ELECTRICAL-PROPERTIES OF CELL PELLETS AND CELL ELECTROFUSION IN A CENTRIFUGE

A new approach is proposed for studying cell deformability by centrifu gal force, electrical properties of cell membranes in a high electric field, and for performing efficient cell electrofusion. Suspensions of cells (L929 and four other cell types examined) are centrifuged in sp ecial chambers, thus forming compact cell pellets in the gap between t he electrodes. The setup allows measurement of the pellet resistance a nd also the high-voltage pulse application during centrifugation. The pellet resistance increases sharply with the centripetal acceleration, which correlates with reduction of the cell pellet porosity due to ce ll compression and deformation. Experiments with cells pretreated with cytochalasin B or colcemid showed that cell deformability depends sig nificantly on the state of cytoskeleton. When the voltage applied to t he cell pellet exceeds a 'critical' value, electrical breakdown (porat ion) of cell membranes occurs. This is seen as a deflection in the I(V ) curve for the cell pellet. The electropores formed during the breakd own reseal in several stages: the fastest takes 0.5-1 ms while the who le process completes in minutes. A novel effect of colloid-osmotic com pression of cell pellets after electric cell permeabilization is descr ibed. Supercritical pulse application to the cell pellet during intens ive centrifugation leads to massive cell fusion. The fusion index grow s with the increase of centripetal acceleration, and drops drastically when the pulse is applied after the centrifuge is stopped. The colloi d-osmotic pellet compression enhances the fusion efficiency. No fusion occurs when cells are brought in contact after the pulse treatment. T he data suggest that tight intermembrane contact formed prior to pulse application is a prerequisite condition for efficient cell electrofus ion. The capacities of the technique proposed and the mechanism of mem brane electrofusion are discussed.