(526e) A 3D Bulk Microelectrode Array for High Throughput Cell Electrofusion

Cell electrofusion is a state-of-the-art technology that applies electric field to induce two or more cells to merge into a hybrid cell in an asexual way. Since the hybrid combines genetic and epigenetic information of two cells, it can be used for gene expression analysis, reprogramming somatic cells, developing antibody, cloning mammals, and cancer immunotherapy, among other important applications. In this work, a micirofluidic chip which integrates a novel 3D bulk microelectrode array is used for cell electrofusion under a low applied voltage. On this chip, insulating bulk is used to fill the space between two adjacent protruding microelectrodes on the same side of the microchannel. With this design, cell trapping in undesirable spaces reported previously can be avoided. The 3D bulk microelectrode array is fabricated on a silicon-on-insulator (SOI) wafer by lithography technology with 4 masks. The 35 mm thick highly doped Si layer is used for the microelectrode array and the wall of the microchannel. Insulating gap with the same depth is fabricated between the two adjacent protruding microelectrodes. A smooth flow thus is formed, and the protruding microelectrode structures is used to generate non-uniform electric field distribution.  The gaps between any two adjacent protruding microelectrodes on the same side of the microchannel are filled by polycrystalline silicon after high temperature oxidation of the surface of the side walls. The highly doped Si layer is covered by a thin aluminum film to improve the conductivity. A SiO₂ film is subsequently deposited on the top of these microelectrodes for better biocompatibility, resistance to oxidation, and corrosion. In cell alignment and pairing research, almost all cells (>99%) were driven by the dielectrophoretic (DEP) force to the high electric field strength area to be aligned as cell-cell pairs. After the alignment, low voltage pulses were loaded on the microelectrode to generate high strength electric filed to induce reversible electroporation of the cytomembrane. Reconstructing cytomembrane, exchanging cytoplasm and forming hybrid can be completed within 3 min. Due to the non-uniform electric-field distribution, two-cell fusion was ensured in this device, even when more than two cells are formed as a multi-cell chain. On this chip, higher fusion efficiency than traditional chemical method (less than 5%) and electrofusion methods (less than 12%) has been achieved.