Dear Colleagues,
I am having problems with resuscitating cryopreserved iPSCs. Upon thawing I get quite a good survival rate, but all my colonies look completely flat, without smooth borders and with fibroblast like appeareance. Can anyone help me troubleshoot please?
My iPSC are derived from PBMC using episomal plasmids. They are cultured on MEF and passaged using Dispase II. My freezing protocol consisted in enzymatically detach colonies and allow them to sediment by gravity. Removed the MEFs from supernatant, washed pellet twice with iPSC medium and resuspended in 500uL of Freezing media A (50% complete iPSC medium +50% KOSR). Then 500uL of Freezing media B (80% iPSC medium and 20% DMSO) were added dropwise, and the whole solution transferred into a cryovial. 10uM ROCK inhibitor was then added and vials placed in a controlled cooling rate container overnight.
The thawing procedure consisted in quickly defrosting the vial in a 37oC waterbath until one ice crystal remained. 1mL of iPSC medium containing 10uM ROCK inhibitor was added in a dropwise manner directly to the cryovial and the whole solution subsequentluy transferred into a 15mL tube. Additional 8 mL of iPSC medium was added to the cells in a dropwise manner. Cell were then spun at 800rpm for 2 minutes, supernatant removed and pellet washed twice in iPSC medium containing ROCK inhibitor. Ultimately cells were resuspended in complete iPSC medium supplemented with ROCK inhibitor and transferred onto a MEF coated dish.
Attachment was observable after 24h, but at 48 colonies appeared extensively differentiated. After 3 days colonies looked completely differentiated. See pictures attached.
Any comments or suggestion?
Many thanks,
Dario
I think your freezing medium may be a problematic one. Use commercially available freezing medium or try with 90% of FBS and 10% of DMSO.
Good luck!
I know it looks a very elaborate way to freeze cells overcomplicating everything when I could have just used 90% FBS and 10%DMSO. This was a recommendation of Life Technologies, which they also teach at their workshops. It was meant to reduce exposure time of cells to DMSO while providing a good cryoprotectant. I wasn't too sure to be honest but now that you think this may be the problem I am starting to trust my initial thoughts about this formula. But I thought if the freezing media was a problem I would have had a problem with survival, why would you think that this medium is causing differentiation? Many people use the 50% medium 40%KOSR and 10% DMSO combination yet they don't have issues? Many thanks for your help
The intention of the recommendation to add half volume media followed by 20% DMSO has nothing to do with reducing DMSO exposure time. The purpose is to allow the gradual addition of the DMSO in order to reduce osmotic shock. Hopefully you are adding the 2xDMSO slowly and with mixing.
There is no benefit to using 90% FBS unless your intention is to waste money.
Are you using cell culture grade DMSO?
In order to eliminate the question of whether or not your cryopreservative is at fault, go ahead and buy a commercial version, such as mFreSR, to compare with what you currently are using.
When you thaw the cryovial, immediately transfer the contents to the 15ml tube and then begin the slow dropwise addition of the media. Your version of adding a ml to the vial is not very helpful because it is almost impossible to adequately mix the added media with the contents of the vial.
In general, I think you are doing way too many washes before and after the freeze. If you learn to time it right, you can remove the dispase, gently wash it once in the plate, remove the PBS, and add iPSC media prior to scraping or pipetting for removal from plate. Only add the volume of media that will represent your half volume of final cryopreservation media volume. After thaw, one wash is sufficient. Centrifugation post thaw is very traumatic for these cells.
There is no need to wash out the feeder cells prior to the freeze, though I do agree that your plated feeders are too dense.
I am concerned that you get "normal" colonies that survive the thaw but then differentiate. That is odd and I don't have a good explanation for that, and for that reason it is possible that nothing I have suggested will make a difference.
Hi Dario,
I culture feeder-free hiPSC but what I see compared to my initial experience on the feeders is that they look too dense. As far as I know, iPSC grown on feeders are supposed to push away the feeder layer and grow in between them. Yet from your pictures, it looks more like the iPSC is on top of the feeders (because they are too dense to be pushed away?). This might cause iPSC not to get enough chemokines or some signals from the feeders. So I would suggest decreasing the feeder intensity, and maybe using the commercial freezing media mFeSR (which I use and have no problems so far). Good luck.
There seems to be a flaw in logic in this answer.
Dario, Did you culture the cells BEFORE the freeze in the same manner as you did AFTER the freeze? I cannot imagine that you would do otherwise. Have you done iPSC culture with more than one line? For how many passages did you culture them?
Your feeders certainly are too dense. They do not get pushed out of the way but instead, for the most part, colonies grow on top of the feeders. However, normally low density feeders yield flat colonies like yours, while high density feeders normally lead to colonies that have more of a 3D nature. Your colonies do not look "right" for the feeder density.
You are going to have to use a basic scientific approach with controls to determine if the problem is the culture conditions or the freeze.
Thanks Ron. Yes absolutely, nothing has changed from before and after. I do culture more than one line. In fact the non-patient cotrol lines are fine after thawing. Obviously even then I see some differentiation but its expected, and after a few weeks in culture and manual selection they recover ok. These patient cells were cultured up to passage 5 before freezing, but then so were the control cells. I know that MEFs look dense but to be honest this is what I found to be the optimal density for my iPSC. With lower counts they don't cope as well. At the moment I am culturing at 50,000 cells/cm2. I really cannot think of anything that could have gone wrong. I will try again without centrifugation and without the extra wash as you suggested, but apart from this everything is pretty much standard. Besides I applied methods I have been thaught by Life Tech experts which I have seen working. In past I used 90%FBS and 10% DMSO to freeze them and manual scraping without dispase to lift them, and they were absoltely fine. Now that I have changed protocol to make it more efficient things started to behave unexpectedly for my patients' cells. Really don't know what else to try
PS: I also checked for mycoplasma contamination and cultures appear to be negative
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