073 Interactions between Zebrafish embryos and ice crystals during isothermal freezing and rapid cooling

Abstract

Cryopreservation of embryos from zebrafish (Danio rerio) and other teleosts would benefit biomedical research, commercial aquaculture, and ecological conservation, but is not yet feasible. Rapid changes in embryo opacity (e.g., darkening) observed almost immediately upon initial contact between zebrafish embryos and external ice crystals have previously been attributed to intracellular ice formation; the absence of any perceptible time delay between extracellular and intracellular ice formation events is recognized as an obstacle to ice-assisted cryopreservation using slow-cooling protocols. Despite the evident importance of the intraembryonic darkening process, the nature of this phenomenon has never before been elucidated. Using high-speed video cryomicroscopy techniques, we have previously shown that darkening events observed using conventional cryomicroscopy methods do not necessarily represent intracellular ice formation [Stott and Karlsson, Cryobiology 58 (1) (2009) 84–95]. Thus, we used our high-speed imaging system to visualize the response of zebrafish embryos to contact with external ice crystals. All experiments used embryos at 0.5-9 hours post fertilization, frozen in E3 medium without additives. One set of experiments subjected embryos to rapid freezing without controlled seeding of ice. High-speed video recordings acquired at up to 5000 frames/second revealed a sequence of events significantly more complex than the response of mammalian somatic cells during freezing. Through slow-motion playback of n=20 videos, we identified 17 distinct events that occurred within the embryos after the spontaneous formation of external ice. The initial intraembryonic event was the formation of (generally transparent) ice crystals in the perivitelline fluid at −22 ± 1°C, as external ice crystals penetrated the chorion. The second and third events were a rapid growth of a dark (i.e., opaque) area starting from the yolk center, followed by a rapid shrinkage and often complete disappearance of this dark spot, all within 156 ± 31 ms of the initial perivitelline crystallization. Other phenomena that were observed in >75% of the embryos included a secondary yolk darkening process, as well as two distinct darkening events in the blastoderm (usually in conjunction with an interim transient increase in blastomere transparency). Significantly, the initial darkening of both yolk and blastoderm was reversible during cooling, which suggests that these phenomena should not be interpreted as manifestations of ice formation. We also investigated ice-embryo interactions after controlled initiation of the external ice formation. After seeding of ice, embryos were held isothermally at −0.4°C for 20 min. Even though all embryos were fully enveloped in ice, there was no visible damage in 41% of embryos (out of n = 29), which is significantly larger than the hypothesized null viability (p Source of funding: National Science Foundation Grant No. CBET-1066619, awarded to JOMK. Conflict of interest: None declared. jens.karlsson@villanova.edu