Method for culturing natural killer (NK) and/or natural killer T (NKT) cells

A technology of NKT cells and culture medium, applied in the field of NK and/or NKT cell culture, which can solve the problems of complicated operation and low cell yield.

Inactive Publication Date: 2012-09-19
CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, only adding IL-2 and IL-15 to the medium can only expand NK cells by about 10 times, and us

Method used

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  • Method for culturing natural killer (NK) and/or natural killer T (NKT) cells
  • Method for culturing natural killer (NK) and/or natural killer T (NKT) cells
  • Method for culturing natural killer (NK) and/or natural killer T (NKT) cells

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Experimental program
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Effect test

Embodiment 1

[0062] The separation and cultivation of embodiment 1, NK cells

[0063] 1. Protein coating:

[0064] Retronectin was purchased from Baoriyi Biotechnology (Beijing) Co., Ltd., catalog number: T200H;

[0065] Anti-CD3 is an anti-CD3 antibody, which is a monoclonal antibody. It was purchased from Baori Medical Biotechnology (Beijing) Co., Ltd. Imported products, origin: Cuba Molecular Immunology Center, trade name: Aio Mountain, imported drug registration number: S20030084;

[0066] The PBS buffer solution with a concentration of 0.01M and a pH value of 7.2 was prepared as follows: 8g NaCl, 0.2g KCl, 1.44g Na 2 HPO 4 and 0.24g KH 2 PO 4 Dissolve in 1000ml deionized water.

[0067] Each 2ml of coating solution A was prepared according to the following method: 25μg Retronectin, 5μg Anti-CD3 and PBS buffer solution with a concentration of 0.01M and a pH value of 7.2 were added to 2ml with PBS buffer solution;

[0068] Each 2ml of coating solution B was prepared according to ...

Embodiment 2

[0099] Embodiment 2, NK cell killing experiment in vitro

[0100] 1. Preparation:

[0101] 1) Human pancreatic cancer JF305 cells (can be purchased from Shanghai Fuxiang Biotechnology Co., Ltd.) and human colon cancer HCT116 cells were cultured in advance;

[0102] 2) configuring phenol red-free 1640 medium containing 5% (volume percentage) FBS;

[0103] 2. Adjust the cell concentration and add to the 96-well plate:

[0104] 1) The cells were washed 3 times with PBS, centrifuged at 1000 rpm for 5 minutes each time, and the supernatant was discarded. The concentration of tumor cell JF305 was adjusted to 2×10 with phenol red-free 1640 medium containing 5% (volume percentage) FBS. 5 / ml;

[0105] 2) The cells were washed 3 times with PBS, centrifuged at 1200 rpm for 5 minutes each time, and the supernatant was discarded. Adjust the NK-A, NK-B, and NK-C cell concentrations obtained in Example 1 with the phenol red-free 1640 medium containing 5% FBS according to the effect-to-...

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Abstract

The invention discloses a method for culturing natural killer (NK) and/or natural killer T (NKT) cells. The method comprises the following step: inoculating isolated NK and/or NKT cells into a culture system A for culture to obtain propagated NK and/or NKT cells, wherein the culture system A consists of a buffer solution containing CD3 antibody and/or Retronectin and inducing factors. Experiments prove that peripheral blood mononuclear cells (PBMC) extracted from peripheral blood are separated and enriched through magnetic beads, high-purity CD56+ cells are obtained, two proteins, namely Retronectin and CD3mAb are added into an in-vitro culture system for joint stimulation, and IL-2 and IL-5 factors are used for assisting in induction, so that a culture method capable of obtaining massive NK and NKT cells with high killing activity is established.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for culturing NK and / or NKT cells. Background technique [0002] NK cells, bone marrow-derived lymphocytes, can recognize and kill virus-infected cells or transformed malignant cells. NK cells are fast-response cells in the body, which can be quickly recruited to the injury site, and the killing effect can be seen in 1 hour in vitro and 4 hours in vivo. The function of NK cells cannot be replaced by other immune cells, and its effect of killing tumor cells is not limited by MHC, so it is called natural killer activity; moreover, NK cells promote the activation of Th1 T cells by secreting IFN-gamma and interacting with DCs. NK cells cultured in vitro include CD56 weak expression and CD56 strong expression accompanied by CD16 expression or non-expression cell populations. The CD56 strong expression cell population kills tumor cells through a non-MHC-restricted pathway; while...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
Inventor 马洁赵晨赵平
Owner CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI
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