A kind of method for cultivating umbilical cord blood cik cells in vitro

Abstract

The invention discloses a method for culturing umbilical cord blood CIK cells in vitro, which comprises the following steps: (1) separating and obtaining umbilical cord blood mononuclear cells from umbilical cord blood, and preparing a mononuclear cell suspension with a serum-free medium; (2) Add IFN-γ850-950U / mL to the cell suspension in the above step (1), add IL-2 600-700U / mL, monoclonal antibody CD3 20-30ng / mL, tripeptide-1 copper 20-50ng after culturing for 1 day / mL, continue to cultivate; (3) Add stevioside 110-120ng / mL and asiaticoside 80-100ng / mL until the 3rd-4th day, and collect the cells until the 12th day of induction culture. Add serum-free medium every 1-2 days to maintain the cell concentration at 1-2×10 6 per mL, the serum-free medium contains stevioside 35-40ng / mL and asiaticoside 20-30ng / mL. The present invention adopts serum-free medium, adds tripeptide-1 copper, stevioside and asiaticoside during the culture process, induces the number of CIK cells obtained by amplification to be large, and maintains better cell activity, and improves its value in clinical application.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a method for culturing umbilical cord blood CIK cells in vitro. Background technique [0002] Multiple cytokine-induced killer cells (CIK cells) are a group of heterogeneous cells obtained by co-culturing human peripheral blood or umbilical cord blood mononuclear cells with various cytokines for a period of time in vitro. Because this kind of cells express both CD3+ and CD56+ membrane protein molecules, they are also called NK-like T lymphocytes, which have both the strong anti-tumor activity of T lymphocytes and the non-MHC-restricted tumor killing advantages of NK cells. Because of its fast expansion in vitro, high killing activity, broad tumor killing spectrum, and few adverse reactions, it can improve the immune function of patients and eliminate small residues. It is considered to be the first choice for anti-tumor adoptive cell immunotherapy. [0003] Most of the CIK cell prepa...

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Problems solved by technology

However, the CIK cells prepared by this method induced by this method have poor reproductive ability. CIK cells contain a large number of CD4+CD25+ regulatory T cells, and the proportion of effector cells (CD3+CD56+) is generally not high. Cell viability low, poor tumor lethality and other problems, the traditional CIK cell expansion culture method in vitro is mostly cultured by adding bovine serum (fetal bovine serum) or human AB serum, which has the potential of spreading diseases and has Endotoxin residues and the risk of infection with other exogenous diseases, there are certain safety hazards, resulting in great limitations in the clinical application of adoptive cell immunotherapy, and the infusion of allogeneic CIK cells can easily lead to serious graft-versus-host disease (GVHD)

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