US20020115593A1

US20020115593A1 - Organ and biological tissue preservation machine perfusion solution

Info

Publication number: US20020115593A1
Application number: US09/976,785
Authority: US
Inventors: Maximilian Polyak; Ben Arrington
Original assignee: Pike Laboratories Inc
Current assignee: Organ Recovery Systems Inc
Priority date: 2000-10-13
Filing date: 2001-10-12
Publication date: 2002-08-22
Also published as: EP1324658A2; AU2001296836A1; US20020068268A1; WO2002030193A2; WO2002030193A3; CA2424058A1; WO2002030192A3; US7014990B2; AU2002213187A1; WO2002030192A2; CA2424047A1; EP1324657A2

Abstract

Machine perfusion solutions for the presentation of organs and biological tissues prior to implantation, including a cellular energy production stimulator under anaerobic conditions and an oxygen free radical scavenger.

Description

REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser. No. 60/240,024 filed on Oct. 13, 2000, entitled “Organ and Biological Tissue Preservation Machine Perfusion Solution,” which is incorporated herein by reference.[0001]

FIELD OF INVENTION

The invention relates to the field of organ and biological tissue preservation. In particular, the invention relates to machine perfusion solutions for the preservation of organs and biological tissues for implant.

BACKGROUND OF INVENTION

It is believed that the ability to preserve human organs for a few days by cold storage after initial flushing with an intracellular electrolyte solution or by pulsatile perfusion with an electrolyte-protein solution has allowed sufficient time for histo-compatibility testing of donor and recipient. It is also believed that preservation by solution or perfusion has also allowed for organ sharing among transplant centers, careful preoperative preparation of the recipient, time for preliminary donor culture results to become available, and vascular repairs of the organ prior to implantation.

It is believed that the 1990's has been a decade characterized by increasing waiting times for cadaveric organs. In renal transplantation, the growing disparity between available donors and patients on the waiting list has stimulated efforts to maximize utilization of cadaveric organs. An obstacle that may arise in the effort to increase utilization is that maximal utilization may require transplantation of all available organs, including extended criteria donor organs. However, by extending the criteria for suitability of donor organs, transplant clinicians may risk a penalty with respect to graft function, diminishing the efficiency of organ utilization if transplanted organs exhibit inferior graft survival. Consequently, interventions that both improve graft function and improve the ability of clinicians to assess the donor organ may be crucial to achieving the goal of maximizing the efficiency of cadaveric transplantation.

The mechanisms of injuries sustained by the cadaveric renal allograft during pre-preservation, cold ischemic preservation and reperfusion are believed to be complex and not fully understood. However, it is believed that there exists ample evidence to suggest that many of the injurious mechanisms occur as a result of the combination of prolonged cold ischemia and reperfusion (I/R). Reperfusion alone may not be deleterious to the graft, since reperfusion after short periods of cold ischemia may be well-tolerated, but reperfusion may be necessary for the manifestation of injuries that originate during deep and prolonged hypothermia. It is suggested that four major components of I/R injury that affect the preserved renal allograft begin during cold ischemia and are expressed during reperfusion. These include endothelial injury, leukocyte sequestration, platelet adhesion and increased coagulation.

Hypothermically-induced injury to the endothelium during preservation may lead to drastic alterations in cytoskeletal and organelle structures. During ischemic stress, profound changes in endothelial cell calcium metabolism may occur. These changes may be marked by the release of calcium from intracellular depots and by the pathological influx of calcium through the plasma membrane. Hypothermic preservation may disrupt the membrane electrical potential gradient, resulting in ion redistribution and uncontrolled circulation of Ca++. The depletion of ATP stored during I/R may compromise ATP-dependent pumps that extrude Ca++ from the cell and the energy intensive shuttle of organelle membranes, causing a dramatic elevation of intracellular free Ca++.

Alterations in cytosolic Ca++ concentration may disrupt several intracellular functions, many of which may result in damaging effects. Unregulated calcium homeostasis has been implicated in the development of endothelial and parenchymal injury and is believed to be a fundamental step in the sequelae of steps leading to lethal cell injury. Among the most significant damaging effects of increased cytosolic Ca++ are believed to be the activation of phospholipase A1, 2 and C, the cytotoxic production of reactive oxygen species by macrophages, the activation of proteases that enhance the conversion of xanthine dehydrogenase to xanthine oxidase, and mitochondrial derangements.

Solutions for preserving organs are described in U.S. Pat. Nos. 4,798,824 and 4,879,283, the disclosures of which are incorporated herein in their entirety. Despite such solutions, it is believed that there remains a need for organ and tissue preserving solutions that allow for static storage and preservation, while demonstrating superior quality preservation of organ and tissue viability and function.

SUMMARY OF THE INVENTION

The invention provides an organ and tissue preserving solution for machine perfusion preservation that demonstrates superior quality preservation when compared to existing preserving media, in terms of organ and tissue viability and function. The organ and biological tissue preservation aqueous machine perfusion solution includes a cellular energy production stimulator under anaerobic conditions and an oxygen free radical scavenger.

The invention also provides a preserved organ and biological tissue. The preserved organ and biological tissue includes a cadaveric organ or tissue within the machine perfusion solution in a deep hypothermic condition or physiological condition.

The invention also provides a perfusion machine comprising a chamber that mimics a deep hypothermic environment or physiological environment, where the machine perfusion solution continuously circulates through the chamber.

The invention also provides a method for preserving an organ and biological tissue. This method includes pouring the machine perfusion solution into a chamber that mimics a deep hypothermic environment or physiological environment, circulating the machine perfusion solution continuously through the chamber, inserting a cadaveric organ or tissue into the chamber, and flushing the cadaveric or tissue with the machine perfusion solution.

The invention further provides a method of preparing an organ and biological tissue machine perfusion solution. This method includes providing a solution with sterile water, adding sodium gluconate, potassium phosphate, adenine, ribose, calcium chloride, pentastarch, magnesium gluconate, HEPES, glucose, mannitol, and insulin to the solution, and mixing superoxide dismutase into the solution.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the invention, the organ and biological tissue preservation machine aqueous perfusion solution includes a cellular energy production stimulator under anaerobic conditions, and an oxygen free radical scavenger. The organ and biological tissue preservation aqueous machine perfusion solution is intended for infusion into the vasculature of cadaveric and living donor organs for transplantation. Once infused, the donor organs are exsanguinated and blood is replaced by the solution in the native vasculature of the organs to return the organs to a normothermic condition. The solution may be used under deep hypothermic conditions or physiological conditions. The solution remains in the vasculature of the organ as well as envelops the entire organ during the period of cold ischemia. This method of preservation allows for the extended storage of organs, tissues, and all biological substances. When the organ or tissue is returned to normothermic conditions, the solution is replaced with blood or other physiologic media. Variations of this solution may also be used for cold storage solution preservation. The machine perfusion solution of the invention may be used in the same manner and for the same tissues and organs as known machine perfusion solutions.

A machine perfusion solution of the invention includes a cellular energy production stimulator under anaerobic conditions. Insulin, which is a polypeptide hormone, is the primary hormone responsible for controlling the uptake, utilization, and storage of cellular nutrients. Insulin stimulates transport of substrates and ions into cells, promotes translocation of proteins between cellular compartments and activates and inactivates specific enzymes.

A machine perfusion solution of the invention also contains an oxygen free radical scavenger. One example, superoxide dismutase, is known for its potent free oxygen radical scavenging properties. Preferably, the superoxide dismutase is conjugated to polyethylene glycol so that its half-life is extended by a factor of about 100 times. Superoxide dismutase is a potent scavenger of several classes of free oxygen radicals during cold ischemia and upon reperfusion. When conjugated to polyethylene glycol, superoxide dismutase can remain active for several hours in the machine perfusion solution of the invention.

According to a preferred embodiment of the invention, an organ and biological tissue preservation machine perfusion solution containing superoxide dismutase in the preserving solution significantly improves vascular resistance, vascular flow, and calcium efflux during the organ preservation period. The inhibition of calcium efflux over time in kidneys preserved by the proposed solution suggests that, in addition to vasoactive effects, an additional cytoprotective and cryoprotective effect may also be important in ameliorating ischemic injury. These improvements are substantiated ultrastructurally by improved appearance of mitochondria in proximal tubular cells compared to mitochondria from kidneys not exposed to the proposed solution.

A machine perfusion solution of the invention may also contain components typically used in known machine perfusion solutions. See, U.S. Patent Nos. 4,798,824 and 4,879,283. For example, other components that may be utilized in the solution include: sodium gluconate and Mg gluconate, which are impermeant anions that reduce cell swelling, KH ₂PO₄, which provides acid-base buffering and maintains the pH of the solution, adenine, which is a precursor to ATP synthesis, and ribose, which reduces cell swelling during hypothermia. In addition, CaCl₂, which is a calcium-dependent mitochondrial function supplement, HEPES, which is an acid-base buffer, glucose, which is a simple sugar that reduces cell swelling and provides energy stores for metabolically stressed cell, and mannitol and pentastarch, which are oncotic supporters, may also be added. NaCl and KOH may also be used for acid-base buffering and maintenance of the pH of the machine perfusion solution.

In a preferred embodiment, the organ or biological tissue preservation machine perfusion solution includes, but is not limited to:

	TABLE 1


	COMPOSITION	AMOUNT IN 1 LITER

Sodium Gluconate	40-160	mM
KH₂PO₄	10-50	mM
Mg Gluconate	1-15	mM
Adenine	1-15	mM
Ribose	1-15	mM
CaCl₂	0.1-2	mM
HEPES	1-30	mM
Glucose	1-30	mM
Mannitol	10-100	mM
Pentastarch	40-60	g/L
Insulin	10-100	U/L
Superoxide Dismutase	1,000-100,000	U/L
Sterile Water	700-900	mL

In a more preferred embodiment, the organ or biological tissue preservation machine perfusion solution includes, but is not limited to:

	TABLE 2


	COMPOSITION	AMOUNT IN 1 LITER

Sodium Gluconate	60-100	mM
KH₂PO₄	20-30	mM
Mg Gluconate	3-8	mM
Adenine	3-8	mM
Ribose	3-8	mM
CaCl₂	0.3-0.8	mM
HEPES	8-15	mM
Glucose	8-15	mM
Mannitol	15-50	mM
Pentastarch	45-55	g/L
Insulin	30-60	U/L
Superoxide Dismutase	10,000-50,000	U/L
Sterile Water	700-900	mL

In a most preferred embodiment, the organ or biological tissue preservation machine perfusion solution includes, but is not limited to:

	TABLE 3


	COMPOSITION	AMOUNT IN 1 LITER

	Sodium Gluconate	Approx 80 mM
	KH₂PO₄	Approx 25 mM
	Mg Gluconate	Approx 5 mM
	Adenine	Approx 5 mM
	Ribose	Approx 5 mM
	CaCl₂	Approx 0.5 mM
	HEPES	Approx 10 mM
	Glucose	Approx 10 mM
	Mannitol	Approx 30 mM
	Pentastarch	Approx 50 g/L
	Insulin	Approx 40 U/L
	Superoxide Dismutase	Approx 25,000 U/L
	Sterile Water	Approx 800 mL

A machine perfusion solution of the invention may be prepared by combining the components described above with sterile water, such as distilled and/or deionized water. For example, to prepare the solution, approximately 700-900 mL, or preferably about 800 mL, of sterile water is poured into a one liter beaker at approximately room temperature. Although a one liter beaker is used in this example, any other container of any size may be used to prepare the solution, where component amounts would be adjusted accordingly. In the most preferred embodiment, the following are added, in any order, to the solution and each is mixed until dissolved in the solution: approximately 80 mol/L sodium gluconate, approximately 25 mol/L potassium phosphate, approximately 5 mol/L adenine, approximately 5 mol/L of ribose, approximately 0.5 mol/L of calcium chloride, and approximately 50 g modified pentastarch. The modified pentastarch is a fractionated colloid mixture of 40-60 kDaltons in diameter and is modified by infusing the pentastarch under 3 atm of pressure through a dialyzing filter with a bore size of about 40-60 kDaltons. About 5 mol/L magnesium gluconate, approximately 10 mol/L HEPES, approximately 10 mol/L glucose, and approximately 30 mol/L mannitol are also added, in any order, and mixed. Approximately 40 U of insulin is also added. Then, in a second step, approximately 25,000 U of superoxide dismutase, which is conjugated to polyethylene glycol, is added to the solution. The first and second step may also be reversed.

The invention also provides a method for preserving an organ or biological tissue. The method includes pouring the machine perfusion solution into a chamber that mimics a deep hypothermic environment or physiological environment and circulating the machine perfusion solution continuously through the chamber. The machine perfusion solution is infused in a mechanical fashion through the arterial or venous vascular system of cadaveric or living donor organs, or infused over or through an avascular biological substance in order to maintain organ or tissue viability during the ex vivo period. Preferred temperatures range from about 2-10° C. in the deep hypothermic condition and are about 37° C., or room temperature, in the physiological condition. Use of this solution provides for the serial assay of solution over time to determine hydrostatic and chemical changes. These hydrostatic and chemical changes provide a mechanism to determine the functional viability of the organ or tissue once it has been returned to physiologic conditions.

The invention further provides a perfusion machine comprising a chamber that mimics a deep hypothermic environment or physiological environment, where the machine perfusion solution continuously circulates through the chamber. Any perfusion machine that is known in the art may be used with the solution, including machines providing pulsatile, low flow, high flow, and roller flow perfusion. Typically, the perfusion machine includes a unit for the static monitoring or transportation of organs or biological tissues and a cassette, or chamber, used to circulate perfusate through the organs or biological tissues. A monitor displays parameters, such as pulse pump rate, perfusate temperature, systolic, mean, and diastolic pressure, and real-time flow. Once such machine is the RM3 Renal Preservation System manufactured by Waters Instruments, Inc.® As discussed above, preferred temperatures range from about 2-10° C. in the deep hypothermic condition and are about 37° C., or room temperature, in the physiological condition.

The invention is further explained by the following of examples of the invention, as well as comparison examples. In all of the examples, kidneys were procured from heart-beating donors and preserved in a laboratory by cold storage preservation. Randomization was accomplished as an open labeled, sequential analysis. All agents were added immediately prior to vascular flush.

Data Collected

The following donor, preservation, and postoperative recipient outcome data were collected for either Example 1 or 2: donor age (D age, years), final donor creatinine (D Cr, mg/dL), donor intraoperative urine output (U/O, mL), cold ischemic time (CIT, hours), perfusion time (PT, hours), perfusate [Na+] (mM/100 g), perfusate [C1-] (mM/100 g), perfusate [K+] (mM/100 g), perfusate [Ca++] (mM/100 g), perfusate pH, renal flow during MP (FL, mL/min/100 g), renal resistance during MP (RES, mmHg/(mL/min/100 g), recipient age (R age, years), recipient discharge creatinine (R Cr, mg/dL), initial length of recipient hospital stay (LOS, days), immediate graft function (IF, %) defined as urine production exceeding 2000 nL during the first 24 post-operative hours, delayed renal allograft graft function (DGF, %) defined as the need for dialysis within the first 7 days post-transplant, and present function (3 Mo or 1 Yr., %) defined as 3 month or one year post-operative graft status.

Method of Preservation

Kidneys were perfused en bloc at 4° C. and at 60 beats per minute with either 1 liter of UW-MPS (Belzer-MPS, TransMed Corp., Elk River, Minn.), Belzer I-Albumin (Suny-Downstate, Brooklyn, N.Y.), or the Machine Perfusion Solution (Storage) (MPS) of the present invention. The Belzer solution, which is also the Control-Belzer solution, is described in U.S. Pat. Nos. 4,798,824 and 4,879,283. The Albumin solution contained, per liter, 17.5 g sodium bicarbonate, 3.4 g potassium dihydrogen phosphate, 1.5 g glucose, 9 g glutathione, 1.3 g adenosine, 4.7 g HEPES, 200K units penicillin, 8 mg dexamethasone, 12 mg phenosulphathelein, 40 units insulin, 150 mL serum albumin, and 1 g magnesium sulfate. In each of the solutions, the kidneys were perfused on RM3 organ perfusion machines (Waters Instruments, Inc.®, Rochester, Minn.), which provide a fixed-pressure system that allows adjustment to the perfusion pressure, as needed. All kidneys were perfused at a systolic pressure below 60 mmHg. Perfusion characteristics (FL, RES, PT, [Na+], [C1-], [K+], [Ca++], and pH) were measured when the kidneys were placed on the machine perfusion system, every 30 minutes for the first 2 hours of MP, and every hour thereafter throughout the period of MP. All chemical data were compared to a baseline assay of perfusate that had not circulated through the kidneys. All perfusion characteristics were standardized to 100 g of tissue weight.

Analysis

The following biochemical components of perfusate were measured every hour throughout MP with an Omni 4 Multianalyte system (Omni, AVL Medical Instruments, Atlanta, Gg.): [Na+], [C1-], [K+], [Ca++], and pH. All biochemical assays were standardized to 10 g of tissue weight. For each measurement, a 0.5 cc aliquot of perfusate is drawn from the perfusion chamber, analyzed by the Omni, and is available for evaluation within 30 seconds.

Statistical analysis

All data are reported as mean values±SEM unless otherwise noted. Paired and unpaired student's t-tests were used where appropriate. All statistical analyses were performed by Statview 4.5 software (Abacus Concepts, Berkeley, Calif.).

EXAMPLE 1

Comparison of selected donor, preservation, and outcome variables by method and type of preservation (mean +/−SEM) [0034]
n=number of recipients [0035]

ns=not significant



		p value
MPS (n = 65)		(unpaired
(Embodiment	Belzer-MPS	student's
of Table 3)	(n + 71)	t-test)

Donor Characteristics
Donor age (y)	64.1	61.1	ns
Final serum creatinine (mg/dl)	1.2	1.2	ns
Preservation characteristics
Cold ischemic time (h)	29	29	ns
Outcome characteristics
Delayed graft function (%)	15	25	0.04
1 year function (%)	95	94	ns

EXAMPLE 2

Comparison of selected donor, preservation, and outcome characteristics by method of machine perfusion solution (mean +/−SEM) [0037]

SOD=superoxide dismutase-polyethylene glycol (25,000 units/L)



		p value
SOD (n = 57)	Control-	(unpaired
(Embodiment	Belzer-MPS	student's
of Table 3)	(n = 140)	t-test)

Donor Characteristics
Donor age (y)	38.3 +/− 12	44.1 +/− 5	0.72
Final serum creatinine (mg/dl)	1.0 +/− 0.4	0.8 +/− 0.5	0.45
Intraoperative urine	150 +/− 140	240 +/− 60	0.56
output (ml)
Preservation characteristics
Cold ischemic time (h)	23 +/− 6	23 +/− 4	0.61
Perfusion time (h)	18 +/− 4	16 +/− 5	0.33
Outcome characteristics
Immediate function (%)	85 +/− 3	85 +/− 5
Delayed grant function (%)	14 +/− 3	18 +/− 4
3 month function (%)	95 +/− 3	87 +/− 5

While the invention has been disclosed with reference to certain preferred embodiments, numerous modifications, alterations, and changes to the described embodiments are possible without departing from the sphere and scope of the invention, as defined in the appended claims and their equivalents thereof. For example, although the detailed description may refer, at times, to only organs, the terms “organ” and “organs” encompass all organs, tissues, and body parts that may be transplanted. Accordingly, it is intended that the invention not be limited to the described embodiments, but that it have the full scope defined by the language of the following claims. [0039]

Claims

What we claim is:

1. An organ or biological tissue preservation aqueous machine perfusion solution comprising:

a cellular energy production stimulator under anaerobic conditions; and

an oxygen free radical scavenger.

2. The machine perfusion solution of claim 1 wherein the cellular energy production stimulator comprises insulin.

3. The machine perfusion solution of claim 1 wherein the oxygen free radical scavenger comprises superoxide dismutase.

4. The machine perfusion solution of claim 3 wherein the superoxide dismutase conjugates to polyethylene glycol.

5. The machine perfusion solution of claim 1 further comprising sodium gluconate, KH₂PO₄, magnesium gluconate, adenine, and ribose.

6. The machine perfusion solution of claim 1 further comprising CaCl₂, HEPES, glucose, mannitol, and pentastarch.

7. The machine perfusion solution of claim 1 further comprising NaCl and KOH.

8. The machine perfusion solution of claim 1 wherein the cellular energy production stimulator comprises about 10-1000 U/L insulin and the oxygen free radical scavenger comprises about 1,000-100,000 U/L superoxide dismutase, further comprising:

about 40-160 mM sodium gluconate;

about 10-50 mM KH₂PO₄;

about 1- 15 mM magnesium gluconate;

about 1-15 mM adenine;

about 1-15 mM ribose;

about 0.1-2 mM CaCl₂;

about 1-30 mM HEPES;

about 1-30 mM glucose;

about 10-100 mM mannitol; and

about 40-60 g/L pentastarch.

9. The machine perfusion solution of claim 1 wherein the cellular energy production stimulator comprises about 30-60 U/L insulin and the oxygen free radical scavenger comprises about 10,000-50,000 U/L superoxide dismutase, further comprising:

about 60-100 mM sodium gluconate;

about 20-30 mM KH₂PO₄;

about 3-8 mM magnesium gluconate;

about 3-8 mM adenine;

about 3-8 mM ribose;

about 0.3-0.8 mM CaCl₂;

about 8-15 mM HEPES;

about 8- 15 nM glucose;

about 15-50 mM mannitol; and

about 45-55 g/L pentastarch.

10. The machine perfusion solution of claim 1 wherein the cellular energy production stimulator comprises about 40 U/L insulin and the oxygen free radical scavenger comprises about 25,000 U/L superoxide dismutase, further comprising:

about 80 mM sodium gluconate;

about 25 mM KH₂PO₄;

about 5 mM magnesium gluconate;

about 5 mM adenine;

about 5 mM ribose;

about 0.5 mM CaCl₂;

about 10 mM HEPES;

about 10 mM glucose;

about 30 mM mannitol; and

about 50 g/L pentastarch.

11. The machine perfusion solution of claim 1 further comprising at least one of distilled water and deionized water.

12. A preserved organ or biological tissue comprising at least one of a cadaveric organ and tissue within the machine perfusion solution of claim 1 in at least one of a deep hypothermic condition and physiological condition.

13. The preserved organ or biological tissue of claim 12 wherein the machine perfusion solution is infused through vasculature of at least one of a cadaveric organ, living donor organ, and tissue.

14. The preserved organ or biological tissue of claim 12 wherein the machine perfusion solution is infused over or through an avascular biological substance to maintain viability of the at least one of a cadaveric organ and tissue during an ex vivo period.

15. The preserved organ or biological tissue of claim 12 wherein the deep hypothermic condition comprises a temperature of about 2-10° C.

16. The preserved organ or biological tissue of claim 12 wherein the physiological condition comprises a temperature of about 37° C.

17. A perfusion machine comprising:

a chamber that mimics at least one of a deep hypothermic environment and physiological environment; and

the machine perfusion solution of claim 1 that continuously circulates through the chamber.

18. The perfusion machine of claim 17 further comprising:

a unit for static monitoring of at least one of an organ and tissue.

19. An organ or biological tissue preservation aqueous machine perfusion solution comprising:

about 1,000-100,000 U/L superoxide dismutase;

about 10-100 U/L insulin;

about 40-160 mM sodium gluconate;

about 10-50 mM KH₂PO₄;

about 1-15 mM magnesium gluconate;

about 1-15 mM adenine;

about 1-15 mM ribose;

about 0.1-2 mM CaCl₂;

about 1-30 mM HEPES;

about 1-30 mM glucose;

about 10-100 mM mannitol;

about 40-60 g/L pentastarch; and

about 700-900 mL sterile water.

20. A method for preserving an organ or biological tissue comprising:

pouring a machine perfusion solution into a chamber that mimics at least one of a deep hypothermic environment and physiological environment, the machine perfusion solution comprising a cellular energy production stimulator under anaerobic conditions and an oxygen free radical scavenger;

circulating the machine perfusion solution continuously through the chamber;

inserting at least one of a cadaveric organ and tissue into the chamber; and

flushing the at least one of a cadaveric organ and tissue with the machine perfusion solution.

21. The method of claim 20 wherein the flushing comprises:

infusing the solution through vasculature of the at least one of a cadaveric organ and tissue.

22. The method of claim 20 wherein the flushing comprises:

infusing the solution over or through an avascular biological substance of the at least one of a cadaveric organ and tissue to maintain viability during an ex vivo period.

23. The method of claim 20 further comprising:

monitoring parameters of the at least one of a cadaveric organ and tissue.

24. The method of claim 20 further comprising:

exsanguinating the at least one of a cadaveric organ or tissue; and

replacing the machine perfusion solution with at least blood to return the at least one of a cadaveric organ and tissue to a normothermic condition.

25. A method of preparing an organ or biological tissue preservation machine perfusion solution comprising:

providing a solution with at least one of distilled water and deionized water;

adding sodium gluconate, potassium phosphate, adenine, ribose, calcium chloride, pentastarch, magnesium gluconate, HEPES, glucose, mannitol, and insulin to the solution; and

mixing superoxide dismutase into the solution.

26. The method of claim 25 further comprising:

mixing the solution until all components are dissolved.

27. The method of claim 25 further comprising:

infusing the pentastarch under pressure through a dialyzing filter; and

conjugating the superoxide dismutase to polyethylene glycol.