Cryopreservation of Organs

Organ Transplantation

The most spectacular aspect of modern medicine is surely the success, and almost routine nature, of organ transplantation. The vital organs can be removed from a newly dead person and carefully inserted into a person who is about to join the newly dead to postpone that deceasement for (perhaps) decades. It's expensive, it's a difficult operation required exquisite surgical skills, it requires a massive network of communication and transportation, and it doesn't always work. Even if it does work, the immunosuppression may give the patient the same prognosis as someone with HIV. But you have to admit that, despite all this, organ transplantation is magic of the highest calibre!

Most organ transplantation is done immediately after the death of the donor with the time that the organ is ex vivo minimized to reduce anoxic and ischeamic damage. The transplanted organ must then function immediately when the recipient is removed from life-support systems; there is no time for recovery or repair. There is also no time (between donor harvest and transplantation) in which to do tissue typing and cross-matching, despite the significant improvement that such measures would confer on the process. The recipient is obliged to take immunosuppressive drugs for the rest of their natural life to prevent their bodies from rejecting the transplanted organ. It is a good idea not to get sick under these conditions, but it beats being underground.

Many of these problems could be overcome if the time problem could be removed from the equation. If there was some way to store, or bank, the organ after removal so that it would not degrade with time (or even to slow the process down to a more manageable level), then many of the problems and expenses associated with organ transplantation could be reduced or eliminated. This, of course, has been one of the dreams of cryobiology since the time of the first successful organ transplant was carried out. It was about 5 years away at that time and remains about 5 years away (although as the granting process settles on a 3 year term even for large theme grants, organ cryopreservation seems to be getting closer to reality).

Approaches to Organ Preservation

There are three current approaches to organ preservation. The first is storage at hypothermic temperatures (above the freezing point but below 37C). The second approach is freezing and thawing with storage at cryogenic temperatures, and the third is storage at cryogenic temperatures without the formation of crystalline ice during the cooling and warming process.

Hypothermic Injury and Storage

The mechanisms of hypothermic cell injury are just starting to become known. This brief paragraph will introduce the topic but will be outdated before the pixels are even dry. There is an increase in intracellular sodium ion concentration during hypothermic exposure however this is due solely to the hypoxic conditions that usually accompany hypothermic storage. The Na+ uptake occurs identically under warm, hypoxic conditions. Thus Na+ free conditions only prevent hypoxic injury but not hypothermic. Hypothermic injury is associated with the production of reactive oxygen species inducing apoptosis. There is an increase in the pool of free (chelatable) Fe++ intracellularly during cold exposure. This free iron will increase the concentration of hydroxyl radicals and that, combined with the decrease in production of superoxide dismutase, will lead to singlet oxygen injury. The most likely source of iron is from intracellular ferritin however the mechanism for the increase is still unkown. Iron chelating agents prevent the hypothermic injury.

Hypothermic storage is the current method that is used for organ transplantation when the donor and recipient are not situated in the same site. The organ is perfused with a preservation solution by pumping the solution through the vasculature. Often, perfusion is done intermittently to reduce the vascular damage that results from hypothermic perfusion. The preservation solutions have to be administered immediately and are also effective at cooling the organ from 37C to 4C. After about one hour of warm ischemia or four hours of hypothermic ischemia (organ simply placed in saline), the organ will be unfit for transplantation. The composition of storage solutions are designed from a few basic principles. Exogenous substrates such as glucose are replaced by nonmetabolizable agents such as mannitol or sucrose. The ion balance, osmolality and pH are optimally controlled, with the ions usually simulating the composition of the cytoplasm rather than the normal high sodium solution (high potassium solutions are called extracellular solutions). Citrate is added for ion chelation and to reduce oxidative reactions in the mitochondria and to enhance the cycling of substrates in the metabolic energy producing pathways. The maximum and suggested storage times for hypothermic storage of several organs is given in the following chart.

OrganMax (hours)Norm (hours)
Kidney7248
Liver4830
Pancreas7224
Heart126

These times are adequate for air transplantation between major centers within a continent (organ transplantation isn't usually done in minor centers) and some of these times are adequate for transportation anywhere in the world.

Freezing and Thawing

There have been many empirical attempts at cryopreservation of organs by conventional freezing and thawing. These efforts have usually focussed on simply ramping up techniques that have worked for cell preservation to organs, treating the larger structures as great, big cells. So far, these attempts have only resulted in complete, abject failure (with the occasional unreproducible success thrown in for spice). There are signficant problems associated with this approach, including the difficulty of generating heat transfer within a large thermal mass with a geometry that has little malleability. The packing density of cells within an organ can approach 80% (whereas preservation of isolated cells becomes problematic at a cell concentration above 20%). The presence of many different cell types, each with its own requirements for optimal cryopreservation limits the recovery of each when a single thermal protocol is imposed on all of the cells. Extracellular ice can cause mechanical damage to the structural integrity of the organ, particularly the vascular component, where ice is likely to form. Mechanical fractures occur in the vitreous solids that exist between ice crystals when thermal stresses occur at low temperatures. These fractures separate parts of the organ from each other. There are disruptions of the attachments that form between cells and between cells and their basement membranes. There are mechanical stresses caused by the osmotic movement of interstitial water. Each of these is an additional, and formidable, source of damage, over and above those that we already know so well from studies of cells in suspension. In short, the cryopreservation of whole organs by scaling up techniques used for cell suspensions is a hard problem. Too hard for the current crop of cryobiologists (so far).

Vitrification

The most promising approach to the cryopreservation of whole organs lies with the process of vitrification. This is the process of taking an aqueous solution and making it into an amorphous solid. When a liquid is cooled down, it can be taken past its melting point without a phase change occuring. The formation of ice first requires nucleation, a stochastic process by which an ice nucleus (a cluster of water molecules that reaches a critical size) forms spontaneously. With lower temperatures, the critical size of a nucleus becomes smaller and eventually approaches the size of clusters within the liquid. The solution can be cooled down to the homogeneous nucleation temperature (the temperature at which the probability of nucleation equals 1) in a supercooled state but below this crystallization occurs.

Since crystal growth requires translation of molecules within the liquid, there is a kinetic aspect to it. If a liquid is cooled fast enough, then the viscosity might increase to a level where molecular translation is too slow to allow crystal growth, or possibly even nucleation from occuring. The supercooled liquid exists in a metastable state in which until the glass transition temperature is reached. At this temperature, the time required for the molecular translations needed for nucleation or crystal growth tends to infinity, so the amorphous solid is stable below this point.

Vitrification can also be achieved by adding solutes that develop a structure within water that needs to be broken down for crystal growth. The solute molecules impede the process of crystal growth simply by getting in the way of other water molecules and interfering with the hydrogen bonding network necessary for ice formation. Both the kinetic approach and the solute approach are additive, so that as the solute concentration is increased, the cooling rate necessary to achieve vitrification is lowered.

About 20 years ago, Greg Fahy decided to extend Luyet's program of cryopreserving cells through vitrification to whole organs. It was a revolutionary leap that necessitated a whole new approach to the problems of getting biological material to low temperatures. Previously, cryobiologists had focussed on the problems that occured when ice formed in a solution. With a single blow, Fahy solved all of those problems by simply eliminating the ice! There was the problem of how to actually get an organ vitrified that remained, and Greg has worked diligently toward this end. A clever and imaginative researcher, he has followed this path almost completely alone, yet his progress over the past two decades has been remarkable.

The essential problem with vitrifying organs is that about half of the water within an organ has to be replaced with solute molecules for vitrification to occur at realizable cooling rates. Replacement of every second water molecule with something else is not an innocuous process; such concentrations of anything seem to have some toxicity associated with them--poison is the dose.

The difficulties that were faced, at the beginning of this program, were; 1. How to get high concentrations of vitrification solutes (VS) into the organ and then remove them upon thawing, 2. How to prevent fracturing of the organs during cryogenic storage, 3. How to cool the organs fast enough to prevent ice crystal formation, 4. How to warm the organs fast enough to prevent devitrification, and 5. How to prevent "chilling injury" during cooling (a poorly defined injury that occurs without ice formation). Fahy and his collaborators have found many solutions to these problems over the years, however many of these solutions have introduced new problems, or made some of the others on the list more prominent. The task of attempting to solve each of these problems individually has often shown the interdependence of the entire system. A few of the solutions that have been tried:

  1. How to get high concentrations of VS's into an organ
  2. Fracturing of vitrified samples
  3. Rapid cooling of organs
  4. Rapid warming of organs
  5. Chilling injury
Greg Fahy now works for a biotech company called 21st Century Medicine and their recent press releases claim to have finally solved (in principle, at least) all of the problems associated with organ cryopreservation.

The claim is that his new VX Solutions eliminate the toxicity problems as well as the requirement for high pressures during vitrification. Since 21st Century Medicine is heavily involved in developing liquid breathing techniques using perfluorocarbons, it seems likely that Greg has gone to perfluorocarbon perfusion. These compounds seem to be non-toxic even when they completely replace water, and perhaps they don't suffer from the high viscosity (and resulting damage to vascular integrity) that conventional vitrification solutions impose at low temperatures. The cooling rates could be substantially increased with perfluorocarbon perfusion, so perhaps the conventional solutions, at much lower concentrations, are used to get VS's into the organ and then perfluorocarbon perfusion is used to vitrify the organ.

Another scientist from 21st Century Medicine has been "working with methoxylated compounds" and has developed something called "Ice Blocker X1". This compound inhibits macroscopic ice crystals from growing in a vitrification solution, especially during warming. This sounds just like conventional antifreeze-proteins (AFP's), and indeed, this seems like a natural fit. The AFP's would bind to the ice nuclei and greatly slow down their growth during warming. Thus, the warming rates necessary to avoid macroscopic ice crystals could be substantially lower, and therefore technologically feasible.

These are promising avenues and it's encouraging to see this work being supported. We can only wait for the good news. Steve Harris, another scientist working at 21st Century Medicine, has said:

Tantalizing...


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Document last updated Feb. 8, 1999.
Copyright © 1997, Ken Muldrew.