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).
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 37°C to 4°C. 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.
|Organ||Max (hours)||Norm (hours)|
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.
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:
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: