For a while now I've been trying to unfreeze some iPSCs which were growing happily previously.
However, now whenever I unfreeze them, they will attach and seem to form some colonies, but after 2-3 days will start to die.
It used to work perfectly before, so is quite frustrating.
For technical specs: I'm using E8 flex, and using Revitacell during unfreezing, and changing media 1 day and then 3 days after. I'm using 60ul of corning matrigel diluted in 6ml of DMEM/F12 as a basement membrane.
I've tried newly purchased E8, and tried increasing the concentration of matrigel, and also adding in antibiotics, but to no effect.
My biggest suspicion right now is that my matrigel is the issue, although it is only about 6-7months old, and was aliquoted and stored at -80C. I'm currently waiting on a new delivery of geltrex and am hoping this will fix my problem (although delivery times are awful thanks to either Covid or Brexit), but in the meantime does anyone else have experience with this problem?
Any suggestions/input would be greatly appreciated!
Also attached a couple of pictures of the cells, 2 days post-freeze (looking okayish) and then 4 days post-freeze (dying off).
Hi! From your description and pictures it seems that the initial quantity of attached cells is insufficient. With iPSCs, it is always best to plate as many cells per square cm as possible. Try smaller dish, or two vials instead of one. No need to worry about "too many cells", it will not happen, as they don't grow on top of each other. Just unfreeze MORE cells per dish, 2-3 times more.
P.S. If there are really many cells, they may not even require matrigel sometimes. In case of iPSCs, the success of culture really depends on cell density.
Hi Yulia! Thank you very much for your suggestion!
I took your suggestion and tried plating out one vial into one well of a 12 well plate (rather than a 6 well plate), giving me a much higher seeding density.
Unfortunately, I still seemed to encounter the same problem.
Thank you very much for your help anyway! :)
Hi Darius McPhail !
I too have encountered a very similar problem, were you able to solve?
may I kindly ask you where did you get the cells and the material? what concentration do you use the coating? have you never tried Geltrex?
you would recommend Revitacell?
Thank you!
Hi Irene Bissoli
Unfortunately, I was unable to solve the issue. Even after trying a range of coatings at different concentrations, my problem remained the same! I also tried the suggestion made here by Yulia, but this was also not able to fix the problem.
I think that something must have gone wrong during the freezing process, or the cells may be too old, or some other issue that I am not aware of.
These cells were a donation from a collaborator's lab.
To answer your last question, in the past I've found that revitacell is quite good for unfreezing stem cells. It tends to give them slightly better post-thaw viability versus just using y27632 (when talking about stem-cell culture prior to the issue mentioned in this thread).
Sorry I can't help any further! :)
