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 le ad to the permeabilization of their plasma membranes. This electro-ind uced permeated stare of the cell membrane is reversible and leads to c ell fusion; ie., the electropermeabilized state is 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 fibrobl asts and red blood cell ghosts) can be easily obtained. Due to size di fferences, the optimum electric field conditions for their permeabiliz ation and the maintenance of their integrity are observed to be differ ent (10 pulses, 100 mu s duration, 1.2 kV/cm for Chinese hamster ovary [CHO] cells and 10 pulses, 100 mu s duration, 3.6 kV/cm for erythrocy te ghosts). Cells and ghosts are electropermeabilized separately and k ept at low temperature. Their fusion is then induced by the creation o f contact between them by gentle centrifugation. This process takes ad vantage of the long-lived fusogenicity of the electropermeabilized cel l membrane. An immediate consequence is the introduction of macro-mole cules into mammalian cells. This is obtained by fi(sing cells with pre -loaded erythrocyte ghosts. Under optimum conditions, penetration of l arge quantities of FITC-dextran (70 kDa) and beta-galactosidase into 9 0%-95% of CHO cells while preserving their viability has been observed .