Our goal is to construct a prototype chamber with protocols for the manufacture of a new low-voltage electroporation (LVEP) system to transfect suspended and adherent cells under physiologic conditions. Electric field amplification results from embedding the cells into the pores of thin (13 um) insulating filters, enabling all 0.33 million to be simultaneously electroporated using 5-20 V across a 2 cm gap. The use of low voltage/current is the key to this new technique. The large increase in current density in the pores, produced by the reduction of current shunt pathways around each embedded cell, amplifies the localized field across the filter 1000X. LVEP produces minimal Joule heating and electrode products as the current is only 25 - 50 mA versus 25 - 50 A in suspension electroporation. Further, only one side of the cell, at the end of the cell process inside the pore, is electroporated. The transmembrane potential of the membrane hemisphere in the pore is constant, thus, the entire area reaches the same potential. The Phase I study demonstrated that LVEP can transfect mammalian cells with 93-98% cell viability, with transfection efficiencies of 5-80% of embedded cells. In Phase II, we will optimize the chamber characteristics and protocols to transfect genes into both adherent and suspended cells to maximize transfection efficiency, cell survival and ease of use. Different plasmids and cells will be examined, including primary cell lines. Important goals are to transfect cell lines proven difficult and demonstrate capability where high cell survival is necessary.
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