I am totally new to feeder-dependent iPSC culturing. Previously I was doing on feeder-free iPSC culturing. Hence I encountered that the iPSCs which grow on the feeder are sparse and not growing as typical colonies which are compacted. From the attached photos, under 4x magnification some cells are individually apart from each other while some are in close contact with each other. Under 10x magnification, those "individual" cells look big without a defined shape, I am not too sure are they cells differentiated or undergoing apoptosis. Before changing medium iPSCs look a bit spiky in shape, after changing, they are more round in shape and starting to form a slightly more round colonies. Can anyone with experiences provide me some advice on this. Thank you.
Dear Teo Ting,
The iPSCs look differentiated to me, probably due to the low confluency of the feeder layer. We usually test each batch of feeders to adjust confluency. It is very important to have good coverage of the surface of the culture plate before you add the iPSCs on top.
Anyway, I would recommend you to go back to feeder-free conditions :)
Good luck with the experiments! Best regards,
Ignacio
Your feeder cell density is far too low. In the old days when I used feeders, I used 1.8 x 10e6 cells per 100mm plate.
Ignacio Rodriguez Polo Ron Hrstka Thank you for your kind advice. However I wonder, too low confluency of the feeder may lead to the differentiation of iPSCs, what would happen if the confluency of feeder is too high?
Do you prefer feeder-free over feeder-dependent methods? However I have read the attached article Article Feeder Layer Cell Actions and Applications
that the use of feeder is somehow better than feeder-free. What do you think?Anyway, is there any difference between feeder-dependent & feeder-free iPSC. What would actually happen if I used feeder-free iPSC to grow on feeder?
Looking forward your helpful reply. Thank you.
Dear Teo Ting Ting
A too dense feeder layer leads to the detachment of your culture, as mention before, you should adjust the confluency.
Feeder cells were instrumental in maintaining iPSC/ESC in culture undifferentiated and proliferating. Nowadays, most labs made the transition to feeder-free culture. In feeder-free, you don't expose your stem cells to cells with another genetic origin (allo- or xenogeneic cells). Additionally, you have chemically defined culture conditions.
In my opinion, the only disadvantage of feeder-free vs feeder-dependent culture is the cost :). You can find many, more recent, publications exploring different media composition and coating strategies to keep pluripotent stem cells in feeder-free culture.
Good luck!
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