Hi,
The attached picture shows my iPSC after 21 hours seeding. 98% of the cell clumps never attached, and many bubble-like structures of different sizes could be seen (which does not exist at the time of seeding). The very few attached cell clumps also look very unhealthy and are seem to be dying.
The hiPSC cell line have been cultured in TeSR-E8 medium with Vitronectin XF, and was frozen using Cryostor CS10 freezing medium from Stemcell. Rock Inhibitor was added during thawing.
I used to freeze and thaw the iPSC without problem, but since last two weeks, after I came back from a vacation, the cells start to die out after thawing. I tried different vials of cells which are frozen in different batches, the results all turned out to be the same. I also tried to change new bottles of E8 medium and Vitronectin, but made no difference.
I'm wondering what are these "bubbles" and what else I may do for trouble shooting.
Thank you.
Hello Zhaoming.
Bulged membranes are typical sign of cell death caused by swollen membranes due to hypotonic solution (low osmotic pressure in the fluid), ruptured and lost nearly all cytoplasmic contents. They look like translucent membrane bulges.
Set the following experiments
1) Try make a brand new solutions for freezing and washing after thawing.
2) Run the old solutions along with above new solutions with same batches of cell mix.
3) Control standard freezing and thawing procedure with any mammalian cells without using vitronectin.
Run three groups together. The results should give you a clear answer.
Lastly, the rest of objects in the picture, the clumps indicate lots of dirty aggregates formed by damaged cells from freezing thawing procedure and contaminated solution containing microbes and particles, too.
Good luck.
Dear Prof. Lee,
Thank you for your suggestions!
I have tried 1) and 2) together, however no much difference was observed.
I'll prepare some other cell and try 1) and 3) next time.
Many Thanks,
Zhaoming
Hi,
I know it was a while ago but I'm wondering if you were ever able to figure out what the problem was? I am having this exact same issue with my iPSC cultures right now and am struggling to get to the bottom of it. Anything you remember would be very helpful!
Thanks,
Katrina
Hi Katrina,
I'm sorry you are also having this issue. It turned out that the cells were not doing well prior to me thawing them. I didn't figure out what I did wrong causing this, I tried some older cell stocks which worked fine and then I moved on without having this issue again.
iPSC/ESCs are demanding and fragile, I had the cells dying because of the CO2 level low (there was a sensor problem with the incubator), because of my colleague carrying out some weird chemical reaction in the incubator, etc.
If the routine iPSC cultures works fine just the thawing, try get different cryopreserve media, use a Mr. Frosty. If even maintaining iPSC is problematic, try new media/coating reagent, a different incubator etc.
Hope this helps!
Zhaoming
- Badges
- Science topic