Influence of various cooling and warming temperatures on survival after thawing of cryopreserved Plasmodium berghei

Abstract

Heavy concentrations of viable P. berghei in the natural milieu [20% ( v v ) parasitized red blood cells, or 20% ( w v ) homogenate of splenic tissue in which malarial cells sequestered wer suspended in a serum-free, protective medium. Various rates of cooling are designated as low (1.3 °C/min) and intermediate (4 °C/ min) on exposure in cold gas evolving from liquid nitrogen refrigerant to −70 °C, and this followed by direct immersion in the low temperature refrigerant (−196 °C). Cooling designated high was accomplished by abrupt immersion of the sealed vials with the live malaria-bearing tissue in the liquid nitrogen refrigerant. Rates of warming and thawing were designated low (after slow rewarming of frozen tissue in air at 25.5 °C) and high (after rapid rewarming and thawing in a water bath at 40 °C). Strip chart recordings were made of the complete cooling and freezing wave patterns of the suspending medium to −70 ° C. The functional survivals of the freeze-thaw P. berghei malaria were measured by a special infectivity titration method. None of the cooling and freezing treatments adversely influenced the parasite survivals. Our data showed the optimum cooling velocity that maximally protected this highly lethal P. berghei strain within the host erythrocyte matrix was 1.3 ° C/min to −70 to −196 ° C. The functional survivals of two RBC stabilates with P. berghei, after retrieval from 25 days storage in the liquid nitrogen refrigerant, excelled by more than 100-fold the infectivity titer found by viability assay in the pool of the 0-days nonfrozen infected RBC. The precise factors favoring the maximal survivals of the freeze-thaw P. berghei are unclear. Several factors, singly or in combination, may have played key roles in protecting the living P. berghei from the freeze-thaw damage. These factors are: The composition of the suspending medium fortified by additions of bicarbonate, glucose, lactalbumin hydrolysate and yeastolate; the presence of naturally occurring peptide-containing materials surrounding the parasites in the host red cell milieu; and the protective glycerol agent. Any of these constituents singly or combined possess potential for reducing freeze-thaw injury to the parasites to produce maximal survivals.