Background: Some Principles of Cryobiology and of Cryopreservation by Vitrification
Living cells are highly complex and dynamic structures of tremendous subtlety, but most cells also possess amazing resiliency in the face of challenges to their survival. Because of this resiliency, the science of cryobiology, which is the study of life at reduced temperatures, is possible.
Cryobiologists have been probing the low-temperature limits of life at least since 1663 when Henry Power reported the survival of “vinegar eels” frozen by exposure to a “keen frost” outside or to a freezing bath of salt and ice in the laboratory. The literature of cryobiology is vast and covers everything from the freezing of cells to human hypothermia. For the purposes of this overview, we will consider only phenomena that occur below the normal freezing point of pure water, which is defined as 0°C.
The phenomena of cryobiology can be thought of as arising from two contradictory effects of temperature reduction. The most familiar effect of reduced temperatures is a reduction in the rate of deterioration of biological systems. It is this effect that allows us to retrieve foods from the household freezer and enjoy them months or years after they would have otherwise become inedible. But the other effect is destructive in nature, and arises not only because of the transformation of liquid water into ice crystals, but also because living systems optimized for survival at higher temperatures cannot perform self-maintenance functions at lower temperatures and may encounter phenomena such as phase changes in membrane lipids or cold-induced protein denaturation for which they have not evolved specific defenses.
Cryobiology succeeds in preserving living systems at cryogenic temperatures when the destructive effects of low temperatures can be limited sufficiently during cooling to allow the protective effects of even lower temperatures to dominate. The challenge of cryopreservation, in other words, is to pass through a specific temperature range of vulnerability to low-temperature injury and successfully reach the safe harbor of temperatures sufficiently low to prevent any further change over virtually unlimited periods of time. The zone of hazardous subzero temperatures depends on the biological system in question and on the way in which it is protected.
Temperature is a measurement of the internal energy in a physical system. It is this internal energy in fluid systems that allows molecules in fluids to tumble, twist, disassociate from one another, move from place to place in the fluid, and to chemically react with other molecules. As the temperature is reduced, less and less energy exists in the system to drive these kinds of molecular motions. In many systems such as pure water, temperature reduction below a certain point results in an abrupt reorganization of the fluid medium into an organized solid lattice known as a crystal. This is known as freezing. In other systems, this does not happen. Instead, temperature reduction just causes more and more slowing of molecular motions, less and less molecular mobility, and slower and slower chemical reaction rates until a critical temperature is reached below which there is insufficient energy for the most mobile molecules in the fluid to move appreciably over the time scale of a typical laboratory observation. At this temperature, the “glass transition temperature,” the system almost completely loses its fluidity and becomes a “solid liquid,” which is more formally known as a “glass,” and is said to have “vitrified.” A glass is like a snapshot of the liquid state, and is essentially a liquid in which molecular rearrangements are practically arrested. At several degrees below the commonly-observed glass transition temperature, molecular motions are so slow that changes are nil for all practical purposes even over time spans of several hundred years or more.
Although pure water normally freezes and can only be vitrified under extreme conditions, mixtures of water and high concentrations of many water-soluble chemicals can vitrify. Chemicals that can allow water to vitrify include most agents that cryobiologists have traditionally used to protect cells from freezing and thawing damage. These agents are called cryoprotective agents (CPAs) or just cryoprotectants for short. When a cell is permeated by cryoprotectants in concentrations high enough to allow vitrification, all of the cell’s molecular constituents become locked into the glass as it forms and therefore become unable to change over time.
Vitrification prevents damage related to ice formation, which includes mechanical disruption of extracellular structures in organized tissues and organs, cellular osmotic dehydration and shrinkage during slow freezing, intracellular ice formation and destructive intracellular ice re crystallization during rapid freezing and during thawing, and exposure to elevated intracellular and extracellular solute concentrations that can produce harmful effects or precipitate after exceeding their solubility limits. For this reason, non-freezing living systems traversing the temperature zone between 0°C and about -90°C are subject to fewer stresses than are frozen systems and thus are less subject to events that can reverse the increasingly protective effects of temperature reduction.
Not all damage is avoided by vitrification, however. There is also damage caused by low temperature exposure per se, which is called either chilling injury or cooling injury. The nature of this injury is not understood, but could be related to lipid phase transitions, protein cold denaturation, or other phenomena. Interestingly, chilling injury does not pertain to many complex living systems, but it does pertain to mammalian kidneys. Fortunately, 21st Century Medicine recently announced the development of the only known practical methods of blocking most chilling injury in the kidney, and demonstrated the routine recovery of kidneys after cooling to -45°C and rewarming.
Basic Definitions
Cryobiology is the branch of biology that studies life at below-normal temperatures. Usually cryobiology is considered to deal with the effects of freezing and thawing. However, any temperature below what is normal for any given living system falls into the realm of cryobiology, including fields such as hypothermia, hibernation, natural frost hardiness of insects and plants, and medical organ preservation in ice. 21st Century Medicine is a cryobiological research and development company concerned both with cooling living systems to cryogenic temperatures and with liquid state preservation of mammalian organs at 0°C and above.
Cryopreservation is the process of preserving and storing living systems in a viable condition at low temperatures for future use. Traditionally, cryopreservation has meant preservation by freezing, and the word is still used with this meaning in many cases. However, the term can also cover preservation by vitrification, or ice-free cryopreservation.
Cryogenics is the branch of physics that studies the causes and effects of extremely low temperatures.
Cryogenics usually relates to cryotechnologies such as producing liquefied gasses as well as to many other low temperature physical effects such as super fluidity and the behavior of Bose-Einstein condensates.
Cryonics is the practice of keeping a clinically dead human body or brain at an extremely low temperature in the hope of later restoring it to life with the help of future medical technologies. Although our research is of great interest to those who are interested in cryonics, 21st Century Medicine is not involved in cryonics.
Vitrification is preservation at extremely low temperatures without freezing. Freezing involves ice crystal formation, which damages delicate structures such as blood vessels. Vitrification instead involves the formation of a glassy or amorphous solid state which, unlike freezing, is not intrinsically damaging even to the most complicated of living systems.
The act of preparing a living system for vitrification can also induce damage itself. This can occur in several ways, but the biggest problem has always been that vitrification requires high cryoprotectant concentrations, and high concentrations tend to biochemically disturb living systems, producing toxic effects. Fortunately, 21st Century Medicine has recently opened entirely new vistas in this regard, achieving much less toxic solutions than ever before at much higher total concentrations than ever before.
How it Works: Technical Aspects of Vitrifying Complex Systems
According to new theoretical and practical observations published by 21st Century Medicine in the February 2004 issue of Cryobiology, cryoprotectant toxicity can be understood as being related to the strength of interaction between water and cryoprotectants that can penetrate living cells. Agents that interact weakly with water don’t vitrify water very well, but they are so much less toxic that they can be used in higher concentrations and still allow vitrification but with considerably less toxicity. This is the central basis of 21st Century Medicine’s newest vitrification solutions, but it is by no means the only reason for the dramatic superiority of our vitrification technology. We also achieve unprecedented stability against ice formation during both cooling and warming by using our Super cool X-1000 and Super cool Z-1000 antinucleator-type ice blockers to prevent ice crystal nucleation and our proprietary ice growth inhibiting polymers to slow the growth of any ice that may form. These advantages are consolidated using a carrier solution, LM5, that maximizes the effectiveness of X-1000 and Z-1000 but also maintains good physiological preservation of whole organs while also contributing to glass-forming ability of the complete solution.
These advances have allowed us to develop a solution, M22, that does not freeze even during extremely slow cooling and rewarming but that can be per fused through kidneys with subsequent uniform life support by the kidneys after transplantation. In addition, we have been able to build into these solutions features that greatly mitigate chilling injury. We were recently able to show that a kidney per fused with a concentration that was previously 100% fatal now produces no damage whatsoever based on post-transplant renal function.
Vitrification can also be hazardous because of the tendency of glasses to fracture when cooled too far below Tag. The ideal storage temperature for a large transplantable organ or tissue is probably around –135 to -150°C, which is about 10 to 25 degrees below typical values of the glass transition temperature, and sufficient to suppress any possible storage injury for hundreds of years. We recently showed that it is possible to cool kidneys to this temperature range and below without producing any fracturing. Furthermore, we have developed controlled vapor storage systems that will allow a wide variety of living systems to be stored at a known, uniform, and well-documented temperature within the ideal temperature range almost regardless of sample size or geometry.
These almost unthinkable advances are taking 21st Century Medicine and the entire field of cryopreservation to places that were formerly impossible, and have already led to breakthroughs in the vitrification of a number of simpler living systems of considerable medical value.
For the full scientific details of many of the fundamental breakthroughs of 21st Century Medicine, we invite the reader to consult our published papers. Of course, we continue to work on the development of even more advanced and sophisticated cryotechnologies, and look forward to many future announcements over the coming months and years.
