Freezing protocol and Thawing are correct,and viability was 80 percent.
DMSO was diluted and removed.
Cell medium was warm and 37 degree.
Ensuring that the cells are viable, one should investigate whether the culture medium is at fault. Typically, medium that has been left standing for too long, lacking glutamine, can cause cells to exhibit poor adherence. Trypan Blue staining can assist in determining whether the cells are alive after resuscitation. Good luck.
Hello Mahsa Daneshmand
The viability for most cells declines and reaches a nadir at 24 hours post-thaw. Most, if not all, of this decline appears to be due to apoptosis (as opposed to necrosis) induced by the stress of the cryopreservation process. After this time point, cells begin to recover and enter exponential growth. Give the cells a day or two more to recover from stress. Usually, 24 hours should be sufficient for adherent cells to attach to the substratum after thawing.
Also, you mentioned that you have used pre-warmed media, which is right because moving from a sub-zero environment straight to room temperature will put cells under a lot of stress. Therefore, it is important to make sure that your cells are put into pre-warmed media right after rapid thawing.
If you have provided an appropriate culture surface, then the most common cause for failure of cells to attach to the substratum is environmental stress. Stress on cells in culture is mediated by biophysical conditions, or by components that are either present or formed in the media. I hope you are using the right media for the culture of CHO. Ham’s F-12 was originally developed for the growth of CHO cells.
Finally, mycoplasma contamination may also be the cause of the problem because mycoplasma contamination can affect many properties of the cells such as function, growth, metabolism, morphology, attachment, and many other properties.
Best.
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