Hi Felipe,
For protocols related to human ES/iPS culture, I would highly recommend the protocols at the following link:
https://www.wicell.org/home/support/stem-cell-protocols/stem-cell-protocols.cmsx.
You can select the protocol for both feeder-free and feeder independent versions.
To answer your question: In general, you don't treat the cells with Rock inhibitor while passaging. You should treat the cells with ROCK inhibitor (concentration in the protocols) for 2-3 hours only before you plan to freeze them. This inhibition helps them survive "better" as single cells when you thaw them.
Hope this helps.
Good luck!
Utkarsh
HI Utkarsh,
thank you. What if you´re passing in single cell? Would you use?
Best,
Felipe
In that case, I most likely would use ROCKi ! This would increase the probability of their survival. Else, they are more likely to die or differentiate. It is very normal for human ES/iPS to die as single cells; this is the reason why they are passaged as clumps.
I have not done assays where I intentionally plate them as single cells (which I believe people would do for picking over-expression/CRISPRed clones). I don't have more experience regarding this.
I guess people who make human ES cells of a particular genotype (shRNA/over-expression/knock-in etc) would know about this issue. I suggest you get in touch with such companies/facilities who do this for a living.
Best,
Utkarsh
Hi Felipe,
I use ROCK inhibitor each time I split iPSC, and add it into the media for seeing the cells in the new plate. It is sufficient to use it only for the split, to increase the chance of survival (+of single cells) - so you will not need to use it the day after splitting.
Good luck.
Hi Felipe, I use ROCKi when thawing iPSCs and also when plating them as single cells. I only add ROCKi when plating them, not before. Concentration: 10uM.
Hi,
I always add a ROCK inhibitor to the medium per 24 h after thawing iPS cells or if I generate embryoid bodies. At the next day, I change the medium on fresh without ROCK inhibitor. I never use ROCK inhibitor to iPSC passage.
Justyna Augustyniak
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