Cell electrofusion based on nanosecond/microsecond pulsed electric fields.

Traditionally, microsecond pulsed electric field was widely used in cell electrofusion technology. However, it was difficult to fuse the cells with different sizes. Because the effect of electroporation based on microsecond pulses was greatly influenced by cell sizes. It had been reported that the differences between cell sizes can be ignored when cells were exposed to nanosecond pulses. However, pores induced by those short nanosecond pulses tended to be very small (0.9 nm) and the pores were more easy to recover. In this work, a finite element method was used to simulate the distribution, radius and density of the pores. The innovative idea of "cell electrofusion based on nanosecond/microsecond pulses" was proposed in order to combine the advantages of nanosecond pulses and microsecond pulses. The model consisted of two contact cells with different sizes. Three kinds of pulsed electric fields were made up of two 100-ns, 10-kV/cm pulses; two 10-μs, 1-kV/cm pulses; and a sequence of a 100-ns, 10-kV/cm pulse, followed by a 10-μs, 1-kV/cm pulse. Some obvious advantageous can be found when nanosecond/microsecond pulses were considered. The pore radius was large enough (70nm) and density was high (5×1013m-2) in the cell junction area. Moreover, pores in the non-contact area of the cell membrane were small (1-10 nm) and sparse (109-1012m-2). Areas where the transmembrane voltage was higher than 1V were only concentrated in the cell junction. The transmembrane voltage of other areas were at most 0.6V when we tested the rest of the cell membrane. Cell fusion efficiency can be improved remarkably because electroporation was concentrated in the cell contact area.