Cryopreservation and Fracturing

[Update: In June, 2003, Alcor began testing a simple fail-safe system for storage at temperatures higher than liquid nitrogen based on low-power electrical heating of insulated containers in or near liquid nitrogen.]

Cryopatients are currently stored under liquid nitrogen at a temperature of -196°C. The first question that might come to mind is why so cold in the first place? The answer is provided in detail in the Alcor publication How Cold is Cold Enough? by Hugh Hixon [1]. In summary, when tissue is frozen, the freezing point of water is depressed by solutes concentrated between growing ice crystals. Water therefore remains a liquid (albeit a very viscous liquid) even at dry ice temperature (-79°C). Finally, usually at temperatures between -90°C and -130°C, a "glass transition" occurs and any remaining unfrozen water turns into a solid glass. Below this temperature, translational molecular motion is very slow to non-existent. With molecules able to do little more than vibrate in place, chemistry is effectively stopped, and extremely long storage times are possible.

The Fracturing Problem

Vitrification solution before cooling

Vitrification solution fractured during cooling

One problem with cooling to temperatures well below the glass transition temperature is that mechanical stresses due to thermal contraction are no longer easily accomodated. Solids want to contract as they are cooled, but glassy solids cannot contract. If tissue is cooled to liquid nitrogen temperature, which is far below the glass transition temperature, these conflicting forces cause the tissue to fracture, or crack into pieces. Fractures have been observed during post-mortem examination of the bodies of frozen cryopatients that were converted to neuropreservation [2]. The Cryonics Institute uses a very slow cooling protocol (1°C per hour) that appears to prevent fracturing of frozen sheep brains [3]. However cooling at such slow rates results in days of exposure time to toxic concentrations of cryoprotectant at relatively high temperatures, making the cost/benefit tradeoff of this approach uncertain.

Fracturing is a special concern for the new vitrification protocol recently brought online by Alcor for neuropatients. Ideally, vitrification avoids ice formation completely so that the entire tissue mass becomes an undisturbed block of glass. Fracturing ruins the otherwise pristine structural preservation that vitrification can achieve, which in some sense makes fracturing a greater tragedy for vitrification than it is for freezing. An ounce of vitrification solution will tend to fracture if it is cooled more than 20 degrees below the glass transition temperature. (For practical vitrification solutions, the glass transition temperature is usually near -125°C.) Ominously, however, the fracture temperature increases with solution volume. Solution volumes greater than one liter can fracture at temperatures only a couple of degrees below the glass transition [4].

It should be mentioned that if advanced nanotechnology is available for patient recovery, then fracturing per se probably causes little information loss. In pure solutions fractures appear as clean refractive index boundaries, so any microscale damage must be at a scale smaller than the wavelength of light. Fractures might just be simple tissue displacement along a surface that is smooth down to the molecular level.

However the number of fracture lines in a single solution mass can number in the thousands, expecially if facturing is delayed until lower temperatures are reached [4]. Fracturing therefore commits cryopatients to the need for molecular repair at cryogenic temperatures (a highly specialized and advanced form of nanotechnology) whereas unfractured patients may be able to benefit sooner from simpler forms of nanotechnology developed for more mainstream medical applications. Unfractured patients also have the option of being rewarmed without any nanotechnology and perfused with alternative preservation solutions, such as fixatives, if emergencies ever necessitate it. Fractured patients would be unperfusable, and likely suffer irreversible information loss, if they are ever rewarmed above the glass transition without prior nanotechnological repair.

Beyond improving existing cryopreservation methods, there is another reason why developing means to avoid fractures is important. Vitrification is an active area of research in cryobiology, and if people interested in the problem bring enough resources to bear, it is likely that some sort of reversible suspended animation of the brain can be achieved within the natural lifetime of most readers. To be reversible, such technology must necessarily store at non-fracturing temperatures. The prospect of this technology is perhaps the most powerful argument for development of higher temperature storage. Higher temperature storage is thus an essential part of the more general effort to achieve revresible suspended animation in our lifetime.

Long Term Instability at Higher Temperatures

The observation that large masses can fracture only a few degrees below the glass transition temperaturs suggests that safe fracture-free storage must take place very close to the glass transition temperature. The long-term stability of temperatures below the glass transition used to be taken for granted by cryobiologists. However the advent of vitrification has forced a re-examination of this belief because metastable vitrification (vitrification based on rapid cooling) is a non-equilibrium process. Vitrified solutions therefore have strong and unique thermodynamic instabilities capable of driving change at temperatures near the glass transition temperature [5].

The most important instability for cryopreservation purposes is a tendency toward ice nucleation. At temperatures down to 20 degrees below the glass transition temperature, water molecules are capable of small translations and rotations to form nanoscale ice-crystals, and there is strong thermodynamic incentive to do so [6, 7]. These nanoscale crystals (called "nuclei") remain small and biologically insignificant below the glass transition, but grow quickly into damaging ice crystals as the temperature rises past -90°C during rewarming. Accumulating ice nuclei are therefore a growing liability that makes future ice-free rewarming efforts progressively more difficult the longer vitrified tissue is stored near the glass transition temperature. For example, storing a vitrification solution 10 degrees below the glass transition for six months was found to double the warming rate necessary to avoid ice growth during rewarming [6]. The vitrification solution that Alcor uses is far more stable than the solution used (VS41A) in this particular experiment, but Alcor must store its patients far longer than six months.

The Importance of Annealing

If truly stable storage requires temperatures more than 20 degrees below the glass transition, but fracturing can occur only a few degrees below it, what can be done? This is a problem that has recently received attention from cryobiologists seeking to preserve vitrified vascular grafts. Obviously preserved blood vessels cannot be transplanted if they crack into pieces during the preservation process. Fortunately it has been discovered that if during cooling samples are first taken slightly below the glass transition, then above it, and then back down below again, it is possible to go all the way to liquid nitrogen temperature without fracturing [8]. This is a remarkable demonstration of how a modest change in cooling protocol can relax thermal stresses, and permit glasses to be cooled to much lower temperatures without fracturing. These "annealing" protocols, which are still in their research infancy, are obviously critically important to the future of higher temperature storage.

Practical Systems

It is possible that future annealing research may show that it is possible to take even large vitrified masses all the way to liquid nitrogen temperature without fracturing. It is more likely, however, that temperatures closer to -150°C will be found optimum for long-term storage. This is the highest temperature at which ice nucleation is confidently stopped, and a temperature that will be safer against fracturing than lower temperatures (assuming that future annealing research will allow lower temperatures to be reached).

Mechanical freezers able to hold up to 12 neuropatients (but not whole body patients) at -140°C are commercially available. For various reasons, these freezers are not ideal for long-term storage. They are better suited for annealing processes, and temporary storage for perhaps a few years while better systems are developed.

Perhaps the best system for long-term higher temperature storage of cryopatients is a system that has been suggested by Alcor engineer, Hugh Hixon (Appendix). This is a system based the same reliable "Bigfoot" dewars presently used by Alcor for liquid nitrogen storage of cryopatients. For use at intermediate tempertures, a liquid nitrogen reservoir would be maintained at the bottom of the dewar with patients stored in the vapor space above the liquid. A control system would circulate the vapor and draw cold for the liquid reservoir as necessary to maintain the desired target temperature in the vapor. Although much research and development is required, this system may ultimately be cost-competitive with existing liquid nitrogen storage.

In the long run, even larger more ambitious systems for higher temperature storage can be constructed [9]. For both biological, and perhaps even economic reasons, the future of human cryopreservation seems to be at temperatures higher than liquid nitrogen.

Appendix by Hugh Hixon:

The advent of vitrifying cryoprotectants which block ice crystal formation when properly applied has led to a shift in focus to another well-recognized problem; cracking of the perfused organs on a macroscopic scale. To prevent this untoward event, it becomes necessary to store patients at a temperature near the glass transition point of the particular cryoprotectant.

Patient storage at LN2 temperature has been the "gold standard" up to the present time, as it has demonstrated consistent reliability at minimum expense. Any system that stores at some other temperature is not likely to be as reliable as LN2 storage by orders of magnitude, and certain problems (some of which we cannot anticipate) are inevitable. It should be possible to technically overcome most of the recognized problems, and achieve acceptable reliability, without raising storage costs to any great degree, although significant initial technology development costs are inescapable, and storage at or near glass transition points does not assure against cracking or other possible adverse effects of long-term thermal cycling.

The anticipated technical problems are:

• 1) Gas stratification. Between the cold liquid in the bottom of a dewar and the outside temperature at the top, there is a natural convective stagnation of the cold gas in a temperature/density gradient. In order to obtain a uniform temperature in the storage volume it is necessary to vertically circulate the gas by stirring, with fans or other means. Conventional fans are not normally expected to operate at cryogenic temperatures, primarily because the lubricant in the bearings freezes.
  
  
• 2) Difficulty of achieving active control with minimum power. Active systems can require a significant amount of power. Since the proposed high temperature storage systems are expected to operate through periods of commercial power failure, power requirements must be minimized. To achieve the synthesis of close temperature regulation and minimal power requirements, a combination of active and passive regulation is required, with fine active control being superimposed on passive regulation.
  
  
• 3) Gas-liquid mixture on fill. In filling, large quantities of cold gas can be introduced along with the LN2, interfering with control system regulation. Since the system is being designed for minimum power requirements, however, separating the cold gas from the liquid prior to its entering the dewar reservoir is necessary.
  
  
• 4) Lack of reliability. Backup systems and alarms are required for redundancy, thus it is expected that the final product will have no less than two nearly separate systems, each independently able to carry regulation on its own, plus appropriate alarms to notify operators of component failure.
  
  
• 5) Data collection. Programming will be required for computerized monitoring of the demonstrator.

Alcor senior staff is of the opinion that "…the ultimate high temperature storage will be in modified Bigfoot dewars, using some of the volume in the bottom of the dewar for an LN2 supply, while regulating the temperature of the remainder of the Bigfoot volume."

References

1. "How Cold is Cold Enough" CRYONICS, January 1985

2. "Post Mortem Examination of Three Cryonic Suspension Patients" CRYONICS, September 1984.

3. http://www.cryonics.org/research3.html

4. "Physical Problems with the Vitrification of Large Biological Systems" G. M. Fahy, J. S. Saur, and R. J. Williams, Cryobiology 27, 492-510 (1990).

5. "Physical Aging of Glassy State: DSC Study of Vitrified Glycerol Systems" Z. H. Chang and J. G. Baust, Cryobiology 28, 87-95 (1991).

6. "Nucleation and Crystal Growth in a Vitrification Solution Tested for Organ Cryopreservation by Vitrification" P. M. Mehl, Cryobiology 30, 509-518 (1993).

7. "Crystallization of Ice in Aqueous Soluions of Glycerol and Dimethyl Sulfoxide" J. M. Hey and D. R. MacFarlane, Cryobiology 33, 205-216 (1996).

8. A. Baudot, abstract presented at the Annual Meeting of the Society for Cryobiology, 2001. 9.