Simultaneous induction of calcium transients in embryoid bodies using microfabricated electrode substrates |
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Authors: | Takayama Yuzo Saito Atsushi Moriguchi Hiroyuki Kotani Kiyoshi Suzuki Takafumi Mabuchi Kunihiko Jimbo Yasuhiko |
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Affiliation: | 1Graduate School of Information Science and Technology, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan,;2Research Fellow of the Japan Society for the Promotion of Science, 1-8 Ichiban-cho, Chiyoda-ku, Tokyo 102-8472, Japan,;3Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba 277-8563, Japan |
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Abstract: | Precise control of differentiation processes of pluripotent stem cells is a key component for the further development of regenerative medicine. For this purpose, combining a cell-aggregate-size treatment for regulating intercellular signal transmissions and an electrical stimulation technique for inducing cellular responses is a promising approach. In the present study, we developed microfabricated electrode substrates that allow simultaneous stimulation of embryoid bodies (EBs) of P19 cells. Mouse embryonal carcinoma P19 cells can be induced to differentiate into three germ layers and serve as a promising stem cell model. Microcavity–array patterns were fabricated onto indium–tin–oxide (ITO) substrates using a standard photo-lithography technique, and uniform-sized EBs of P19 cells were inserted into each microcavity. Electrical stimulation was applied to the EBs through substrate electrodes and stimulus-induced intracellular calcium transients were monitored. We confirmed that the developed electrode device could simultaneously stimulate smaller (200 μm diameter) and larger (500 μm diameter) EBs inserted in the microcavities and induce specific spatio-temporal patterns of intracellular calcium transients in the EBs with fine reproducibility. We concluded that the developed microcavity array with embedded electrodes could simultaneously and effectively stimulate uniform-sized EBs inserted in it. Therefore, it is a promising experimental tool for precisely controlling cell differentiation processes. |
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Keywords: | Calcium imaging Electrical stimulation Embryoid body Microfabrication P19 embryonal carcinoma cell line |
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