Cold Storage

Cryopreservation techniques include the freezing, storing, and thawing of cells, tissues, or whole organisms so that the end result is viable and resembles the starting material. The motivation may be to maintain a type culture collection for research to ensure availability for reproductive or therapeutic use, or to establish a resource of organisms that are threatened by extinction. In general, liquid nitrogen temperature—approximately −130°C, the temperature at which frozen water no longer sublimates and recrystallizes—is used for such long-term storage. But, in order for cryopreservation to succeed, biologic material requires cryoprotectant agents to prevent damage that can occur from osmotic stress or ice crystal formation during freezing and/or thawing.

Insurance Policy

Mary Hagedorn, senior scientist at the Smithsonian National Zoologic Park in Washington, DC, whose duty station is the Hawaii Institute of Marine Biology, is investigating the use of cryopreservation with aquatic species. She has applied fertility techniques to the preservation of marine fish species, but also has realized the great need to preserve coral species. “We will lose all of our coral reefs due to global warming,” she says. “Global warming is causing huge problems for corals, especially warm water corals in tropical reefs that live at the top of their physiologic range.” Another threat is acidification of the oceans caused by an increase in dissolved carbon dioxide, which affects the ability of corals and other marine life to extract the calcium carbonate they need for shells, bones, and other structures. “We could see all ocean creatures disappear in 50 years, except jellyfish.”

“Most of what people do to save these organisms,” Hagedorn says, “is to restore and protect [habitat] areas. This is great, but other actions are needed.” Hagedorn is developing cryopreservation methods that could be used to reseed coral reefs, since coral sperm can be frozen and used to fertilize fresh oocytes. “I see myself and my science as an insurance broker for the future. I hope it's something we never have to use.” Hagedorn has investigated freezing coral larvae, which she says is as challenging as freezing human oocytes, due to their size and fat content. Additionally, work on reproductive cells—and therefore larvae—is hampered by the animals’ short reproductive season. But, she notes that much work on the physiology of these invertebrates remains to be done. “Where we are going next is really exciting,” she says. “We are taking chips of polyps and freezing them, so we won't be tied to the breeding season.” She hopes that by training people to collect polyps and freeze 50 to 60 pieces for each species, coral reefs could be reseeded in the future if necessary.

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Image 1. (Click to enlarge)

Hagedorn's group is also working on isolating coral stem cells. This would allow researchers to study coral diseases in vitro, which currently is not possible. One of the species she is working with is Elkhorn coral, Acropora palmata, one of the three most important reef-building species in the Caribbean, and which is a threatened species under the Endangered Species Act, according to the United States Fish and Wildlife Service. Hagedorn notes that one problem with this species’ population in the Florida Keys is that, due to such low diversity, it consists almost entirely of clones. She would like to be able to introduce more diversity into this population by hybridizing it with material from related populations, and holds out hope that the Fish and Wildlife Service will allow her to do this.

Empirical Methods

Replacing coral reefs takes more than repopulating coral polyps, notes Jerry Brand, a professor in the School of Biological Sciences at the University of Texas in Austin, Texas. The coral community relies on zooxanthellae, symbiotic dinoflagellates (algae) that provide corals with energy. Although Brand does not work with zooxanthellae, his work with cryopreservation of other algae species is applicable to many microorganisms and other species. Algae, Brand notes, make up an important component of many ecosystems, and if coral reefs are to be replaced, the entire ecosystem may need to be restored.

An advantage of maintaining frozen stocks of microorganisms is the ability to preserve strains whose precise identity is known. Continually passaged strains under the selective pressure of culture are likely to develop genetic or epigenetic changes that may only be advantageous in vitro, leading to the loss of the original, well-characterized strain. In addition, maintaining cultures in vitro is resource-, time-, and labor-consuming. Brand describes the algae collection at the University of Texas as one of the largest and most diverse in the world, containing about 400 strains. About two-thirds of these are cryopreserved. Brand notes that most fresh water strains, including those that exist in soil or as epiphytes, those that live in fresh water, and those that are less than 40 micrometers in diameter, can be frozen. Species that are difficult to cryopreserve are marine species, have large cells, are multicellular, or have large vacuoles.