Dear All,
I am currently performing a stemcell differentiation into neuronal progenitors. However, I`ve realized that the incubator stopped heating yesterday so that my cells were kept under 23 degree for 15h. CO2(5%) and humidity was okay.
There is no cell death, and the cell morphology is still as it is suppose to be. I am in day 6 of differentiation which I need 7 days more.
Only problem was medium was used less than it should be used( color was not orange-yellow enough, indicating cell metabolism was slower).
Do you think that I should trash everything and start differentiation from 0? That will cost me so much time and money, since I am differentiating 9 different colony of stemcell together. Or should I just continue to procedure?
I also had stemcells in the incubator, their morfology was also completely normal, no unspesific differentiation, completely round edges. But less medium usage as well
Best Regards
Yagiz
Don't discard, let's complete the differentiation. As you found no change in morphology or any detachment of undifferentiated or control cells, so no need to worry. Check markers expression after 7 days. Then you will have differentiation results as well as realization that whether CO2 disruption affected your cells or not.
cheers!!!
Temp is important for the enzymatic activity of the cell, reducing or increasing the temp will affect the efficiency of the enzyme.
As you have already noticed, the rate of proliferation (or differentiation in your case) has slowed down. This is because your cells are not functioning at the optimum level.
My personal opinion is you should continue, however you need to take the results with a pinch of salt. You 7 day differentiation schedule will now be around 9 to 10 days (Your cells need to prime again to become efficient).
Depending on the downstream process you can decide if you have to repeat the experiment. I would repeat the protocol with optimum settings again, just to confirm.
It should be Okay to Continue without Trashing them!
Your differentiation sounds like your cells are at neural stem cell stage / neural epithelial induction stage; the meso-endoderm cells at this stage tend survive well as you are nearing the maturation stage for neural differentiation. However, If you are so keen to publish the data from this batch (from IS/WB) I would recommend to just re-check with the specific lines that works now.
Good luck brother!
Regards
Sri
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