Electric-field-induced permeabilization and fusion of embryonic amphibian cells.

Abstract

The technique of electropulsation has been shown to be highly efficient in promoting penetration of exogenous molecules into living cells, transfection, and cell fusion in different animal, vegetal, and bacterial cell systems. Introduction of such exogenous compounds, i.e., plasmids, into living cells is of great interest for embryological studies. Embryonic amphibian ectodermal cells from Pleurodeles waltl gastrulae, either freshly dissociated or cultured for 5 days, can be permeabilized when submitted to an external electric field of sufficient intensity: 500 V/cm for isolated spherical cells and 150-200 V/cm for plated cultured cells. Permeabilization was indicated by both the leakage of metabolites (ATP) from the cells and the uptake of exogenous compounds (pyranin) into the cells. With the use of higher field intensities (600 V/cm for freshly dissociated cells and 300 V/cm for cultured cells) cell fusion and syncytial structures could also be obtained. Isolated spherical cells had 100% viability immediately after being pulsed at intensities up to 600 V/cm. Cell lysis was observed above this value, although the nonlysed cells were observed to spread on a substrate and differentiate normally. For the cultured plated cells, cell viability fell with increasing electric-field strength, and for a given electric field value, cell viability decreased with the age of the culture after pulsing. Nevertheless, for electric-field intensities less than or equal to 300 V/cm, 100% of the cells remained attached to the substrate and differentiated normally over the following 5 days.