Trehalose improves survival of electrotransfected mammalian cells

Background Electropermeabilization is widely used for introduction of DNA and other foreign molecules into eukaryotic cells. However, conditions yielding the greatest molecule uptake and gene expression can result in low cell survival. In this study, we assessed the efficiency of trehalose for enhancing cell viability after excessive electropermeabilization. This disaccharide was chosen because of its capability of stabilizing cell membranes under various stressful conditions, such as dehydration and freezing. Materials and Methods Various mammalian cell lines were electropermeabilized by single exponentially decaying electric pulses of few kV/cm strength and of several‐microsecond duration. Propidium iodide (PI) and a plasmid encoding green fluorescent protein (GFP), respectively, served as reporter molecules. The effects of trehalose on PI‐uptake, GFP gene expression, transfection yield, and short‐ and long‐term viability were analyzed by flow cytometry and electronic cell counting. Results The substitution of inositol by trehalose in pulse media protected cells against field‐induced cell lysis. The protection effect saturated at about 40–50 mM trehalose. Transfection yield and gene expression were not significantly affected by trehalose. But the transfection efficiency was generally higher in the presence of trehalose, mainly because of the increased cell survival. Conclusions We demonstrated that trehalose‐substituted media are superior to standard trehalose‐free pulse media for improving cell survival and achieving higher electrotransfection efficiency. Cytometry 45:161–169, 2001. © 2001 Wiley‐Liss, Inc.