Major Progress Toward
Vitrification of Human Cryopatients

by Brian Wowk

For more than a decade, cryobiology has tantalized us with the hope of eliminating freezing damage via vitrification. While a variety of cell types and small tissue samples can be successfully vitrified, there have been daunting technical obstacles to the vitrification of large organs or whole human beings.

Now, however, a fresh approach has opened up exciting new possibilities.

How Ice Damage Occurs

At room temperature, water molecules are arranged randomly and can mingle with other molecules. When the water freezes, however, its molecules align themselves in a rigid structure that has no room for other substances. This means that when ice forms inside human tissue, the ice squeezes vital ions and proteins out of the tissue, forcing them into shrinking pockets of residual unfrozen water. Even the fabric of cells themselves is crushed into these tiny spaces among the ice crystals.

As cooling continues, more than eighty percent of tissue volume can become converted to ice, and the cells are crushed beyond recovery.

Vitrification eliminates the formation of ice during cooling. It is a way of stopping biological time without disturbing the natural order inside living cells.

During vitrification, ice formation is completely inhibited by cryoprotectants (chemicals that prevent water from freezing). As a result, liquid water molecules maintain their natural random arrangements during deep cooling. There is no disturbance of other chemicals or cell components. Everything stays exactly where it belongs while cooling proceeds. At a temperature below about 100 degrees Celsius, molecules cease to move relative to each other, and biological time is stopped. This is called the "glass transition" temperature.

Partial Vitrification: the Story so Far

Partial vitrification has been the basis of every successful cryopreservation experiment during the past fifty years. When modest concentrations of a cryoprotectant such as glycerol are used to protect cells during freezing, this reduces the amount of ice that forms, because the cryoprotectant becomes progressively concentrated, lowering the freezing point of the remaining unfrozen liquid. Eventually the cryoprotectant becomes so concentrated (around 70% of solution) that no more ice can form. With further cooling, the remaining solution vitrifies. Cells that can survive being squeezed into these narrow vitrified spaces with 70% cryoprotectant concentration will survive the freezing process.

A problem still exists, however. Collections of individual cells can move in response to growing ice, but cells in organs cannot move without disrupting the normal cell-to-cell relationships that organs require for integrated function. This is a primary reason why successful organ preservation by freezing has eluded cryobiologists for decades.

Complete Vitrification

There are obvious advantages to vitrifying an entire mass of tissue instead of just narrow spaces between ice crystals. If ice is eliminated, there is zero physical displacement or physical damage to cells, and no progressive concentration of toxic chemicals in residual solution during ice growth. Instead, everything stays "in place" like a movie slowing down and then stopping.

The problem is that completely stopping ice growth requires cryoprotectant concentrations near 70%. All known cryoprotectants are intensely toxic at this concentration, often dissolving cell membranes and obliterating the very biological structures we seek to preserve. (As we have seen, pockets of 70% concentrated cryoprotectant form during ordinary freezing with glycerol. However, these pockets do not form until deep sub-zero temperatures, where toxic effects are diminished.)

In 1981 cryobiologist Gregory Fahy working at the American Red Cross suggested a way around this problem. Fahy noted that highly concentrated (but non-lethal) solutions of cryoprotectant were capable of supercooling (cooling below their freezing point without freezing) if cooled rapidly. In particular, Fahy showed that cryoprotectant concentrations near 50% could supercool all the way to the glass transition temperature without any ice formation (achieving complete vitrification) if cooled at a rate near 10 degrees Celsius per minute.

Vitrification in Cryonics

Bearing all this in mind, there are two primary obstacles to successful tissue vitrification. The first obstacle is the design of cryoprotectant mixtures that are sufficiently penetrating and non-toxic to replace 50% of water in cells without injuring them. Dr. Fahy has introduced cryoprotectants at progressively lower temperatures to reduce toxicity, so that the final "vitrifiable" concentration is not reached until the temperature falls below -20 degrees Celsius. Unfortunately, glycerol is too viscous and non-penetrating to be usable at such low temperatures in cryonics patients. Consequently, this cryoprotectant is unsuitable for human vitrification.

Researchers at 21st Century Medicine, in collaboration with BioPreservation (CryoCare's cryopreservation service provider) have been developing new cryo-protectant mixtures that can be used at low temperatures. Concurrently, they have completed construction of a unique sub-zero operating room in which patients can be perfused with the new cryoprotectants. Full results of this work will be presented in a future issue of CryoCare Report.

The second obstacle to vitrification of large organs is more formidable, and for years has appeared insurmountable. The cooling rate of 10 degrees Celsius per minute, which is required for vitrification by supercooling, is 100 times faster than the fastest cooling rates ever achieved for human cryopatients. Even direct immersion of patients in ultra-cold silicone oil only results in cooling at 0.1 degrees Celsius per minute, due to the large volume-to-surface-area ratio of the human body. Cryoprotectant concentrations that are required to vitrify at such slow cooling rates are devastatingly toxic.

Radical Cooling Technology

The cooling-rate obstacle to human vitrification has now been overcome. Researchers at 21st Century Medicine have developed a radical new method of cooling organs and cryopatients by replacing the blood with a fluorocarbon substitute that remains a freely flowing liquid down to -100 degrees Celsius. The compound is completely non-toxic and biologically inert, and functions as a heat-exchange fluid that allows cooling hundreds of times faster than could be achieved previously. Initial results suggest that cooling rates near 10 degrees Celsius per minute can be achieved for whole bodies, and 50 degrees Celsius per minute for brains. This opens for the first time the possibility of vitrifying humans with survivable concentrations of cryoprotectant.

The Road Ahead

Work at 21st Century Medicine over the next year will focus on optimizing the hardware and software necessary for sub-zero cryoprotective perfusion and rapid fluorocarbon cooling. Development of cryoprotective solutions suitable for human vitrification will proceed in parallel. If this work goes well, a cryopatient vitrification capability may be in place as soon as 1998, heralding the end of ice in cryonics.

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CryoCare Report #10 Online Edition