Characterization of PEG-mediated electrofusion of human erythrocytes

Polyethylene glycol (PEG) and electrofusion were applied together in a simple and highly efficient cell fusion method. PEG (8000 M(r)) was used to bring human erythrocytes into contact, and a single 4.4 kV/cm, 80 microseconds duration pulse was applied to cell suspensions. The fusion yield (FY) is PEG concentration-dependent. A maximum FY (50%) was found at about 10% PEG. Higher PEG concentrations (> 10%) suppressed FY caused by colloid osmotic shrinkage. Morphological changes, such as colloidal osmotic swelling and shrinking, and the expanding and contraction of fusion lumen, when suspension media were changed from PBS to isotonic 15% dextran solutions, was examined by microscopy. FY was found to depend on both simple osmotic and colloidal-osmotic swelling. From the swelling behavior, we propose two types of electropores: the pre-fusion sites between cell pairs, and electropores on each individual cell connecting intracellular and extracellular space. The latter type is responsible for the colloidal osmotic swelling and shrinking of cell which, together with simple osmotic swelling, is responsible for expanding the pre-fusion sites into fusion lumens. Resealing of electropores resulted in reducing FY, but the FY can be restored by simple osmotic shock. Apparently, PEG plays two opposite roles in this fusion method; one is to promote pre-pulse and post-pulse cell-cell contact, protecting pre-fusion sites, and the other suppresses FY by colloid osmotic shrinkage of cells after pulsing, especially when high PEG concentration is used. 10% PEG 8000 represents the optimal combination of these properties.