Method for culturing odontogenic stem cells and separating exosomes
A separation method and exosome technology, applied in cell dissociation methods, cell culture active agents, animal cells, etc., can solve the problems of high cost, difficulty in extracting exosomes, easy contamination, etc., and achieve simple operation and saving Material and financial resources and energy consumption, the effect of saving reagents
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example 1
[0043] Exosomes from the culture supernatant were extracted from DPSCs.
[0044] In this example 1, exosomes were extracted when the DPSCs that had been passaged to the fourth generation were cultured until the cell confluence reached 90%, such as figure 1 shown. The main steps are as follows:
[0045] 1) Prepare the medium α-MEM+0.5%BSA for exosome isolation and store at 4°C;
[0046] 2) Replace with the medium for exosome isolation one day before exosome collection;
[0047] 3) After 24 hours of culture, collect the cell supernatant. For maximum recovery, you can wash the culture dish twice with sterile PBS, and use the PBS washing solution + cell supernatant as the object of exosome isolation;
[0048] 4) The cleaning solution from the previous step was centrifuged at 4°C at a differential speed of 3000*g for 20 minutes to remove cell debris and dead cells. If the amount of liquid was small, it could be frozen to -80°C refrigerator and extracted after the liquid was suff...
example 2
[0053] The shape, size and surface marker proteins of the exosomes prepared in Example 1 were detected.
[0054] Experimental method: Exosome photos were taken by transmission electron microscope, NTA was used to detect the size of extracted particles, and weston blot was used to detect surface marker proteins.
[0055] Experimental process: 1) Drop 20 µl of the exosomes prepared in Example 1 onto the sample-loading copper grid (pore size 2nm) and let it stand at room temperature for 3 minutes, then absorb the liquid with filter paper on the side of the copper grid, and add 3% tungsten phospholipid dropwise Negatively stain the sample with 30 µL of acid solution (room temperature, 5 minutes). Use filter paper to absorb the negative dye solution, transfer the copper grid to a transmission electron microscope (TEM), observe and take pictures. 2) The nanoparticle size tracer experiment was completed in Guangzhou Ruibo Company. The steps are as follows: adjust the parameters of t...
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