Culture system and culture method for efficient amplification in vitro of autologous NK cells

Abstract

The invention provides a culture system and a culture method for efficient amplification in vitro of autologous NK (Natural Killer) cells. The culture system comprises a basic culture medium containing GT-T551, interleukin IL-2, interleukin 1L-12, interleukin IL-15, interleukin IL-18, interleukin 1L-17, interleukin IL-22 and natural traditional Chinese medicine substances. The culture method comprises the following steps of: collecting and separating autologous 40ml peripheral blood mononuclear cells; resuspending the cells by a culture medium containing 10 percent of self plasma; adding the cells into a cell culture bottle coated by CD3 antibodies or CD56 antibodiees; performing stimulation by a cell factor combination A; putting the cells in a 37 DEG C and 5-percent-CO<2> culture tank to perform incubation and shaking; after 6 to 7 days, transferring the cells into a cell culture bag to be cultured; complementally adding a culture medium containing a cell factor combination B; in the culture period, culturing the cells in a basic culture medium containing IL-2 every 3 to 4 days; and obtaining high-purity NK cells after the total culture time of 17 to 19 days. The culture system and the culture method have the advantages that the NK cells can be amplified by more than 1000 times in vitro; and the high-purity NK cells exceeding 5*10<9> can be obtained.

Description

technical field [0001] The invention relates to the field of in vitro culture of immune cells, in particular to a culture system for efficiently expanding autologous NK cells in vitro, and at the same time relates to a culture method for efficiently expanding autologous NK cells in vitro. Background technique [0002] Natural killer cells (NK) originate from bone marrow hematopoietic progenitor cells and mature in the bone marrow microenvironment. They are important natural immune system effector cells for the body to resist pathogen invasion and normal cell malignant transformation. In 2007, ZamaiLorisⅢ wrote an article pointing out that NK cells play a "controlling" role in the proliferation and metastasis of tumors in the body. Tumor immunotherapy based on NK cells has good clinical application prospects, and has been a hot research topic for domestic and foreign scholars in recent years. However, as a rare cell subset in lymphocytes, NK cells only account for about 5% to...

AI Technical Summary

Problems solved by technology

However, most of the in vitro stimulation and expansion cultures so far can only make NK cells expand tens to hundreds of times in vitro, and the purity is not ideal

[0004] In addition, there are also research reports using the trophoblast cell line method (such as using K562 cells themselves as the trophoblast cell line). This method can obtain NK cells with high purity, but K562 itself is a kind of tumor cell, which has potential safety hazards.

It has also been reported that embryonic stem cells are induced to differentiate in vitro to form NK cells. The in vitro induction of stem cells is uncertain and has certain safety hazards, so it cannot be applied clinically for the time being.

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