I'm using porcine bone marrow-derived mesenchymal stem cells and when I have excess cells, I freeze them down. When I thaw them again for the purpose of conducting experiments like differentiation, culturing in scaffolds, etc., must I allow them to recover first before using them? Or is it okay to use them immediately? And how do you advice me to allow them to "recover"? Thank you very much,
To summarise the different points that myself and others have made: part of the reason for not using cells directly is because it is necessary to remove DMSO and dead cells that might affect your seeding density calculation, and the other part of the reason is to allow cells to re-establish normal cell cycle. It's strongly advised not to use them directly, but to passage them at least twice.
Thank you Jianhua for the answer. Actually the cells I freeze down are at passage 2-5. In this case, when I thaw them, can I use them already? Thank you.
I agree with Kevin, it's not advisable to use them until they have been passaged at least twice, since they need to recover and resume their normal cell cycle. Using them before recovery will affect their rate of metabolism and proliferation.
I prefer to split cells at least 2 times before to use them, so they can adapt and growth better
I agree, passaging twice is a good practice. But it also depends on whether you can afford this given the limited passage number available.
Another important often neglected factor is to thaw them at sufficient high density in he first plating. You do not want to select for cells specialised in growing at low density (We study non-genetic phenotype heterogeneity and this is a property that varies from cell to cell greatly)
Sometimes I used the cells after thawing after 2 days in medium (it was the time for recovering), but I didnt wait for splitting (I wanted to use cell with the lowest passaging number as possible) - it was ok for me, it worked, but like others say - it probably could affect metabolism of cells in some way, and experiments in some cases.
one more problem if you directly use thawed cells for experiment is there might be a high percentage of dead cells, which is not good for controlling cell seeding density.
Hi Christine,
You have already cultured your cells 2-5 passages before freezing them down, it is important to use these cells at low passages in any experiments, specially for differentiation, because primary bone marrow-derived mesenchymal stem cells undergo senescence at higher passages . About your question; Culture your cells 2-3 days after thawing and then start you experiments, please remember negative control (and positive control if you have). I have used this methods for many years. Good luck.
I agree with previous comments. Immediately after thawing cells are still stressed with all consequences (many dead cells, uneven reaction to any kind of manipulation including stimulation of differentiation, etc.). Let them recover
To summarise the different points that myself and others have made: part of the reason for not using cells directly is because it is necessary to remove DMSO and dead cells that might affect your seeding density calculation, and the other part of the reason is to allow cells to re-establish normal cell cycle. It's strongly advised not to use them directly, but to passage them at least twice.
Thank you so much everyone for all your valuable insights and answers. I appreciate them all very much.
A little known fact with sparse but existing evidence in the literature is that a proportion of thawed cells undergo apoptosis within 48 hours post-thaw. This is likely caused by minor ruptures of mitochondrial membranes causing a leak of cytochrome c triggering apoptotic events.
Bone Marrow derived MSCs lose thier multipotentiality with increasing passage, and have to be kept subconfluent. Like all cells, they need at least 1 passage to recover from the freezing process. This means you need to freeze the cells down at passage 1. If you need to go to P3 or 4 because of required cell numbers, its better to do these passages after freeing and not before.
- Badges
- Science method