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Peripheral blood-based LCL-NK cell co-culture method, cell and product

A combined culture and peripheral blood technology, applied in the direction of cell culture active agents, blood/immune system cells, animal cells, etc., can solve the problems of unsatisfactory clinical effect, insufficient NK cell number and killing activity, etc.

Active Publication Date: 2021-05-28
JIANGSU MOBILI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In hematologic malignancies, NK cell infusion is mainly used in clinical research for the treatment of leukemia and lymphoma, but the clinical effect of NK cell adoptive immunotherapy is often unsatisfactory. The possible important reason is that the number and killing activity of NK cells are insufficient

Method used

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  • Peripheral blood-based LCL-NK cell co-culture method, cell and product
  • Peripheral blood-based LCL-NK cell co-culture method, cell and product
  • Peripheral blood-based LCL-NK cell co-culture method, cell and product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] A kind of specific implementation of the scheme of the present invention comprises the following steps:

[0029] PBMC Peripheral Blood Mononuclear Cell Isolation

[0030] Take 5ml of human peripheral blood and put it in a 10ml centrifuge tube, then add the same amount of PBS and mix well. Take another 10ml centrifuge tube and add 5ml of lymphocyte separation medium. Use a dropper to slowly drop the above-mentioned mixture of peripheral blood and PBS along the tube wall onto the liquid surface of the lymphocyte separation medium, and be careful not to damage the layered liquid surface. Centrifuge horizontally at 800g for 20min. After centrifugation, the centrifuge tube was separated into 3 layers. Use an aspirator to draw the white cloud layer between the upper and middle layer interface into a new centrifuge tube, try not to bring other layers of cells. Add more than 5 times of PBS to this centrifuge tube, centrifuge at 300g for 5min, and discard the supernatant. R...

Embodiment 2

[0053] The difference between this example and Example 1 is that in the preparation of LCL cells, the method of radiation inactivation is used for radiation inactivation, and the radiation dose is 40Gy.

[0054]In this example, compared with groups A and B, the proportion of NK cells cultured by this culture method was significantly increased to about 55±5.2%, and the amplification factor of NK cells was about 1000±58 times. After culture, NK cells still expressed a high level of CD16; while the inhibitory receptors (CD158a and CD158b) on the surface of NK cells did not change significantly (P>0.05), while the activating receptors included NCRs (NKp30, NKp44 and NKp46) and NKG2D was significantly increased (P<0.05). The killing ability to K562 was significantly increased (p<0.01). Similarly, compared with the cytotoxicity of NK cells cultured with IL-2 alone, it was found that the killing ability of NK cells cultured by this experimental method to K562 was also significantly ...

Embodiment 3

[0056] The difference between this example and Example 1 is that in the preparation of LCL cells, the method of radiation inactivation is used for radiation inactivation, and the radiation dose is 50Gy.

[0057] In this example, compared with groups A and B, the proportion of NK cells cultured by this culture method was significantly increased to about 82±6.2%), and the amplification factor of NK cells was about 700±45 times. After culture, NK cells still expressed a high level of CD16; while the inhibitory receptors (CD158a and CD158b) on the surface of NK cells did not change significantly (P>0.05), while the activating receptors included NCRs (NKp30, NKp44 and NKp46) and NKG2D was significantly increased (P<0.05). The killing ability to K562 was significantly increased (p<0.01). Similarly, compared with the cytotoxicity of NK cells cultured with IL-2 alone, it was found that the killing ability of NK cells cultured by this experimental method to K562 was also significantly...

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Abstract

The invention provides a peripheral blood-based LCL-NK cell co-culture method, whichcomprises the following steps: preparing and obtaining the PBMC from the peripheral blood, and preparing the LCL cells from the PBMC, wherein the LCL cells are obtained by culturing PBMC (peripheral blood mononuclear cells) in supernate containing EBV (electron barr virus); the EBV supernate is obtained by carrying out culture passage on B958 cells, then carrying out starvation lysis and then separating; and co-culturing the cells in a culture system formed by PBMC, LCL cells, cell factors and a culture solution. According to the culture method provided by the scheme, the purity of a cell product is effectively improved, and the culture efficiency, the NK cell killing property and the like are remarkably improved.

Description

technical field [0001] The invention belongs to the technical field of biology and biomedicine, and in particular relates to a peripheral blood-based LCL-NK cell joint culture method, cells and products. Background technique [0002] The immune response protects the body from pathogens, and the immune system is composed of numerous immune-related cells and cytokines. White blood cells, especially lymphocytes, play an important role in the immune system. Representative cells constituting lymphocytes include cells of the innate immune system and cells of the acquired immune system. Natural killer cells (NK cells) are a representative innate immune cell. It is known that natural killer cells can kill tumors in a non-specific way, recognize and kill viruses, bacteria, etc., and can kill with enzymes such as perforin and granzyme Pathogens, or kill pathogens through Fas-FasL interaction. According to reports, for tumor patients, the reduction of the ability of NK cells to kill...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078C12N5/0783C12N13/00
CPCC12N5/0634C12N5/0646C12N13/00C12N2500/70C12N2501/2302C12N2501/2315C12N2501/2321C12N2501/26C12N2501/125C12N2501/2307C12N2501/2312C12N2501/2318C12N2502/1192
Inventor 沈健欧阳效晴葛永杨淑青
Owner JIANGSU MOBILI BIOTECH CO LTD
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