OSMOTIC RESPONSE OF MAMMALIAN-CELLS - EFFECTS OF PERMEATING CRYOPROTECTANTS ON NONSOLVENT VOLUME

The nonsolvent volume, b, of a cell permits calculation of cell water volume from measurements of total cell volume, and, consequently, it i s used extensively in the determination of membrane permeability coeff icients for water and solutes and also in simulations of water and sol ute fluxes during freezing of cells. The nonsolvent volume is most com monly determined from the ordinate intercept of plots of cell volume a s a function of the reciprocal of extracellular nonpermeating solute c oncentration (so-called Boyle-van't Hoff plots). Once derived, b is of ten assumed to be constant even under conditions that may differ marke dly from those under which it was determined. Our aim was to investiga te whether this assumption was valid when cells were exposed to the cr yoprotectants glycerol, dimethyl sulphoxide (Me(2)SO), or propane-1,2- diol. Rabbit corneal keratocytes, a fibroblastic cell type, were expos ed to 10% (v/v) cryoprotectant for 30 min at 22 degrees C in solutions containing a range of nonpermeating solute concentrations. Cell volum es were determined by an electronic particle sizer and mode volume plo tted as an inverse function of the concentration of nonpermeating solu te. The cells behaved as osmometers under all conditions studied, but we found no evidence to suggest that the nonsolvent volume of cells wa s altered by Me(2)SO or propane-1,2-diol. Glycerol, however, reduced t he slope of the Boyle-van't Hoff plot, but this could be ascribed to t he failure of the cells to equilibrate fully with the glycerol over th e 30 min exposure time; thus, b was unaffected by glycerol. It may be assumed, therefore, that the nonsolvent volume was not influenced by t he presence inside cells of any of these nonelectrolyte cryoprotectant s. (C) 1996 Wiley-Liss, Inc.