Design and Analysis

Zebrafish Embryos Successfully Cryopreserved with Gold Nanotechnology

14 July 2017

Scientists have been trying to cryopreserve an embryo from zebrafish for over 60 years. According to a new study, scientists have achieved full cryopreservation of a zebrafish embryo. The zebrafish is an important medical model for human health in health science research. The research team at the University of Minnesota and the Smithsonian Conservation Biology Institute (SCBI) have been able to produce the first-ever evidence for the first cryopreservation of the fish embryos.

Researchers use gold nanotechnology and lasers to warm the embryo. Warming and freezing the embryo has been the stumbling block in previous studies. The results of this experiment have profound implications for human health, wildlife, conservation and aquaculture.

The zebrafish (YourGenome)The zebrafish (YourGenome)

"There's no doubt that the use of this technology, in this way, marks a paradigm shift for cryopreservation and the conservation of many wildlife species," said Mary Hagedorn, an SCBI research scientist, and paper co-author. Hagedorn has been working on cryopreserving zebrafish embryos since 1992.

"To get anything to work at such cold temperatures, you usually have to get creative. Here we take a unique approach by combining biology with an exciting engineering technology to do what has been impossible previously: to successfully freeze and thaw a fish embryo so that the embryo begins to develop, rather than falls apart," Hagedorn added.

By freezing the sperm, eggs, and embryos conservationists can protect at-risk species and their genetic diversity. This makes it possible to bolster the genetic pool and improve the health of the wild population for years. Cryopreserving the embryos of some mammals and sperm from many species of fish has already been achieved in the medical field. But freezing fish embryos is much more complicated.

Successful cryopreservation of embryos starts with cooling the embryo to a cryogenically stable state. The next step is to warm the embryo faster than it was cooled. The scientists then used antifreeze to stop the growth of ice crystals, which could pop the membrane and cause the embryo to fall apart. Fish embryos are very large, which makes it difficult to thaw them quickly and avoid ice crystals. Because aquatic animals need to survive in harsh environments, the embryonic membranes are mostly impenetrable, which blocks the cryoprotectants out.

That’s where the laser gold nanotechnology steps in. Laser gold nanotechnology is rapidly growing technology being developed for cryopreservation applications by University of Minnesota Mechanical Engineering professor John Bischof. This technology was critical to the success of this study and other biomedical applications.

"Lasers have the exciting ability to act like a "light switch" that can turn biological activity on and off within gold nanoparticle laden biomaterials," said Bischof, senior author of the study. "In this case, by careful engineering and deployment of gold nanoparticles within a cryogenically stored and biological inactive embryo, we can use a laser pulse to quickly warm the embryo back to ambient temperatures and switch biological activity, and therefore life, back on."

Gold nanorods are tiny cylinders of gold which convert absorbed light into heat. The authors of the study inject the cryoprotectant and nano gold particles into the embryos. The gold particles transfer heat uniformly through the embryo when the laser hits the embryo. The laser warms the embryo from -196 degrees C to 20 degrees C in one thousandth of a second.This fast warming rate combined with the cryoprotectant prevented the formation of ice crystals.

Embryos that went through this process went on to at least the 24-hour stage of development. At this stage, the fish have developed a heart, gills, tail musculature and the ability to move. This proves the post-thaw viability.

The next step for the researchers is to fine-tune the process to increase embryo survival rate. Also, they will investigate the use of automation to increase the number of embryos they can thaw simultaneously.

The embryos of other aquatic animals are very similar to the zebrafish, so this technology is applicable to more than just the zebrafish embryos. The technology could be customized to cryopreserve reptile and bird embryos, as well as enhance the process of cryopreserving mammalian embryos, including animals like giant pandas. The technology could also help aquaculture farms become more efficient and cost effective. This would put less pressure on wild populations.

Zebrafish are used as important diseases models to study melanoma, heart disease and blood disorders another health issues. Cryopreserved zebrafish embryos will prevent scientists from losing entire research lines and gives them the flexibility to bring the lines back when they need.

A paper on this research was published in the ACS Nano journal.

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