I am working on MC3T3-E1 cell line and isolating exosomes from cell culture media by using standard ultracentrifuagtion method. I have characterized with TEM and checked the size and charge with DLS. But most of the TEM grids were empty with no particles, indicating that the exosome yield is very less. Any suggestions to increase the yield of exosomes without affecting their natural property?
Thanks in advance!
Hi Sasmita,
Before you take your precious exosome samples to TEM and have a 'surprise' moment, it might be better to measure the exosome concentration (particles/mL) by Nano-particle tracking analysis (Nanosight, Marvern). If you do not have access to this piece of equipment, you can measure the protein content of your EV by microBCA. These two methods should give you confidence in identifying high-yield batches that can be used later for TEM.
Several things I may consider
(a) Stimulate the cell line with cytokines. Activated cells in general produce more exosomes, you may or may not want to take this step depending on your research question.
(b) Delay supernatant collection before ultracentrifugation. You need to make sure the cell line did not enter apoptosis because of lack of nutrients in the media.
(c) You may want to use a multi-layer culture flask to simply have higher cell densities to begin with.
(d) Extend ultracentrifugation by another hour and see if you might get bigger pellets or better readouts on BCA or NTA.
Good luck!
Hello Tommy Lu, Thank you for your reply. We don't have access to NTA, Nanosight, but yes we have BCA kit. I will check the protein content. But regarding the 4th point you mentioned, I have not seen any pellet after performing two ultracentrifugations or may be I could not distinguish. Do you see pellets each time when you isolate exosomes?
Thanks in advance!
Sasmita Samal
I could see tiny pellets when I polled EVs from a total 200mL of supernatant of actively dividing lymphoma cells. The fact that I used suspension cells easily allowed me to scale up the level of EV production in terms of cell number and media volume. It might be tricky for you since you are growing adherent cells and they tend to stop growing at full confluency due to contact inhibition, hence, you are more likely to obtain low EV concentration. This is why I thought the multilayered flask may be helpful for your experiment. In addition, use microBSA kit not the BSA. MicroBSA is more sensitive to low protein content.You might not get meaningful readouts with BSA.
Purification could be a tricky business at the beginning but you will get there once you know your cells better.
Tommy Lu
Thanks for your valuable suggestion. I had also started with 200ml culture supernatant. But before isolating exosomes, I incubated cells with serum free media for 24 hrs, to avoid contamination from FBS derived exosomes. Do you think that it might be hampering exososme yield? Because with this condition cells must be starving.
I will also try culturing cells with multilayered flasks.By actively dividing cells do you mean cells at 60-70% confluency?
According to Article Human Melanoma-Derived Extracellular Vesicles Regulate Dendr...
some cell lines produce more EVs under hypoxic conditions. They use a very simple method for hypoxia induction.
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