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Immune cell in-vitro induction amplification, cryopreservation and resuscitation method

An immune cell and cryopreservation technology, applied in the field of immune cell expansion in vitro, can solve the problems of low cell growth efficiency, poor NK cell performance, and high production cost, and achieve the goal of promoting immune cell expansion, improving activation efficiency, and ensuring efficiency. Effect

Inactive Publication Date: 2021-07-16
THE SECOND HOSPITAL OF HEBEI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, the problems of existing NK cell culture in vitro mainly lie in the slow expansion speed of NK cells in vitro, and a large number of T cells mixed together, and the performance of the cultivated NK cells is poor, and the biological activity is insufficient. The application number is CN201611233042.5 " A method for inducing and expanding immune cells in vitro", which improves the induction and expansion efficiency of immune cells by adding substances such as interleukin-2 to the medium, but the growth efficiency of cells is still low in the actual production process, and the production cost also higher

Method used

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  • Immune cell in-vitro induction amplification, cryopreservation and resuscitation method

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Effect test

Embodiment 1

[0040] Place the mononuclear cells in No. 1 medium for activating culture in the coated culture container for 24 hours, then use No. 4 medium, add EPO at 1.2 ng / ml after 2 hours of culture, and then culture until the differentiation of immune cells When the expression of CD56 exceeds 90%, use No. 7 medium to continue the culture, add IL-2 at 500IU / ml after the secondary expansion culture for more than 2 hours, and then continue the culture; record it as the 0th day, observe the cells every day, according to the culture Change the medium every 2-3 days to subculture once.

Embodiment 2

[0042] Place the mononuclear cells in No. 2 medium for activation and culture in the coated culture container for 24 hours, then use No. 5 medium, add EPO at 2.0 ng / ml after 2 hours of culture, and then culture until the differentiation of immune cells When the expression of CD56 exceeds 90%, use No. 8 medium to continue the culture, add IL-2 at 600IU / ml after the secondary expansion culture for more than 2 hours, and then continue the culture; record it as the 0th day, observe the cells every day, according to the culture Change the medium every 2-3 days to subculture once.

Embodiment 3

[0044] Place the mononuclear cells in No. 3 medium for activation and culture in the coated culture container for 24 hours, then use No. 6 medium, add EPO at 1.6 ng / ml after 2 hours of culture, and then culture until the differentiation of immune cells When the expression of CD56 exceeds 90%, use No. 9 medium to continue culturing, add IL-2 at 550IU / ml after the secondary expansion culture for more than 2 hours, and then continue culturing; record it as day 0, observe the cells every day, according to the culture Change the medium every 2-3 days to subculture once.

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Abstract

The invention provides an immune cell in-vitro induced amplification, cryopreservation and resuscitation method, and relates to the technical field of immune cell in-vitro amplification. The immune cell in-vitro induced amplification, cryopreservation and resuscitation method mainly comprises the following steps: activating and culturing mononuclear cells by using an immune cell activation culture medium, performing primary induced amplification culture by using a primary amplification culture medium, performing secondary amplification culture by using a secondary amplification culture medium, performing cryopreservation treatment by using a cryopreservation solution, and mixing and unfreezing the cryopreservation solution. According to the present invention, the defects in the prior art are overcome, the induction efficiency of immune cells is effectively improved, the in-vitro amplification speed is high, the safety is high, and the cells still have good cell characteristics after being stored for a long time.

Description

technical field [0001] The invention relates to the technical field of in vitro expansion of immune cells, in particular to a method for inductive expansion, cryopreservation and recovery of immune cells in vitro. Background technique [0002] Cancer is one of the most threatening diseases to human beings in modern society, and the treatment of cancer has always been the focus of modern medicine. Tumor immunotherapy is recognized as the fourth major tumor treatment method by domestic and foreign medical circles, among which the autologous immune cell (T cell, NK cell) treatment technology is the first batch of third-class medical technology allowed to be used clinically by the Ministry of Health. In recent years, natural killer cell (NK) immunotherapy technology is becoming a reliable anti-cancer therapy worldwide, which is suitable for the clinical treatment of various malignant tumors with few side effects and no damage to normal tissues. NK cells came into people's sight...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/078A01N1/02
CPCA01N1/02C12N5/0634C12N5/0646C12N2500/84C12N2501/14C12N2501/2302C12N2501/231C12N2501/24C12N2501/37
Inventor 安雯婷郭丽娜
Owner THE SECOND HOSPITAL OF HEBEI MEDICAL UNIV
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