Hi everyone,
I've been recently culturing NIH3T3 cells. After a round of trypsinization and freezing, the thawed cells seem a bit unhealthy. I see random black clusters of cells approximately after 24h of culture, which seem to be dead cell clumps. While I change the medium frequently (like every day), the black clusters are reduced in frequency but a few still seem to be there even after extensive washing (3cc PBS for a T25 flask, x2). They again accumulate after a few hours.
I assume there must have been a problem with trypsin, despite double checking the concentration which was 0.25%. I also kept the centrifugation speed for thawing quite low (about 200 g for 10 minutes); I am pretty confused.
Has anybody had the same problem following trypsinization? And what is your suggestion to revive the living cells in culture and eliminate the dead ones?
Do not centrifuge. Add complete media and then after a day when the cells are adhered wash with 1XPBS and add new media. In this way you may minimise the cells which are already under a lot of stress due to freezing/thawing.
Also be careful how you add the media - don´t add the cells to the media, add the media to the cells slowly in single drops. As for the serum, I found the cells preferred calf serum as opposed to foetal serum.
Thank you very much for your reply V H Krishna Prasad.
Wouldn't the remaining DMSO hurt the living cells then?
Thank you very much for your reply Diana Baxter.
I did the exact same thing while thawing the cells, but thanks for the point regarding calf serum.
By the way, does anybody think this might be a contamination problem? Like yeast contamination?
Unfortunately, I could not recover the cells, and after about 36h of changing the medium, almost all of the cells died. I'm worried if this has been a contamination problem I might as well lose my other cell batches.
Hello Mina Hosseini
After the cells have properly adhered ( that would take around 12-20 hrs depending on the cell line), then we may remove the media from the flask and wash it with 1X PBS 3-4 times to remove all the dead cells and DMSO.
DMSO has very low toxicity to cells. However, its effect increases according to its concentration.
I agree with VH Krishna & Diane’s advise as to handling freshly thawed cells.
Is it possible there is a problem with your freezing media? You could check viability after thaw with Trypan Blue. Also, I have noticed NIH3T3 cells are sensitive to less that 37C temps, so make sure your liquids are entirely warm. Best of luck!
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