC).

[0076] 3. Use BD FACSCanto II flow cytometer and BD FACSDiva software for flow detection, set gates reasonably and obtain >5×10 4 The number of cells was collected and analyzed.

[0077] The results of flow cytometry show that the purity of the NK cells cultured by the method of the present invention can reach 98.5%, which is obviously better than that of the NK cells cultured by the prior art, which is 38.9% pure. See figure 1 .

experiment example 2

[0078] Experimental example 2. Experimental scheme of NK cell killing ability

[0079] 1. Resuscitate target cells:

[0080] Resuscitate a K562 cell, culture it with 1640+10% FBS, and observe the cells after 2-3 days of culture. If the cells are in good condition, carry out the target cell number confirmation experiment (the total number of K562 required should be determined after confirming the optimal target cell number).

[0081] 2. Confirm the number of target cells

[0082] 2.1. Preparation of target cell suspension: pipetting K562 cells into a uniform cell suspension, count, and use target cell culture medium to adjust the density of the cell suspension to 2 × 10 6 pcs / ml, make equal dilutions on this basis to prepare 1×10 6 pcs / ml, 0.5×10 6 pcs / ml, 0.25×10 6 pcs / ml, 0.125×10 6 cells / ml of cell suspension;

[0083] 2.2. Take 100 μL of the target cell suspension and add it to a 96-well plate to prepare target cell dilution, 3 wells per concentration; split the cells...

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Abstract

The invention discloses a high-purity allogeneic NK cell culture medium and an in vitro expansion method. Mononuclear cells were isolated from placental blood by lymphocyte separation medium, and the isolated mononuclear cells were added to a serum-free immune cell culture medium system containing CD314 antibody, IL-2, inactivated autologous plasma, etc. for 3 days; 10% inactivated autologous plasma and 1000IU / mL IL-2 were added once on the 4th and 5th days, respectively. After 7 days of continuous culture, a large number of cells were seen to expand, and the culture was continued for 2 days, and supplementary factors were added as needed. The present invention does not need to pre-coat the culture flask, and does not need to use feeder layer cells. After 14-16 days of culture, the prepared NK cell CD3-CD16+ / CD56+ cells have an expression rate of more than 90%, and the in vitro antitumor activity is strong, which can solve the problem of current In the NK culture system and technology, there are problems of low purity and poor killing activity of NK cells.

Description

technical field [0001] The invention belongs to the technical field of NK cell culture, and relates to a high-purity allogeneic NK cell culture medium and an in vitro amplification method. Background technique [0002] Natural killer cells (NK cells) are important members of the innate immune system. They are derived from bone marrow lymphoid stem cells and are mainly distributed in bone marrow, peripheral blood, liver, spleen, lung and lymph nodes. They have the ability to rapidly produce effector cytokines and The ability to kill viral infections or tumor cells. NK cells can non-specifically kill tumor cells and virus-infected cells without pre-sensitization, and they can recognize and lyse tumor cells by rapidly activating a series of NK-activating receptors. NK cells have a rapid onset of action, do not require tumor-specific recognition, are not limited by the major histocompatibility complex (MHC) inhibitory activity on the cell surface, and have broad-spectrum anti-t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2500/90C12N2501/59C12N2501/599C12N2500/72C12N2501/2302C12N2501/2315C12N2501/2318C12N2509/00
Inventor 张旭辉胡向军李胜华修冰水徐绍梅
Owner 圣至润合(北京)生物科技有限公司
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