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Method for in-vitro amplification of NK cells

A technology for NK cells and in vitro expansion, which is applied in the field of NK cell culture and expansion, and can solve the problems of high cost and unsatisfactory purity

Inactive Publication Date: 2013-03-27
SHANGHAI CLAISON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In order to solve the problems of unsatisfactory purity and high cost of NK cells after expansion in the prior art, the present invention provides a new method for in vitro expansion of NK cells, which can obtain a large number of high-purity, high-purity NK cells under in vitro culture conditions. NK cells with strong killing activity

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  • Method for in-vitro amplification of NK cells

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Embodiment 1

[0026] 1. Isolation of peripheral blood mononuclear cells (PBMC) and culture of NK cells

[0027] Collect peripheral blood mononuclear cells (PBMC) from the patient through a blood cell separator, transfer the collected blood sample to a centrifuge tube; 700g, centrifuge for 10 minutes, and absorb the upper layer of plasma for later use; use 0.9% normal saline blood sample to restore the sample to its original volume, Mix well; slowly add the diluted blood to Ficoll, 900g, and centrifuge for 20min; absorb the milky white mononuclear cell layer at the interface of the separation solution; centrifuge and wash twice and count; resuspend PBMC with medium, and adjust the cell concentration to 1.5×10 6 / ml,

[0028] Transfer to a 75cm cell line pre-coated with CD3McAb and CD226McAb 2 In the culture flask, add 500IU / ml IL-2 and 10ng / ml IL-18, culture for 72 hours,

[0029] The cells were collected by centrifugation, counted, and mixed with the same number of K562 cells killed by 1...

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Abstract

The invention relates to a method for in-vitro amplification of NK cells, and in particular relates to a method for massive in-vitro amplification of NK cells, wherein the method comprises the following steps of: a, inoculating a peripheral blood mononuclear cell in a CD3McAb and CD226McAb pre-coated culture bottle for coculture; b, adding 1L-2 and 1L-18, coculturing for 72hours to stimulate amplification of NK cells; c, transferring the NK cells, K562 cells after lethal treatment and a serum-free medium containing 1L-2 and 1L-18 in a cell culture bag for coculture; and d, collecting the NK cells. According to the method for in-vitro amplification of the NK cells, two antibodies CD3McAb and CD226McAb are simultaneously coated, so the cell factor synthesis and ADCC effect are promoted, and killing toxicity of the NK cells is remarkably improved; the activation and amplification on the NK cells are achieved just by the 1L-2 and 1L-18 cell factors, so the amplification multiple and cell toxicity of the NK cells are guaranteed, and the cost of cell culture is reduced.

Description

[technical field] [0001] The invention relates to a method for cultivating and expanding NK cells, in particular to a method for massively expanding NK cells in vitro. [Background technique] [0002] Natural killer cells (NK) are cytotoxic lymphocytes belonging to the lymphocyte lineage that contain perforin and granzyme granules. Its recognition of target cells is not restricted by MHC, it can directly kill tumor cells without prior sensitization, and can also secrete cytokines to regulate the functions of other immune cells. It is the main bearer of the body's natural immunity and the core of acquired cellular immunity. Regulatory cells play an important role in tumor immunity, anti-viral infection and removal of non-self cells. Studies have proved that natural killer cells are not only an important component of the natural immune system, but also have some characteristics of acquired immune cells. In the immune system, NK cells respond faster than T cells or B cells, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
Inventor 叶永清杨雪晶苏国新
Owner SHANGHAI CLAISON BIOTECH
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