Comparison of fast (one-step) and interrupted slow cooling methods using a range of intracellular and extracellular cryoprotectants for the freeze-preservation of Plasmodium yoelii-infected mouse erythrocytes.

Several cryoprotectants were compared using two different cooling procedures for the cryopreservation of both normal and Plasmodium yoelii-infected mouse erythrocytes. Fast cooling to -196 degrees C by direct plunge into liquid nitrogen followed by rapid thawing in a 37 degree C water bath protected uninfected and parasitized erythrocytes against freeze-thaw damage significantly better than an interrupted slow cooling procedure in which cells were frozen to -70 degrees C at a rate of -1 degree C/min followed by storage in liquid nitrogen. Using the former procedure, the highest percentage erythrocyte recoveries (over 85%) and shortest pre-2% parasitaemia times (equivalent to unfrozen cells) in mice challenged with thawed parasitized blood were observed with the intracellular (penetrating) cryoprotectants glycerol or dimethylsulphoxide (Me2SO). At the concentrations used in this study, the extracellular (non-penetrating) cryoprotectants, hydroxyethylstarch, dextran or polyvinyl pyrrolidone were significantly less effective at protecting against freeze-thaw damage. Some evidence of freeze-thaw damage was obtained in cells fast frozen at low cell density (less than or equal to 10(4)/ml) in glycerol or Me2SO. This effect was masked when higher cell densities (10(7) to 10(8)/ml) were used. Addition of the anti-oxidant and membrane stabilizing molecules, vitamin E, sodium selenite or selenomethionine to infected erythrocytes just before and during cryopreservation, did not improve, and in one case inhibited, subsequent development in challenged mice.