High efficiency electrotransfection with aluminum electrodes using microsecond controlled pulses

Electropermeabilization (electroporation) has been used widely for incorpor ation of xenomolecules (up to the size of DNA) into eukaryotic cells withou t loss of cellular functions. Frequently, low-cost equipment is used which delivers exponentially decaying field pulses of low intensity and milliseco nd duration time. This together with the use of disposable cuvettes equippe d with aluminum plate electrodes can considerably reduce the survival of th e cells and the incorporation of xenomolecules because of the detrimental s ide effects of long-duration pulses and the toxicity of Al3+ ions on permea bilized cells. Atomic absorption spectroscopy showed that substantial amoun ts of Al3+-ions (up to 1 mM) were solubilized from the electrodes upon puls ing. Solubilization of Al3+-ions occurred because of the changes of the pH close to the electrodes induced by electrolysis of water. The local pH-chan ges could be visualized by the addition of pH-indicators together with agar ose to the pulse medium. In contrast, when using short pulses of 40 mu s to 100 mu s duration, small amounts of Al3+-ions were only solubilized from t he electrodes upon pulsing. Electroinjection of the plasmid pEGFP-Cl (encod ing for Enhanced Green Fluorescence Protein) in four different (murine and human) cell lines showed that relatively high survival rates of cells and s pecies-specific transfection yields (of up to 50-70% as measured by FAGS an alysis) could be obtained by using aluminum electrodes provided that short- duration pulses were applied. These yields were in the range obtained by us ing stainless steel electrodes. The required short-duration pulses and fiel d strengths for field-induced incorporation of pEGFP-Cl were achieved using a novel power supply (maximum output voltage of 1.2 kV, pulse durations be tween 15 mu s and 500 mu s) in combination with strongly hypo-osmolar pulse media. (C) 1998 Elsevier Science S.A. All rights reserved.