Studies of cell pellets: II. Osmotic properties, electroporation, and related phenomena: membrane interactions.
Using the relations between pellet structure and electric properties derived from the preceding paper, the responses of rabbit erythrocyte pellets to osmotic or colloidal-osmotic effects from exchanged supernatants and from electroporation were investigated. Changing the ionic strength of the supernatant, or replacing it with dextran or poly(ethylene glycol) solutions, caused changes of Rp according to the osmotic behavior of the pellet. Rp was high and ohmic before electroporation, but dropped abruptly in the first few microseconds once the transmembrane voltage exceeded the membrane breakdown potential. After the initial drop, Rp increased as a result of the reduction of intercellular space. Rp increased regardless of whether the pellets were formed before or immediately after the pulse, indicating that porated cells experienced a slow colloidal-osmotic swelling. The intercellular or intermembrane distances between cells in a pellet, as a function of osmotic, colloidal-osmotic, and centrifugal pressures used to compress rabbit erythrocyte pellets, were deduced from the Rp measurement. This offered a unique opportunity to measure the intermembrane repulsive force in a disordered system including living cells. Electrohemolysis of pelleted cells was reduced because of limited swelling by the compactness of the pellet. Electrofusion was observed when the applied voltage per pellet membrane exceeded the breakdown voltage. The fusion yield was independent of pulse length greater than 10 microseconds, because after the breakdown of membrane resistance, voltage drop across the pellet became insignificant. Replacing the supernatant with poly(ethylene glycol) or dextran solutions, or coating pellets with unporated cell layers reduced the colloidal-osmotic swelling and hemolysis, but also reduced the electrofusion yield. These manipulations can be explored to increase electroloading and electrofusion efficiencies.
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Membrane Biophysics Laboratory, Roswell Park Cancer Institute, Buffalo, New York 14263.
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D000818:Animals; D002498:Centrifugation; D003102:Colloids; D003911:Dextrans; D004558:Electric Stimulation; D018274:Electroporation; D004910:Erythrocyte Membrane / Q000502:physiology*; D004912:Erythrocytes / Q000502:physiology*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D006461:Hemolysis; D007700:Kinetics; D008561:Membrane Fusion; D009994:Osmolar Concentration; D011092:Polyethylene Glycols; D011817:Rabbits; D013997:Time Factors
D003102:Colloids; D003911:Dextrans; C015219:fluorescein isothiocyanate dextran; D011092:Polyethylene Glycols; D016650:Fluorescein-5-isothiocyanate