I am using mTeSR1 feeder free system for my iPSCs. They generally grow beautifully in that particular media from Stemcell Technologies but for some reason the level of spontaneous differentiation differs greatly between passages. I am trying to do my splits as consistently as possible, sometimes my colonies are perfect morphology but sometimes they have weird patterns of differentiation with different cell types. I have tried manual passaging using a decreasing tipped Pasteur pipette, dispase (1mg/ml from Stemcell Technologies), and I tried using one of the commercial rollers from Life technologies (cat#23181010).
I attached a pic, of the differentiated colonies. The cluster circled in blue is the type of morphology I need... as said before there are times when 95% of the plate looks like what I circled, and there are times when 95% of the colonies look like all that other surrounding garbage!!! The image looks a bit too confluent, but even at the start of the new passage (2+ days after splitting) there are sometimes already differentiated pieces. So far I have only tried growing 3 different cell lines in mTeSR1 feeder free conditions, and all 3 have random differentiation and random instances where they feel like staying pluripotent.
I am also using matrigel (BD #354277), ROCK inhibitor (Sigma #Y0503) in my media at 5uM growing them on 60mm and 100mm plates. Please, if you can give me any insight on what I might be doing wrong? Any tips or advice on techniques? I need these cultures to be as pure as possible, so any notes you have on what causes spontaneous differentiation would be appreciated.