Human granulocytes are injured when returned to isotonic conditions after exposure at 0 degree C to hyperosmotic solutions of NaCl or sucrose with osmolalities above 0.6 osmolal. The damage was expressed as a loss of membrane integrity [fluorescein diacetate (FDA) assay] only after 60-90 min incubation at 37 degrees C. Survival after exposure to a 1.4-osmolal solution at 0 degree C was dependent on the extent of subsequent dilution. Dilution to below 0.6 osmolal was damaging, but cells could be returned to near-osmotic conditions provided that the solute concentration was increased again to 0.64 osmolal before the cells were incubated at 37 degrees C. Granulocyte cell volumes were measured under various osmotic conditions by computer-assisted micrometry. The cells did not display a minimum volume but behaved as osmometers over the observed range of 0.2-1.4 osmolal. Granulocyte volume at a given osmolality was independent of whether the cells had first been exposed to a strongly hyperosmotic medium, indicating that no solute loading occurred in hyperosmotic sucrose solutions. Even though the cells did not survive sequential exposure to greater than 0.6 osmolal solutions, subsequent return to isotonicity, and incubation at 37 degrees C, neither cell lysis nor loss in FDA-positive cells occurred after the first two steps. This finding is not consistent with the critical-surface area-increment theory of freezing injury. The mechanism of cell injury in hyperosmotic solutions is thus not known. However, the results show that osmotic stress is potentially a major damaging factor both in the equilibration of cells with protective additives and during freezing and thawing.
Effects of Pressure-shift Freezing on the Structural and Physical Properties of Gelatin Hydrogel Matrices
