Hi,

You should add ROCK inhibitor or RevitaCell Supplement (100X) to fresh E8 medium for a period of 24h after thawing hiPSC cells.

Good luck!

ROCK inhibitor exposure for one hour prior to the freeze helps thaw survival. It has very little effect on survival when added at the time of thawing.

You have two potential sources for problems. Either your geltrex coating or your thawing is at fault. If this is your first contact with iPS work, then you have no iPS cells available to test your geltrex. Is there anyone else nearby who has iPS cells in culture? If you could obtain a fresh stock from someone else then you could use that to determine if your geltrex coating is good. Are you keeping it cold right up until it is transferred to the plate? Not doing that seems to be the most common mistake.

I cannot really assess your thawing procedure without knowing what it is.

Hi Anirudh,

Thawing can definitely be a challenge sometimes. I’m not sure which protocol you are following, but make sure to add the medium to the thawed cells slowly so as to not cause osmotic shock the cells. Also, in general, we recommend a different version of Geltrex for iPSC culture with Essential 8 Medium (Catalog # A1413302) or Vitronectin (Catalog #A14700). For improved survival, you may also try adding RevitaCell Supplement (Catalog # A2644501) and/or even replace the Geltrex with the more robust rhLaminin-521 matrix (Catalog # A29248), at least during thawing. What cryomedium was used during cryopreservation of the cells and what medium system were they grown in ahead of cryopreservation?

Best,

Rhonda

Hello everyone and I really appreciate your answers for my question.

Hi Ron, this was the first time I thawed the cells which I obtained from the vendor so I have no idea if it was supplemented with RevitaCell before cryopreservation. The problem was that there is no other lab in my institution working with iPSC's. Yes, I have maintained the geltrex solution and also did the coating procedure on ice.

Hi Rhonda, thank you very much for your recommendation of alternatives to ready-to-use geltrex solution. I followed the thawing procedure as mentioned by catalog supplemented with GIBCO human episomal line (Catalog# A18945). Also I have supplemented the essential-8 medium with RevitaCell (1X FINAL) as mentioned in the protocol. I think my problem was specifically due to the basement membrane because not even a single cell adhered to the coated well. I will definitely try your suggested alternatives and hope it works. Also, as an extended question, do you want me to follow the thin gel method if I use the geltrex which you suggested (Catalog # A1413302).

Further comments and suggestions would be much appreciated!

Thanks,

Anirudh

I use that version of Geltrex as well. The density that I use most frequently is 1mg per 100mm plate. This of course would translate to 1mg per 3 60mm plates or 1 tissue cluster whether 6 well, 12 well, etc.

If the freeze or thaw was done incorrectly, then complete failure to attach also can happen and no improvement in the matrix will change that.