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High-purity allogeneic NK cell culture medium and in-vitro amplification method

An allogeneic and NK cell technology, applied in cell dissociation methods, cell culture active agents, and culture processes, can solve the problems of poor killing activity and low purity of NK cells, and achieve low cost, high-purity amplification, Effects in simple steps

Active Publication Date: 2021-04-09
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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  • High-purity allogeneic NK cell culture medium and in-vitro amplification method
  • High-purity allogeneic NK cell culture medium and in-vitro amplification method
  • High-purity allogeneic NK cell culture medium and in-vitro amplification method

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Experimental program
Comparison scheme
Effect test

Embodiment

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

[0047]1. Separation and plasma preparation of peripheral blood peripheral blood monocytes (PBMC) of peripheral blod Mononuclear Cell, PBMC

[0048]1.1. Transfer 30ml placenta blood sample to T75 bottles. The sample is a voluntary donation and signing the corresponding informed consent books, the entire process meets ethical requirements.

[0049]1.2. With a 50 ml centrifuge tube, 15 ml of human lymphocyte isolation is added to the bottom of the centrifuge tube.

[0050]1.3. On 15 ml of human lymphocyte separation, 15 to 30 ml of blood sample was slowly added with a pipette.

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

[0052]1.5. After the centrifugation, the plasma layer was sued into the plasma layer without disturbing the white film layer, and transferred to 50 ml of centrifuge tube.

[0053]1.6. Separate the white film layer (PBMC layer) and transferred to another 50 ml of centrifuge tube. ...

experiment example 1

[0073]Experimental Example 1. NK Cell Purity Detection

[0074]1. Cell staining: 2 μl of cell suspension added 2 μl of CD56 and CD3 antibody, slightly oscillation, and retaining for 20 minutes.

[0075]2. Upload detection: Add 400 μL of PBS and mix, the color scheme is CD56 (APC-CY7-A), CD3 (FITC).

[0076]3. Flow detection, reasonable door and acquisition with BD FacScanto II streaming cytometry and BD FACSDIVA software>5 × 104The number of cells was collected.

[0077]Flow cell test results show that the purity of NK cells cultured in the process of the present invention can reach 98.5%, which is significantly better than that of the prior art cultured NK cell purity of 38.9%. seefigure 1 .

experiment example 2

[0078]Experimental example 2. NK cell killing ability Experiment

[0079]Resuscitation target cell:

[0080]The recovery of K562 cells were used to cultivate 1640 + 10% FBS, and the cells were observed after 2 to 3 days. If the cellular state of the cells was carried out (the total number of K562 required to confirm the maximum number of target cells).

[0081]2. Confirm the number of target cells

[0082]2.. Preparation of target cell suspension: blowing K562 cells into uniform cell suspension, count, using target cell culture medium to adjust cell suspension density to 2 × 106One / ml, based on this, dilution, prepare 1 × 106One / ml, 0.5 × 106One / ml, 0.25 × 106One / ml, 0.125 × 106Suspension of a / ml;

[0083]2.2. Take 100 μL of target cell suspension, in a 96-well plate, the target cell dilution is prepared, and each concentration is 3 wells; the cells are cracked with a lysate. 250g centrifugal plate for 4 minutes. 50 μl of the supernatant was transferred to the enzyme analysis plate, and ...

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Abstract

The invention discloses a high-purity allogeneic NK cell culture medium and an in-vitro amplification method. Mononuclear cells are separated from placenta blood through a lymphocyte separation liquid, and the separated mononuclear cells are added into a serum-free immune cell culture medium system containing a CD314 antibody, IL-2, inactivated autologous plasma and the like to be cultured for 3 days; and 10% inactivated autologous plasma and 1000 IU / mL IL-2 are added once on the fourth day and the fifth day of culture respectively for culture, massive amplification of cells can be seen after continuous culture is performed for 7 days, continuous culture is performed for 2 days, and addition factors are supplemented as required. A culture bottle does not need to be coated in advance, feeder layer cells do not need to be used, the expression rate of the prepared NK cells CD3-CD16+ / CD56+ reaches up to 90% or above after 14-16 days of culture, the in-vitro tumor killing activity is high, and the problems that in an existing NK culture system and technology, the purity of the NK cells is low, and the killing activity is poor can be solved.

Description

Technical field[0001]The present invention belongs to the field of NK cell culture techniques, and relates to a high purity allogeneic NK cell culture medium and in vitro amplification method.Background technique[0002]Natural Killer Cell, NK cells) is an important member of the natural immune system, from bone marrow lymphoid stem cells, mainly distributed in bone marrow, peripheral blood, liver, spleen, lung and lymph nodes, and rapidly generate effect cytokines and The ability to kill viral infections or tumor cells. NK cells do not need pre-sensitizing to kill tumor cells and viral infection cells, which can identify and lyse tumor cells by rapid activation of a series of NK activation receptors. NK cells are fast and do not require tumor-specific identification, and have a broad-spectrum anti-tumor effect, which is not required to inhibit active limitation (MHC) inhibitory activity. Thus, allogeneic NK cells play an important role in tumor adverse sexual immunotherapy. However, ...

Claims

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Application Information

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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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