Fish embryos successfully cryopreserved, thawed and reanimated 10% of them

Scientists report for the first time the ability to both deep freeze and reanimate zebrafish embryos. The method, appearing in the journal ACS Nano, could potentially be used to bank larger aquatic and other vertebrate oocytes and embryos, too, for a life in the future.

In the trials, only about 10 percent of the embryos survived to 24 hours. At this point, survivors started squirming and wiggling as their hearts, eyes, and nervous systems developed, proving their viability, yet none survived to day five, the final time point the team used.

The advance is important for the field of genetics, cryobiologist Amir Arav of the Israeli company FertileSafe tells The Scientist. Zebrafish have become an important model organism for studying the genetics of vertebrates and humans, especially when it comes to cancers including melanoma, pancreatic cancer, and certain types of leukemia. Being able to preserve the different genetic lines of zebrafish generated in these studies means researchers wouldn’t need to maintain live populations or run the risk losing irreplaceable research lines. It is also the most cost-effective method for this kind of research.

Clemson University bioengineer Kelvin Brockbank says the fish also serve as a model for conservation. “Cryopreservation of an organism such as a zebrafish is also important because it is representative of other fish species and other organisms in which cryoconservation is needed due to the encroachment of mankind on their habitats and global warming,” he writes in an email to The Scientist. “We are losing species every day and if we don’t actively establish banks of organisms of all types even more will be lost.”

The team is continuing to work on the technique to improve the viability of the embryos. “The fish aren’t swimming away yet,” Bischof says. Tweaks to the laser, gold nanoparticles, and even the cryoprotectant could make the method more suitable for embryos with a diameter of a millimeter or smaller. That would mean there would be one way of cryopreservation for all organisms with embryos of that size.

Cryoprotectant and gold nanoparticles injected into zebrafish embryos allowed them to be frozen and reanimated. It is the first time this has been done with zebrafish embryos

Cryopreservation has been used to save sperm, oocytes and even embryos of many species, including humans, cattle and lab animals. Preserving the embryos of most fishes, however, has remained an elusive goal. The embryos are relatively large with big yolks and are divided by multiple compartments. These traits make the embryos difficult to cool and warm uniformly without damage and ice formation. A few techniques, including microinjection of cryoprotectants and laser irradiation for re-warming, have shown promise toward achieving this long-sought goal. John Bischof and colleagues wanted to tweak the methods to see if they could finally make cryopreserving fish a reality.

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

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 (14 million °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. 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.

The researchers injected a cryoprotectant, along with plasmonic gold nanoparticles to serve as a laser absorber, directly into zebrafish embryos. Plunging the embryos in liquid nitrogen rapidly cooled them to a cryogenically stable state in less than a second, according to modeling results. The researchers then used laser irradiation to heat up the nanoparticles, which were uniformly distributed inside the embryos, at an ultra-fast rate (1.4 x 107 degrees Celsius per minute). Not all of the embryos made it, but many were revived —a feat that is currently not possible by other techniques. Their hearts, eyes and nervous systems developed through at least the next 28 hours — and they started to wiggle. As more fish populations shrink and become threatened, the researchers say the cryopreservation method could help establish banks of frozen fish germ cells and embryos that could one day help replenish the oceans’ biodiversity. The technique could also be applied to amphibian, reptile and bird species with similar embryonic sizes and structures.