Preparation of neural stem cell-derived Exosomes, and application of neural stem cell-derived Exosomes in nervous system diseases
A technology for nervous system diseases and neural stem cells, which is applied in the fields of nervous system diseases, nervous system cells, cardiovascular system diseases, etc., to achieve the effects of short time consumption, easy storage and transportation, and high yield
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Embodiment 1
[0035] Example 1: Umbilical cord-derived mesenchymal stem cells are induced to differentiate into neural stem cells
[0036] Under aseptic conditions, take the umbilical cord of a healthy term cesarean section newborn and process it within 4 hours, wash it with phosphate solution (PBS) and remove the arteries and veins, cut them into pieces to a diameter of about 1~2mm, and divide them into T- Add 5-6ml of complete medium (α-MEM medium containing 10% fetal bovine serum, 200ug / mL penicillin, and 200ug / mL streptomycin) to the 25 culture flask, and shake until the tissue pieces are evenly distributed in the culture flask. At 37℃, 5%CO 2 Culture in a cell incubator, and change the medium in half on the 4th day, and then change the medium every 3 days. When the cells have grown to 80% confluence, they are subcultured ( figure 1 -A).
[0037] The P3-P5 generation mesenchymal stem cell suspension was inoculated in a plastic culture dish with a diameter of 40mm, and the cells grew t...
Embodiment 2
[0038] Example 2: Rat neural stem cell culture
[0039] Take SD rat embryos (pregnancy 14-16 days) under aseptic conditions. After fully rinsing with D-Hanks solution, peel off the meninges under a dissecting microscope to accurately separate the hippocampus, cut the hippocampus with ophthalmic scissors, and transfer to DMEM / F12(1:1), B27, bFGF, EGF serum-free medium, pipette and blow mechanically to separate single cell suspension. The fluid was changed in half every 3 days, and the cytokines bFGF and EGF were added. Take good P3~P5 generation cells, and use them for the preparation of Exosomes when the cultured cell fusion rate reaches 70%-80%. ( figure 2 )
Embodiment 3
[0040] Example 3: Preparation of Exosome by Ultracentrifugation and Filter Membrane
[0041] Under aseptic conditions, collect the complete medium supernatant of 3-5 generations of various types of stem cells, centrifuge at 20 000g*20min at 4℃, discard the precipitate, collect the supernatant, and filter through a 0.22 μm sterile filter membrane To remove impurities, the obtained filtrate was centrifuged again, 120 000g*60min, the supernatant was discarded, the precipitate was resuspended by pipetting with PBS, and the precipitate suspension was collected to obtain Exosomes.
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