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Preparation method of PB (Peripheral Blood) immature DC (Dendritic Cell) exosome

A technology of peripheral blood and exosomes, which is applied in the directions of blood/immune system cells, animal cells, vertebrate cells, etc., to achieve the effect of good exosome yield, increased ratio, and simple and easy method.

Inactive Publication Date: 2019-04-26
药鼎(北京)国际细胞医学技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, exosomes from immature DC cells have broad application prospects in various autoimmune diseases, but there is a lack of a perfect method for preparing exosomes from immature DC cells in the prior art

Method used

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  • Preparation method of PB (Peripheral Blood) immature DC (Dendritic Cell) exosome
  • Preparation method of PB (Peripheral Blood) immature DC (Dendritic Cell) exosome
  • Preparation method of PB (Peripheral Blood) immature DC (Dendritic Cell) exosome

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preparation example Construction

[0022] Specifically, the present invention relates to a method for preparing exosomes from peripheral blood immature DC cells, comprising:

[0023] 1) culturing PBMC cells isolated from peripheral blood using medium A to obtain isolated immature dendritic cells;

[0024] 2) After culturing the isolated immature dendritic cells in culture medium B, centrifuging the culture solution at low temperature to remove dead cells and debris, and filtering to remove vesicles; performing ultracentrifugation on the obtained liquid, discarding the supernatant to obtain Immature DC exosomes;

[0025] Wherein, the medium A is an AIM-V medium supplemented with the following components: 0.8-1.2v / v% autologous plasma, GM-CSF 40-60ng / mL, IL-4 10-30ng / mL and PEG- 2 0.4~0.6μg / mL;

[0026] The medium B is an AIM-V medium supplemented with the following components: 0.8-1.2v / v% exosome-free autologous plasma, GM-CSF 30-50ng / mL, IL-4 30-50ng / m, TGF-β 10-20ng / mL, VEGF 40-50ng / mL.

[0027] In some em...

Embodiment 1

[0068] The main purpose of the present invention is to establish a method for isolating exosomes from immature DC cells in peripheral blood.

[0069] 1. Peripheral blood DC cell culture medium

[0070] 1) DC common culture: AIM-V medium (containing 0.8% autologous plasma), GM-CSF concentration 60ng / mL, IL-4 concentration 40ng / mL, PEG-2 0.4μg / mL.

[0071] 2) DC exosome-free medium: AIM-V medium (containing 0.8% exosome-free autologous plasma), GM-CSF 30ng / mL, IL-4 50ng / mL, TGF-β 10ng / mL, VEGF 50ng / mL.

[0072] 2. Isolation method of exosomes from immature DC cells in peripheral blood

[0073] 1) Preparation of exosome-free autologous plasma: Pour the autologous plasma into special ultracentrifuge tubes under sterile conditions, 38.6mL per tube, 4 tubes, after confirming the balance, put it into an ultracentrifuge, and centrifuge at 110,000g for 16h.

[0074] 2) The next day, carefully draw the upper layer of clarified serum into a new sterile centrifuge tube, filter it with...

Embodiment 2

[0090] The main purpose of the present invention is to establish a method for isolating exosomes from immature DC cells in peripheral blood.

[0091] 1. Peripheral blood DC cell culture medium

[0092] 1) DC common medium: AIM-V medium (containing 1.2% autologous plasma), GM-CSF concentration 40ng / mL, IL-4 concentration 60ng / mL, PEG-2 0.6μg / mL.

[0093] 2) DC exosome-free medium: AIM-V medium (containing 1.2% exosome-free autologous plasma), GM-CSF 50ng / mL, IL-4 30ng / mL, TGF-β20ng / mL, VEGF 40ng / mL mL.

[0094] 2. Isolation method of exosomes from immature DC cells in peripheral blood

[0095] 1) Preparation of exosome-free autologous plasma: Pour the autologous plasma into special ultracentrifuge tubes under sterile conditions, 38.6mL per tube, 4 tubes. After confirming the balance, put it into an ultracentrifuge and centrifuge at 130,000g for 8h.

[0096] 2) The next day, carefully draw the upper layer of clarified serum into a new sterile centrifuge tube, filter it with a...

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Abstract

The invention relates to a preparation method of a PB (Peripheral Blood) immature DC (Dendritic Cell) exosome. The preparation method comprises the following steps: (1) culturing PBMCs (Peripheral Blood Mononuclear Cells) separated from PB by using a culture medium A, thus obtaining in-vitro immature DCs; (2) after culturing the in-vitro immature DCs in a culture medium B, carrying out low-temperature centrifugation on a culture solution for removing dead cells and debris, and removing vesicae by filtering; carrying out ultracentrifugation on an obtained solution, and removing supernatant, thus obtaining the immature DC exosome, wherein autologous plasma, a GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor), IL-4 (Interleukin 4) and PEG-2 (Polyethylene Glycol 2) are additionally added in the culture medium A on the basis of an AIM-V culture medium; exosome-free autologous plasma, the GM-CSF, the IL-4, a TGF (Transforming Growth Factor)-beta and a VEGF (Vascular Endothelial Growth Factor) are additionally added in the culture medium B on the basis of the AIM-V culture medium. The preparation method disclosed by the invention is not only capable of effectively inhibiting DC maturity, but also has better exosome yield.

Description

technical field [0001] The invention relates to the technical field of exosome isolation, in particular to a method for preparing exosomes from immature DC cells in peripheral blood. Background technique [0002] Dendritic cells (DCs) are the most effective antigen-presenting cells in the body, express B7, MHC-II molecules, ICAM-1 and other co-stimulatory molecules on the surface, activate helper T cells, and prompt them to secrete IL- 2 and other cytokines transmit specific antigen signals and finally activate cytotoxic T cells. Therefore, DC cells play a key role in initiating a series of immune responses in the body, and are of great significance in the immune function of the body and the immune management of tumors. [0003] Most DC cells originate from the bone marrow, enter the peripheral blood from the bone marrow, and then distribute to various tissues throughout the body. DCs are widely distributed in all organs of the body except the brain, but the number is very s...

Claims

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Application Information

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IPC IPC(8): C12N5/0784
Inventor 周海涛
Owner 药鼎(北京)国际细胞医学技术有限公司
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