I have an easy answer on that.... it depends if you are talking about freezing cells on culture plates directly or bulk freezing of split cells.... for the first it is better to have feeders... eliminate media, add a drop of recombinant trypsin with rock inhibitor and wait a minute... aspirate this carefully and add half of the volume of the well of freezing medium, which is cold 90% serum and 10% tissue culture DMSO... directly to -80 and recovery is above 70%...  recombinant trypsin is not inactivated with serum and if you are using KO SR you don't need to wash it before adding trypsine...

if you are bulking up, no feeders and cells from matrigel cultures are better.... trypsinise as above or scrape or use collagenase... any method will work with viability over 90% if the slow freezing method is applied...(with a mr frosty or similar...)

Thanks but I don't understand some points. First, the serum in freezing medium are normal? Because than you speak about KSR .  Second, this recipe is used for both case:hIPSC with feeders and hIPSC on matrigel?  Third if you use trypsin are you forced to  thaw out on feeders? Because IPSC doesn't live at single cell on matrigel... thanks

I agree with Maria Vicenta Camarasa , she provided  a full answer .

see the attachment

Good Luck

Thanks, but  I don't care commercial products.  I prefer a cheap freezing medium. So I will try  90% serum and 10 % of DMSO.

Just as a follow up from the previous notes, although I guess you did some freezing by now.

You can freeze iPS or ES cells in 10% DMSO combined with any serum or serum containing medium (FBS or KO-SR or hESm). I personally would recommend to use KO-SR or hESm as they are more defined and less variable than just FBS. This is the same for cells grown on feeders or feeder-free.

In terms of which combination is best, I should make a few extra notes. You will find that the freezing medium by itself is not making a major difference, but what really makes a big difference is the following: 1) How you dissociate your cells - best get smaller clumps as the freezing is more homogenous. 2) How you thaw your cells when they are small clumps - add Rock inhibitor during thawing. 3) Keep them at -150C and don't leave them at -80C for more than a day.

A paper that we published a while back might help:

1. Martin-Ibanez R, Unger C, Stromberg A, Baker D, Canals JM, Hovatta O. Novel cryopreservation method for dissociated human embryonic stem cells in the presence of a ROCK inhibitor. Hum Reprod. 2008;23(12):2744–2754. doi:10.1093/humrep/den316.

Again, the freezing medium works best on small clumps or single cells, but since they do not like to be single cells you need Rock-inhibitor for thawing.

Another thing that makes a big difference is seeding density. For the same reasons. If you thaw them and seed them at a very high density (10^6 cells/cm2), they will immediately have neighboring cells and have a much higher survival rate. A very nice technical note by a Japanese group.

Reference: 1. Miyazaki T, Nakatsuji N, Suemori H. Optimization of slow cooling cryopreservation for human pluripotent stem cells. genesis. 2014;52(1):49–55. doi:10.1002/dvg.22725.

Hope that helps.

Thank so much!! Unfortunately I left some days at -80°C I hope not to compromise everything...

"I prefer a cheap freezing medium. So I will try 90% serum and 10 % of DMSO."

I cannot imagine why anyone would consider a freeze medium consisting of 90% serum to be cheap. I never have found a case in which straight DMSO and serum was necessary.