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NK cell in-vitro amplification system and culture method

An in vitro expansion and NK cell technology, which is applied in the direction of cell culture active agent, cell culture support/coating, animal cells, etc., can solve the problems of difficult quality control of product stability and high economic cost of NK cell culture medium, and achieve activation The effect of fewer components, less manpower, and simplified culture methods

Pending Publication Date: 2021-08-24
江苏豪科生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This combination of multi-component culture medium first led to the high economic cost of the NK cell culture medium; secondly, it was difficult to control the stability and quality of its products due to the large number of components; thirdly, the activating medium added OK432, and OK432 is an indispensable and necessary ingredient

Method used

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  • NK cell in-vitro amplification system and culture method
  • NK cell in-vitro amplification system and culture method
  • NK cell in-vitro amplification system and culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Preparation method of anti-CD52 immunomagnetic beads

[0070] 1. Place the tube containing the magnetic microspheres on the vortex shaker, turn on the instrument, shake, and mix the magnetic microspheres thoroughly.

[0071] 2. Pipette 100 μl of magnetic microspheres into a sterile EP tube.

[0072] 3. Add 100 μl of pH7.4 PBS buffer to the EP tube, and vortex to resuspend. The EP tube was placed in the magnet, and stood for 30 Sec, so that the magnetic beads were adsorbed on the magnet side of the EP tube. Aspirate the supernatant. PBS was added and the washing was repeated 3 times. Go to supernatant.

[0073] 4. Add 100 μl of pH7.4 PBS buffer and 30 μl of purified Anti-CD52 (IgG, 10-30ug) to the EP tube.

[0074] 5. Mix well and incubate at 4°C for 30 minutes with stirring.

[0075] 6. Place the EP tube in the magnet, and after the magnetic beads are completely adsorbed to one side of the magnet, remove the supernatant. Additional PBS was added and the...

Embodiment 2

[0084] Example 2 Anti-CD52 Immunomagnetic Bead Amplification, Culture and Flow Analysis of NK Cells

[0085] 1. Preparation of PBMC cells. Peripheral blood was collected from healthy volunteers, anticoagulated with heparin, and human peripheral blood mononuclear cells (PBMC) were separated by Ficoll density centrifugation.

[0086] 2. NK cell culture. PBMCs were adjusted to 2 × 10 in serum-free medium (i.e. Lonza X-VIVO 15 serum-free medium). 6 cells / ml, the anti-CD52 immunomagnetic beads (4×10) prepared in Example 1 were added to Lonza X-VIVO 15 serum-free medium. 5 cells / ml), anti-CD3 antibody (1ng / ml) and IL-2 (200U / ml) to form an activation medium, and cultured at 37°C for 48 hours in 5% CO2.

[0087] 3. According to the cell growth state, change the medium every 2-3 days with the proliferation medium consisting of 200U / ml IL-2 and basal medium (i.e. Lonza X-VIVO 15 serum-free medium) until the 14th day. sky.

[0088] 4, flow cytometry detection. On day 14 of cell cult...

Embodiment 3

[0093] Example 3 Comparison of expansion efficiency of NK cells cultured with different Anti-CD52 combinations

[0094] 1. The peripheral blood of 2 healthy volunteers (Volunteer 1 and Volunteer 2) were collected respectively, anticoagulated with heparin, and human peripheral blood mononuclear cells (PBMC) were separated by Ficoll density centrifugation.

[0095] 2. Use serum-free medium (i.e. Lonza X-VIVO 15 serum-free medium) for PBMC to adjust the cell concentration to 2×10 6 cells / ml.

[0096] 3. Culture of NK cells.

[0097] Culture method 1. According to Example 2, anti-CD52 and anti-CD3 were pre-coated and activated in combination with IL-2 (200U / ml) to culture PBMC to induce NK cell differentiation and expansion.

[0098] Cultivation method 2, adopts the activation medium and the proliferation medium of the present invention to cultivate:

[0099] The activation medium consists of an activator and a basal medium (ie, Lonza X-VIVO 15 serum-free medium), and the activ...

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Abstract

The invention provides an NK cell in-vitro amplification system. The NK cell in-vitro amplification system comprises an activation culture medium for cell activation and a proliferation culture medium for cell proliferation, the activating culture medium is composed of an activating agent and a basic culture medium, and the activating agent is a combination of solid-phase anti-CD52 and IL-2; the proliferation culture medium is composed of a proliferation agent and a basic culture medium, and the proliferation agent is IL-2. Compared with the prior art, the NK cell in-vitro amplification system disclosed by the invention has the following technical effects: 1) the culture medium contains the immobilized anti-CD52, and the NK cells can be efficiently stimulated to enter an activated and proliferated state by combining the use of IL-2, so that a large number of activated NK cells can be easily obtained; moreover, no trophoblasts are used in the culture process, and the prepared NK cells can be used for clinical research and treatment; and 2) the NK cell culture method is simplified, few NK cell activation components are used, few material resources and manpower are used, the production cost of NK cell preparation is obviously reduced, and the method is particularly suitable for large-scale production.

Description

technical field [0001] The invention relates to a high-efficiency expansion system and a culture method of NK cells in vitro, and belongs to the field of biotechnology. Background technique [0002] NK cells (natural killer, NK) are a kind of natural immune cells with killing function, their phenotype is CD3-CD56+, and their proportion in peripheral blood is relatively low, about 5-15%. After activation, NK cells can efficiently kill tumor cells non-antigen-specifically by secreting effector molecules such as granzyme and perforin. In recent years, with the promotion and application of cell therapy technology in clinic, NK cells have been paid more attention in tumor cell immunotherapy because of their non-antigen-specific killing characteristics. NK cells can not only be used directly as tumoricidal effector cells themselves, but also as carrier cells for CAR-NK cells. In the in vitro expansion and preparation of NK cells, bone marrow cells rich in hematopoietic stem cell...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2501/2302C12N2501/599C12N2500/90C12N2533/50
Inventor 袁正隆
Owner 江苏豪科生物工程有限公司
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