Is it possible to wake up iPSC stock in liquid nitrogen directly to feeder free culture or do I have to first wake it up on feeder dependent culture, and then change to a feeder free system?
Yes - buy our product at CET. We have feed-free IPS growth media.
http://celleng-tech.com/index/ipsgrowthmedia.html
You can directly culture the thawed iPSCs to the dish coated with Matrigel.
I would suggest trying to grow them on a variety of coated plates (i.e collagen, Matrigel, fibronectin etc.) and see what works best.
Collagen is dirt cheap so why not try it out, see if it works.
It all depends on how the cells were cultured before freezing, and how well they were frozen and transported. If you only have one vial I would be inclined to thaw it on feeders and then put in feeder-free system. If it was previously grown on feeder-free then there should not be a problem. I recommend the Life Technologies Cellstart-StemPro combo, but I know it is not cheap.
I don't know if you are working with human cells or not, but I suggest you to check this manuscript: Increased Reprogramming of Human Fetal Hepatocytes Compared
With Adult Hepatocytes in Feeder-Free Conditions. Hansel et al., Cell Transplant DOI: http://dx.doi.org/10.3727/096368912X662453
add Rocki in the medium at the Day0 and replace with the ES medium after 24 hours, you will find your ES growing fine in the matrigel.
You should be able to thaw them and put directly in the feeder-free culture, especially, if the cells were cultured in these conditions before freezing. However, they should feel a lot better initially on the feeders, then (in 5 days to a week, when they are growing robustly), you could switch them to feeder-free.
After using multiple methods, Vitrification has been incredibly successful in thawing hESC/hiPSCs with higher efficiency and less risk for karyotypic abnormalities than traditional DMSO and mFrSR methods. However the cells were cultured prior to freezing (MG or other, even on MEFs), are the conditions by which they should be thawed. Although I will say that the safest, (or most guaranteed for some type of recovery) is still using feeders, although i favor feeder-free conditions whenever possible. I will note that if I must use feeders, human Nuff feeders are a great sybarite for MEFs.
DEAR SIR, my suggestion is to use both the system if ur study include to compare. in feeder free system u can use gelatin, fibronectin, collagen and matrigel. gelatin is cost effective and reproduce good result.
u can follow this publication.Feeder- and serum-free establishment and expansion
of human induced pluripotent stem cells
MEHDI TOTONCHI1,2,#, ADELEH TAEI1,#, ALI SEIFINEJAD1, MOHAMMADSHARIF TABEBORDBAR1,
HASSAN RASSOULI1, ALI FARROKHI1, HAMID GOURABI2, NASSER AGHDAMI1,
GHASEM HOSSEINI-SALEKDEH1, and HOSSEIN BAHARVAND*,1,3
Int. J. Dev. Biol. 54: 877-886 (2010)
doi: 10.1387/ijdb.092903mt
iPSC culture can be directly revived on feeder free culture. I use E8 supplemented media
instead matrigel
I also forgot to mention, please use rock inhibitor with essential8 supplanted media for thawing. But It has to be replaced on second day with essential 8. I don't use mTser.
I always put the matrigel coated plates in 37degree for2hrs before I use them
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