Fish-eye view into cryopreservation

Mandarin fish

These results can go towards a model for the cryopreservation of endangered species with more challenging embryos, such as some coral fishes like this Mandarin fish. Credit: Emily Mobley
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Scientists from the ISIS Neutron and Muon Source have solved the mechanism behind the long-term freeze storage technique ‘cryopreservation’ that is used to preserve the embryos of species that are in danger of extinction. The study, published in Royal Society Open Science, opens the door to protecting endangered species that have never before been able to be preserved.

Despite global conservation efforts, many marine, fresh-water and land animals may become extinct in the next few decades. In an effort to protect endangered species, conservationists can freeze embryos in the early stage of their development and these can be stored for thousands of years - a technique known as cryopreservation. Some species’ embryos, however, are more difficult to cryopreserve than others. For the process to be successful, it is essential that ice crystals do not form as they act as tiny knives, puncturing cell membranes and killing the embryo.

Now for the first time, the mechanism behind cryopreservation has been cracked at the molecular level. These results will make it possible to cryopreserve embryos of endangered species which are currently unachievable. In mammals the freezing of embryos is common place, whereas in some fish species it is more difficult; fish eggs have strong outer membranes that are impermeable to cryoprotectants - the chemicals used to preserve the embryo and protect it from ice crystals.

In neutron diffraction experiments at ISIS, scientists ‘watched’ the freezing process take place and were able to see what was happening to individual components in the mix of cryoprotectants and water.

Dr. Alan Soper and Dr. Oleg Kirichek took three different mixtures of cryoprotectants in water and quenched cooled, or rapidly cooled them in liquid nitrogen to -196 degrees celsius in seconds. They then performed neutron diffraction experiments on SANDALS to study the molecular structure of each mixture after freezing.

One mixture in particular had previously led to a perfectly conserved fish egg of a Common Carp. The mixture, which produced diffraction patterns typical of a glass structure, consisted of 23 vol% 1,2-propanediol, 17 vol% methanol and 20 vol% dimethylsulfoxide in water.  

As neutrons are very sensitive to hydrogen, a key element of water, scientists were able to ‘see’ at the molecular level what happened to the mixture after quench cooling.

They found that in mixtures with a particular concentration of cryoprotectants, the chemicals formed long chains and acted like a mesh or ‘sponge’ that locked the smaller water molecules in to pockets. In normal freezing, water molecules link up to form ice crystals, however the cryoprotectant mesh meant the water molecules were isolated in small clusters.

Simulation showing the water molecules confined to clusters

A 1.5 nm slab of the EPSR simulation box for samples containing 23 vol% PD, 17 vol% methanol, 20 vol% DMSO and 40 vol% water, showing only the water molecules. Notice how the water congregates in percolating clusters. It is proposed that it is this inability of the water to form an extended, three-dimensional network that prevents it from forming ice crystals. Credit: Alan Soper
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Dr. Alan Soper, an ISIS scientist and world expert in the structure of water and water-based solutions said:

"Neutrons were crucial for this study because they instantly highlighted any ice that did develop in some samples, and also, by virtue of the ability to perform hydrogen/deuterium substitution on the water molecules, allowed us to access the water structure independently of the contributions from the other components in these solutions.”

The study confirms observational experiments by scientists in the Czech Republic, who after several years of trial and error experiments, noticed that a particular mixture of cryoprotectants led to a perfectly preserved fish egg from a Common Carp.

Now the mechanism of cryopreservation has been found, these results can go towards a model for the cryopreservation of endangered species with more challenging embryos, such as some coral fishes.

Professor William Holt, former Head of Reproductive Biology at the Zoological Society of London (ZSL) said this work will contribute to conservation efforts:

"This represents a significant step in the development of a technique for fish oocyte cryopreservation. If eggs could be frozen and stored without losing viability, they would provide a valuable resource for conservation programmes. By providing genetic support to endangered populations, these techniques would help reduce the damaging effects of inbreeding, enhancing animal welfare."

 

Emily Mobley

Dr Alan Soper and Dr Oleg Kirichek

Research date: March 2016

Further Information

For further information, the paper is available online

Kirichek, O., Soper, A.K., Dzyuba, B. and Holt, W.V. (2016) Segregated water observed in a putative fish embryo cryopreservative. R. Soc. open sci. 3: 150655

DOI: 10.1098/rsos.150655

The Zoological Society of London (ZSL)

Founded in 1826, the Zoological Society of London (ZSL) is an international scientific, conservation and educational charity whose mission is to promote and achieve the worldwide conservation of animals and their habitats. Our mission is realised through our ground-breaking science, our active conservation projects in more than 50 countries and our two Zoos, ZSL London Zoo and ZSL Whipsnade Zoo.

For more information visit www.zsl.org

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