Ensemble stimulation of embryoid bodies using microfabricated ITO substrates

Precise control of differentiation processes of pluripotent stem cell is key component for realization of regenerative medicine. Electrical stimulation is one of the promising techniques, particularly for regulation of neuronal regeneration. In the present study, we developed substrates with embedded electrodes, which allowed ensemble electrical stimulation of embryoid bodies (EBs) of stem cells. Microcavity-array patterns were fabricated on substrates with embedded electrodes using standard photo-lithography. Uniform-size EBs of P19 cells were prepared, inserted one by one in each microcavity, and electrical stimulation was applied through substrate electrodes. Stimulus-induced intracellular calcium transients were successfully monitored by fluorescence imaging. The results suggested that this method would be useful for applying precisely-controlled electrical stimulation to a large number of EBs of stem cells.