State of the Science Stories: Cryobanking | Assemble+

State-of-the-Science Stories: Cryobanking Marine Organisms

 

What is Cryobanking?

Crybanking is the process of cooling and storing cells, tissues, or organs at very low or freezing temperatures to preserve them for future use. Cryobanking allows one to use the preserved material for a much longer period of time, allowing for a broader range of R&D and industrial uses. 

ASSEMBLE Plus is working on the design of cryopreservation protocols of various marine genetic and biological resources (e.g. genetically-modified metazoans, macroalgae and microrganisms) for bio-banking. These organisms are strategically important resources for academia and industry (e.g. with bio-actives for food, feed, pharma and cosmetics), but cryobanking protocols for them are not yet sufficient or do not exist. We are also exploring processes to extend the reproduction window of commercial species.

 

What is ASSEMBLE Plus doing that is different?

Aquaculture

ASSEMBLE Plus is working on the developing the protocols that will allow the cryopreservation of sea urchins and mussels. Sea urchins only reproduce for four to six months of the year, and this is inefficent for research purposes! While it is possible to force their reproduction by tricking them into believing that it is spring (hence creating a stock of out-of-season reproducers), this is expensive. Cryopreservation of the sperm, embryos, or larvae in a biobank is a cheaper and better alternative, allowing one to work with sea urchins all year around. Cryopreservation has also proven to be an incredibly useful technique for the land animal breeding sector, and it can equally be useful for sea urchin seed production.

As mussels also reproduce only twice a year (in spring and autumn), this is inefficient for research and for the aquaculture industry. It is believed that cryopreservation here can also offer an opportunity for extending the mussel window, but until now this had not been achieved. The research team at the Universidade de Vigo obtained this year, for the first time, seed from cryopreserved mussel larvae (M. galloprovincialis). These were then grown to juvenile stage and then put through the ropes in a marine environment to reach maturation. A proof of concept that cryopreservation of mussel larvae can be used to improve market seasonality was thus achieved! A protocol for mussel spawning was developed and the results are published. As IFREMER has already done the same for oysters (C. gigas), it is quite clear that cryopreservation of molluscs has a great potential in the aquaculture industry.

Biodiversity

One of the most interesting applications of cryopreservation and biobanking is its role in preserving genetic biodiversity, and specifically in the preservation of the genetic material of endangered species. CCMAR is working on with the cryopreservation of the Portuguese oyster (C. angulata) and of species of biomedical interest such as different zebrafish strains.

This biodiversity preservation is important and useful for culture collections and research facilities. Roscoff are investigating the cryopreservation of species of macroalgae, e.g. Laminaria sp. and Prophyra sp. SAMS have developed a method for cryopreserving gametophytes of the brown seaweed, Saccharina latissima, and this has resulted in a paper (Visch et al., 2019). NIOZ has successfully developed a cryopreservation protocol for hypersaline microbial mats (a multi-layered sheet of different microorganisms of a few centimetres thick that grow in moist areas), which can now be preserved at -150 ºC and can be grown again for 120 days!

In total, ASSEMBLE Plus has developed cryopreservation approaches for over 200 algal species (micro and macroalgae), thanks to the combined efforts on standardising and training among several institutions: SAMS, Roscoff Marine Station (SU), MBA, UPV-EHU, ECIMAT-Uvigo). Cryopreserved samples can now be shipped using one-use dry shippers between institutions in a very simple and comfortable manner.

Products and outputs

 

What next?

  • The CCMAR team has been working on the development of improved cryopreservation protocols for Chamelea gallina and Crassostrea angula sperm and larvae.
  • UPV are working on sperm cryopreservation for the thicklip grey mullet (Chelo labrosus), which are used as sentinels of environmental health in pollution monitoring campaigns on the Basque coast.
  • Roscoff, SAMS, MBA and ECIMAT are working on the identification of recalcitrant microalgae species (species that are difficult to cryopreserve). Experiments for benthic-filter feeder, amphioxus, are scheduled for next spawning season to test a number of cryopreservation techniques on embryos, and then to evaluate using markers and/or sequencing techniques.

 

Potential Impact

Research conducted at the University of Vigo on sea urchin embryo cryopreservation was patented in 2015, and as yet no-one has shown an interest in buying the patent. Interest in land-based seed production is renewed, and it is clear that cryobiology can be used as an out-of-season tool for seed storage.

In general molluscs and other marine invertebrate fisheries and aquaculture companies rely on the intake of seed from the ocean. This is the case of mussels in Spain where seed is extracted from the rocky shores of selected locations. Cryopreservation can mitigate the impact that collecting seed from the natural environment can have, and can allow for the species to be grown year-round. 

Many traditional fisheries such as of clams have been approaching culturing in a sustainable way, producing seed inland that is later transferred to the beach to increase natural production and ensure annual collection rates without over-exploitation of the resources. They are always restocking. This approach means that they are better prepared for the future market, and are mitigating risks related to climate change.

This approach can be also useful for sea urchins, with worldwide decline of stocks and many EU fisheries reporting overfishing. However, attempts to grow sea urchins either inland or on-shore have not been successful. Cryopreservation of seed would make it possible to to seed the ocean in known areas, to re-stock natural populations and to allow a healthy production-extraction balance. This provides a cheaper, more efficient, and functional way to allow the continued fishing of sea urchins.

Cryopreservation has been use in land animals husbandry for decades now and has boosted the industry in many directions. It is being used for animal conservation efforts and repopulation. In the marine field, cryobanking is still under development. Thanks to Assemble Plus, a critical mass of knowledge now exists, allowing us to make a leap in development. We aim to make researchers aware of the multiple applications that exist right now and that will exist in the near future.

 

References

Diogo, P., Martins, G., Quinzixo, I., Nogueira, R., Gavaia, P.J. and Cabrita, E. (2018) Electric ultrafreezer (− 150 °C) as an alternative for zebrafish sperm cryopreservation and storage. Fish Physiol. Biochem., 44(6): 1443-1455
Diogo, P., Martins, G., Eufrásio, A., Silva, T., Cabrita, E. and Gavaia, P. (2019) Selection criteria of zebrafish male donors for sperm cryopreservation. Zebrafish, 16(2): 182-196
Heres, P., Rodriguez-Riveiro, R., Troncoso, J. and Paredes, E. (2019) Toxicity tests of cryoprotecting agents for Mytilus galloprovincialis (Lamark, 1819) early developmental stages. Cryobiology, 86: 40-46
Paredes, E. and Bellas, J. (2019) The Use of Cryopreserved Biological Material for Water Quality Assessment. Frontiers in Marine Science, doi: 10.3389/fmars.2019.00454
https://patentscope.wipo.int/search/en/detail.jsf?docId=ES154529708&recNum=50&docAn=201400536&queryString=FP:(Universidade%20de%20Vigo)&maxRec=140
Riesco, M.F., Félix, F., Matias, D., Joaquim, S., Suquet, M. and Cabrita, E. (2019) Comparative study on cellular and molecular responses in oyster sperm revealed different susceptibilities to cryopreservation. Aquaculture, 498: 223-229
Rodriguez-Riveiro, R., Heres, P., Troncoso, J. and Paredes, E. (2019) Long term survival of cryopreserved mussel larvae (Mytilus galloprovinciallis). Cryobiology, 512, doi: 10.1016/j.aquaculture.2019.734326
Visch, W., Rad-Menéndez, C., Nylund, G.M., Pavia, H., Ryan, M.J. and Day, J. (2019) Underpinning the development of seaweed biotechnology: Cryopreservation of brown algae (Saccharina latissima) gametophyte. Biopreservation and Biobanking, 17(5), doi: 10.1089/bio.2018.0147

 

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