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.
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[0022] Specifically, the present invention relates to a preparation method of peripheral blood immature DC cell exosomes, including:
[0023] 1) PBMC cells isolated from peripheral blood are cultured in medium A to obtain immature dendritic cells in vitro;
[0024] 2) After culturing the isolated immature dendritic cells in medium B, centrifuge the culture solution at low temperature to remove dead cells and debris, and filter to remove vesicles; the obtained liquid is subjected to ultracentrifugation, and the supernatant is discarded to obtain Immature DC cell exosomes;
[0025] Wherein, the medium A is an AIM-V medium additionally containing 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 embodiments, ...
Example Embodiment
[0067] Example 1
[0068] The main purpose of the present invention is to establish a method for separating immature DC exosomes from peripheral blood.
[0069] 1. Peripheral blood DC cell culture medium
[0070] 1) DC general 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. Immature DC cell exosomes isolation method from peripheral blood
[0073] 1) Preparation of exosomes-free autologous plasma: Pour autologous plasma into a special ultracentrifuge tube under aseptic conditions, each tube is 38.6mL, 4 tubes, after confirming the balance, put it in an ultracentrifuge, and centrifuge at 110000g for 16h.
[0074] 2) The next day, carefully pipet the upper clear serum into a new sterile centrifuge tube, filter it with a 0.20μm filter into ...
Example Embodiment
[0089] Example 2
[0090] The main purpose of the present invention is to establish a method for separating immature DC exosomes from 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-β 20ng / mL, VEGF 40ng / mL.
[0094] 2. Immature DC cell exosomes isolation method from peripheral blood
[0095] 1) Preparation of exosome-free autologous plasma: Pour autologous plasma into a special ultracentrifuge tube under aseptic conditions, 38.6mL per tube, 4 tubes, after confirming the balance, put it in an ultracentrifuge, centrifuge at 130,000g for 8h.
[0096] 2) On the second day, carefully pipet the upper clear serum into a new sterile centrifuge tube, filter it with a 0.24μm filter into anothe...
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