Gold Nanorod Induced Warming of Embryos from the Cryogenic State Enhances Viability

Abstract

Zebrafish embryos can attain a stable cryogenic state by microinjection of cryoprotectants followed by rapid cooling, but the massive size of the embryo has consistently led to failure during the convective warming process. Here we address this zebrafish cryopreservation problem by using gold nanorods (GNRs) to assist in the warming process. Specifically, we microinjected the cryoprotectant propylene glycol into zebrafish embryos along with GNRs, and the samples were cooled at a rate of 90 000 °C/min in liquid nitrogen. We demonstrated the ability to unfreeze the zebrafish rapidly (1.4 × 10⁷ °C/min) by irradiating the sample with a 1064 nm laser pulse for 1 ms due to the excitation of GNRs. This rapid warming process led to the outrunning of ice formation, which can damage the embryos. The results from 14 trials (n = 223) demonstrated viable embryos with consistent structure at 1 h (31%) and continuing development at 3 h (17%) and movement at 24 h (10%) postwarming. This compares starkly with 0% viability, structure, or movement at all time points in convectively warmed controls (n = 50, p < 0.001, ANOVA). Our nanoparticle-based warming process could be applied to the storage of fish, and with proper modification, can potentially be used for other vertebrate embryos.

Keywords: cryopreservation; gold nanoparticles; laser warming; zebrafish.