Cryobiology is the branch of biology that studies the effects of low temperatures on living things. The word cryobiology is derived from the Greek words "cryo" = cold, "bios" = life, and "logos" = science. In practice, cryobiology is the study of biological material or systems at temperatures below normal.

Materials or systems studied may include proteins cells, tissues, organs, or whole organisms. Temperatures may range from moderately hypothermic conditions to cryogenic temperatures.

Cryopreservation of cells is guided by the "Two-Factor Hypothesis" of American cryobiologist Peter Mazur, which states that excessively rapid cooling kills cells by intracellular ice formation. Excessively slow cooling kills cells by either electrolyte toxicity or mechanical crushing. During slow cooling ice forms extra-cellularly, causing water to osmotically leave cells, thereby dehydrating them. Intracellular ice can be much more damaging than extracellular ice.

For red blood cells the optimum cooling rate is very rapid (nearly 100 °C per second), whereas for stem cells the optimum cooling rate is very slow (1 °C per minute). Cryoprotectants, such as DMSO (dimethyl sulfoxide) and glycerol, are used to protect cells from freezing. A variety of cell types are protected by 10% DMSO. Cryobiologists attempt to optimize cryoprotectant concentration (minimizing both ice formation and toxicity) as well as cooling rate. Cells may be cooled at an optimum cooling rate to a temperature between −30 °C and −40 °C before being plunged into liquid nitrogen.

Slow cooling methods rely on the fact that cells contain few nucleating agents, but contain naturally-occurring vitrifying substances that can prevent ice formation in cells that have been moderately dehydrated.

Some Cryobiologists are seeking mixtures of cryoprotectants for full vitrification (zero ice formation) in preservation of cells, tissues and organs. Vitrification methods pose a challenge in the requirement to search for cryoprotectant mixtures that can minimize toxicity.

CRYOBIOLOGY, (publisher: Elsevier) is the foremost scientific publication in this area, with approximately 60 refereed contributions published each year. Articles concern any aspect of low temperature biology and medicine (e.g. freezing, cold tolerance and adaptation, medical applications of reduced temperature, cryosurgery, hypothermia, perfusion of organs and cryoprotective compounds)


A cryoprotectant is a substance that is used to protect biological tissue from freezing damage (i.e. that due to ice formation). Arctic and Antarctic insects, fish and amphibians create cryoprotectants (antifreeze compounds and antifreeze proteins) in their bodies to minimize freezing damage during cold winter periods. Insects most often use sugars or polyols as cryoprotectants. One species that uses cryptopectant is Polistes exclamans. In this species the different levels of cryoprotectant can be used to distinguish between morphologies.[1] Arctic frogs use glucose, but Arctic salamanders create glycerol in their livers for use as a cryoprotectant. Cryoprotectants operate simply by increasing the solute concentration in cells. However, in order to be biologically viable they must (1) easily penetrate cells, and (2) not be toxic to the cell.

Conventional cryoprotectants

Conventional cryoprotectants are glycols (alcohols containing at least two hydroxyl groups), such as ethylene glycol, propylene glycol, and glycerol. Ethylene glycol is commonly used as automobile antifreeze, and propylene glycol has been used to reduce ice formation in ice cream. Dimethyl sulfoxide (DMSO) is also regarded as a conventional cryoprotectant. Glycerol and DMSO have been used for decades by cryobiologists to reduce ice formation in sperm and embryos that are cold-preserved in liquid nitrogen.


Mixtures of cryoprotectants have less toxicity and are more effective than single-agent cryoprotectants. A mixture of formamide with DMSO (dimethyl sulfoxide), propylene glycol, and a colloid was for many years, the most effective of all artificially created cryoprotectants. Cryoprotectant mixtures have been used for vitrification (i.e. solidification without crystal ice formation). Vitrification has important applications in preserving embryos, biological tissues, and organs for transplant. Vitrification is also used in cryonics in an effort to eliminate freezing damage.

Glass transition temperature

Some cryoprotectants function by lowering the glass transition temperature of a solution or of a material. In this way, the cryoprotectant prevents actual freezing, and the solution maintains some flexibility in a glassy phase. Many cryoprotectants also function by forming hydrogen bonds with biological molecules as water molecules are displaced. Hydrogen bonding in aqueous solutions is important for proper protein and DNA function. Thus, as the cryoprotectant replaces the water molecules, the biological material retains its native physiological structure and function, although they are no longer immersed in an aqueous environment. This preservation strategy is most often utilized in anhydrobiosis.

Freezable tissues

Generally, cryopreservation is easier for thin samples and small clumps of individual cells, because these can be cooled more quickly and so require lesser doses of toxic cryoprotectants. Therefore, cryopreservation of human livers and hearts for storage and transplant is still impractical.

Nevertheless, suitable combinations of cryoprotectants and regimes of cooling and rinsing during warming often allow the successful cryopreservation of biological materials, particularly cell suspensions or thin tissue samples. Examples include:

Semen in semen cryopreservation


     o Special cells for transfusion

     o Stem cells. It is optimal in high concentration of synthetic serum, stepwise equilibration and slow cooling.

     o Umbilical cord blood Further information: Cord blood bank#Cryopreservation

• Tissue samples like tumors and histological cross sections

• Eggs (oocytes) in oocyte cryopreservation

Embryos that are 2, 4 or 8 cells when frozen, in embryo cryopreservation

Ovarian tissue in ovarian tissue cryopreservation

Plant seeds or shoots may be cryopreserved for conservation purposes.

Additionally, efforts are underway to preserve humans cryogenically, known as cryonics.

How Cryonics Works

by Stephanie Watson

The year is 1967. A British secret agent has been "frozen," awaiting the day when his arch nemesis will return from his own deep freeze to once again threaten the world. That day finally arrives in 1997. The agent is revived after 30 years on ice, and he saves the world from imminent destruction.

You'll probably recognize this scenario from the hit movie, "Austin Powers: International Man of Mystery" (1997). Cryonics also shows up in films like "Vanilla Sky" (2001), "Sleeper" (1973) and "2001: A Space Odyssey" (1968). But is it pure Hollywood fiction, or can people r-eally be fr¬ozen and then thawed to live on years later?

The science behind the idea does exist. It's called cryogenics -- the study of what happens to materials at really low temperatures. Cryonics -- the technique used to stor¬e human bodies at extremely low temperatures with the hope of one day reviving them -- is being performed today, but the technology is still in its infancy.

In this article, we'll look at the practice of cryonics, learn how it's done and find out whether humans really can be brought back from the deep freeze.

In an operating room at Alcor Life Extension Foundation, a cryonics patient is cooled in a vat of dry ice as part of the "freezing" procedure.

Photo courtesy Alcor Life Extension Foundation

What is Cryonics?

Cryonics is the practice of preserving human bodies in extremely cold temperatures with the hope of reviving them sometime in the future. The idea is that, if some¬one has "died" from a disease that is incurable today, he or she can be "frozen" and then revived in the future when a cure has been discovered. A person preserved this way is said to be in cryonic suspension.

To understand the technology behind cryonics, think about the news stories you've heard of people who have fallen into an icy lake and have been submerged for up to an hour in the frigid water before being rescued. The ones who survived did so because the icy water put their body into a sort of suspended animation, slowing down their metabolism and brain function to the point where they needed almost no oxygen.

Cryonics is a bit different from being resuscitated after falling into an icy lake, though. First of all, it's illegal to perform cryonic suspension on someone who is still alive. People who undergo this procedure must first be pronounced legally dead -- that is, their heart must have stopped beating. But if they're dead, how can they ever be revived? According to scientists who perform cryonics, "legally dead" is not the same as "totally dead." Total death, they say, is the point at which all brain function ceases. Legal death occurs when the heart has stopped beating, but some cellular brain function remains. Cryonics preserves the little cell function that remains so that, theoretically, the person can be resuscitated in the future.

How is Cryonics Performed?

Operating room at Alcor Life Extension Foundation

Photo courtesy Alcor Life Extension Foundation

If ¬you decide to have yourself placed in cryonic suspension, what happens to you? Well, first, you have to join a cryonics facility and pay an annual membership fee (in the area of $400 a year). Then, when your heart stops beating and you are pronounced "legally dead," an emergency response team from the facility springs into action. The team stabilizes your body, supplying your brain with enough oxygen and blood to preserve minimal function until you can be transported to the suspension facility. Your body is packed in ice and injected with heparin (an anticoagulant) to prevent your blood from clotting during the trip. A medical team awaits the arrival of your body at the cryonics facility.