Preparation and separation method of human skin fibroblast exosome
A fibroblast, human skin technology, applied in artificial cell constructs, epidermal cells/skin cells, biochemical equipment and methods, etc. Skin repairing effect, secretion promoting effect, and yield enhancing effect
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Embodiment 1
[0040] Embodiment 1, a method for preparing and separating exosomes from human skin fibroblasts, comprising the following steps:
[0041] S1, in the ultra-low attachment flask (75cm 2 ), adding DMEM medium and low serum medium, adding 1 million (magnitude) human skin fibroblasts, and culturing them in a 5% carbon dioxide incubator at 37° C. for three days to form suspended cell spheres.
[0042] S2, replace with serum-free DMEM medium, continue to cultivate for 12h, then transfer to an anaerobic incubator with a partial pressure of carbon dioxide of 5%, and cultivate for 2h, then pass into the anaerobic incubator to account for a total partial pressure of 0.05%. Nitric oxide, continue to culture for 12h.
[0043] S3, extracting the culture supernatant in step S2, and removing cells and cell debris therein with a 0.22 μm filter membrane.
[0044] S4. The exosomes are separated by ultracentrifugation, and the following steps are specifically adopted:
[0045] S4-1. Centrifuge...
Embodiment 2
[0048] Example 2, a method for preparing and separating exosomes from human skin fibroblasts, differs from Example 1 in that in step S1, human skin fibroblasts are cultured by means of suspension culture, as follows:
[0049] S1, to common adherent culture bottle (bottom area 175cm 2 ) was added with DMEM medium and low serum growth agent, and 1 million human skin fibroblasts were added, and cultured in a 5% carbon dioxide incubator at 37° C. for three days.
Embodiment 3
[0060] Example 3, a method for preparing and separating human skin fibroblast exosomes, differs from Example 1 in that in step S2, the following method is specifically adopted:
[0061] S2. Replace with serum-free DMEM medium, continue to cultivate for 8 hours, then transfer to an anaerobic incubator with a partial pressure of carbon dioxide of 5%, and cultivate for 2 hours, then pass into the anaerobic incubator to account for a total partial pressure of 0.05%. Nitric oxide, continue to cultivate for 10h.
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