Method for preparing NK by using freeze-dried feeder layer cells

Abstract

The invention provides a method for preparing NK by using freeze-dried feeder layer cells. In the NK preparing method, engineering cells expressing cytokines and membrane proteins are constructed, andthe engineering cells expressing the cytokines and membrane proteins are freeze-dried. According to the method, after freeze-drying of feeder layer cells, the stimulation effect of the engineering cells as feeder layer cells is retained, the costs for cell inactivation and preservation process are also reduced, the costs for laboratories and clinical use are reduced, and the use convenience is improved. Compared with an existing feeder layer using technology, NK cells quantity for clinical treatment can be provided within 12-21 days, the cell viability is high, and the cells have high killingeffect on K562 and A549; The NK medium method has obvious advantages in proliferation ratio and quantity compared with a factor method and a special NK culture medium method; the cell quantity and purity obtained through amplification after stem cells are induced and split up into NK are poor compared with the method.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to NK cell culture technology. Background technique [0002] Natural killer cells (NK) are important immune cells in the body, not only related to anti-tumor, anti-viral infection and immune regulation, but also involved in the occurrence of hypersensitivity and autoimmune diseases in some cases, and can recognize Target cells, killing mediators. [0003] Existing NK amplification culture techniques are roughly divided into four types: [0004] 1. Factor method: extract peripheral blood or use a single cell collection machine combined with lymphocyte separation fluid to obtain PBMC (peripheral blood mononuclear cells), then directly culture or sort NK cells in PBMC, and separate them in ordinary lymphocyte culture medium NK expansion using cytokines or co-feeder cells. [0005] 2. Antibody coating method: PBMC or sorted NK cells are expanded and cultured in the antibody-coated culture ...

AI Technical Summary

Problems solved by technology

[0008] The factor method and antibody coating method (publication number: CN105238754B) are difficult to meet the clinical needs in terms of NK amplification quantity, purity and NK cell viability when feeder layer cells are not used, while the feeder layer used in the traditional scheme needs to use mitomyces After inactivation, it needs to be stored in liquid nitrogen, which increases the use cost of the feeder layer and is not conducive to the commercialization of the feeder layer. The slight residue of mitomycin may make NK especially difficult to use in the subsequent use. It has adverse effects on the human body in clinical use; in addition, if NK is first sorted from PBMC (public number: CN103620022B), the operation steps and costs are increased, and the heterologous components in the sorting may be added in the culture system to bring safety risk

Although the dedicated NK medium (publication number: CN104894065B) simplifies the steps of NK amplification, it has no obvious advantages in terms of NK amplification purity and efficiency compared with traditional methods, and the cost of the dedicated NK medium is relatively high

The method of inducing stem cells to differentiate into NK (public number: CN101801374B; public number: CN104711225B) has potential safety risks in the process of stem cell source acquisition and differentiation induction, and the expansion efficiency is not high

Images