Vitrification-Based Cryopreservation of Drosophila Embryos

Abstract

Currently, over 30,000 strains of Drosophila melanogaster are maintained by geneti-cists through regular transfer of breeding stocks. A more cost effective solution is to cryopreserve their embryos. Cooling and warming rates> 1O,000°C/min. are required to prevent chilling injury. To avoid the lethal intracellular ice normally produced at such high cooling rates, it is necessary to use ~50% (w/w) concentrations of glass-inducing solutes to vitrify the embryos. Differential scanning calorimetry (DSC) is used to develop and evalu-ate ethylene glycol and polyvinyl pyrrolidone based vitrification solutions. The resulting solution consists of 8.5M ethylene glycol + 10% polyvinylpyrrolidone in D-20 Drosophila culture medium. A two stage method is used for the introduction and concentration of these solutes within the embryo. The method reduces the exposure time to the solution and, consequently, reduces toxicity. Both DSC and freezing experiments suggest that, while twelve-hour embryos will vitrify using cooling rates >200°C/min., they will devitrify and be killed with even moderately rapid warming rates of -1 ,900°C/min. Very rapid warming (-IOO,OOO°C/min.) results in variable numbers of successfully cryopreserved embryos. This sensitivity to warming rate is typical of devitrification. The variability in survival is reduced using embryos of a precisely determined embryonic stage. The vitrification of the older, fifteen-hour, embryos yields an optimized hatching rate of 68%, with 35 -40% of the resulting larvae developing to normal adults. This success rate in embryos of this age may reflect a reduced sensitivity to limited devitrification or a more even distribution of the ethylene glycol within the embryo.