Hello
I have human iPS cells that are being thawed and cultured in mTeSR1 media. I want to transition them to E8 media. In the past we have had very limited success with this type of media transition.
What is the best method to do this? What level of growth should the cells be when this is done? Please share experience.
Thank you.
RW
In general, it is best to transition media comparing original medium (T) with successive dilutions with the desired new medium (E). For example, compare T/E medium (100/0) T/E (80/20), T/E (60/40), T/E (40/60), T/E (60/40), T/E (80/20), and then T/E (0/100). This step down procedure may or may not work. If that does not work, then something beneficial may be lacking or something detrimental may be in E medium.
Oops! T/E should be 100/0, 80/20, 60/40, 40/60, 20/80, 0/100 in the sequence.
Thank you for the prompt response, Jane. What is the typical culture confluency when this should be done?
Hi Ranjuna- I agree with this method above if you are planning to use matrigel with both TeSR and E8. You could split your cells into TeSR a bit more confluent than you normally would (maybe 1:4 instead of 1:6), then the next day switch to T/E 80/20, then the next day 60/40, etc. When you split your cells for the first time in E8 100% you could try use ROCK inhibitor. I would switch your cells into E8/matrigel first before trying E8/vitronectin. This is all assuming you are using EDTA to passage cells.
You could try to thaw straight into E8/matrigel + RI, when a culture has been frozen in TeSR.
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