| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | February 13, 2001 |
| PATENT TITLE |
Method for preparing organs for transplantation after cryopreservation |
| PATENT ABSTRACT |
The invention relates to the field of organ and tissue perfusion. More particularly, the present invention relates to a method for preparing organs, such as the kidney and liver, for cryopreservation through the introduction of vitrifiable concentrations of cryoprotectant into them. To prepare the organ for cryopreservation, the donor human or animal, is treated in the usual manner and may also be treated with iloprost, or other vasodilators, and/or transforming growth factor .beta.1. Alternatively, or additionally, the organ which is to be cryopreserved can be administered iloprost, or other vasodilators, and/or transforming growth factor .beta.1 directly into its artery. The invention also relates to preparing organs for transplantation by a method for the removal of the cryoprotectant therefrom using low (such as raffinose, sucrose, mannitol, etc.), medium (such as agents with intermediate molecular weights of around 600-2,000) and high (such as hydroxyethyl starch) molecular weight agents osmotic buffering agents. The invention is also directed to new post-transplantation treatments such as the use of transforming growth factor .beta.1, N-acetylcysteine and aurothioglucose. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | April 28, 1998 |
| PATENT REFERENCES CITED |
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Fahy et al., "Some Emerging Principles Underlying the Physical Properties, Biological Actions, and Utility of Vitrification Solutions," Cryobiology 24:196-213 (1987). Fahy et al., "Physical Problems with Vitrification of Large Systems," Cryobiology 26:569-570 (Abstract No. 100) (1989). Fahy et al., "Equipment Solutions, Perfusion Techniques and Medications Permitting Survival of Kidneys Perfused with Vitrifiable Media," Cryobiology 28:511-512 (Abstract No. 2) (1991). Farrant, J., "Mechanism of Cell Damage During Freezing and Thawing and Its Prevention," Nature 205:1284-1287 (1965). Fuller et al., "Studies on Cryoprotectant Equilibration in the Intact Rat Liver Using Nuclear Magnetic Resonance Spectroscopy: A Noninvasive Method to Assess Distribution of Dimethyl Sulfoxide in Tissues," Cryobiology 26:112-118 (1989). Halasz et al., "Studies in Cryoprotection II: Propylene Glycol and Glycerol," Cryobiology 21:144-147 (1984). Hill et al., "The Renal Haemodynamic and Excretory Actions of Prostacyclin and 6-OXO-PGF.sub.1.alpha. in Anaesthetized Dogs," Prostaglandins 17(1):87-98)(1979). Hooper et al., "The Use of a Prostacyclin Analog, Iloprost, as an Adjunt to Pulmonary Preservation with Euro-Collins Solution," Transplantation 49(3):495-499 (1990). Isenberg et al., "Prevention of Ischemic Renal Damage with Prostacyclin," Mount Sinai J. Med. 49(5):415-417 (1982). Jacobsen, I.A., "Distribution and Removal of Glycerol by Vascular Albumin Perfusion in Rabbit Kidneys," Cryobiology 15:302-311 (1978). Jacobsen, I.A., "An Introduction to the Problems of Organ Cryopreservation," in: The Biophysics of Organ Cryopreservation (Pegg et al., eds.) Plenum Press, New York, NY, pp. 15-21 (1987). Jacobsen et al., Transplantation of Rabbit Kidneys Perfused with Glycerol Solutions at 10.degree.C, Cryobiology 15:18-26 (1978). Jacobosen et al., "Effect of Cooling and Warming Rate on Glycerolized Rabbit Kidneys," Cryobiology 19:668 (Abstract No. 60) (1982). Jacobsen et al., "Cryopreservation of Organs: A Review," Cryobiology 21:377-384 (1984). Jacobsen et al., "Introduction and Removal of Cryoprotective Agents with Rabbit Kidneys: Assessment by Transplantation," Cryobiology 25:285-299 (1988). Jutte et al., "Cryopreservation of Mouse, Monkey and Human Islets of Langerhans for Transplantation Purposes," Netherlands J. Surg. 39:15-18 (1987). Khirabadi et al., "Comparison of Euro-Collins Solution and RPS-2 as Potential Carriers from Vitrification Solutions Using the Rabbit Renal Autograft Model," Cryobiology 26:593-594 (Abstract No. 251) (1989). Khirabadi et al., "Organ Cryopreservation: Protective Effects of a Prostacylin Analogue (Iloprost) on Nephrotoxic Injuries of Cryoprotective Agents (CPAs) in Rabbits," presented as a poster at the American Red Cross on the afternoon of May 14, 1990, in a conference titled "Blood Services Scientific Conference". Khirabadi et al., "Life Support Function of Rabbit Kidneys Cooled to -30.degree.C," Cryobiology 29:721-722 (Abstract No. 52) (1992). Khirabadi et al., "Life Support Function of Rabbit Kidneys Exposed to Extreme Hydrostatic Pressure," Cryobiology 29:722 (Abstract No. 53) (1992). Khirabadi et al., "Life Support Function of Rabbit Kidneys Perfused with 8 Molar Cryoprotectant," Cryobiology 30:612 (Abstract No. 9) (1993). Khirabadi et al., "Perfusion of Rabbit Kidneys with 8 Molar Cryoprotectant (V52)," Cryobiology 30:611-612 (Abstract No. 8) (1993). Khirabadi et al., "Cryopreservation of the Mammalian Kidney, I. Transplantation of Rabbit Kidneys Perfused with EC and RPS-2 at 2-4.degree.C," Cryobiology 31:10-25 (1994). Kootstra et al., "A New Device Towards Intermediate Term Kidney Preservation--An Experimental Study," Scand. J. Urol. Nephrol. 54 (Suppl.):86-89 (1980). Langkopf et al., "Improvement in the Preservation of Ischemically Impaired Renal Transplants of Pigs by Iloprost," Prostaglandins Leukotrienes and Medicine 21:23-28 (1986). Levin, R.I., "Optimum Methods for the Introduction or Removal of Permeable Cryoprotectants: Perfused Tissues and Organs," Cryobiology 18:617-618 (Abstract No. 22) (1981). Levin, R.L., "The Osmotic Behavior of Perfused Organs," Cryobiology 18(6):37 (1981). MacFarlane et al., "Homogeneous Nucleation and Glass Formation in Cryoprotective Systmes at High Pressures," Cryo-Letters 2:353-358 (1981). Mehl et al., "Nucleation and Crystal Growth in a Vitrification Solution Tested for Organ Cryopreservation by Vitrification," Cryobiology 29:725 (Abstract No. 61) (1992). Mundy et al., "Experimental Assessment of Prostacyclin in the Harvesting of Kidneys for Transplantation," Transplantation 30(4):251-255 (1980). Okouchi et al., "Comparison of cryoprotectant toxicities at and below Cv using rat liver slices," Cryobiology 30:627 (1993). Okouchi et al., "Liver Cryopreservation: UW Solution as a Vehicle for Vitrification Solution," Cryobiology 30:613 (Abstract No. 12) (1993). Pegg, D.E., "Banking of Cells, Tissues, and Organs at Low Temperatures," in: Current Trends in Cryobiology (Smith, A.U., ed.) Plenum Press, NY, NY, pp. 175-177 (1970). Pegg, D.E., "Perfusion of Rabbit Kidneys with Cryoprotective Agents," Cryobiology 9:411-419 (1972). Pegg, D.E., "The Mechanism of Cryoinjury in Glycerol-Treated Rabbit Kidneys," Cryobiology 16:618 (Abstract No. 108) (1979). Pegg, D.E., "Mechanism of Cryoinjury in Organs," Cryobiology 18:617 (Abstract No. 19) (1981). Pegg et al., "Renal Preservation by Hypothermic Perfusion, 1. The Importance of Pressure Control," Cryobiology 10:56-66 (1973). Pegg et al., "Hypothermic Perfusion of Rabbit Kidneys with Solutions Containing Gelatin Polypeptides," Transplantation 24(1):29-38 (1977). Pegg et al., "Perfusion of Rabbit Kidneys with Glycerol Solutions at 5.degree.C," Cryobiology 14:168-178 (1977). Pegg et al., "Analysis of the Introduction and Removal of Glycerol in Rabbit Kidneys Using a Krogh Cylinder Model," Cryobiology 23:150-160 (1986). Pegg et al., "Optimization of a Vehicle Solution for the Introduction and Removal of Glycerol with Rabbit Kidneys," Cryobiology 23:56-63 (1986). Pegg et al., "Perfusion of Rabbit Kidneys with Solutions Containing Propane-1,2-diol," Cryobiology 24:420-428 (1987). Perry, R.M., "Mathematical Analysis of Recirculating Perfusion Systems, with Application to Cryonic Suspension," Cryonics 9:24-38 (1988). Rall et al., "Ice-free Cryopreservation of Mouse Embryos at -196.degree.C by Vitrification," Nature 313:573-575 (1985). Rebmann et al., "Die Verlangerung der Lagerungskonservierung von Schweinenieren auf 72 Stunden durch Einsatz von Iloprost," (see English Summary on p. 616), Z. Urol. Nephrol. 78:611-617 (1985). Rijkmans et al., "Intermediate ex-vivo and in-vitro Perfusion to Prolong Hypothermic Kidney Preservation up to 6 Days," in: Organ Preservation, Basic and Applied Aspects, (Pegg et al., eds.) MTP Press, Lancaster, UK, pp. 267-272 (1982). Rijkmans et al., "Six-Day Canine Kidney Preservation, Hypothermic Perfusion Combined with Isolated Blood Perfusion," Transplantation 37(2):130-134 (1984). Ruggera et al., "Rapid and Uniform Electromagnetic Heating of Aqueous Cryoprotectant Solutions from Cryogenic Temperatures," Cryobiology 26:568 (Abstract No. 96) (1989). Sadri, F., "Organ Perfusion Systems: An Evaluation Criteria," T.O.P.S. Medical Corporation, Bellevue, WA (8 pages) (1987). Schabel et al., "Renal Storage Preservation at -5.degree.C for 7 Days," Cryobiology 25:513 (Abstract No. 16) (1988). Schror et al., "Dissociation of Antiplatelet Effects from Myocardial Cytoprotective Activity During Acute Myocardial Ischemia in Cats by a New Carbacyclin Derivative," J. Cardiovascular Pharmacol. 4(4):554-561 (1982). Segal et al., "Function of Rabbit Kidneys in vitro at Normothermia Following Equilibration with 3.0 M Me.sub.2 SO and Removal by Hypertonic Washout at 10.degree.C," Cryobiology 19:50-60 (1982). Segal et al., "Kinetics of Permeation and Intracellular Events Associated with Me.sub.2 SO Permeation of Rabbit Kidneys During Perfusion at 10.degree.C," Cryobiology 19:41-49 (1982). Sherwood et al., "Engineering Aspects of Equipment Design for Subzero Organ Preservation," in: Organ Preservation (Pegg, D.E., ed.) pp. 152-174, Churchill, Livingstone, London, UK (1973). Skaer et al., "Non-Penetrating Polymeric Cryofixatives for Ultrastructural and Analytical Studies of Biological Tissues," Cryobiology 15:589-602 (1978). van Gilst et al., "Improved Functional Recovery of the Isolated Rat Heart After 24 Hours of Hypothermic Arrest with a Stable Prostacyclin Analogue (ZK 36 374)," J. Mol. Cell, Cardiol. 15:789-792 (1983). Van Der Wijk et al., "Successful 96- and 144- Hour Experimental Kidney Preservation: A Combination of Standard Machine Preservation and Newly Developed Normothermic ex Vivo Perfusion," Cryobiology 17:473-477 (1980). Van Der Wijk et al., "Six-Day Kidney Preservation in a Canine Model," Transplantation 35(5):408-411 (1983). Waters Instruments Medical Group, Renal Preservation System, Water Instruments Inc., Rochester, NY, (7 pages) (1982). Conscience, J.F., et al., "An Improved Preservation Technique for Cells of Hemopoietic Origin," Cryobiology 22(5):495-498 (1985). Lefer "Mechanisms of the Protective Effects of Transforming Growth Factor--B in Reperfusion Injury", Biochem. Pharm. 42:1323-7 (1991). Turker et al., "Iloprost Preserves Function Against Anoxia," Prostoglandins Leukot Essent Fatty Acids 31:45-52 (1988). |
| PATENT GOVERNMENT INTERESTS |
RIGHTS OF THE UNITED STATES GOVERNMENT IN THIS INVENTION This invention was made with United States Government support under National Institutes of Health Grant Nos. GM 1759, BSRG 2507 and RR 05737. The United States Government has certain rights in this invention. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A method for preparing an organ for transplantation after its cryopreservation with a cryoprotectant, comprising: (a) warming said organ to a temperature which permits reperfusion of said organ, wherein damage to said organ is minimized; (b) perfusing said organ with a composition comprising a cryoprotectant and a non-penetrating osmotic buffering agent having a molecular weight of no more than 500,000, wherein said composition has a non-vitrifiable concentration of cryoprotectant that is less than the concentration of cryoprotectant used for said cryopreservation; and (c) perfusing substantially all of said cryoprotectant out of said organ while concurrently increasing the temperature of said organ to render said organ suitable for transplantation. 2. The method of claim 1, wherein said cryopreservation is by vitrification. 3. The method of claim 1, wherein said cryopreservation is by freeing. 4. The method of claim 1, wherein said osmotic buffering agent is a low molecular weight osmotic buffering agent having a molecular weight of less than 1000 daltons. 5. The method of claim 4, wherein said low molecular weight osmotic buffering agent is selected from the group consisting of maltose, potassium and sodium fructose 1,6-diphosphate, potassium and sodium lactobionate, potassium and sodium glycerophosphate, raffinose, maltopentose, stachyose, sucrose and mannitol. 6. The method of claim 4, wherein said low molecular weight osmotic buffering agent is sucrose. 7. The method of claim 6, further comprising perfusing said organ with a composition comprising mannitol after substantially all of said cryoprotectant is perfused out of said organ. 8. The method of claim 4, wherein said low molecular weight osmotic buffering agent is mannitol. 9. The method of claim 4, wherein the concentration of the low molecular weight osmotic buffering agent is gradually reduced to a nonzero value while the concentration of said cryoprotectant is also being reduced to less than 200 millimolar. 10. The method of claim 9, wherein the concentration of said low molecular weight osmotic buffering agent is reduced to between 250 mM and 1,000 mM. 11. The method of either claim 9, wherein the concentration of said cryoprotectant is reduced to zero. 12. The method of claim 4, wherein the concentration of said low molecular weight osmotic buffering agent is reduced after the concentration of cryoprotectant has been reduced to less than 200 millimolar. 13. The method of claim 4, wherein said osmotic buffering agent has a molecular weight of 100 to less than 1000 daltons. 14. The method of claim 4, wherein said osmotic buffering agent has a molecular weight of no more than 400 daltons. 15. The method of claim 1 wherein said organ is a kidney. 16. The method of claim 1, wherein said temperature in step (a) of said claim is -3.0.degree. C. when said organ is a kidney. 17. The method of claim 1, wherein said non-vitrifiable concentration of cryoprotectant is from 20-40% weight/volume. 18. The method of claim 1, wherein, in step (b), said organ is perfused with said composition for a time sufficient to permit the approximate osmotic equilibration of said organ. 19. The method of claim 1, wherein said osmotic buffering agent has a molecular weight of from 360 to 10,000 daltons. 20. The method of claim 19, wherein said composition contains 2-15% weight/volume of said osmotic buffering agent. 21. The method of claim 19, wherein said osmotic buffering agent is at least one osmotic buffering agent selected from the group consisting of maltose, raffinose, potassium and sodium fructose 1,6-diphosphate, potassium and sodium lactobionate, maltotriose, maltopentose, stachyose, potassium raffinose undecaacetate, Ficoll and hydroxyethyl starch. 22. The method of claim 19, wherein said osmotic buffering agent has a molecular weight of from 360 to 2000 daltons. 23. The method of claim 1, wherein, in step (c), the cryoprotectant is removed at a rate of -31 to -75 mM/minute. |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION This invention relates to the field of organ perfusion. More particularly, it relates to a computer controlled apparatus and method for perfusing isolated animal, including human, organs. Still more particularly, this invention relates to an apparatus and methods for introducing vitrifiable concentrations of cryoprotective agents into isolated organs or tissues in preparation for their cryopreservation and for removing these agents from the organs and tissues after their cryopreservation in preparation for their transplantation into an animal, including into a human. BACKGROUND OF THE INVENTION Cryopreservation (that is, preservation at very low temperatures) of organs would allow organ banks to be established for use by transplant surgeons in much the same way that blood banks are used by the medical community today. At the present time, cryopreservation can be approached by freezing an organ or by vitrifying the organ. If an organ is frozen, ice crystals form within the organ which mechanically disrupt its structure and hence damage its ability to function correctly when it is transplanted into a recipient. Vitrification, by contrast, means solidification, as in a glass, without ice crystal formation. The main difficulty with cryopreservation is that it requires the perfusion of organs with high concentrations of cryoprotective agents (water soluble organic molecules that minimize or prevent freezing injury during cooling to very low temperatures). No fully suitable equipment or method(s) has been developed to date for carrying out this perfusion process. This has prevented the establishment of viable organ banks that could potentially save lives. Devices and methods for perfusing organs with cryoprotectant have been described in the literature since the early 1970's. See, Pegg, D. E., in Current Trends in Cryobiology (A. U. Smith, editor) Plenum Press, New York, N.Y., 1970, pp. 153-180, but particularly pages 175-177; and Pegg, D. E., Cryobiology 9:411-419 (1972). In the apparatus initially described by Pegg, two perfusion circuits operated simultaneously, one with and one without cryoprotectant. Cryoprotectant was introduced and removed by abruptly switching from the cryoprotectant-free circuit to the cryoprotectant-containing circuit, then back again. The pressure was controlled by undescribed techniques, and data was fed into a data logger which provided a paper tape output which was processed by a programmable desk-top Wang calculator. The experimental results were poor. The equipment and technique described were considered inadequate by Pegg and his colleagues, who later modified them considerably. In 1973, Sherwood et al. (in Organ Preservation, D. E. Pegg, ed., Churchill Livingstone, London (1973), pp. 152-174), described four potential perfusion systems, none of which are known to have been built. The first system consisted of a family of reservoirs connected directly to the organ via a multiway valve, changes being made in steps simply by switching from one reservoir to another. The second system created changes in concentration by metering flow from a diluent reservoir and from a cryoprotectant concentrate reservoir into a mixing chamber and then to the kidney. No separate pump for controlling flow to the kidney was included. Total flow was controlled by the output of the metering pumps used for mixing. A heat exchanger was used before rather than after the filter (thus limiting heat exchanger effectiveness), and there was an absence of most arterial sensing. As will become readily apparent below, the only similarity between this system and the present invention was the use of two concentration sensors, one in the arterial line and one in the venous line of the kidney. Organ flow rate was forced to vary in order to minimize arteriovenous (A-V) concentration differences. The sensing of concentration before and after the kidney in the circuit is analogous to but substantially inferior to the use of a refractometer and a differential refractometer in the present invention. The present inventors' experience has shown that the use of a differential refractometer is necessary for its greater sensitivity. The concept of controlling organ A-V gradient by controlling organ flow is distinctly inferior to the system of the present invention. The third system described by Sherwood et al. also lacked a kidney perfusion pump, relying on a "backpressure control valve" to recirculate perfusate from the filter in such a way as to maintain the desired perfusion pressure to the kidney. As with the second Sherwood system, the heat exchanger is proximal to the filter and no bubble trap is present. The perfusate reservoir's concentration is controlled by metered addition of cryoprotectant or diluent as in the second Sherwood system, and if flow from the organ is not recirculated, major problems arise in maintaining and control-ling perfusate volume and concentration. None of these features is desirable. The fourth system was noted by Pegg in an appendix to the main paper. In this system, perfusate is drained by gravity directly from the mixing reservoir to the kidney through a heat exchanger, re-entering the reservoir after passing through the kidney. Concentration is sensed also by directly and separately pumping liquid from the reservoir to the refractometer and back. Modifications and additional details were reported by Pegg et al. (Cryobiology 14:168-178 (1977)). The apparatus used one mixing reservoir and one reservoir for adding glycerol concentrate or glycerol-free perfusate to the mixing reservoir to control concentration. The volume of the mixing reservoir was held constant during perfusion, necessitating an exponentially increasing rate of diluent addition during cryoprotectant washout to maintain a linear rate of concentration change. The constant mixing reservoir volume and the presence of only a single delivery reservoir also made it impossible to abruptly change perfusate concentration. All components of the circuit other than the kidney and a pre-kidney heat exchanger were located on a lab bench at ambient temperature, with the reservoir being thermostated at a constant 30.degree. C. The kidney and the heat exchanger were located in a styrofoam box whose internal temperature was not controlled. Despite this lack of control of the air temperature surrounding the kidney, only the arterial temperature but not the venous temperature or even the kidney surface temperature was measured. The use of a styrofoam box also did not allow for perfusion under sterile conditions. The only possible way of measuring organ flow rate was by switching off the effluent recirculation pump and manually recording the time required for a given volume of fluid to accumulate in the effluent reservoir, since there was no perfusion pump which specifically supplied the organ, unlike the present invention. Pressure was controlled, not on the basis of kidney resistance, but on the basis of the combined resistance of the kidney and a manually adjustable bypass valve used to allow rapid circulation of perfusate through the heat exchanger and back to the mixing reservoir. The pressure sensor was located at the arterial cannula, creating a fluid dead space requiring manual cleaning and potentially introducing undesired addition of unmixed dead space fluid into the arterial cannula. Pressure control was achieved by means of a specially-fabricated pressure control unit whose electrical circuit was described in an earlier paper (Pegg et al., Cryobiology 10:56-66 (1973)). Arterial concentration but not venous concentration was measured. No computer control or monitoring was used. Concentration was controlled by feeding the output of the recording refractometer into a "process controller" for comparison to the output of a linear voltage ramp generator and appropriate adjustment of concentrate or diluent flow rate. Glycerol concentrations were measured manually at 5 minute intervals at both the mixing reservoir and the arterial sample port: evidently, the refractometer was not used to send a measurable signal to a recording device. Temperature and flow were recorded manually at 5 minute intervals. Arterial pressure and kidney weight were recorded as pen traces on a strip chart recorder. None of these features is desirable. Further refinements were reported by Jacobsen et al. (Cryobiology 15:18-26 (1978)). A bubble trap was added, the sample port on the kidney bypass was eliminated (concentration was measured at the distal end of the bypass line instead), and temperature was recorded as a trace on a strip chart recorder rather than manually every 5 minutes. Additionally, these authors reported that bypass concentration lagged reservoir concentration by 5 min (v. 3 min or less for arterial concentration in the present invention) and that terminal cryoprotectant concentration could not be brought to less than 70 mM after adding 5 liters of diluent to the mixing reservoir (v. near-zero terminal concentrations in the present invention using less than 3 liters of diluent and using peak cryoprotectant concentrations approximately twice those of Jacobsen et al., supra). A variation on the system was also reported the same year by I. A. Jacobsen (Cryobiology 15:302-311 (1978)). Jacobsen measured but did not report air temperatures surrounding the kidney during perfusion. He reduced the mixing reservoir volume to 70 ml, which was a small fraction of the 400 ml total volume of the circuit. No electronic-output refractometer appears to have been used to directly sense glycerol concentration and control addition and washout. Instead, the calculated values of concentrate or diluent flow rate were drawn on paper with India ink and read by a Leeds and Northrup Trendtrak Programmer which then controlled the concentrate/diluent pump. Despite the low circuit volume, the minimum concentration of cryoprotectant which could be achieved was about 100 mM. Additional alterations of the same system were reported by Armitage et al. (Cryobiology 18:370-377 (1981)). Essentially, the entire perfusion circuit previously used was placed into a refrigerated cabinet. Instead of a voltage ramp controller, a cam-follower was used. Again, however, it was necessary to calculate the required rates of addition of glycerol or diluent using theoretical equations in order to cut the cam properly, an approach which may introduce errors in the actual achievement of the desired concentration-time histories. Finally, a modification was made in which an additional reservoir was added to the circuit. This reservoir was apparently accessed by manual stopcocks (the mode of switching to and from this reservoir was not clearly explained), and use of the new reservoir was at the expense of being able to filter the perfusate or send it through a bubble trap. The new reservoir was not used to change cryoprotectant concentration; rather, it was used to change the ionic composition of the medium after the cryoprotectant had been added. The volume of the mixing reservoir was set at 500 ml, allowing a final cryoprotectant concentration of 40 mM to be achieved. To the best of the inventors' knowledge, the devices and methods described above represent the current state of the art of cryoprotectant perfusion as practiced by others. An approach to organ preservation at cryogenic temperatures previously described by one of the Applicants involved vitrifying rather than freezing organs during cooling (see, for example, Fahy et al., Cryobiology 21:407-426 (1984); and U.S. Pat. No. 4,559,298). "Vitrification" means solidification without freezing and is a form of cryopreservation. Vitrification can be brought about in living systems, such as isolated human or other animal organs, by replacing large fractions of the water in these systems with cryoprotective agents (also known as cryoprotectants) whose presence inhibits crystallization of water (i.e., ice formation) when the system or organ is cooled. Vitrification typically requires concentrations greater than 6 molar (M) cryoprotectant. However, using known techniques, it has not been possible to use sufficiently high cryoprotectant concentrations to vitrify an organ without killing it. The limiting concentration for organ survival was typically just over 4 M. One type of damage caused by cryoprotectants is osmotic damage. Cryobiologists learned of the osmotic effects of cryoprotectants in the 1950's and of the necessity of controlling these effects so as to prevent unnecessary damage during the addition and removal of cryoprotectants to isolated cells and tissues. Similar lessons were learned when cryobiologists moved on to studies of whole organ perfusion with cryoprotectants. Attention to the principles of osmosis were essential to induce tolerance to cryoprotectant addition to organs. Despite efforts to control the deleterious osmotic effects of cryoprotectants, limits of tolerance to cryoprotectants are still observed. There appear to be genuine, inherent toxic effects of cryoprotectants that are independent of the transient osmotic effects of these chemical agents. Studies by the present inventors and others have examined methods of controlling the non-osmotic, inherent toxicity of cryoprotective agents. The results indicate that several techniques can be effective alone and in combination. These include (a) exposure to the highest concentrations at reduced temperatures; (b) the use of specific combinations of cryoprotectants whose effects cancel out each other's toxicities; (c) exposure to cryoprotectants in vehicle solutions that are optimized for those particular cryoprotectants; (d) the use of non-penetrating agents that can substitute for a portion of the penetrating agent otherwise needed, thus sparing the cellular interior from exposure to additional intracellular agent; and (e) minimization of the time spent within the concentration range of rapid time-dependent toxicity. Means by which these principles could be applied to whole organs so as to permit them to be treated with vitrifiable solutions without perishing, however, have not been clear or available. Some of these techniques are in potential conflict with the need to control osmotic forces. For example, reduced temperatures also reduce the influx and efflux rate of cryoprotectants, thereby prolonging and intensifying their osmotic effects. Similarly, minimizing exposure time to cryoprotectants maximizes their potential osmotic effects. Thus, there must be a balance reached between the control of osmotic damage and the control of toxicity. Adequate means for obtaining this balance have not been described in the literature. In some cases, intensifying the osmotic effects of cryoprotectants by minimizing exposure times to these agents can be beneficial and complementary to the reduced toxicity that results, but safe means for achieving this in whole organs have not been described. Organ preservation at cryogenic temperatures would permit the reduction of the wastage of valuable human organs and would facilitate better matching of donor and recipient, a factor which continues to be important despite the many recent advances in controlling rejection (see, Takiff et al., Transplantation 47:102-105 (1989); Gilks et al., Transplantation 43:669-674 (1987)). Furthermore, most techniques now being explored for inducing recipient immunological tolerance of a specific donor organ would be facilitated by the availability of more time for recipient preparation. One major limitation in organ cryopreservation studies has been the lack of suitable equipment for controlling perfusion parameters such as cryoprotectant concentration-time history, pressure, and temperature. Previously described standard perfusion machines are not designed for this application and are unable to meet the requirements addressed here. Patented techniques heretofore known are described in: U.S. Pat. No. 3,753,865 to Belzer et al.; U.S. Pat. No. 3,772,153 to De Roissart et al.; U.S. Pat. No. 3,843,455 to Bier, M. U.S. Pat. No. 3,892,628 to Thorne et al.; U.S. Pat. No. 3,914,954 to Doerig, R. K.; U.S. Pat. No. 3,995,444 to Clark et al.; U.S. Pat. No. 4,629,686 to Gruenberg, M. L.; and U.S. Pat. No. 4,837,390 to Reneau, R. P. Equipment described for cryopreservation applications in the past has permitted only relatively simple experimental protocols to be carried out, and has often been awkward to use. Only Adem et al. have reported using a computer for organ perfusion with cryoprotectant (see, for example, J. Biomed. Engineering 3:134-139 (1981)). However, their specific design has several major flaws that limit its utility. The present invention overcomes substantially all of the deficiencies of known apparatus and methods. SUMMARY OF THE INVENTION In one embodiment, the present invention is directed to a computer-controlled apparatus and methods for perfusing a human or other animal organ, such as a kidney, liver, heart, etc., with a perfusate, and may include preparing the organ for such perfusion. The perfusion of the organ may be done for any one of a number of reasons including, but not limited to, for example: to prepare the organ for cryopreservation; to prepare the organ for transplantation after its cryopreservation; to preserve it by conventional means above 0.degree. C.; to keep it alive temporarily at high temperatures to study its physiology; to test the organ's viability; to attempt resuscitation of the organ; and to fix the organ for structural studies. The apparatus and methods may also be used to superfuse an organ or tissue slice. In another embodiment, this invention is directed to the treatment of the donor animal and/or the about-to-be donated organ with iloprost and/or other drugs to prepare it for perfusion. In another embodiment, this invention is directed to an apparatus and method which is used to prepare the organ for cryopreservation, such as by vitrification. In another embodiment, this invention is directed to an apparatus and methods for preparing an organ for transplantation into an appropriate host after its cryopreservation. In one embodiment, this invention is directed to a method of preparing a biological organ for cryopreservation, comprising the steps of: (a) perfusing the organ with gradually increasing concentrations of cryoprotectant solution to a first predetermined concentration while concurrently reducing the temperature of the organ; (b) maintaining the concentration of the cryoprotectant for a sufficient time to permit the approximate osmotic equilibration of the organ to occur; and (c) increasing the cryoprotectant concentration of the solution to a higher second predetermined concentration and maintaining the cryoprotectant concentration of the solution at the second concentration for a time sufficient to permit the approximate osmotic equilibration of the organ to occur. The organ is then removed from the perfusion apparatus and is cryopreserved using an appropriate method or is further prepared for cryopreservation. After cryopreservation the organ is warmed in an apparatus which is not the apparatus of this invention. In preparation for the organ's transplantation into a recipient, the organ is then reattached to the perfusion apparatus of this invention. In another embodiment, this invention is directed to a method of preparing an organ for transplantation after its cryopreservation and subsequent warming, comprising: (a) warming the organ to a temperature which permits reperfusion of the organ, wherein damage to the organ is minimized; (b) perfusing the organ with a non-vitrifiable concentration of cryoprotectant for a time sufficient to permit the approximate osmotic equilibration of the organ to occur; and (c) perfusing substantially all of the cryoprotectant out of the organ while concurrently increasing the temperature of the organ to render the organ suitable for transplantation. In another embodiment, this invention is directed to a method of preparing an organ for transplantation further comprising perfusing the organ with a reduced concentration of cryoprotectant in combination with: a low molecular weight (LMW) "nonpenetrating" osmotic buffering agent (OBA); or a high molecular weight (HMW) "nonpenetrating" OBA; or a combination of LMW and HMW OBAs which are added and removed in an orchestrated fashion which is appropriate for, and may vary from, organ to organ. In the case of the liver, osmotic buffers (OB) do not have to be used at all. In the case of most other organs, the organ is perfused with the appropriate cryoprotectant solution containing a first OBA concentration for a time sufficient to permit approximate osmotic equilibration of the organ to occur. Substantially all of the cryoprotectant is then washed out (to a final cryoprotectant concentration of less than 200 millimolar) while decreasing the concentration of the OBA to a second, nonzero level substantially below the first buffering agent concentration level and while concurrently increasing the temperature of the organ. Finally, the organ is perfused to remove the OBA sufficiently to render the organ suitable for transplantation. Exemplifications include the rabbit kidney, the rat liver, and the human kidney. The apparatus of the invention comprises a computer operated perfusion circuit containing a plurality of fluid reservoirs, a means for raising and lowering concentrations and an organ container. A first fluid flow path is defined as a loop from the plurality of reservoirs to necessary sensors and temperature conditioning means and back to the plurality of reservoirs. The reservoirs are selectively connectable to the first fluid flow path. Pump means are interposed in a second fluid flow path for pumping fluid from the first fluid flow path to a second fluid flow path. The organ container is located in this second fluid flow path. Pump means may also be included in the second fluid flow path for pumping fluid from the organ container to one or more of the reservoirs or to waste. One or more sensors are interposed in the fluid flow paths for sensing at least one of the concentration, concentration differential, temperature, pressure, and pH of the fluid flowing in the first and/or second fluid flow paths. Measuring means are interposed in the first and second fluid flow paths for measuring concentration and temperature differences between the upstream and downstream sides, in the fluid flow direction, of the organ container. The sensor(s) and the measuring means are connected to a programmable computer for providing a continuous information stream from the sensor(s) to the computer. Finally, the computer is coupled to the selection means and the pump means to continuously selectively control (a) the flow of fluid from each of the reservoirs individually to the fluid flow paths, (b) the flow of fluid from each of the fluid flow paths individually to each of the reservoirs, and (c) at least one of the concentration, temperature, pressure and pH of the fluid flowing in the first and/or second fluid flow path, in accordance with a predetermined computer program without substantial operator intervention. Additional features of the apparatus of this invention may include a heat exchanger interposed in the first fluid flow path for conditioning the temperature of fluid flowing in this fluid flow path. A second heat exchanger may be interposed in the second fluid flow path for conditioning the temperature of fluid flowing in the second fluid flow path. In describing the apparatus and methods of this invention, many of the various aspects of the same have been numbered. This numbering has been done to create a conceptual organization and structure for this application. This numbering should not be interpreted to necessarily mean or imply that the particular steps in this invention must be performed in the sequences in which they are presented. |
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