High-efficiency electrofusion between cells of different sizes was achieved by application of fusing electric pulses to cells in centrifuged pellets. Larger target cells (Chinese hamster ovary or L1210 cells) were stacked among smaller human erythrocytes or erythrocyte ghosts by sequential centrifugation at 700 g to form five-tier pellets in a specially designed centrifugation-electrofusion chamber. The membranes of erythrocytes and ghost were labeled with fluorescent membrane dye (1,1' dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine (Dil)), and the contents of ghosts were loaded with water-soluble fluorescent dye (42-kDa fluorescein isothiocyanate dextran (FITC-dextran)), to monitor heterogeneous cell fusion. Fusion efficiency was assayed by the extent of either membrane dye mixing or contents (FITC- dextran) mixing with target cells. Four rectangular electric pulses at 300 V and 80 μs each were found to give the optimal fusion results of ~80% heterogeneous fusion by the content-mixing assay and ~95% by the membrane- dye-mixing assay. Cell viability remained greater than 80% after electrofusion. Because of the electric breakdown of cell membranes at the beginning of the pulse, the pellet resistance and hence the partial voltage across the pellet reduced rapidly during the remaining pulse time. This voltage redistribution favored the survival of fused cells. The limited colloidal-osmotic swelling of cells in pellets enhanced cell-cell contact and increased the pellet resistance after each pulse. As a result, the partial voltage across the pellet was restored when the next pulse was applied. This redistribution of pulse voltage in the pellet system permitted the breakdown of cell membranes at a lower applied voltage threshold than that required for electrofusion of cells in suspension or in dielectrophoretic cell chains. The cell viability and soluble dye retention within cells (FITC-dextran) remained at the same high levels for 3 h when the cells were incubated in respective culture media with serum at 37°C. Viability and dye retention decreased significantly within 30 min when cells were incubated in phosphate-buffered saline without serum. The pellet technique was applied to form hybridomas by fusion of larger SP2/0 murine myelomas with smaller naive mouse lymphocytes. An optimum of 173 ± 70 hypoxanthine aminopterin thymidine (HAT)-selected clones of the hybridomas was obtained from 40,000 SP2/0 cells and 1.5 x 106 lymphocytes used in each trial. This high-efficiency fusion technique may be adapted to mediate drug and gene transfer to target cells ex vivo as well as to form hybrid cells with limited cell sources.
Li, L. H., Hensen, M. L., Zhao, Y. L., & Hui, S. W. (1996). Electrofusion between heterogeneous-sized mammalian cells in a pellet: Potential applications in drug delivery and hybridoma formation. Biophysical Journal, 71(1), 479–486. https://doi.org/10.1016/S0006-3495(96)79249-4