Study Of The Cell-Electrofusion Method Based Microelectrode Array Chips

Cell fusion has been widely used in crossbreeding, monoclonal antibody preparation, drug screening, and other fields since its invention due to its advantages such as strong controllability, easy operation, and non-toxic to cells. Cell fusion is gradually becoming an important tool in modern bioengineering technology. With the development of the microfluidic technology and the microfabrication technology, chip-based cell electrofusion technology is developing rapidly in recent years. In addition to the advantages of the traditional electrofusion technology, chip-based cell electrofusion is receiving more and more attention because it has the advantages of more accuracy and efficiency, high integration, easy to experimental observation, and less sample consumption.In this paper, a series of cell electrofusion chips based on microelectrode array were fabricated using the modern microfabricated technology. Dielectrophoresis and cell electroporation method were used for cell alignment and electrofusion. Cell alignment and electroporation are the two most important steps in the cell electrofusion process, so a thorough discussion on their mechanism and model was carried on in this paper. Based on this discussion, the finite element method was used to analyze the effects of the distribution of electric field on the cell alignment and the transmembrane potential distribution. According to this, the structure of microelectrode and the design of microelectrode array chip were optimized to obtain an electric field environment that was beneficial to cell electrofusion. Based on the simulation analysis, a discrete coplanar microelectrode and micro-cavity microelectrode structure were designed, and the chip fabrication, material and packaging technology were studied. Cell electrofusion chips based on thin film microelectrode array, discrete coplanar microelectrode array, and micro-cavity microelectrode array were developed. Finally, a wide variety of cell fusion experiments were carried out on the cell electrofusion platform to verify the effectiveness of the new method. Specifically, the study in this paper mainly included the following points:①Theory and simulation research of cell electrofusionBased on the studies of dielectrophoresis, cell electroporation, and relevant theoreies, in order to further optimize the structure of microelectrode, the finite element analysis software COMSOL Multiphysics® was used to analyze the cell movement under dielectrophoresis to establish an optimized cell alignment method. At the same time, a single cell electroporation model based on the transmembrane potential was established to analyze the electric field distribution, the transmembrane potential distribution, the distribution of electroporation area and pore density, etc.; Based on the electric field constriction effect, the electric field distribution situation was analyzed in the discrete electrode, the arc-shaped cavity electrode, the rectangular cavity electrode, and the transmembrane potential distribution was discussed and the optimal micro-cavity structure was chosen finally. A microfluidic cell-electrofusion chip was developed based on this structure. Thus the process of the electroporation/electrofusion was optimized, and it can effectively avoid multiple cell fusion and improve fusion rate and so on.② Development of cell-electrofusion microfluidic chipsOptimized cell electrofusion chips were designed according to the theoretical analysis and simulation calculation. By considering the structure, material, processing technology, packaging, etc. of the chip, three kinds of chips were developed: cell electrofusion microchip based on the thin film microelectrode, discrete coplanar microelectrode, and micro-cavity microelectrode. As to the selection of materials, silicon, gold and aluminum were adopted as the electrode material. The polyimide polymer, Si O2-Polysilicon-Si O2 “insulation” + “floating silicon” structure were used to fill the “dead area” between theadjacent protruding sidewall microelectrodes; somematerials, such as quartz glass and SOI were chosen as the chip substrate. Some advanced manufacturing process, such as soft photolithography and IC process were chosen as the chip manufacturing process. In the experiments, this series of chips showed considerable advantages such as good biological compatibility, chemical corrosion resistance, electrical properties, and so on.③ Establishment of a microfluidic cell-electrofusion microsystemUsing the cell-electrofusion chips and an electrofusion signal generator as the main equipment, cell electrofusion chip platform based on electrode microarray was set up. The experiment platform has several advantages such as simple operation, good controllability, good experimental results, etc.. Through an image observation and collection system constructed by microscope and CCD, the whole process of cell electrofusion can be clearly observed and recorded. The fusion parameters can be adjusted to obtain the ideal cell alignment efficiency and fusion efficiency.④ Experimental research on the microfluidic cell-electrofusion chipsOn the cell-electrofusion experimental platform, several cells have been used to study the experimental condition and parameters of the cell alignment and fusion, including K562 cells, NIH3T3 cells, Myoblast cells, etc. The problem that cells cannot be fused in the “dead area” has been solved by using the newly developed cell-electrofusion chips. In addition, the new design of microelectrodes made the electric fields more concentrated, thus the alignment and fusion voltage were reduced. By repeating experiments, the required electrical parameters of cell alignment and cell fusion were obtained, and the high-efficiency cell alignment and cell fusion were achived. Compared to the PEG method and the traditional electrofusion method, the cell alignment efficiency and the fusion efficiency have been significantly improved on the new microelectrode chips. On the thin film electrode chip, cell-cell alignment efficiency of K562 cells was higher than 70.7%, and the electrofusion efficiency was about 43.1%. On the discrete coplanar electrode chip, almost 100% of NIH3T3 cells were aligned to the edge of the microelectrodes, and cell-cell alignment efficiency reached 69.8%. The electrofusion efficiency was above 40% of the total cells loaded into the device. On the micro-cavity electrode chip, 98% of NIH3T3 cells were attracted into the cavities; and cell-cell alignment efficiency was about 67.9%. The electrofusion efficiency reached 50.3%.In conclusion, through the optimization of cell electrofusion chip design, this paper developed a series of cell electrofusion chips with high cell alignment efficiency and high fusion efficiency, and established a cell electrofusion microsystem experimental platform including cell electrofuison chips and electrofusion signal generator. In the electrofusion experiments of variety of cells, good experiment results have been achieved. This study set up good foundation for the realization of the miniaturization of a cell fusion system, building efficient and automatic cell fusion chip system, and could be further applied in the actual research, such as crossbreeding studies and the epigenetic reprogramming of somatic cells, etc.