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.
Bibliographical noteFunding Information:
J.B. received support from the Kuhrmeyer Chair and the Institute for Engineering in Medicine at University of Minnesota. M.H. received support from the Anela Kolohe Foundation, the Cedarhill Foundation, the Skippy Frank Translational Medicine Fund, the Roddenberry Foundation, the Paul M. Angell Family Foundation, the Hawaii Institute of Marine Biology, and the Smithsonian Institution. The manuscript was approved for publication by the Hawaii Institute of Marine Biology as contribution no. 1672. We thank the Zebrafish Core and College of Science and Engineering research shop at the University of Minnesota for access to zebrafish and the 1064 nm laser, respectively. We also thank Mr. Marc Tye for microinjection training, Dr. Zhe Gao for helping with transmission electron microscopy, and University Imaging Centers at the University of Minnesota for imaging.
© 2017 American Chemical Society.
- gold nanoparticles
- laser warming
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