This paper presents the use of electric-field constriction created by a microfabricated structure to realize high-yield electrofusion of biological cells. The method uses an orifice on an electrically insulating wall (orifice plate), whose diameter is as small as that of the cells. First a. c. voltage is applied between the electrodes on each side of the orifice plate, and the cells are dielectrophoretically trapped so that the contact point is placed in the orifice. Then a pulse voltage is applied. Due to the field constriction created by the orifice, we can induce controlled magnitude of membrane voltage selectively around the contact point, regardless of the cell size. The field constriction also ensures 1:1 fusion even when more than two cells are forming a chain at the orifice. A device for electrofusion has been made with a standard SU-8 lithography and PDMS molding, and real-time observation of the electrofusion process is made. Experiments using plant protoplasts or mammalian cells show that the process is highly reproducible, and the yield higher than 90% is achieved.
展开▼
