I am passaging my hiPSCs with EDTA and E8 medium. However, I have noticed the presence of spontaneously differentiated cells in the final passage.
What other techniques are available to improve the purity of the final passage, and is there any literature on this?
Hello Joshua,
Generally, Essential 8 media suppresses the differentiation. I am not clear what you mean by final passage. In my experience you can keep culturing hiPSC in undifferentiated state for years in E8 media and using EDTA.
Here are somethings you may attempt:
1. Revive a new passage of hiPSCs which were previously frozen and were undifferentiated
2. You may have to remove some differentiated colonies prior to passaging in your existing culture
3. Add 10-20ng/mL of bFGF to existing E8 media in the current passage
4. Try making fresh E8 media, as if you make small volumes of complete E8 media prior to use and not use E8 media made more than a week before
I suggest you revive new hiPSC vial and avoid using E8 media that was made more 7 days ago.
All the best.
Thanks.
Prasad.