Hi,
We are looking to culture our mES cell lines in 2i media. We currently use either classic ES cell culturing conditions (FBS, LIF, GSK inhibitor and MEFs) or serum free KnockOut cell culturing conditions (KOSR, LIF, GSK inhibitor and MEFs). Our previous attempts to get our mES cells to grow in 2i have not been successful. They look fine for the 1st 48 hrs but once passaged practically everything died. We've tried a couple of different adaption protocols, but to no avail. Can anyone recommend any protocols they have used successfully. Do we have to deplete the MEFs first before swapping to 2i media or can these processes been run in parallel? Most of the papers i've read just say adapt your cells to 2i, but they don't elaborate on how this was achieved. At least one of our ES cell lines will grow in classic media without MEFs, but the morphology is poor and after ~ 5 passages we no longer have anything that resembles an ES cell colony. Any suggestions greatly appreciated.
Natalie
Hi, you could refer: https://crem.bu.edu/wp-content/uploads/2022/01/2i-Media-Feeder-free-Serum-free.pdf
We successfully cultured mES cells using this protocol. Following our experiences, mES cells could be died if the 2i media is stored for a period of time.
I hope you can solve the problem.
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