(August 2004)

To the scientific community, biotechnology is a field of study aimed at understanding how things work at the cellular or biochemical level; with the ultimate goal of deploying that knowledge to control or manipulate life. One topic in biotechnology that swings between the practical and the conceptual is the field of cryobiology.

Mike Myers in Austin Powers: The Spy who Shagged Me, Tom Cruise in Vanilla Sky and Mel Gibson in Forever Young, have all introduced us to the idea of “cryonics.” In fact, the concept of freezing and reanimating organisms is not only accepted but believed to be real. The popularity and actual existence of cryogenic companies add to the confusion on whether this technology actually exists and works. If you follow their marketing, cryogenic companies will eagerly collect your money to give you a chance at their “life-extending” possibilities. But will this be just a very frigid burial? Before you sign over your life savings, read this article and learn what applications of cryonics are practical at this time.

Defrosting a Mystery: Cryobiology Defined

Cryobiology comes from three Greek words: “kryos” meaning cold, “bios” meaning life and “logos” meaning discourse of study. Cryobiology is the science of studying the effects of very low temperature on life. Living organisms all share basic building blocks. These blocks include the genetic library held by the DNA double helix that is transferred to RNA in order for macromolecules like proteins, polysaccharides and lipids, to be manufactured. Despite their importance it is H₂0, aka water, that is the most fundamental building block to all life. Water is the key to life because it is the primary solvent in all living creatures. In cryobiology the nature of water’s transformation from a liquid solvent to a solid structure during freezing provides for the ability to preserve or destroy. Understanding how to manipulate the freezing process is the cryobiologist’s ultimate goal.

Through evolution, certain organisms have adapted to survive at low temperatures well below the freezing point of water (0°C). These organisms have the ability to create biomolecules that act as anti-freeze, lowering the temperature at which intracellular and to some extent, extracellular ice forms. In Figure 1, the scale of standard physiological temperatures for several species is shown. At -20°C, the Himalayan midge is still biologically active and is therefore able to prevent water from freezing in its cells.

Just as food is stored in the deep freezer, low temperatures have the ability to preserve life. Intact DNA and proteins from the woolly mammoth can be still be found some 50,000 years later in the frozen tundra of Siberia. On the other hand, these temperatures also have the ability to destroy. Frozen crops or frostbite on ears or toes are examples where the cold has damaged cells so badly that they are destroyed. It is this dual nature of the cold to preserve and destroy that has created its own special field of cryobiology in the entire subject of biotechnology.

Out of the Ice Age: History of Cryobiology

Cryobiology may sound like a brand new field created in the 21st century, but in fact the first documented study can be traced back to Sir Robert Boyle in his 1683 monograph “New Experiments and Observations Touching Cold”, in which this famous physicist documented the effects of freezing on living animals. Further research was continued through the 18th and 19th centuries, but it was not until the mid 1900’s that real progress was reported. In the 1940s, scientist Chris Polge of the University of Cambridge, accidentally discovered the cryoprotective abilities of glycerol through the mislabeling of reagent bottles. Using glycerol, the scientist Peter Mazur in 1963 conducted experiments to model the mechanisms of freezing within cells. These early studies on the cold have underscored the recent developments in cryobiology today.

Frozen Solid: Challenges and Obstacles in Cryobiology

From the fundamental research on the modeling of cells during the freezing process conducted by the scientist Mazur, it was discovered that it is the rate of cooling that determines the survival rate and type of damage that cells experience. To prevent irreparable damage, cells must not be cooled too slowly, or too quickly.

When cells are cooled too slowly, the outside environment of the cell freezes first and extracelluar ice forms. Extracellular ice creates a chemical potential difference across the membrane of cells creating osmosis of water which dehydrates and shrinks the cell. The slower the cells are cooled, the longer cells are dehydrated causing irreparable damage termed “solution injury” (Figure 2B). On the other hand, when cells are cooled too quickly, the cell retains water within the cell. The water expands when frozen and intracellular ice damage forms (Figure 2F). The abrasive ice crystals physically destroy the cell itself, termed “intracellular ice injury”. Both mechanisms, if balanced perfectly would achieve a maximum survival rate where the total cell damage from both mechanisms is minimized (Figure 2D). Specific cell properties such as membrane permeability to water and initial intracellular water concentration will determine the precise rate of cooling as shown in Figure 3.

Fun with Refrigeration: Current Topics and Research in Cryobiology

Understanding how the cold works on the cellular level is the basis of several specialized cryobiology focus areas: cryosurgery, cryopreservation and cryonics (as known as cyrobiosis).

In cryosurgery, the power of the cold to destroy cells, rather than preserve, is used to surgically operate on cells. Low temperature exposure (usually -20°C) can kill undesirable cells like cancer cells. In addition to the two types of cellular damage discussed earlier (solution injury and intracellular ice injury), exposure to the cold can eliminate blood circulation in the undesirable cells, thereby preventing the delivery of nutrients and oxygen as well as the removal of wastes and CO2. Finally, when the body thaws, the body’s immune system naturally detects and quickly removes the damaged cells.

In cryopreservation, the power of the cold to preserve cells is used to store or keep desirable cells in a state of suspended animation. Cell and tissue banks of frozen cells help scientists manufacture cellular products and conduct experiments on cells. So far only thin tissue cells like skin cells can be successfully frozen and unthawed. Extending this success to larger tissues and organs is a challenge in future cryopreservation research.

In cyronics or cryobiosis, the power of the cold is harnessed to preserve whole organisms, beyond just cells and tissue. This is the most well-known application of cryopreservation, but also the most controversial and so far unproven. Despite the scientific shortfalls such as the lack of a revival method, there are believers in this technology and have signed up as participants for their own preservation. In April 22, 2001, an article from the New York Times Magazine reported that in the United States about 90 people are “suspended” nationwide and the number continues to increase as more and more believers are frozen post-mortem.

Putting it on Ice: Conclusion

Both cryosurgery and cryopreservation are technologies with promising success given our understanding of the science of cryprotectants and freeze-thaw protocols while cryonics remains a meeting of the “high-tech” and the “sci-fi” which should be dreamed rather than tried. However cryonic service companies promote promises of life extension past the current technological limitations in hopes of a future remedy for our current level of technology. It is a risky proposition to invest in a conceptually appealing technology that lacks scientific validation. The impracticality of these scientific claims may be only dispelled over time with further innovative research.

(Art by Jiang Long)