Contributions of Cooling and Warming Rate and Developmental Stage to the Survival of Drosophila Embryos Cooled to -205°C

Abstract

Because of their high susceptibility to chilling injury, permeabilized Drosophila embryos can not be cryobiologically preserved by slow freezing at rates low enough to prevent the formation of intraembryonic ice. Calculations indicated that to outrun the chilling injury they must be cooled and warmed rapidly at an estimated 20,000°C/min or faster. Ordinarily, such cooling rates would inevitably produce lethal intracellular ice. To prevent this, embryos must contain and be surrounded by sufficiently high concentrations of glass-promoting solutes to induce vitrification on cooling and prevent devitrification on warming. Like Steponkus et al. ( Nature 345, 170, 1990) we have used ethylene glycol as the solute and have exposed permeabilized 12-h embryos to it in two steps. (Permeabilization was effected by exposing dechorionated embryos to a mixture of 0.3% 1-butanol in n-heptane for 90 or 110 s.) The two steps were (i) a 30-min exposure to 2 M ethylene glycol at 23°C and (ii) a 5-min exposure to 8.5 M ethylene glycol [±10% polyvinylpyrrolidone (PVP)] at 5°C. The volumetric response to the first step indicates that full permeation of the 2 M glycol has been approached by 30 min. The point of the second step is to raise the intraembryonic concentration of ethylene glycol to near 8.5 M ethylene glycol by osmotic dehydration. Survival based on hatching is some 45% at this point. When 12-h embryos in 8.5 M glycol containing 10% PVP are then cooled to -205°C at ∼ 100,000°C/min and warmed at about that rate, an average of about 12% survive (hatch), although in about half the runs 15-29% survive. Survivals in the absence of PVP are usually poorer but have been as high as 40%. Currently, 5% of the surviving larvae develop to adult flies (Steponkus et al. reported 18% hatching and 3% development to adult). Embryos that develop but do not hatch show readily detectable abnormalities in mouth parts and dorsal closure. Very high warming rates are much more critical to survival than are very high cooling rates; for example, none survive when warming is 2000°C/min. The deleterious effect of slow warming is exerted between - 80 and - 40°C. The lack of reciprocity between the effects of time spent cooling and time spent warming argues against ascribing death to chilling injury. Rather, it and other data argue for ascribing death to the devitrification during warming of cytoplasm that vitrified during cooling. Various lines of evidence including calorimetry indicate that a warming rate of ∼ 100°C/min is high enough to prevent devitrification of 8.5 M ethylene glycol + PVP (54 wt% glycol). The fact that survival requires much higher warming rates strongly suggests that that concentration of glycol is not being attained in the embryos as a whole or in compartments within the embryos. Superimposed on the effect of warming rate is an effect of embryo age. The survival of 14- to 15-h embryos is more than double that of 12-h embryos.