I am wondering if anyone has problems thawing embryonic or induced pluripotent stem cells. In particular, I am finding that after two or three days post-thaw that the feeder layer becomes so dense that it chokes out the colonies that would have developed. At the moment, I freeze down with feeders as these cell lines are not yet adapted to a feeder-free system. If I get such high survival of feeders from my thawed vial, do I need to thaw on feeders? Any advice?
THANKS!
Hi Leigh,
You saw feeder cells grow? Did you mitotically inactivate your feeder cells? As if you dnt like you saw, feeder cells will overgrow the ES cells. Try inactivating the feeder cells with mitomycin C and then seed your ES cells.
All the best. Hope this helps.
Regards,
Prasad.
Hi Leigh,
You saw feeder cells grow? Did you mitotically inactivate your feeder cells? As if you dnt like you saw, feeder cells will overgrow the ES cells. Try inactivating the feeder cells with mitomycin C and then seed your ES cells.
All the best. Hope this helps.
Regards,
Prasad.
First you should inactive your MEF cells then thaw and seed your ES cells
Agreed. You should inactivate your feeders. When splitting your cells, you could also let your pieces sink, while Mefs float. If you do this for a few passages, the MEFs will exhaust Hayflick's limit. If I were in your position I'd use irradiated or MC treated Mef, while using 1/2 Mef CM (F12/KSR), with 1.5x bFGF. Moving to a feeder free system is great if you can afford it. I grow hESCs in several systems, but one that is looking good is Lonza's L7 system. Adaptation from feeders is robust. Cells grow very well, and differentiation is not affected In my hands. If you are not in a position to pay Lonza's high prices, you can always use Mef conditioned medium. It's a very common way to grow hESCs and works quite well. Matrigel and mTeSR are a good standard in the field. In the end, it really depends on what you are trying to do with your cells. What are your basic goals? Differentiation, molecular mechanisms of pluripotency...etc? If you give us an idea about your work and budget, we can make some suggestions to help you make a decision.
Regards
Thanks everyone.
I am using inactivated MEFs but I seem to get a high MEF survival when I thaw out the ES cells which is compounded by two issues as far as I can see. The thawed MEFs add to the fresh MEFS creating a dense feeder layer and ES cells may be differentiating to fibroblast-like cells.
I am able to get some iPS lines to a feeder free system (matrigel + mTESR) but other iPS lines are challenging to get to that point especially if I cannot thaw them first! We get these iPS lines made through a facility that hands them to us as frozen stocks so it is imperative I can grow these from frozen.
Has anyone thawed ES cells (originally frozen on MEFs) into MEF conditioned media?
Thanks again!
We usually thaw/unthaw our pluripotent cells in MEF conditioned media with acceptable results. May I ask you how you are managing to freeze your cells right now?
Just use E-8 or m-Tesr1 medium on matrigel coated plates directly to thaw iPS- no need to use Rock inhibitor and try to minimize the pipetting during the thawing process. We never ever had problem in thawing human iPS lines made in our lab.
good luck
Hi Leigh,
When you freeze the cells, do you use trypsin or collagenase? I would suggest you use collagenase to get your colonies, specifically use 1mg/mL in basal media, filter and add enough to cover your cells, leave in incubator for 45 minutes to an hour. Only the ES colonies will be floating. Pick up your ES colonies gently, do not pipette the collagenase, just aspirate the colonies. Use this for your transfer/ freeze.
Also, generally, if you have Mitomycin treated or irradiated feeders they do not grow, even in transfers they generally will float and are eliminated during washes. I strongly feel that it is your ES cells that are differentiating. Can you put up a picture. The only time that I have seen a culture with a thick feeder layer is when it was an irradiated feeder layer, wherein the irradiation was not sufficient.
Also, as suggested by Ioaniss and Ashutosh, you can use matrigel and mTeSR. If you really want to just grow with MEF conditioned media, you have to coat your plate with first matrigel or porcine gel, seed your cells and add half fresh media and half conditioned media everyday.
Hope something works!
-Kavita
Hi Leigh
There´s a simple test you can do to test your feeder´s inactivation. In my lab we usually seed 250-300 thousand in a 10cm dish and wait 4 days. If the feeder is not completely inactivated you´ll see it. I think the standard for mitC treatment is 0,05mg/ml for 3h. You can go lower in time or concentration but you might get some variability in the inactivation.
When you freeze your cells you can also pick them up mechanically (like 1 grown colony to 8-10 pieces) and freeze it like that. It usually increases survival.
It can also depend on the freezing method. Are you using a Frosty (isopropanol method) or a cryofreezer?
Hope It helps.
Best
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line