Dear all,
Does anyone know if freezing exosomes in -80 affects their structure? I mean, whether closed vesicle structure will not be damaged and will be fully preserved with bilayer tightness and without any leakage?
many thanks for help.
I would say that depends on what you want to do. In the field the discussion about exosomes definition and isolation is still controversial. How do you isolate it? what you want to do with them after you freeze them down? If you want to study their surface i think that you should just try to do the experiment in which you test the same sample fresh and after you freeze your exosomes down and see if you obtain the same results. If you want to study the proteins contained inside them you should not have problems freezing them. Luca.
Basically, I plan to isolate serum from blood, freeze it in -80 and subsequently obtain exosomes. Finally, miRNA from inside of them will be isolated. The question is if keeping serum in -80 affects exosomes tight structure?
thank you. Damian.
miRNA is conserved in the -80. Yes, you will be able to isolate you exosomes after froze down the serum and isolate the miRNA from them. If you want to make sure try do an experiment as i suggested where you test it on the same sample one aliquot fresh and one aliquot frozen (same size aliquots). Cheers.
ideally the exosomes should be processed fresh. keep in mind- many things damage EVs: ultracentrifugation, repeated freeze/thaw cycles, preparation for EM... But a single -80 freezing should not be too bad
Again Alexander we don't have any idea how he is sorting out the exosomes. There are many different approaches, some of them they don't even use the ultracentrifuges. But in any case the ultracentrifuges damages both fresh and frozen exosomes! But you will see them if you stain them and you analyze them with surface markers. Again a double experiment on same sample fresh and frozen will allow him to understand the performance of his technique.
At first, please accept my thanks for your help and information you have provided. I do plan to obtain exosomes by ultracentrifugation. Unfortunately, I need undamaged exosomes, because I am interested in their inside content. Is there any other not as invasive as ultracentrifugation method for exosomes isolation?
Freeze-thaw may induce clumping and flattening - processing for traditional TEM will induce cup-shaped morphology, whereas cryo-TEM preserves shape. Serum may contain platelet-derived EVs (up to 50% total) so EDTA plasma may be better depending on your concerns. You might also consider size exclusion HPLC to obtain your final EV for TEM for structural preservation - see pubs attached.
Thank you very much for your help. I will study these article later.
Well, I heard from experienced exosome researchers that you should freeze them at -70oC in presence of DMSO to preserve them. But then DMSO may be a problem for downstream applications. Still if you are needing intact EVs and you want to freeze them that would be an option.
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