Mechanistic studies of gene delivery into mammalian cells by electrical short-circuiting via an aqueous droplet in dielectric oil.

We have developed a novel methodology for the delivery of cell-impermeable molecules, based on electrical short-circuiting via a water droplet in dielectric oil. When a cell suspension droplet is placed between a pair of electrodes with an intense DC electric field, droplet bouncing and droplet deformation, which results in an instantaneous short-circuit, can be induced, depending on the electric field strength. We have demonstrated successful transfection of various mammalian cells using the short-circuiting; however, the molecular mechanism remains to be elucidated. In this study, flow cytometric assays were performed with Jurkat cells. An aqueous droplet containing Jurkat cells and plasmids carrying fluorescent proteins was treated with droplet bouncing or short-circuiting. The short-circuiting resulted in sufficient cell viability and fluorescent protein expression after 24 hours' incubation. In contrast, droplet bouncing did not result in successful gene transfection. Transient membrane pore formation was investigated by uptake of a cell-impermeable fluorescence dye YO-PRO-1 and the influx of calcium ions. As a result, short-circuiting increased YO-PRO-1 fluorescence intensity and intracellular calcium ion concentration, but droplet bouncing did not. We also investigated the contribution of endocytosis to the transfection. The pre-treatment of cells with endocytosis inhibitors decreased the efficiency of gene transfection in a concentration-dependent manner. Besides, the use of pH-sensitive dye conjugates indicated the formation of an acidic environment in the endosomes after the short-circuiting. Endocytosis is a possible mechanism for the intracellular delivery of exogenous DNA.

Fundamental study on a gene transfection methodology for mammalian cells using water-in-oil droplet deformation in a DC electric field.

We have developed a gene transfection method called water-in-oil droplet electroporation (EP) that uses a dielectric oil and a liquid droplet containing live cells and exogenous DNA. When a cell suspension droplet is placed between a pair of electrodes, an intense DC electric field can induce droplet deformation, resulting in an instantaneous short circuit caused by the droplet elongating and contacting the two electrodes simultaneously. Small transient pores are generated in the cell membrane during the short, allowing the introduction of exogenous DNA into the cells. The droplet EP was characterized by varying the following experimental parameters: applied voltage, number of short circuits, type of medium (electric conductivity), concentration of exogenous DNA, and size of the droplet. In addition, the formation of transient pores in the cell membrane during droplet EP and the transfection efficiency were evaluated.

Novel Parallelized Electroporation by Electrostatic Manipulation of a Water-in-Oil Droplet as a Microreactor.

Electroporation is the most widely used transfection method for delivery of cell-impermeable molecules into cells. We developed a novel gene transfection method, water-in-oil (W/O) droplet electroporation, using dielectric oil and an aqueous droplet containing mammalian cells and transgene DNA. When a liquid droplet suspended between a pair of electrodes in dielectric oil is exposed to a DC electric field, the droplet moves between the pair of electrodes periodically and droplet deformation occurs under the intense DC electric field. During electrostatic manipulation of the droplet, the local intense electric field and instantaneous short circuit via the droplet due to droplet deformation facilitate gene transfection. This method has several advantages over conventional transfection techniques, including co-transfection of multiple transgene DNAs into even as few as 103 cells, transfection into differentiated neural cells, and the capable establishment of stable cell lines. In addition, there have been improvements in W/O droplet electroporation electrodes for disposable 96-well plates making them suitable for concurrent performance without thermal loading by a DC electric field. This technique will lead to the development of cell transfection methods for novel regenerative medicine and gene therapy.