The University of Minnesota has made a highly-touted new technology for “cryo-preserving” living biomaterials such as fish embryos available for licensing. Ultimately, the public institution is seeking a commercialization partner for the method hailed as a breakthrough for wildlife conservation and human health research.
In 2017, they provided first-ever reproducible evidence for the successful cryopreservation of zebrafish embryos.
This was the first time a frozen fish embryo had survived to grow after being thawed out from a cryogenic state. Zebrafish are particularly valuable to health researchers because their genomes approximate those of humans closely enough to be used for modelling diseases in lab experiments.
For the health researchers, the ability to cryopreserve zebrafish embryos would be valuable because it would make their studies on human diseases such as muscular dystrophy and melanoma easier to conduct and replicate — they wouldn’t have to work around the fishes’ spawning schedules or contend with “genetic drift” of subsequent generations.
And for wildlife preservations, they can freeze sperm, eggs and embryos. They can safeguard at-risk aquatic species and their genetic diversity, making it possible to bolster the genetic pool and therefore guarantee the health of wild populations years — or even centuries — later.
They overcome a previous roadblock in cryopreserving fish embryos: Due to their large size, traditional ways of thawing them out are too slow and result in the formation of ice crystals which damage them and prevent viability. The key innovation of the U technology addresses that problem with the use of gold nanoparticles, or cylindrical “nanorods,” which are injected into the embryo before freezing.
They essentially act as a “distributed network” of ultra-efficient heaters that generate very fast warming rates when illuminated with an infrared laser, thus avoiding the creation of ice crystals. The nanorods also have low toxicity levels.
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. A well-calibrated millisecond laser pulse can suddenly heat up an embryo by way of the gold distributed throughout it, reheating it at the astonishing rate of 1.4 x 107 °C per minute, an almost unfathomable temperature that is manageable in the quick bursts that the researchers employ.
The results were hot enough—and widely distributed enough—to successfully reheat an entire zebrafish embryo at once.