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Method for in vitro culture of high-proliferation and high-mortality NK cells

A technology of NK cells and cell culture, applied in the field of cellular immunity, to achieve the effect of high proliferation and high lethality

Inactive Publication Date: 2016-01-13
SHANDONG XINRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the previous work of the inventor, the four efficient NK cell expansion schemes reported in the literature were studied from the aspects of expansion multiple, cell phenotype and killing activity, and it was believed that the cytokine combination of IL-2 and IL-15 was In vitro amplification can efficiently and stably obtain amplified products, but there is still a certain gap with the inventor's ideal effect, and further research is needed

Method used

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  • Method for in vitro culture of high-proliferation and high-mortality NK cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: The method for expanding NK cells in vitro provided by the present invention

[0023] Add coating solution containing anti-human CD3 and anti-human CD16 monoclonal antibodies to the cell culture flask in advance, incubate at room temperature for 1 h, and the final concentrations of anti-human CD3 and anti-human CD16 monoclonal antibodies are both 0.15 mg / ml.

[0024] Collect the patient's peripheral blood mononuclear cells through a blood cell separator, transfer the collected blood sample to a centrifuge tube; centrifuge at 700g 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 and mix it; Slowly add the diluted blood to Ficoll, centrifuge at 900g 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 serum-free medium, and adjust the cell concentration to ...

Embodiment 2

[0027] Embodiment 2: The method for expanding NK cells in vitro provided by the present invention

[0028] Add the coating solution containing anti-human CD3 and anti-human CD16 monoclonal antibodies to the cell culture flask in advance, and incubate at room temperature for 6 hours. The final concentrations of anti-human CD3 and anti-human CD16 monoclonal antibodies are both 0.01 mg / ml.

[0029] Collect the patient's peripheral blood mononuclear cells through a blood cell separator, transfer the collected blood sample to a centrifuge tube; centrifuge at 700g 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 and mix it; Slowly add the diluted blood to Ficoll, centrifuge at 900g 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 serum-free medium, and adjust the cell concentra...

Embodiment 3

[0032] Embodiment 3: The method for expanding NK cells in vitro provided by the present invention

[0033] Add coating solution containing anti-human CD3 and anti-human CD16 monoclonal antibodies to the cell culture flask in advance, incubate at room temperature for 0.5 h, and the final concentrations of anti-human CD3 and anti-human CD16 monoclonal antibodies are both 3.0 mg / ml.

[0034] Collect the patient's peripheral blood mononuclear cells through a blood cell separator, transfer the collected blood sample to a centrifuge tube; centrifuge at 700g 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 and mix it; Slowly add the diluted blood to Ficoll, centrifuge at 900g 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 serum-free medium, and adjust the cell concentration to...

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Abstract

The invention discloses a method for in vitro culture of high-proliferation and high-mortality NK cells. The culture method comprises the following steps: (1) performing coating of anti-human CD3 monoclonal antibodies with a concentration of 0.01-3.0 mg / ml and anti-human CD16 monoclonal antibodies with a concentration of 0.01-3.0 mg / ml in a cell culture bottle, causing the coating conditions to be room temperature, causing the incubating time to be 0.5-6 h; (2) causing mononuclear cells obtained through separation out of peripheral blood to be inoculated in the cell culture bottle through serum-free culture solution, and performing stimulation for 12-24 h; (3) adding serum-free culture solution for NK cell culture, IL-2 with a concentration of 20-50 ng / ml, IL-15 with a concentration of 20-50 ng / ml and PedinophyllolB with a concentration of 30-80 ng / ml, performing stimulation for 12-84 h, and then transferring the solution into a cell culture bag to go on performing culture; and (4) obtaining NK cells. According to the method, the high-proliferation and high-mortality NK cells can be obtained, and the method can be used for tumor cell immunotherapy.

Description

technical field [0001] The invention belongs to the field of cellular immunity and relates to a tumor immune cell, in particular to an in vitro culture method for NK cells with high proliferative power and high lethality. Background technique [0002] Adoptive cellular immunotherapy (ACI) is a biological treatment method for tumor treatment by infusing lymphocytes back into tumor patients after being stimulated and cultured in vitro, directly killing tumor cells or stimulating the body's immune response to kill tumor cells. In recent years, with the development of medical technology, NK cell-based adoptive cellular immunotherapy for tumors has become more and more widely used. NK cells are mainly distributed in the bone marrow, peripheral blood and spleen, and are the main effector cells of the innate immune system, accounting for 10% to 20% of peripheral blood lymphocytes. It is mainly involved in the process of anti-tumor, anti-viral infection and immune regulation of the...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/17A61P35/00
Inventor 赵顺英
Owner SHANDONG XINRUI BIOTECH CO LTD
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