I used differential ultracentrifugation to isolate exosomes from cell culture medium(osteosarcoma 143B cultured with 10%exosomes-depleted FBS,24h). But after 100,000gx70min(beckman 70Ti rotor,almost 40mL condition medium) ,I cannot see the pellet. and finally I get low yeild exosomes.
What can I do to increase production of exosomes???
The pellet depends on the cell line used to isolate exosomes. You can't really see a pellet for cell lines such as HEK293T, HUVEC or HDMEC. However, I saw a huge pellet when I isolated exosomes for B16 melanoma cell line. Even if you don't see the pellet, the exosomes are still there.
If you want to increase the production of the exosomes, you should mark a side on the bottom of the ultracentrifuge tube, and face that side outwards prior to ultracentrifugation. Once centrifugation is done, you can focus resuspending around the marked area where the exosomes are present. You could also try adding 1XPBS on the ultracentrifuge tube and let it sit overnight at 4°C in order to increase your yield. Hope this helps.
Use more cells or collect conditioned medium for a longer period of time.
You almost never see a pellet using the ultracentrifugation approach. The only times I see pellets (and get higher yields) is with precipitation based techniques. However, I have seen literature stating that exosome production can be enhanced when cells are cultured in more acidic conditions.
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