Preparation method for DC cells

A cell and monocyte technology, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve the problem of low number of DC cells

Inactive Publication Date: 2016-11-16
SINOBIOWAY CELL THERAPY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, DC cells are cultured in vitro mainly by using two cytokines, GM-CSF and IL-4, in conjunction with DC cell culture medium. The number of DC cells prepared by this meth

Method used

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  • Preparation method for DC cells

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Embodiment 1

[0018] A method for preparing DC cells, comprising the steps of:

[0019] S1. Incubating human monocytes in a medium to obtain a solution of immature DC cells, wherein the medium is a DC-GROW medium containing IL-4 and GM-CSF, wherein the concentration of IL-4 is 1000 U / ml, The concentration of GM-CSF is 800U / ml;

[0020] S2. Take the immature DC cell solution obtained in S1, add IL-1β, IL-6, TNF-α, PGE2, GM-CSF, IL-4, and IL-7 to obtain a mixed solution; adjust the temperature to 36°C, and incubate 50h, collect cells, wherein, in the mixture, the concentration of IL-1β is 20ng / ml, the concentration of IL-6 is 200ng / ml, the concentration of TNF-α is 20ng / ml, the concentration of PGE2 is 2μg / ml, GM - The concentration of CSF is 800 U / ml, the concentration of IL-4 is 1000 U / ml, and the concentration of IL-7 is 20 ng / ml.

Embodiment 2

[0022] A method for preparing DC cells, comprising the steps of:

[0023] S1. Add human mononuclear cells to DC-GROW medium and mix well, and culture to obtain solution A; adjust the temperature to 35°C, incubate for 4.5h, remove non-adherent cells, add medium to adherent cells, and continue to incubate. When cell colonies are formed, continue to incubate for 23h to obtain immature DC cell solution, wherein the medium is DC-GROW medium containing IL-4 and GM-CSF, wherein the concentration of IL-4 is 1000U / ml, GM -The concentration of CSF is 800U / ml, and in solution A, the concentration of human mononuclear cells is 1×10 6 a / ml;

[0024] S2. Take the immature DC cell solution obtained in S1, add IL-1β, IL-6, TNF-α, PGE2, GM-CSF, IL-4, and IL-7 to obtain a mixed solution; adjust the temperature to 37°C, and incubate 48h, collect the cells, wherein, in the mixture, the concentration of IL-1β is 20ng / ml, the concentration of IL-6 is 200ng / ml, the concentration of TNF-α is 20ng / m...

Embodiment 3

[0026] A method for preparing DC cells, comprising the steps of:

[0027] S1. Add human mononuclear cells to DC-GROW medium and mix well, and culture to obtain solution A; adjust the temperature to 37°C, incubate for 3.5 hours, remove non-adherent cells, add medium to adherent cells, and continue to incubate. When cell colonies are formed, continue to incubate for 25 hours to obtain immature DC cell solution, wherein the medium is DC-GROW medium containing IL-4 and GM-CSF, wherein the concentration of IL-4 is 1000U / ml, GM -The concentration of CSF is 800U / ml, and in solution A, the concentration of human mononuclear cells is 1×10 6 a / ml;

[0028] S2. Take the immature DC cell solution obtained in S1, add IL-1β, IL-6, TNF-α, PGE2, GM-CSF, IL-4, and IL-7 to obtain a mixed solution; adjust the temperature to 35°C, and incubate 52h, collect cells, wherein, in the mixture, the concentration of IL-1β is 20ng / ml, the concentration of IL-6 is 200ng / ml, the concentration of TNF-α is ...

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Abstract

The invention discloses a preparation method for DC cells. The preparation method comprises the following steps: S1, carrying out incubation of human monocytes in a medium so as to obtain an immature DC cell solution; S2, adding IL-1beta, IL-6, TNF-alpha, PGE2, GM-CSF, IL-4 and IL-7 into the immature DC cell solution prepared in the step S1 so as to obtain a mixed solution; and S3, adjusting a temperature to 35 to 37 DEG C, carrying out incubation for 48 to 52 h and collecting cells. The prepared DC cells is great in number; and the preparation method is simple to operate and suitable for industrial production.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing DC cells. Background technique [0002] Dendritic cells, referred to as DC cells, are the most powerful antigen-presenting cells found so far. It has been confirmed that DC cells are the only antigen-presenting cells that can significantly stimulate the proliferation of initial T cells. DC cells can effectively induce the proliferation and activation of antigen-specific T cells. They are the main initiators and participants of the body's tumor immune response, and they are promising therapeutic tools. At present, DC cells are cultured in vitro mainly by using two cytokines, GM-CSF and IL-4, in conjunction with DC cell culture medium. The number of DC cells prepared by this method is not high, and it cannot meet people's needs for DC cells. Therefore, it is necessary to A new method for preparing DC cells is provided to increase the number of prepared DC cells....

Claims

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Application Information

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IPC IPC(8): C12N5/0784
CPCC12N5/0639C12N2501/22C12N2501/2301C12N2501/2304C12N2501/2306C12N2501/2307C12N2501/25C12N2501/999
Inventor 王维吴斌许国贞刘振云凌发忠熊武平程丰伟
Owner SINOBIOWAY CELL THERAPY CO LTD
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