I was asking myself why we usually change the media just the day after thawing the cells. It seems that the two reasons most of the people exposed are:
1. To get rid of the DMSO leftovers.
2. To remove the dead cells floating in the media as soon as possible.
Without entering to discuss whether the small amount of DMSO in our media could be affecting cell proliferation or not, I was wondering about the necessity of getting rid of the dead cells. Apoptotic cells are known to release factors that increase wound healing and cell proliferation both in vivo and in vitro.
Do we have any research that supports that these dead cells could not be beneficial for the first days of culture after thawing?
Hi Julián González Rubio . It would make sense to change media if there was a massive die off after thawing. I can't imagine that it benefits live cells having chunks of cell debris sticking to them.
Hello Julián González Rubio
It is necessary to replace the medium with fresh medium the day after thawing for the two reasons you mentioned above. In addition, I would like to mention that presence of dead cells in culture would in fact harm the viable cells who are already in stress due to the process of thawing. The dead cells would release their DNA into the medium and DNA being sticky in nature would cause the formation of cell clumps. The viable cells would not be exposed to nutrients in the culture medium. Moreover, the metabolic waste released from dead cells would make the viable cells difficult to survive who are already in stressful condition. This would cause further loss in percentage viability.
So, I would suggest you to follow the usual protocol of changing the culture medium the day after thawing so that the cells recover from stress and proliferate.
Best,
One additional point is that the apoptotic process releases nucleases, and proteolitic enzymes into the media. They also make it more acid.
It is usually recommended to spit the cells ( 3000 rpm, 3 min for cancer cells) from -80 degree, discard the supernatant and resuspended the pellets into the fresh media to remove the DMSO that affect the cells viability. Moreover it is really necessary to replace the medium to remove the debris and dead cells because the presence of dead cells would definitely affect the viability (decrease the percentage of viability)and consequently could induce the apoptotic effects on cells and affect the negative control during your experiment.
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