Pulse-first heterofusion of cells by electric field pulses and associated loading of macromolecules into mammalian cells.
Exposing cells to brief, high-intensity electrical field pulses can lead to the permeabilization of their plasma membranes. This electro-induced permeated state of the cell membrane is reversible and leads to cell fusion; i.e., the electropermeabilized state if fusogenic. The size of cells intended for fusing, however, limits the obtention of viable hybrids when there is a wide range of cell sizes. We report here that electrofusion of cells of different origins and sizes (hamster fibroblasts and red blood cell ghosts) can be easily obtained. Due to size differences, the optimum electric field conditions for their permeabilization and the maintenance of their integrity are observed to be different (10 pulses, 100 microseconds duration, 1.2 kV/cm for Chinese hamster ovary [CHO] kV/cm for erythrocyte ghosts). Cells and ghosts are electropermeabilized separately and kept at low temperature. Their fusion is then induced by the creation of contact between them by gentle centrifugation. This process takes advantage of the long-lived fusogenicity of the electropermeabilized cell membrane. An immediate consequence is the introduction of macromolecules into mammalian cells. This is obtained by fusing cells with pre-loaded erythrocyte ghosts. Under optimum conditions, penetration of large quantities of FITC-dextran (70 kDa) and beta-galactosidase into 90%-95% of CHO cells while preserving their viability has been observed.
Rols
M P
MP
Centre National de la Recherche Scientifique, Toulouse, France.
Dahhou
F
F
Teissié
J
J
D000818:Animals; D016466:CHO Cells; D002459:Cell Fusion; D006224:Cricetinae; D003911:Dextrans / Q000378:metabolism; D004560:Electricity; D004910:Erythrocyte Membrane / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000378:metabolism; D010539:Permeability; D001616:beta-Galactosidase / Q000378:metabolism
D003911:Dextrans; C015219:fluorescein isothiocyanate dextran; D001616:beta-Galactosidase; D016650:Fluorescein-5-isothiocyanate