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Method for pure culture of natural killer cells in vitro

A natural killer cell, pure culture technology, applied in the field of natural killer cell in vitro pure culture, can solve problems such as high safety risks, achieve high amplification multiples, reduce damage, and high safety.

Active Publication Date: 2020-06-16
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 only 30-70%.
In addition, there is also a method of using trophoblast cells to stimulate the growth of NK cells. However, since the trophoblast cells are generally inactivated cancer cells in this method, the safety risk of clinical use is relatively high.

Method used

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  • Method for pure culture of natural killer cells in vitro
  • Method for pure culture of natural killer cells in vitro
  • Method for pure culture of natural killer cells in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0051] S1, collect peripheral blood, separate PBMC:

[0052] 50mL of peripheral blood was drawn from the venous blood vessels and placed in heparin sodium anticoagulation blood collection tubes. Subsequently, the peripheral blood was transferred to a 50 mL centrifuge tube and centrifuged at 580 g 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 the cell layer.

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

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

Embodiment 2

[0064] Example 2 Effect of medium composition on the expansion efficiency of NK cells

[0065] In this example, the method of in vitro pure culture of natural killer cells in Example 1 was used to explore the influence of the composition of the culture solution on the expansion efficiency of NK cells. Among them, the composition of the control medium is shown in Table 1 below:

[0066] Table 1 Composition of control medium

[0067]

[0068]

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

[0070] by Figure 4 It can be seen that under the same OK432 concentration condition, a higher concentration of IL-2 has a significant effect on the proliferation of NK cells. Under the condition of 0.5-2μg / mL OK432 in the culture medium, the addition of 600 IU uni...

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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 present 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) are a type of human immune cells, derived from bone marrow lymphocytes. In the human body, NK cells are mainly distributed in peripheral blood and spleen. At the same time, they are also found in a small amount in other tissues such as lymph nodes. NK cells mainly exist in peripheral blood and spleen, but the proportions of distribution in different populations are different, roughly accounting for 5% to 15% of peripheral blood lymphocytes. [0003] NK cells are generally identified and screened by the protein molecules expressed on their surface. Since this cell expresses CD56 but lacks CD3, the combination of markers 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 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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