Hello,
I am growing R1-ESCs in high glucose DMEM, 15%FBS, sodium pyruvate, non-essential amino acids and L-glutamine. Can these cells be grown in 2i+LIF medium?
How many passages do I need to grow them in this medium before I can proceed to apply some differentiation protocols?
What is the exact composition of the 2i+LIF medium? Do the ESCs have to be grown on feeders when growing in this medium?
Yes! R1 ESCs adapt well to 2i+LIF on gelatin coated plates (see recipe below). Switching media should help with the reproducibility of your differentiation. It usually takes 4-5 passages. Best of luck!
Karl
Here's our recipe. Taken from Koehler and Hashino Nat Protocols 2014:
LIF-2i ES cell maintenance medium To prepare 100 ml of basal LIF-2i medium, combine 48.75 ml of Advanced DMEM/F12, 48.75 ml of Neurobasal medium, 1 ml of B27 supplement (minus Vitamin A), 500 μl of N2 supplement and 1 ml of GlutaMax in a sterile 250-ml bottle. This medium can be used for 3–4 weeks. Prepare complete LIF-2i medium immediately before use by adding 100 μl of penicillin-streptomycin, 10 μl of LIF, 1 μl of PD-0325901 (1 µM final conc.) and 3 μl of CHIR99021 (3 µM final conc.) to 10 ml of the basal LIF-2i medium. Complete LIF-2i medium can be used for up to 1 week if stored at 4 °C.
One thing to remember is that it is important to use B27 that does not contain vitamin A for ES cell culture. Vitamin A can be converted into retinoic acid in culture and can cause spontaneous neural induction. Serial passaging can eliminate any neuronal cells that may arise while adapting ES cells to LIF-2i medium.
Yes! R1 ESCs adapt well to 2i+LIF on gelatin coated plates (see recipe below). Switching media should help with the reproducibility of your differentiation. It usually takes 4-5 passages. Best of luck!
Karl
Here's our recipe. Taken from Koehler and Hashino Nat Protocols 2014:
LIF-2i ES cell maintenance medium To prepare 100 ml of basal LIF-2i medium, combine 48.75 ml of Advanced DMEM/F12, 48.75 ml of Neurobasal medium, 1 ml of B27 supplement (minus Vitamin A), 500 μl of N2 supplement and 1 ml of GlutaMax in a sterile 250-ml bottle. This medium can be used for 3–4 weeks. Prepare complete LIF-2i medium immediately before use by adding 100 μl of penicillin-streptomycin, 10 μl of LIF, 1 μl of PD-0325901 (1 µM final conc.) and 3 μl of CHIR99021 (3 µM final conc.) to 10 ml of the basal LIF-2i medium. Complete LIF-2i medium can be used for up to 1 week if stored at 4 °C.
One thing to remember is that it is important to use B27 that does not contain vitamin A for ES cell culture. Vitamin A can be converted into retinoic acid in culture and can cause spontaneous neural induction. Serial passaging can eliminate any neuronal cells that may arise while adapting ES cells to LIF-2i medium.
Dear Karl,
Thank you so much for the response; it was very helpful. I do have a couple of more questions:
At what passage number of the ESCs should I start to adapt them? Currently, I have cells that are beyond passage 30.
Could you also kindly give me the company name from where you buy the above reagents or preferably the cat nos (if you have time) since it seems that I have to be very careful about the B27, etc.
Waiting for your reply
I want to thank Karl Koehler to is quick answer and I do not have to add any furthe information.
Best wshes
Horst Spielmann
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