Natural killer cell (NK cell) culture medium and preparation method thereof

A technology of natural killer cells and NK cells, applied in the field of biomedicine, can solve the problems of low cell purity, low amplification multiple, and long culture cycle, and achieve the effects of low culture cost, simple culture operation, and short culture cycle.

Active Publication Date: 2018-01-12
BIOCELLS BEIJING BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these techniques generally have problems such as high c...

Method used

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  • Natural killer cell (NK cell) culture medium and preparation method thereof
  • Natural killer cell (NK cell) culture medium and preparation method thereof
  • Natural killer cell (NK cell) culture medium and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1: Isolation of Peripheral Blood Mononuclear Cells (PBMC) and Culture of NK Cells

[0080] A coating solution containing an anti-CD16 antibody (biolegend, clone number 3G8) was added to the cell culture flask in advance, and coated overnight at 4° C., and the antibody concentration of the coating solution was 1.0 μg / mL.

[0081] Peripheral blood was collected from volunteers using a blood collection bag. Transfer the collected blood sample to a 50mL centrifuge tube; centrifuge at 3000rpm for 8 minutes, absorb the upper layer of plasma, put it in a 56°C water bath to inactivate for 30 minutes, and prepare it for cultivation; use 0.9% normal saline to restore the blood sample to its original volume, and mix well; Slowly add the diluted blood sample to 15mL Ficoll, centrifuge at 800g for 15min; absorb the buffy coat mononuclear cell layer at the interface of the lymphocyte separation solution; centrifuge at 1600rpm for 8min, wash twice and count; Resuspend and mix...

Embodiment 2

[0086] Example 2: Immunophenotypic detection of cultured NK cells

[0087] Take the cells on the 0th, 8th, 14th and 21st day of culture respectively, add them to 1.5mL EP tubes, and each tube contains about 1.0×10 6 cells. After centrifuging at 2500rpm for 5 minutes, the supernatant was discarded and washed once with PBS. The cells were resuspended in 100 μL of PBS, and 5 μL of fluorescently labeled antibodies (biolegend, CD3-FITC, CD56-pc5.5, CD16-APC, CD314-PE) were added, in which each fluorescent antibody was a mouse anti-human antibody, 4 Incubate at ℃ for 30 minutes in the dark; wash with PBS twice, discard the supernatant; resuspend the cells in 0.2mL PBS, and use cytoflex flow cytometry for detection and analysis.

[0088] The results are shown in figure 1 Middle: On day 8 of culture, NK(CD3 - CD56 + ) was 81.53%; on day 14, 94.3%; on day 21, 91.04%. The above results indicate that the phenotype of NK cells can reach the peak of culture on day 13, and NK (CD3 - ...

Embodiment 3

[0089] Example 3: Determination of NK cell IFN-γ

[0090] The cells were divided into experimental group and control group, and cultured according to different culture schemes.

[0091] Experimental group culture program: the same as in Example 1.

[0092] Culture program for the control group: use blood collection bags to collect peripheral blood from volunteers, transfer the collected blood samples to 50mL centrifuge tubes; centrifuge at 3000rpm for 8 minutes, absorb the upper layer of plasma, put it in a 56°C water bath for 30 minutes, and use it for later use during cultivation; Restore the blood sample to its original volume with 0.9% normal saline, and mix well; slowly add the diluted blood sample to 15mL Ficoll, and centrifuge at 800g for 15min; absorb the buffy coat mononuclear cell layer at the interface of the lymphocyte separation solution; centrifuge at 1600rpm for 8min, wash twice and Count; resuspend the peripheral blood mononuclear cells with Gtt551 lymphocyte ...

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Abstract

The invention provides a culture medium for culturing a natural killer cell (NK cell). The culture medium comprises IL-2 and an antitumor drug. The invention also provides a culture medium for culturing a NK cell, and the culture medium comprises IL-2 and lentinan. The invention also provides a method of using the culture medium to culture a NK cell, a NK cell prepared thereby, and an applicationof the NK cell.

Description

technical field [0001] The present application generally relates to the field of biomedicine, and in particular, the present application relates to a medium for culturing natural killer cells (NK cells), a method for cultivating NK cells using it, NK cells obtained using the method, and the obtained NK cells use. Background of the invention [0002] Natural killer cells (NK cells) are considered to be the body's first line of defense against infection and tumor, and are important immune cells of the body, which are closely related to anti-tumor, anti-viral infection and immune regulation. NK cells originate from bone marrow-derived CD34 + Hematopoietic progenitor cells, accounting for about 5%-15% of peripheral blood lymphocytes, are immunophenotypically characterized by CD3 - CD16 + CD56 + , mainly distributed in peripheral blood, lymph nodes, spleen and bone marrow, and can also migrate to inflammatory sites under the action of different chemokines. [0003] NK cells ...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/17A61P35/00A61P31/00A61P37/02
Inventor 王军伟韩化敏
Owner BIOCELLS BEIJING BIOTECH CO LTD
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