Hello everybody,
Two days ago I performed an isolation of adipose-derived stem cells. I think almost sure I did everything properly. But I observe the cells after more than 24 hours (let's say 36 hours more or less) and I did not see anything attached (I do not know if when I change the medium after 72h I will see something, but I do not think they go in a good term). However It is the first time that I do this isolation and I do not have idea.
My principal doubt is about the transport, because the samples cannot been procesed directly. What we did is taking them in 4ºC vials with DMEM 3%P/S, However I believe the biggest mistake that we made is the thermal shock (But I read 4ºC for their transport/preservation). So I think personally, the best way I guess is keep the vials at room temperature for the moment of the surgery, then I have several doubts how to proceed, I thought 3 options:
- Take the samples at room temperature, storage them at 4ºC (in this way they will get colder slowly) and then perform the extraction
- Take the samples at room temperature and transport them at room temperature to make the isolation when they arrive directly.
- Take the samples at room temperature, storage them at 4ºC (in this way they will get colder slowly) and leave them again getting warmer at room temperature 24h before the extraction. (However in this point I am worried about the time).
The transport last around 5-6 hours.
Thank you very much in advance,
Sergio.
Dear Sergio,
in my opinion the temperature of 4°C from the obtaining up to culture beginning may be the best, because the conditions in the tissue before individual cells isolation are strongly hypoxic. Was the temperature in your experiment really 4°C? The freezing or the higher temperature may be detrimental.
Best regards
What we did exactly was: put the fat inside a vial at 4°C (the vial was already cold previously) and then in the lab I performed the extraction. I had all the time the doubt about the temperature... for washing after arriving at the lab under the hood...etc because the collagenase digestion is at 37°C and it is another great change (of course after this I used always 37°C tempered media for resuspension, centrifuges... until the culture).
If everything was ok, my other concern is the mince because maybe I performed it too strong.
Thank you for your answer :)
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