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Autologous NK cells and culture method and application thereof

A culture method and technology for NK cells, which can be applied to cell culture active agents, animal cells, and culture processes, etc., can solve the problems of unsatisfactory autologous NK cytotoxicity, low culture expansion times and purity, and unsatisfactory tumor treatment effect. To achieve the effect of strong tumor killing activity, high purity, maintaining activity and proliferation ability

Inactive Publication Date: 2016-08-17
SHENZHEN RUI XIANG YUAN SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art, and provide a kind of autologous NK cell and its culture method, to solve existing autologous NK cell existence toxicity is not ideal, and its cultivation has low amplification factor and purity, high cost etc. question
[0007] Another object of the present invention is to provide a cell therapy tumor drug, so as to overcome the unsatisfactory toxicity of other existing cell therapy drugs and the low amplification factor and tumorigenic risk in the culture method, resulting in unsatisfactory tumor treatment effect and high cost. technical problem

Method used

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  • Autologous NK cells and culture method and application thereof

Examples

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Effect test

Embodiment 1

[0055] This example provides a method for in vitro preparation of autologous NK cells with high cytotoxic activity and autologous NK cells cultured by the method. The autologous NK cell body culture method comprises the following steps:

[0056] S11. Antibody coating:

[0057] Use PBS to prepare a mixed coating solution containing 40 μg / ml anti-human HER2 monoclonal antibody and 20 μg / ml fibronectin, coat the T-75 culture bottle, put it in the refrigerator at 4°C overnight, and wash the culture bottle twice with PBS the next day to wait use;

[0058] S12. PMBC preparation:

[0059] Collect 50-100ml of peripheral blood with sterile sodium heparin blood collection tubes, transfer the peripheral blood to a centrifuge tube; centrifuge to obtain the upper layer of plasma, inactivate at 56°C and centrifuge again, take the upper layer of serum, and store it in a 4°C refrigerator for later use; centrifuge the lower layer of plasma Add 0.9% normal saline to make up to the whole bloo...

Embodiment 2

[0066] The autologous NK cell body culture method (without adding fibronectin) comprises the following steps:

[0067] S11. Antibody coating:

[0068] Prepare a T-75 culture flask containing 40 μg / ml anti-human HER2 monoclonal antibody coated with PBS, put it in a refrigerator at 4°C overnight, and wash the culture flask twice with PBS the next day for use;

[0069] S12. PMBC preparation:

[0070]Collect 50-100ml of peripheral blood with sterile sodium heparin blood collection tubes, transfer the peripheral blood to a centrifuge tube; centrifuge to obtain the upper layer of plasma, inactivate at 56°C and centrifuge again, take the upper layer of serum, and store it in a 4°C refrigerator for later use; centrifuge the lower layer of plasma Add 0.9% normal saline to make up to the whole blood volume, mix well and slowly add to the human lymphatic separation fluid, centrifuge at 2000rpm for 20min, take the buffy coat cells, wash twice and count to get PBMC;

[0071] S13. Inducti...

Embodiment 3

[0077] The autologous NK cell body culture method (without adding autologous serum) comprises the following steps:

[0078] S11. Antibody coating:

[0079] Use PBS to prepare a mixed coating solution containing 40 μg / ml anti-human HER2 monoclonal antibody and 20 μg / ml fibronectin, coat the T-75 culture bottle, put it in the refrigerator at 4°C overnight, and wash the culture bottle twice with PBS the next day to wait use;

[0080] S12. PMBC preparation:

[0081] Collect 50-100ml of peripheral blood with sterile sodium heparin blood collection tubes, transfer the peripheral blood to a centrifuge tube; centrifuge to obtain the upper layer of plasma, inactivate at 56°C and centrifuge again, take the upper layer of serum, and store it in a 4°C refrigerator for later use; centrifuge the lower layer of plasma Add 0.9% normal saline to make up to the whole blood volume, mix well and slowly add to the human lymphatic separation fluid, centrifuge at 2000rpm for 20min, take the buffy ...

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Abstract

The invention discloses autologous NK cells and a culture method and application thereof. The culture method of the autologous NK cells comprises the following steps of collecting peripheral blood mononuclear cells of a patient, combining the mononuclear cells, anti-HER2 monoclonal antibodies or HER2 monoclonal antibodies and fibronectin for use and adding cell factors into a serum-free medium for induction and multiplication culture. According to the culture method of the autologous NK cells, the anti-HER2 monoclonal antibodies and the fibronectin are adopted for improving the irritating activity of irritation for inducing an individual cell; the cell factors are adopted for inducing the autologous NK cells, the synergistic effect is generated on multiplication of the NK cells by means of combined application of the cell factors, the killing activity of the autologous NK cells is improved, and toxic and side effects caused by applying a large dosage of single factors are reduced. In addition, by means of the culture method, the culture time is shortened, the culture cost is reduced, and the purity of the autologous NK cells is guaranteed.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an autologous NK cell, a culture method thereof and an application thereof. Background technique [0002] In the early 1970s, when studying the specific killing of target cells by T cells, NCI researchers found that the splenocytes of normal control mice could kill some tumor cells like the splenocytes of immunized mice, and then found that normal human peripheral blood lymphocytes could also kill certain tumor cells. Can naturally kill certain cancer cells. The killing function of these cells does not require prior immunization or sensitization, so they are named natural killer cells (NKCells). The discovery of NK cells caused a sensation in the world. Scholars then proposed that NK cells are the main bearer of the body's In-nate Immunity, and found that NK cells are not only effective against cancer cells, but also against viruses, intracellular parasites and aging mut...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/17A61P35/00
CPCC12N5/0646A61K35/17C12N2500/84C12N2501/2302C12N2501/2315C12N2501/2318C12N2501/998
Inventor 张文张诺琳陈伟雄
Owner SHENZHEN RUI XIANG YUAN SCI & TECH CO LTD
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