The Prometheus Project

Organ Cryopreservation

by Gregory M. Fahy, PhD

This article has been made available to the Prometheus Project by Nanothinc -- the web's premier site for nanotechnology and nanoscale-related information, education, entertainment, products, and services.

Introduction

It is possible to live a long time as long as nothing in particular kills you, even without major advances in anti-aging medicine. It is also possible to die despite complete abolition of aging if something in particular does come along to kill you. Given the prominence of heart attacks as the leading cause of death, it is clear that organ failure is a life-limiting condition for older individuals. The ability to replace a failing heart, liver, kidney, or other vital organ could add many good, healthy years to life. Fortunately for the aged, transplant surgeons are more open-minded than ever about transplanting older individuals, since it has recently been shown that they tend to live just as long with their transplants as do younger individuals.

Obstacles to Life Extension Through Organ Transplants

One problem with transplanting hearts is the short lifetime of the heart outside the donor's body -- on the order of 4 to 6 hours. Only 50% of kidney donors are heart donors, and the limiting "shelf life" of the heart is probably one significant reason for the failure to transplant the "missing" hearts. In Europe, one fifth of all collected, healthy human livers expire before the logistics of getting them transplanted can be overcome. Another problem is rejection. Even though tissue matching between donor and recipient has a large effect on the outcome of a heart transplant in terms of the severity of rejection and the longevity of the transplant, tissue matching is impossible for hearts and livers because there is not enough time to get it done before the organs must be transplanted.

Conquering Time Through Cryopreservation

For these reasons and others, my laboratory has been attempting to remove time as a consideration from the transplantation arena. To do this, we have been attempting to cool organs to cryogenic temperatures without harming them, so that they can be warmed up and made available whenever an ideal recipient is available. In many cases, the availability of organs in a bank could save lives by making organs available within 2 to 3 hours of a traumatic incident, a prospect that cannot be imagined without true organ banking. At cryogenic temperatures, biological time comes to an end, and preservation times become indefinite. Storage times of months to years should be no problem. But first, it is necessary to be able to cool the organ to and warm the organ from cryogenic temperatures with minimal damage, and that is the problem that will be discussed here. We have not as yet solved this problem, but our research to date has, we believe, brought us to the brink of success. This paper provides an informal overview of our work, and the interested reader can find more details by consulting the bibliography at the end of this paper.

Vitrification as an Approach to Cryopreservation

Normally, cooling to very low temperatures causes ice to form in the biological system being cooled. Ice may not kill the individual cells in an organ -- indeed, freezing is very successful at preserving most human and animal cells -- but it can damage the noncellular components of an organ, such as the molecular meshes that support fine blood vessels or the molecular docking devices that hold cells in tight contact with each other. The result is an organ without adequate blood circulation on warming and with other severe problems that prevent the organ from surviving after transplantation.

In 1980, I decided to begin looking at a radical alternative. In this alternative, which is called vitrification, no ice forms in the organ regardless of how deeply it is cooled. The formation of ice is prevented by the presence of added chemicals that interact strongly with water and therefore prevent water from interacting with itself. A familiar illustration of this concept is the use of antifreeze in a car's radiator in the winter. The more antifreeze you use, the lower the temperature at which the radiator freezes. If you use very high amounts of antifreeze, it is literally possible to prevent freezing entirely, regardless of how low the temperature drops.

The prevention of freezing means that the water in an organ remains a liquid during cooling. However, as cooling proceeds, the thermal energy within the liquid becomes less and less. Temperature is essentially a measure of internal energy in a system, this internal energy being the factor that drives molecular motions. Consequently, as temperature goes down, molecular motions in the liquid permeating the organ slow down. It turns out that there is a minimum amount of thermal energy required to allow molecules to move from place to place in a liquid (translational motion). When this minimum energy becomes unavailable due to cooling, the liquid "locks up" into a solid state. This "arrested liquid" state is known as a glass, and the conversion of a liquid into a glass is known as vitrification. A glass is, on the macroscopic level, a liquid that is too cold to flow. The nice thing about vitrification is that there is nothing about it that should be biologically damaging. A vitrified liquid is not different from the ordinary liquid except that it does not possess most molecular motions and, therefore, it does not permit any appreciable deteriorative changes with time.

Special Problems Raised by Vitrification

To vitrify organs for later transplantation, about half of the water in the organs must be replaced with chemical agents, or "biological antifreeze agents." Scientifically, these chemicals are known as cryoprotective agents (CPAs) or cryoprotectants. Although CPAs are nontoxic in comparison with other chemicals or drugs, the need to use them in such enormous concentrations does pose a number of challenges. They can kill cells by direct chemical toxicity or indirectly, by being added or removed improperly. My laboratory has been working on the problems of introducing and removing the required concentrations of cryoprotectants since 1981.

Another problem is "cooling injury." This is injury associated with cooling in the absence of freezing. It may be partly a result of additional exposure time to cryoprotectant due to the time required for cooling and warming, but is also a direct injury caused by cooling per se. The biological nature of "cooling injury" remains speculative at best. As such, it has been particularly difficult to combat this form of injury.

The final problem is devitrification. Devitrification is not the opposite of vitrification: it is not just the liquification of a glass as temperature is raised. For historical reasons, "devitrification" is the name given to the freezing of a formerly vitrified solution. It turns out that devitrification tends to occur during warming from the vitrified state, and it is rapid. To prevent devitrification with current technology, the vitrified organ must be warmed uniformly at about 300'C per minute so that ice simply does not have time to form in injurious quantities. Stated in another way, the organ must be warmed from near the banking temperature to the normal freezing point of water in about 20 seconds or less.

Overcoming the Problems of Vitrification

Toxicity
Since 1986, my colleagues and I have been using a computer-operated organ perfusion machine to introduce and remove concentrations of cryoprotectant that are sufficient to allow organs to vitrify upon cooling. The primary organ studied to date has been the rabbit kidney, but significant work has also been accomplished on rat livers, and work on both tracks is continuing.

Our first successful kidney transplants were accomplished circa 1989. Our initial survival rates were poor, and the quality of survival was dismal. Most of the renal mass was found to be replaced with scar tissue when the animals were followed up a month or more after their transplants. Still, this was very encouraging, because the results were good enough to give us feedback on whether changes in procedure were beneficial or detrimental. Within a short time, we had converted our marginal success to 100% survival with apparently 100% recovery of renal function.

These results were obtained using the lowest possible concentrations of cryoprotectant needed for vitrification. With these concentrations, vitrification was only possible by combining the cryoprotectant with 1,000 atmospheres (atm) of hydrostatic pressure. Unhappily, we found that these high pressures could not be tolerated. We therefore raised the concentration to a concentration that required only 500 atm of applied pressure, resulting in a drop in survival from 100% to virtually 0%. Given a couple of years of effort, this survival rate was boosted to 75%, but when we combined this concentration with 500 atm, the survival rate again fell to zero. Raising concentration still more, so that no pressure would be needed, also resulted in a 0% survival rate.

It turned out that the problem with toxicity could only be successfully addressed by simultaneously addressing the problem of cooling injury.

Cooling injury
When kidneys permeated with the lowest CPA concentrations were cooled, without freezing, to -30'C, initially only about 30% of them survived. Using the refinements in procedure discovered in learning how to induce survival at the higher concentrations, survival at the lower concentrations was raised to about 60% after cooling to -30'C -- an improvement, but still clearly inadequate. When the kidneys perfused with the higher concentration were similarly cooled, 0% survived.

These results were depressing, yet they also suggested a possible solution. It appeared that cooling injury was becoming more severe as concentration was being elevated. This in turn suggested that cooling injury would be less severe at lower concentrations than we had studied. The new approach, then, would be to perfuse the kidneys with a relatively nontoxic concentration, drop the temperature to as close to -30'C as possible (which should not be harmful due to the lower concentration of cryoprotectant present), and then, at the lower temperature, add the rest of the cryoprotectant needed to vitrify. Results from tissue slice and red blood cell experiments suggested that further cooling, after addition of all of the cryoprotectant needed to vitrify, would not produce further cooling injury, so that the way to vitrification would be open.

Fortunately, these hypotheses were proven correct. At the lowest concentrations needed for vitrification, 100% survival resulted, and at the next higher concentration 100% survival was attained after cooling to -32'C. These results emboldened us enough to try concentrations that will vitrify with no applied pressure. The results of the first two experiments in our most recent series were very encouraging: 2 of 2 such kidneys survived, with good life support function after transplantation. When one of the kidneys in this group was further cooled to -46'C, it also survived, with good renal function after long-term follow-up. Tissue slice experiments suggest that cooling to temperatures below -46'C will induce little or no additional injury beyond the injury caused by cooling to -46'C. If this result holds for whole kidneys, it means that it is possible, now, to vitrify rabbit kidneys without lasting harm. Proving this, however, will require successful warming techniques for avoiding devitrification.

Devitrification
In 1990, Ruggera and Fahy (see Bibliography) reported success in warming test CPA solutions at rates of up to about 200'C/min. Further tests have been successful at rates of over 400'C/min. Given that success will require warming at about 300-350'C/min according to our best current estimates, there is good reason to hope that appropriate electromagnetic warming methods can be applied successfully to intact rabbit kidneys. At the time of this writing, plans are underway to install the necessary equipment for these studies and to begin collaborative work to finalize and further develop the heating technique.

The Future

Vitrification has been successfully applied to human islets of Langerhans, human monocytes, human red blood cells, human liver cells in culture, certain kinds of plants and plant tissues, and to animal embryos and egg cells. It has been applied with partial success to human corneas. It is also clear that, if earlier researchers had understood the scientific issues as well as we do today, they could have successfully vitrified whole organs (particularly guinea pig uteri and adult frog hearts) in 1965. Our laboratory has obtained encouragement that it may be possible to vitrify both rabbit kidneys and rat livers. These observations illustrate the universal nature of cryopreservation by vitrification and imply that success with one type of major organ can be followed by success with virtually any other type of organ or human tissue desired for banking.

The implications may be larger than the field of organ transplantation as it is now understood. As the problems of organ rejection are overcome, it will become possible to transplant organs and tissues that are not strictly necessary for life. These organs and tissues include, for example, thyroid glands, intestines, bladders, hands, and replacement breasts after radical mastectomies. The aggregate need for these nonvital organs may greatly exceed that for vital organs, and banking is likely to be a necessity to overcome the otherwise overwhelming logistic difficulties of matching donation with transplantation. All of these applications may be of considerable value in improving the quality and even the quantity of life for older individuals. Another more radical possibility is the removal of organs from an individual on a temporary basis, followed by return of these organs to the donor at a later time. This might be done, for example, if nephrotoxic chemotherapeutic agents (such as cisplatin) or heavy radiation in the area of the kidneys would otherwise have to be withheld during intensive anticancer therapy to prevent irreversible kidney damage: By banking the kidneys, the patient could be cured of cancer and then receive his own kidneys back, none the worse for wear.

Whatever the final results may be, we are encouraged to think that the problem of organ cryopreservation, which has been the object of study for the past 40 years or so, can indeed be solved, and solved in the foreseeable future. I believe that, in addition to being of direct medical use, organ cryopreservation may have significant benefits on a psychological level. In particular, by showing that another seemingly impossible medical problem can be solved, successful organ cryopreservation will encourage medical researchers to stretch their minds and their ambitions in pursuit of additional "impossible" goals, with benefits to patients in general and to life extension enthusiasts in particular that can scarcely be foretold.

Bibliography

Full papers:
1. Fahy G.M., Hirsh, A. Prospects for organ preservation by vitrification. In: Pegg, D.E., Jacobsen, I.A., Halasz, N.A., Eds. MTP Press, Ltd., pp. 399-404, 1982.

2. Fahy, G.M., MacFarlane, D.R., Angell, CA., Meryman, H.T. Vitrification as an approach to cryopreservation. Cryobiology 21:407-426, 1984.

3. Rall, W.F., Fahy, G.M. Ice-free cryopreservation of mouse embryos at - 196'C by vitrification. Nature 313:573-575, 1985.

4. Fahy, G.M., Takahashi, T., Meryman, H.T. Practical aspects of ice-free cryopreservation. In: Smit-Siblinga, C.T., Das, P.C., Eds. Future Developments in Blood Banking Boston: Martinus Nijhoff, pp. 111-122, 1986.

5. Fahy, G.M. Vitrification: A new approach to organ cryopreservation. Prog Clin Biol Res 224:305-35, 1986.

6. Fahy, G.M., Levy, D.I., Ali, S.E. Some emerging principles underlying the physical properties, biological actions, and utility of vitrification solutions. Cryobiology 24:196-213, 1987.

7. Fahy, G.M. Biological effects of vitrification and devitrification. In: Karow, A.M., Pegg, D.E., Eds. Biophysics of Organ Cryopreservation New York: Plenum Press, pp. 265-293, 1987.

8. Fahy, G.M. Vitrification. In: McGrath, J.J., Diller, K.R., Eds. Low Temperature Biotechnology: Emerging Applications and Engineering Contributions New York: ASME, pp. 113-146, 1988.

9. Ruggera, P.S., Fahy, G.M. Rapid and uniform electromagnetic heating of aqueous cryoprotectant solutions from cryogenic temperatures. Cryobiology 27:465-478, 1990.

10. Fahy, G.M., Saur, J., Williarns, R.J. Physical problems with vitrification of large systems. Cryobiology 27:492-510, 1990.

11. Fahy, G.M. Organ perfusion equipment for the introduction and removal of cryoprotectants. Biomed Instr Technol 28:87-100, 1994.

12. Khirabadi, B.S., Fahy, G.M. Cryopreservation of the mammalian kidney. I. Transplantation of rabbit kidneys perfused with EC and RPS-2 at 2-4 degrees-C. Cryobiology 30:10-25, 1994.

Abstracts:
1. Fahy, G.M. Prospects for vitrification of whole organs. Cryobiology 18:617, 1981.

2. Fahy, G.M., MacFarlane, D.R., Angell, C.A. Recent progress toward vitrification of kidneys. Cryobiology 19:668-669, 1982.

3. Rall, W.F., Fahy, G.M. Vitrification: A new approach to embryo cryopreservation. Theriogenology 23:220, 1985.

4. Fahy, G.M., Kittrell, J.L., Severns, M. A fully automated system for treating organs with cryoprotective agents. Cryobiology 22:607-608, 1985.

5. Rall, W.F., Fahy, G.M. Cryopreservation of mouse embryos by vitrification. Cryobiology 22:603, 1985.

6. Fahy, G.M. Vitrification of multicellular systems and whole organs. Cryobiology 24:580-581, 1987.

7. Khirabadi, B.S., Ali, S., Fahy, G. Rabbit kidney autotransplantation: Attempts to develop a model for evaluation of organ preservation techniques. Cryobiology 25:512, 1988.

8. Smith, S., Fahy, G.M., Khirabadi, B.S., Mehl, P. Ultrasonic imaging of ice and fractures in cryoprotectant-water glass and in vitrified organs. Cryobiology 27:687, 1990.

9. Stoiancheva, K.N., Tsonev, L.I., Fahy, G.M., Khirabadi, B.S., Tsvetkov, T.D., Meryman, H.T. Low Toxicity of 2,3-butanediol for dog and rabbit kidney parenchyma. Cryobiology 28:581-582, 1991.

10. Fahy, G.M., Khirabadi, B.S. Unequivocal demonstration of cryoprotectant toxicity neutralization. Cryobiology 28:581, 1991.

11. Fahy, G.M. The potential future utility of organ cryopreservation. Cryobiology 28:513, 1991.

12. Fahy, G.M., Khirabadi, B.S., Mehl, P. Equipment, solutions, perfusion techniques, and medications permitting survival of kidneys perfused with vitrifiable media. Cryobiology 28:511-512, 1991.

13. Khirabadi, B., Fahy, G., Saur, J., Brandeis, J. Organ cryopreservation: protective effects of a prostacyclin analogue (Iloprost) on nephrotoxic injuries of cryoprotective agents (CPAs) in rabbits. Cryobiology 28:597-598, 1991.

14. Khirabadi, B.S., Fahy, G.M., Antonovich, T., Sabnis, S., Saur, J. Life support function of rabbit kidneys cooled to -30'C. Cryobiology 9:721-722, 1992.

15. Khirabadi, B.S., Fahy, G.M., Saur, J., Matthes, J. Life support function of rabbit kidneys exposed to extreme hydrostatic pressure. Cryobiology 9:722, 1992.

16. Mehl, P.M., Fahy, G.M. Nucleation and crystal growth in a vitrification solution tested for organ cryopreservation by vitrification. Cryobiology 29:725, 1992.

17. Khirabadi, B.S., Fahy, G.M., Saur, J., Meryman, H. Perfusion of rabbit kidneys with 8 molar cryoprotectant (V52). Cryobiology 30:611-612, 1993.

18. Khirabadi, B.S., Fahy, G.M., Nannini, P., Saur, J., Meryman, H. Life support function of rabbit kidneys perfused with 8 molar cryoprotectant. Cryobiology 30:612, 1993.

19. Okouchi, Y., Saur, J., Fahy, G.M. Liver cryopreservation: UW solution as a vehicle for vitrification solution. Cryobiology 30:613, 1993.

20. Okouchi, Y., Fahy, G.M. Comparison of cryoprotectant toxicities at and below Cv using rat liver slices. Cryobiology 30:627, 1993.

21. Fahy, G.M., Ali, S.E., Levy, D.I. Physiology of rabbit kidneys after perfusion with VS4. Cryobiology 31:573, 1994.

22. Khirabadi, B.S., Fahy, G.M., Saur, J., Ewing, L., Meryman, H.T. Failure of rabbit kidneys to survive chilling to -30'C after perfusion with 8M cryoprotectant at -3'C. Cryobiology 31:596-597, 1994.

23. Khirabadi, B.S., Fahy, G.M., Ewing, L., Saur, J., Meryman, H.T.100% survival of rabbit kidneys chilled to -32'C after perfusion with 8M cryoprotectant at -22'C. Cryobiology 31:597, 1994.

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