Cryopreservation of Plasmodium chabaudi. II. Cooling and warming rates

Various cooling and warming rates were investigated to determine the optimum conditions for cryopreserving the intraerythrocytic stages of Plasmodium chabaudi. Infected blood, equilibrated in 10% v/v glycerol at 37 degrees C or in 15% v/v Me2SO at 0 degree C for 10 min, was cryopreserved using cooling rates between 1 and 5100 degrees C min-1. After overnight storage in liquid nitrogen the samples were warmed at 12,000 degrees C min-1. Warming rates between 1 and 12,000 degrees C min-1 were investigated using samples previously cooled at 3600 degrees C min-1. After thawing, the glycerol and Me2SO were removed by dilution in 15% v/v glucose-supplemented phosphate-buffered saline. Survival was assayed by inoculation of groups of five mice each with 10(6) infected cells and the time taken to reach a level of 2% parasitemia estimated. The optimum cooling rate was 3600 degrees C min-1 for parasites frozen using either 10% glycerol or 15% Me2SO; the pre-2% patent periods were 0.90 and 1.01 days above control values (representing survival levels of 21 and 17.5%, respectively). The optimum warming rate was 12,000 degrees C min-1; the pre-2% patent periods were 1.01 and 1.32 days above control values, respectively (18 and 10% survival), for glycerol and Me2SO. With ethanediol (5% v/v) and sucrose (15% w/v) as cryoprotectants the optimum warming rates were also 12,000 degrees C min-1 while the optimum cooling rates were 330 and 3600 degrees C min-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)