In-vitro pure culture method for natural killer cells

A technology of natural killer cells and pure culture, applied in the field of pure culture of natural killer cells in vitro, can solve problems such as high safety risks, and achieve the effects of high amplification multiples, high safety, and clear medium components

Active Publication Date: 2019-04-23
IREGENE THERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this method, since the initial PBMC cells are a cell mixture with NK cell content of only 5-15%, contamination of other types of cells cannot be avoided during the subsequent culture process. Generally, the content of NK cells in the final culture is

Method used

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  • In-vitro pure culture method for natural killer cells
  • In-vitro pure culture method for natural killer cells
  • In-vitro pure culture method for natural killer cells

Examples

Experimental program
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Embodiment 1

[0050] Please combine figure 1 , the present embodiment provides a method for in vitro pure culture of natural killer cells, comprising the following steps:

[0051] S1, collecting peripheral blood and separating PBMC:

[0052] 50 mL of peripheral blood was drawn from a venous vessel and placed in a sodium heparin anticoagulant blood collection tube. Subsequently, the peripheral blood was transferred to a 50mL centrifuge tube and centrifuged at 580g for 30 minutes. After centrifugation, the liquid in the centrifuge tube was divided into two layers, the upper layer was plasma, and the lower layer was cell layer.

[0053] Carefully draw the upper layer of plasma, put it in a 50mL centrifuge tube, inactivate it in a dry bath at 56°C for 30 minutes, then centrifuge it at 800g for 20 minutes, remove the supernatant, and store in a refrigerator at -20°C.

[0054] Resuspend the lower cell layer with about 25mL PBS buffer to obtain a uniform cell mixture. Take two new 50mL centrifu...

Embodiment 2

[0064] The influence of embodiment 2 culture medium composition on NK cell expansion efficiency

[0065] In this example, the influence of the composition of the culture medium on the expansion efficiency of NK cells was explored by adopting the method of in vitro pure culture of natural killer cells in Example 1. Wherein, the composition of control medium is shown in Table 1 below:

[0066] Table 1 Composition table of control culture medium

[0067]

[0068]

[0069] In this example, the cells under different culture conditions are counted, and the proliferation rate is compared with the statistical basis of "the concentration of OK432 is 0 μg / mL, and the concentration of IL-2 is 300 IU", and the statistical results are shown in Figure 4 .

[0070] Depend on Figure 4 It can be seen that under the same concentration of OK432, the effect of higher concentration of IL-2 on the proliferation of NK cells is obvious. Under the condition that the culture medium contains...

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Abstract

The invention relates to an in-vitro pure culture method for natural killer cells. The method includes the following steps that peripheral blood is collected, single karyocyte is separated, magnetic beads combined with specific antibodies are adopted to remove the NK cells, and NK cell populations are obtained; the NK cell populations are put into a culture bottle coated with multiple antibodies,and are subjected to activation culture with a complete medium containing IL-2, IL-7, IL-15, anti-CD16, OK432 and inactivated autologous serum, and inactivated NK cell populations are obtained; the inactivated NK cell populations are put into a culture bottle free of antibody coating and continue to carry out multiplication culture, and the natural killer cells are obtained. Any heterologous serumingredients and trophoblast cells are not used in the in-vitro pure culture method, and high amplification efficiency can be wholly obtained; meanwhile, the purity of the amplified NK cells reaches 90% or above, in-vitro efficient pure culture is achieved, and the clinical application requirement is conveniently met.

Description

technical field [0001] The invention relates to the field of cells, in particular to a method for in vitro pure culture of natural killer cells. Background technique [0002] Natural killer cells (natμral killer, NK) is a kind of human immune cells, derived from bone marrow lymphocytes. In the human body, NK cells are mainly distributed in peripheral blood and spleen, and at the same time, a small amount exists in other tissues such as lymph nodes. NK cells mainly exist in peripheral blood and spleen, but the distribution ratio varies among different populations, accounting for roughly 5% to 15% of peripheral blood lymphocytes. [0003] NK cells are generally identified and screened by the protein molecules expressed on their surface. Since the cells express CD56 and lack CD3, the marker combination CD3-CD56+ is usually used to define NK cells. In addition, NK cells also have surface molecular markers such as CD-16, CD-2, and NKP46 (Hadad et al., Front Immμnol., 2015, 6:4...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/17A61P35/00
CPCA61K35/17A61P35/00C12N5/0646C12N2501/06C12N2501/2302C12N2501/2307C12N2501/2315C12N2501/50C12N2501/505C12N2501/515C12N2501/53C12N2501/599
Inventor 魏君蔡萌
Owner IREGENE THERAPEUTICS LTD
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