I have passage 1 neurospheres revived from LN2 storage less that 24 hours ago, and have found a peculiar contaminant in the culture. The attached images are of the odd feature under phase contrast and bright field. The contaminant is most often spherical but a few are irregular in shape, all consist of a large vacuole like structure, some appear to be filled or surrounded by granular material. The vacuole structures range from 20-100 m in diameter. Any suggestions would be helpful.
Dear Aaron Gilmour,
Maybe it is apoptotic vesicle! The medium is very clean!
Dear Aaron,
Your neurospheres are looking pretty normal, the medium is clear as Katherine mentioned, it is not contamination. This vacuoles might be the air bubbles in the medium.
Bests,
Amir
Hi Aaron, yes I agree with the others - its not contamination but rather the spheres/cells that did not survive the freeze/thaw process and are in the process of apoptosizing/dying etc. Its normal to see some of this this after freeze thawing and they should clear up after a few days of growth.
Hope this helps!
Hi Aaron,
I do agree with the other comments here. I do not think there is contamination. However, I would like to ask if the neurospheres survive beyond day 2. If they do, you are fine. If they do not, you may have to revisit your freezing protocol.
Hi Aaron
I believe that what you see it is not a contamination. It resembles cellular debries and apoptotic cells...dying cells. It could be possible that cells did not like the freeze/thaw process and now they are suffering for that. So the "contamination" you see in your culture are just apoptotic or death cells, that will be cleared with medium changes. Try to be careful during next cell-thawing process.
Hi all, thanks for all the responses. It is a freeze/thaw issue, after passaging at 3 days post thawing, I have good sphere formation without issues.
Cheers
Aaron
definitely not contamination.
If you used the same media (DMEM F12 + bFGF+ EGF+ etc) then this is only due to the thawing process!
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