Maintaining a WiCell fibroblast-derived line of iPSC in monolayer with laminin coating and E8 media, then using Wnti directed protocol (Gsk3i day 0, Wnti with conditioned media day 3, insulin day 6; RMPI basal media). I am getting consistent differentiation (confirmed with spontaneous beating) but only in the middle two wells, B2 and B3, of a 12 well plate. Even when I account for possible pipetting error by filling wells out of order with cell suspension, this still occurs. The other wells fail to differentiate or even lose confluency and die off. Any ideas why?
These are called "edge effects". This is a gas exchange issue. Most likely it's caused by faster evaporation of water in the edge wells, which concentrates the salts from the medium, and harms the cells by osmotic stress. Can you measure the humidity in your incubator? Do you have plenty of standing water, to saturate the atmosphere? Increase the frequency of your media changes to mitigate this problem. Another trick is to fill the empty space between your wells with water.
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