Organ Preservation 

Updated: Nov 07, 2020
  • Author: Erik B Finger, MD, PhD; Chief Editor: Ron Shapiro, MD  more...
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Practice Essentials

The first successful organ transplantation was performed by John Merrill and Joseph Murray at the Peter Bent Brigham Hospital, between two identical twins, in December 1954. [1] After that, in 1963, the first human liver [2]  and lung [3] transplants were performed, followed by pancreas [4] and heart [5] transplantations soon thereafter in 1966 and 1967, respectively. As of June 2020, 809,655 transplants had been reported to the United Organ Sharing (UNOS) network since its creation of national database in 1988. [6] With improved surgical techniques and immunosuppressive medications, outcomes after kidney and extrarenal (liver, pancreas, lung, heart) transplantation have continued to improve (see Table 1, below).

Table 1. Transplant outcomes (Open Table in a new window)

Transplants

Patient Survival (%)

Graft Survival (%)

1 year

3 year

5 year

1 year

3 year

5 year

Heart

90.9

85.6

78.6

90.5

84.9

77.7

Lung (Single)

86.7

64.5

47.3

85.3

62.1

45.5

Lung (Double)

87.7

71.8

58.6

87.3

70.4

55.7

Heart / Lung

80.9

58.3

50.2

80.9

58.3

49.2

Liver (Living Donor)

92.3

88.4

83.3

88

82

77.3

Liver (Deceased Donor)

91.2

82.8

75

89.1

80

71.9

Kidney (Living Donor)

98.8

96.1

92.1

94.7

87.8

78.6

Kidney (Deceased Donor)

96.3

91.3

83.3

93.2

85.1

74.4

Pancreas

90.9

87.5

79.6

81.8

71.4

60.1

Simultaneous Pancreas-Kidney

97.5

94.7

88.8

95.7

89.5

81.4

Intestine

82.8

68.9

58.9

77.2

60.7

50.6

Solid-organ transplantations have become the therapeutic option of choice for end-stage organ failure. In many cases, transplantation offers the only definitive treatment for a given disease entity. As a result, the list of indications for solid-organ transplantation has expanded considerably, placing increasing pressure on an already limited supply of donor organs.

The growth in the number of patients wanting or waiting for a transplant has outpaced the supply of available organs. As of June 2020, 111,111 candidates were on the waiting list for organ transplantation in the United States, compared with 72,571 in April 2011; of the patients on the 2020 list, 93,730 were waiting for a kidney. [6, 7] Shortage of organs for transplantations prolonged the patients waiting time, as a result, there was an increase in the mortality and morbidity rate during the waiting time. [8] For instance, waiting time for a deceased-donor kidney transplant varies by candidate age: in 2011-2014, median wait time was 936 days for those 1-5 years old, 920 days for those 6-10 years old, and 680 days for those 11-17 years old. [6]

Each year, more patients are placed on the waiting lists than receive transplants, causing the waiting time to increase. In 2015, more than 6,500 candidates for transplantation died while on the waiting list. [6] With such constraints, preservation of organs for transport between centers becomes crucial in order to facilitate broader sharing of these limited-resource items.

The technology of organ preservation has improved considerably. Several organ-preservation solutions are available, and these are being constantly modified to provide improved organ storage and outcomes. This article discusses the pathophysiology and techniques of organ preservation and describes various preservation solutions currently used for kidney, liver, pancreas, small-bowel, lung, and heart transplantation.

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Pathophysiology of Organ Preservation

The removal, storage, and transplantation of a solid organ from a donor profoundly alters the homeostasis of the interior milieu of the organ. These effects manifest in the degree to which the return of normal organ function is delayed or prevented after transplantation is completed. The injury an organ sustains during recovery, preservation, and transplantation occurs primarily as a result of ischemia and hypothermia. Techniques for organ preservation serve to minimize this damage to promote optimal graft survival and function.

Phases of Organ Damage During Transplantation

Damage to organs during transplantation occurs in 2 phases: first, the warm ischemic phase, then the cold ischemic phase.

The warm ischemic phase includes the time from the interruption of circulation to the donor organ to the time the organ is flushed with hypothermic preservation solution. In multiorgan recovery, the organs are cooled before they are removed. However, in living-donor organ recovery, organs are removed first, then cooled with organ preservation solutions on a back table. Also, warm ischemic time (WIT) is measured by the organ implantation period, which starts from the removal of the organ from cold preservation solution until its reperfusion with warm blood. Prolonged WIT is caused by the long organ extraction time and other donor and recipient factors such as the following [9, 10, 11, 12, 13] :

  • Donor after circulatory death (DCD), anatomical variation, vascular disease, high body mass index, poor vasculature, previous transplantation history and poor health condition.

The cold ischemic phase, occurs when the organ is preserved in a hypothermic state prior to transplantation into the recipient. Acceptable cold ischemic time (CIT) limits vary from organ to organ—typically, 4-6 hours for heart, < 12 hours for liver and pancreas, and < 24 hours for kidney. Shortening the CIT may reduce risk for post-transplantation graft failure and patient mortality, and longer hospital stay. [14, 15]

Mechanisms of Tissue Injury

Integrity of the cell structure

The cell membrane plays a structural role for the cell and provides an active interface with the extracellular environment. Receptors, ion regulation, and enzyme systems linked to the cell-membrane complex contain extracellular, transmembrane, and intracellular components essential to their function.

The stability of the membrane to chemical and water permeability depends on the integrity of the lipid bilayer and on tight control of temperature, pH, and osmolarity. [16]  Organ ischemia and preservation disrupt all of these relationships. Lowering the temperature causes a phase transition of lipids and results in profound changes in membrane stability. In addition, it drastically alters the function of membrane-bound enzymes. [16]  Hypothermia-induced structural changes in the membrane increase permeability, which contributes to cell swelling. Therefore, organ-preservation solutions are hypertonic to minimize these alterations.

Ionic composition of the cell

The sodium-potassium adenosine triphosphatase (Na-K ATPase) pump functions to maintain the ionic composition of the cell. The pump is disrupted by the lack of adenosine triphosphate (ATP) production and by excessive production of hydrogen ions because of anaerobic metabolism during ischemia. When the Na-K ATPase pump is paralyzed, potassium moves out of the cell and diffuses down its concentration gradient to the extracellular space, whereas sodium, which is normally kept at a low concentration in the cell, pours in. This ionic shift causes cell swelling and disruption of the cell if unchecked. Current preservation solutions have electrolyte compositions similar to the milieu inside the cell, with high potassium and low sodium concentrations to minimize the osmotic gradients.

Hydrogen-ion production continues in ischemic organs and causes intracellular pH to decrease without replenishment of buffering capabilities. Under conditions requiring a switch from aerobic to anaerobic glycolysis, the production of lactic acid also increases. The liver appears to be especially susceptible to injury from byproducts of anerobic metabolism.

Calcium ion permeability is increased with ischemia, and a rapid influx of calcium overpowers the intracellular buffering capacity. Calmodulin activity increases and, in turn, causes upregulation of phospholipases. Subsequent production of prostaglandin derivatives results in mitochondrial and cell-membrane injury. Increased cellular calcium concentrations also initiate myofibrillar contraction of the vascular smooth muscle, causing vasospasm and subsequent ischemic damage.

Endothelin, a 21–amino acid peptide with potent vasoconstrictor properties, is another factor that plays a major role in ischemia by inducing vasospasm, which delays recovery of organ function after revascularization. [17, 18] Investigators have evaluated the use of endothelin A and B receptor antagonists to improve results after solid-organ transplantation, reporting favorable results.

ATP generation

A combination of the anaerobic enzymatic breakdown of glucose (glycolysis) and aerobic cellular respiration provides for the energy requirements of aerobic cells. These processes encompass the transfer of electrons from organic molecules to molecular oxygen. Hypothermia decreases the metabolic rate and slows enzymatic degradation of cellular components, but metabolism is not completely suppressed. Cooling from 37°C to 0°C reduces cellular metabolism 12-fold. [16] Although metabolism and utilization of cellular energy stores are slowed, ATP and adenosine diphosphate (ADP), the major sources of cellular metabolic energy, are gradually depleted during hypothermia. This depletion is due to residual energy requirements of the cell that exceed the capacity of the cell to regenerate ATP.

During ischemia and organ preservation, the glycolytic pathway is diverted towards lactate production as a byproduct of ATP generation, as the oxidative Krebs tricarboxylic acid cycle (TCA) cycle and mitochondrial respiration are impaired. Therefore, mitochondrial dysfunction is responsible for most of the changes in cellular energy associated with ischemia and organ preservation.

Hypothermic preservation reduces the activity of mitochondrial enzymes. [19] Cellular respiration, which requires adenine nucleotide substrates, is also reduced. Transport of ADP into the mitochondrion, where acts as a substrate for conversion to high-energy ATP, requires a membrane adenine nucleotide translocase. Hypothermia impairs the function of the translocase, decreasing the supply of ADP available in the mitochondria for conversion to ATP. [16]  Phospholipid hydrolysis by phospholipases increases levels of free fatty acids, which also affect function of the translocase. Adenylate kinase converts excess ADP accumulating in the cytoplasm to ATP, and adenosine monophosphate accumulates as a by-product. This effect reduces purine synthesis and results in the loss of ATP precursors from the cell.

Vascular renal resistance during hypothermic machine perfusion has been found to be an independent risk factor for 1-year graft failure. Renal resistance must be taken into account when evaluating graft quality and predicting a successful outcome; however, renal resistance on its own cannot be used to precisely predict the outcome, [20]

Reperfusion injury

Much of the injury to transplanted organs occurs not during ischemia, but during reperfusion. This finding has led to many advances in organ preservation aimed at preventing this type of injury. Furthermore, some of the events that occur during reperfusion may result in enhanced immunogenicity of the graft. [21] Oxygen free-radicals generated during reperfusion are the main cause of the reperfusion injury, but cytokines and nitric oxide also play a role. [21, 22]

Oxygen free-radicals are among the most important mediators of reperfusion injury and act by various mechanisms. During ischemic states, increased intracellular calcium levels activate cytosolic enzymes, resulting in the conversion of xanthine dehydrogenase to xanthine oxidase. [21] Both xanthine dehydrogenase and xanthine oxidase catabolize hypoxanthine and xanthine to uric acid, and xanthine oxidase uses molecular oxygen as an electron acceptor, forming superoxide as a result. [21] Superoxide anion rapidly reacts with itself to form hydrogen peroxide, a potent oxidant capable of injuring the cell by oxidizing lipid membranes and cellular proteins. Hydrogen peroxide then produces a cascade of oxygen free radicals, including hydroxyl radical and singlet oxygen, that are even more potent than the others. The damaging effects of oxygen free-radicals begin on reperfusion of the organ.

During ischemic conditions, tissue oxygen levels fall below the threshold needed to allow xanthine oxidase to metabolize xanthine and hypoxanthine. This decrease allows the intracellular concentration of these metabolites to rise. On reperfusion, oxygen is suddenly available, and metabolism proceeds rapidly, resulting in a sudden production of reactive oxygen intermediates. The cellular pathways to scavenge oxygen free-radicals are overwhelmed, and cellular injury ensues.

Allopurinol, an inhibitor of xanthine oxidase, has protective effects when it is used before the ischemic insult, as shown in various experimental systems. Lipid peroxidation is another consequence of free radical generation. [22] In this process, interaction of highly reactive oxygen species with polyunsaturated fatty acids in the cell membrane starts a chain reaction that may ultimately destroy cellular integrity and result in cell death. The magnitude of lipid peroxidation appears to be inversely related to levels of glutathione, which functions as an endogenous free-radical scavenger. [22] Therefore, glutathione and other agents that protect against peroxidation are useful in organ preservation solutions to attenuate reperfusion injury.

Production of oxygen free radicals also initiates production of prostaglandins, including leukotriene B4, by means of direct activation of phospholipase A210. This chemoattractant causes leukocyte adherence to vascular endothelium. These neutrophils may contribute to local injury by plugging the microcirculation and by degranulation with resulting proteolytic damage to the organ.

Cytokines are intercellular messenger molecules that may be produced in various normal and pathophysiologic states. Ischemia and reperfusion are associated with marked release of tumor necrosis factor (TNF)-alpha, interferon-gamma, interleukin-1, and interleukin-8. [23] These cytokines cause upregulation of adhesion molecules and cause leukocyte adherence and platelet plugging after revascularization, resulting in graft failure and rejection.

Nitric oxide (NO) is an extremely labile autacoid generated by nitric oxide synthase from L-arginine. Reports indicate that NO production is induced by inflammatory cytokines, such as TNF-alpha, interferon-gamma, and interleukin-1. Increased NO synthesis also correlates with acute rejection. [24]

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Preservation Solutions and Their Pharmacology

Various solutions are used for organ preservation. Each differs in composition, but the purposes of each are similar: to prevent cellular edema, to delay cell destruction, and to maximize organ function after perfusion is reestablished. [25, 26] Organ preservation solutions are classified, based on their components, as intracellular-like or extracellular-like. See Tables 2 and 3, below.

Table 2. Composition of common organ preservation solutions (Open Table in a new window)

Components

Intracellular-like Solutions

Extracellular-like Solutions

 

EC

UW

HOC

PBS

HTK

Celsior

Kyoto

IGL-1

Electrolytes

  Sodium

10

25

84

120

15

100

100

120

  Potassium

115

125

79

-

9

15

44

25

  Calcium

-

-

-

-

0.015

0.25

-

0.5

  Chloride

15

20

-

-

50

28

-

20

  Magnesium

5

5

-

-

4

13

-

5

  Magnesium sulfate

-

-

40

-

-

-

-

5

  Phosphate

50

25

-

60

-

-

25

25

  Sulfate

5

5

-

-

-

-

-

-

  Bicarbonate

10

-

-

-

-

-

-

-

Colloids

  HES (g/l)

-

50

-

-

-

-

30

-

  PEG-35

-

-

-

-

-

-

-

0.03

Impermeant/Buffers

  Citrate

-

-

80

-

-

-

-

-

  Glucose

195

-

-

-

-

-

-

-

  Gluconate

-

-

-

-

-

-

100

-

  Glutamate

-

-

-

-

-

20

-

-

  Histidine

-

-

-

-

200

30

-

-

  Ketoglutarate

-

-

-

-

1

-

-

-

  Lactobionate

-

100

-

-

-

80

-

100

  Mannitol

-

-

185

-

30

60

-

-

  Sucrose

-

-

-

140

-

-

-

-

  Tryptophan

-

-

-

-

2

-

-

-

  Trehalose

-

-

-

-

-

-

41

-

  Raffinose

-

30

-

-

-

-

-

30

Others

 

 

 

 

 

 

 

 

  Adenosine

-

5

-

-

-

-

-

5

  Glutathione

-

3

-

-

-

3

-

3

  Allopurinol

-

1

-

-

-

-

-

1

  Glutamic acid

-

-

-

-

-

20

-

-

pH

7

7.4

-

7.4

7.2

7.3

-

7.4

Osmolality (mOsm/l)

375

320

-

310

310

320

-

320

EC, Euro-Collins; UW, University of Wisconsin; HOC, hypertonic citrate/Marshalls solution, PBS, phosphate-buffered sucrose; HTK, histidine-tryptophan-ketoglutarate; IGL-1, Institute George Lopez; HES, hydroxyethyl starch; PEG-35, polyethylene glycol with an average MW of 35kDa; Note: All units are shown as mmol/L unless otherwise indicated

Table 3. Preservation solution constituents and their functions (Open Table in a new window)

Constituent

Function

Example

Osmotic active agents

Prevent cell swelling

Lactobionate, raffinose, citrate, gluconate

Electrolytes

Exert an osmotic effect

Na+, K+, Ca+, Mg+

+  ion buffers

Regulate extracellular H+

Phosphate, histidine, N -(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid (HEPES) buffer

Colloid

Initial vascular flush out and perfusion

Albumin, HES

Metabolic inhibitors

Suppress degradation of cell constituents

Allopurinol, antiproteases, chlorpromazine

Metabolites

Facilitate restoration of metabolism on reperfusion

Adenosine, glutathione

Antioxidants

Inhibit oxygen free-radical injury

Amino steroids, vitamin E, deferoxamine (Desferal)

Intracellular-like solutions

Euro-Collins solution

The development of solutions with ionic composition similar to the intracellular environment allowed organ preservation to be attempted. These early solutions, called Collins solutions, contained high concentrations of potassium, magnesium, phosphate, sulphate, and glucose. Euro-Collins (EC) solution was developed as a modification of the original Collins solution and contained high concentrations of potassium (110 mM), phosphate (60 mM), and glucose (180 mM). Magnesium was eliminated, without any negative effect on organ preservation. Organ preservation improved with the use of EC solution. When it was used for kidney preservation, delay in kidney function after implantation was significantly reduced. The solution was adequate for use in preserving the heart, liver, and lung.

University of Wisconsin solution

Although University of Wisconsin (UW) solution was initially developed in 1986 for pancreas preservation, currently it is widely used for all abdominal organ preservations. [27, 28] It has been considered the standard for kidney, pancreas, and liver preservation, effectively extending the ischemic time for those organs and allowing them to be transported considerable distances to waiting recipients. UW solution has also been successfully applied to small-bowel, lung, and heart preservation.

The composition of the solution is complex. Analysis of its various components has shown that some may be omitted or replaced with results similar to that of the original solution. By example, hydroxyethyl starch (HES) which is present in the original formulation, conveys no distinct advantage to the solution when used for cold storage. In fact, HES adds to the viscosity of the solution as well as to the unit expense. HES-free derivatives of the solution have shown similar, if not better, clinical results than those of the original formulation. Lactobionate is a major effective component of the solution. Its insoluble nature maintains the colloid oncotic pressure of the solution, delaying or preventing equilibration of the solution across the cell membrane, and thus delaying the development of cellular edema.

Potential disadvantages of UW solution are its higher potassium concentration, which increases risk of cardiovascular arrest, ischemic-type biliary complications and microcirculatory disturbances due to its particle formation, [29] and increased viscosity, which may impair organ perfusion during recovery. However, transplant recipients do not display an overt rise in serum potassium following reperfusion. [30, 31]

During cold ischemia, organs (and especially livers) lose glutathione rapidly, resulting in delayed graft function or organ failure after transplantation. [19] Glutathione is unstable in solution and effective as an oxygen free-radical scavenger only if it is added to UW solution immediately before use. Adenosine and allopurinol help in this function.

UW solution can protect from cold ischemic damage for a longer time than EC or other Collins solutions. Although it was proven to be able to preserve liver for 24-48 hours and kidney for up to 72 hours, the recommended ischemia time limits for liver, kidney, and pancreas are typically much shorter. [32, 33]

Ross-Marshall citrate solutions

Ross-Marshall citrate solutions were developed as alternatives to the Collins solutions. Their electrolytic compositions are similar, except that citrate replaces phosphate and mannitol replaces glucose. The citrate acts as a buffer and chelates with magnesium to form an impermeable molecule that helps stabilize the extracellular environment. These solutions are not commonly used in clinical practice.

Phosphate-buffered sucrose solution

This solution contains sucrose 140 mmol/L and is buffered by phosphate. In experimental studies, it preserved dog kidneys for 3 days. It is not commonly used today.

Extracellular-like solutions

Bretschneider histidine tryptophan ketoglutarate solution

Initially developed as a cardioplegia solution for the use in open heart surgery, Bretschneider histidine tryptophan ketoglutarate (HTK) solution was found to be effective in liver and kidney preservation. Its contents include histidine (200 mM), mannitol (30 mM), tryptophan, and alpha-ketoglutaric acid. Histidine maintains the osmolarity and enhances the solution’s buffering capacity during ischemia-induced acidosis. Histidine, amino acid tryptophan, and alpha-ketoglutarate help to stabilize cell membranes, improve production of ATP during reperfusion, and inhibit the production of lactate through glycolysis. [34] Tryptophan, histidine, and mannitol act as oxygen free-radical scavengers.

The advantages of this solution are its reduced viscosity; low potassium, sodium, magnesium concentrations; and lower unit cost compared with UW and Celsior solutions. As this solution diffuses easily, cools quickly, and poses limited risk of hyperkalemia in patients, it is often used in abdominal and thoracic organ preservation. Use of the solution improved kidney function after transplantation compared with EC solution. It has become increasingly popular in recent years, but some concern exists regarding an increased risk for pancreatitis when used for preservation of pancreas grafts. However, this concern has not been confirmed by a randomized study. Outcomes of transplants using HTK have been comparable to those using UW in many organ systems. [35, 36, 37]

Celsior solution

Celsior (CS) is a recently developed extracellular-type, low-viscosity (due to the absence of HES) preservation solution that couples the impermeant, inert osmotic carrier from UW solution (by using lactobionate and mannitol) and the strong buffer from Bretschneider HTK solution (by using histidine). The reduced glutathione in Celsior solution is one of the best antioxidants available. The solution was specifically designed for heart transplantation, but is being currently used in clinical lung, liver, kidney, and small bowel preservation, and is under investigation for pancreas transplantation. [38, 39] In liver transplantation, primary dysfunction graft dysfunction was lower with Celsior solution than with UW solutions (7.4% vs 9.8%, respectively). However, this difference was not borne out in a meta-analysis (relative risk [RR]=0.68; 95% confidence interval [CI]=0.22-1.97). [40]

A meta-analysis comparing patient survival and donor heart dysfunction outcomes found that use of UW solution for organ preservation was associated with a significantly better survival at 30 days and 90 days compared with Celsior and HTK. Hearts preserved with UW solution exhibited less ischemic necrosis than those preserved with Celsior (RD = -0.07, 95% CI = -0.08 to 0.05, P <  0.00001) but there was no statistical difference in outcomes when comparing HTK with Celsior solution. [41]

ET-Kyoto solution

Researchers at Kyoto University developed a solution that contains trehalose and gluconate in a high sodium–low potassium formulation. [42] The solution is chemically stable at room temperature and is effective for skin and lung preservation in animal models. Extracellular-type trehalose-containing (ET)–Kyoto solution is also being actively investigated in clinical trials for transplantation of the lungs, heart, and other organs.

Institute George Lopez (IGL-1) solution

IGL-1 solution was developed a group from Lyon, France and is used clinically for hypothermic flushing and storage of kidney, liver, and pancreas. The composition of IGL-1 solution is similar to that of UW solution, but it contains less potassium, higher sodium, and replaces HES with polyethylene glycol (PEG) as an impermeant colloid. The colloid balances hydrostatic pressure and prevents the creation of tissue edema, which is also an important factor in maintaining organ viability during prolonged cold ischemia. Polyethylene glycol is a nontoxic water-soluble compound that decreases lipid peroxidation and protects against cell swelling during cold temperature, and reduces the early inflammation and injury related to ischemic reperfusion. [43, 44, 45]

A meta-analysis that compared IGL-1, UW, CS, and HTK solutions in liver transplantation found no significant difference in the risk of primary non-function (PNF) and 1-year post-transplantation graft survival. [46] However, in kidney transplantation it has shown an improved early function compared with that of UW solution, but similar rates of graft and patient survival. [47] Although few studies have been conducted on the use of IGL-1 for transplantation of pancreas, patient and graft survival rate 1 month after pancreas transplantation were shown to be reasonably good, at 95.7% and 93.6%, respectively. [48]

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Techniques of Organ Preservation

Because most transplanted organs are from deceased donors, the organ must inevitably be stored after its removal from the donor until it can be transplanted into a suitable recipient. The donor and recipient are often in different locations, and time is needed to transport the donor organ to the hospital where the recipient is being prepared for transplantation. Effective, safe, and reliable methods are needed to preserve the organ ex vivo until transplantation can be performed. Acceptable preservation times vary with the organ, as discussed above.

Normothermic preservation

The demand for organ transplantation is increasing year by year, increasingly outpacing the potential supply. To expand the donor pool, organs are being harvested from donors who were considered less ideal in years past. These include Donation after Cardiac Death (DCD) donors, older donors, those with more medical comorbidity, and even those with increased risk for, or known presence of, HIV and hepatitis C infection. In these extended- or expanded-criteria donors, post-transplantation primary graft dysfunction (PGD) and ischemic biliary complications are common. [49, 50, 51, 52]  

Use of DCD donors is one method for increasing the pool of available donors. Under defined circumstances, a patient who has suffered debilitating injury but has not been determined as deceased by brain death assessment may still become a donor. After suitable consent is obtained from the patient’s family, withdrawal of mechanical support is performed in a method consistent with standard care practice. If cardiac activity ceases, death is diagnosed by cardiac death criteria and rapid organ recovery can be performed. The result of this process is a period of warm ischemia that may further exacerbate the preservation injury seen for more routine DBD donation.

Several strategies have been employed to reduce or recover from the warm ischemic damage caused during the DCD process. One approach uses normothermic regional perfusion (NRP) of the donor after declaration of death to allow the organ to function normally by restoring the blood flow and prevent warm ischemic injury. [53]

NRP utilizes extracorporeal membrane oxygenation (ECMO). Depending on a country or center’s protocol, cannnulation may be done pre- or post-mortem. The femoral artery or aorta and femoral vein or inferior vena cava (IVC) are cannulated. Blood is collected from the vein cannula, enriched with oxygen and restored to body temperature (35.5-37º C) in the ECMO device, and flushed back via the artery cannula. After 2-4 hours of NRP, the organ is flushed with cold preservation solution and transported to the recipient hospital after the standard organ retrieval procedure.

Kidney transplantation using NRP has shown lower rates of delayed graft function (DGF) compared with uncontrolled DCD (46.6% vs 73.4%; P< 0.01). Liver transplantation using NRP has resulted in a significantly lower rate of biliary complications (overall 8% vs 31%; P< 0.001) and graft loss (12% vs 24%; P< 0.008) compared with DCD. [54, 55]

Normothermic perfusion may also extend acceptable preservation times to enable sharing of organs across a broader geographic area. In some areas, organs may be discarded for lack of an appropriate recipient in close proximity. Ex-vivo normothermic machine perfusion (EVNMP) prevents irreversible cellular injury by restoring oxygen delivery for normal cellular respiration and ATP production. Perfusion of the organ with oxygenated and nutrient-rich blood or blood substitute solutions restores respiratory function. This is frequently performed using mobile perfusion/oxygenation devices for transport of organs from donor location to the recipient hospital.

Currently, EVNMP is performed in heart, lung, and liver transplantations. Heart transplantation with EVNMP is considered to be a safe and effective method, resulting in the same short-term, intermediate, and long-term clinical outcomes as conventional transplantation. [56, 57, 58] In lung transplantation, use of EVNMP has yielded the same long-term survival and pulmonary function as conventional lung transplantation, and has been proven to reduce rates of primary graft dysfunction (PGD) in the first 72 hours post-transplantation. [59, 60, 61] Compared with conventional static cold stored preservation, liver EVNMP resulted in 74% less early allograft dysfunction (EAD), 50% less graft injury, and 54% longer mean preservation time for the EVNMP group. However, no significant differences in patient and graft survival or biliary complication rates were found. [61, 62]

Sub-normothermic preservation

In sub-normothermic perfusion, organs are perfused with oxygenated, nutrient-rich, cell-free perfusate at temperatures below normal body temperature (typically ~21°C or room temperature). Under these conditions, the metabolic demand is reduced and ATP production can occur (albeit less efficiently). In preclinical studies using discarded livers, this approach was shown to preserve hepatocellular and biliary function for several hours. [63, 64]

Hypothermic preservation

Hypothermic (or static cold storage at 0-4°C) is the standard technique of organ preservation because it is simple, does not require sophisticated expensive equipment, and allows ease of transport. Hypothermia is beneficial because it slows metabolism. [16, 65, 66]

As above, organs exposed to normothermic ischemia remain viable for relatively short periods, usually less than 1 hour. During warm ischemia, the absence of oxygen leads to a rapid decline in ATP levels in the cells, to a redistribution of electrolytes across the cell membrane, and to a decrease in biosynthetic reactions. However, biodegrading reactions continue; these include the accumulation of lactic acid, a decrease in intracellular pH, proteolysis, lipolysis, and lipid peroxidation.

With hypothermia, the degradative reactions are considerably slowed but not halted. A 10º C decrease in temperature slows the metabolic rate approximately by a factor of 2. Cooling an organ from 37º C to 0º C slows metabolism by a factor of 12-13. However, hypothermic cold storage is not sufficient to prevent organ damage, and perfusion with stabilizing organ preservation solutions is required.

Two techniques for hypothermic preservation are employed: (1) simple static cold storage and (2) hypothermic machine perfusion. With simple cold storage, the organ is flushed with cold preservative solution, immersed in the same solution, and stored in a sterile container on ice. Advantages of static cold storage include universal availability and ease of transport.

With continuous hypothermic perfusion, developed by Belzer in 1967, a machine is used to pump perfusion fluid through the organ. [67] Perfusion may be pulsatile or continuous, depending on the organ system (pulsatile perfusion is often used for arterial perfusates in liver and kidney whereas continuous perfusion is used for portal venous flow). The ideal temperature for hypothermic perfusion is 0-12º C, depending on the organ and the perfusate solution. One major advantatge of hypothermic machine perfusion is that if the pump fails, the system defaults to static cold storage, whereas failure during normothermic machine perfusion results in ischemia.

For kidneys, machine perfusion offers superior results compared with simple cold storage. [68] With simple cold storage, approximately 25-30% of transplanted kidneys have delayed graft function, but with machine perfusion, the rate can be less than 10%. [69] Hypothermic perfusion usage resulted a lower serum creatinine level for the first 14 days after transplantation and lower risk of graft failure for the first year post-transplantation. [70] The perfusate used is similar to the UW solution, except for the impermeant. For continuous perfusion, gluconate is used in place of lactobionic acid. [68] However, when a liver harvested from a DCD donor was performed using hypothermic oxygenated perfusion for 1.7-2.7 hours, the results were the same with that of a liver harvested from a DBD donor, resulting a good function in the early post-transplantation period. [71]

High sub-zero preservation

Storage of organs below the freezing point of water (< 0°C) has the potential to further reduce metabolic activity and extend acceptable preservation times. For this method to be effective, however, the system must avoid (or limit) ice formation. The phase transition of liquid to solid results in cellular injury and gross alteration of the tissue architecture.

One strategy to achieve low temperature storage without ice formation is to copy what happens in nature. Many species of insects, fish, reptiles, and even mammals have adapted to life in very cold environments. For example, polar fish live in waters below the freezing point of their blood. In order to avoid ice, they produce ice-inhibiting proteins that surround developing ice crystals and prevent the growth of these nascent destructive particles. Taking notice of these nature inspired processes, several groups are investigating methods for storage under sub-zero conditions without ice formation. A group at Massachusetts General Hospital has published several reports in human and animal models that use preservation solutions and modified sugar compounds to inhibit ice and allow liver storage for up to 4 days before transplantation, [72, 73] a potentially transformative approach.

Cryopreservation

Cryopreservation, or storage of biologic materials at ultralow (ie, in liquid nitrogen at -160°C temperatures, results in cessation of all metabolic activity. Storage under these conditions is theoretically indefinite [74] and could enable true organ banking. However, one of the main limiting problems in cryopreservation is minimizing ice-related damage that can kill cells and disrupt tissues. [75, 76] Conventional cryopreservation relies on the use of cryoprotective agents (CPAs) to stabilize cells and allow cooling to, and subsequent rewarming from, liquid nitrogen temperatures. While this approach does allow cryopreservation of cells in suspension and some small aggregates, such as mammalian embryos, it is only effective for these very small biospecimens. Attempts at cryopreserving larger tissues and organs have uniformly failed due to injury from ice formation.

An alternative strategy to conventional cryopreservation is the use of vitrification, or storage of organs in a glass-like amorphous state at cryogenic temperatures. This approach takes advantage of the knowledge of the physics of ice formation. As a liquid is cooled below the melting point of ice (Tm = 0°C for water) there is a thermodynamic drive to form ice. For a finite region below the melting point, liquids can exist in a metastable supercooled state. That is, the liquid wants to form ice, but has not overcome the activation energy required for phase transition from liquid to solid. As temperature drops even lower the activation energy is minimal and ice will spontaneously form.

However, if the system is cooled very rapidly there is a counterbalancing force: the increase in viscosity of cold liquids. As viscosity increases, the ability of water molecules to rearrange to form ice is reduced. If a liquid is cooled very rapidly, the viscosity will increase to a level that these molecules cannot rearrange to form ice and they system enters a stable glasslike state. The fluid behaves like a solid but does not develop ice (similar to glass). Unfortunately the rate at which water needs to be cooled to achieve a vitrified state is practically unachievable (> 107° C/min). [77, 78]

New developments in stabilizing CPA solutions have reduced the cooling rate considerably and have allowed limited successful vitrification of mouse embryos, [79] and later were applied for vitrification of rabbit kidneys. [80, 81] Despite these advances in tissue and organ vitrification, a further problem remained: the ability to rewarm them—a process that needs to be extremely fast and uniform in order to avoid ice damage or organ cracking. Recent developments using radiofrequency waves to heat organs perfused with iron oxide nanoparticles [82, 83, 84, 85] offer the promise of finally achieving vitrification and rewarming of organs for transplant, or for true organ banking.

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Current Preservation Techniques

Kidney, liver, and pancreas

Until relatively recently, the primary solution used for cold-storage preservation of the kidneys was the Euro-Collins solution. [86] Its formulation provides a hyperosmolar environment with an intracellular electrolyte composition intended to reduce cellular swelling. In combination with hypothermia, kidneys can be safely stored in this solution for up to 36-48 hours before transplantation. [86]

In the 1980s, the advent of new immunosuppressive agents, such as cyclosporine, meant that for the first time, organs other than the kidneys could be transplanted with good success rates. With this development, the need for effective preservation became apparent.

The limitations of cold ischemia to approximately 8 hours for the liver and pancreas meant that donor and recipient teams had to be closely coordinated. [87] Complex recipient operations that required a multitude of ancillary support services had to be organized in the middle of the night, and all personnel involved in the procedure, including the surgeons, were starting the operation in a suboptimal fatigued condition.

Researchers at the University of Wisconsin developed a solution that simplified the practice of hepatic and pancreatic transplantation at most centers in North America and Europe. [88] The solution, UW cold-storage solution, is based on lactobionate, raffinose, HES, and a host of other ingredients designed to provide high-energy phosphate precursors, hydrogen-ion buffering capacity, and antioxidant properties. [87]

Lactobionate, an impermeant anion to prevent cellular swelling, was used in place of the glucose contained in Euro-Collins solution. Raffinose, a naturally occurring trisaccharide of fructose, glucose, and galactose, provides additional osmotic activity, while HES is a colloid intended to prevent an increase in the extracellular space, though it makes the solution viscous.

Most teams that perform hepatic and pancreatic transplantation prefer UW solution as the preservation method of choice. [87] Both the liver and pancreas can reliably be stored for 12-18 hours, and isolated clinical cases with total cold ischemia times in excess of 30 hours have been reported. [89] However, evidence is accumulating that cold ischemia times longer than 12 hours may be associated with a high incidence of biliary strictures. [89]

Whether the UW cold-storage solution has any significant advantages over Euro-Collins solution for the preservation of kidneys is unclear. In a large, randomized, multicenter European trial, patient and graft survival rates were similar among recipients of the 2 solutions, but the incidence of delayed graft function requiring dialysis was approximately one third lower in the UW group. Results of ongoing trials are needed before a definitive statement can be made about the relative merits of the 2 solutions in kidney preservation.

New preservative solutions, including Celsior solution, are emerging and may have advantages over UW solution, [89, 90] though results are conflicting. [91] The Kyoto solution was evaluated for kidney storage and found to be as good as UW solution. Furthermore, the Kyoto solution was less viscous and stable at room temperature for as long as 3 years, and it was cost effective as compared with UW solution. [92]

Another solution that is being increasingly used presently is Bretschneider's HTK solution (Custodiol). [93] Originally developed for cardioplegia, HTK is routinely used for liver, kidney, and heart transplantation. HTK solution has been shown to be superior tonEuro-Collins solution in prospective trials. [94, 95, 96, 97] Advantages include lower viscosity and less leucocyte adhesion. In the Eurotransplant multicenter trial, rates of delayed graft function were lower with HTK solution than with UW solution and the two solutions were otherwise comparable. [98] Other reports have shown better delayed graft function rates with UW solution. [96]

A randomized study comparing the efficacy of UW solution versus HTK in 102 liver transplant recipients reported that both solutions were equally effective in graft preservation. [99] In a single-center study of liver transplantation from extended-criteria donors, HTK and UW were clinically indistinguishable but biliary complications were less common with HTK. [100] However, in a retrospective single-center study from Korea of living-donor liver transplantation, the incidence of preservation injury was greater in grafts treated with HTK versus UW solution. [101]

UW solution has proven to be effective in pancreas preservation, leading to clinically successful whole-pancreas transplantation. In addition, human pancreatic grafts can be preserved for longer than 24 hours by using UW solution. However, in clinical islet transplantation, prolonged cold storage in UW solution before islet isolation significantly reduced recovery of viable islets. [102]

A 2-layer cold-storage method using perfluorochemical (PFC) and UW solution has been evaluated for whole-pancreas preservation. This method continuously supplies sufficient oxygen to a pancreas during preservation and reduces cold ischemic injury by producing ATP, which maintains cellular integrity and controls ischemic cell swelling. [103] A prospective randomized study comparing HTK versus UW in 68 pancreas transplant recipients reported that the solutions were equally suitable for perfusion and organ preservation. In this study, 6-month patient survival was 96.3% (HTK) versus 100% (UW) (P = 0.397). [104]

Heart and lungs

Cardiac preservation has changed relatively little in recent years. Hyperkalemic, crystalloid cardioplegia solution is used at 4°C, and 4 hours is the generally accepted limit of cold ischemia. [8] For this reason, donor and recipient operations must be finely coordinated. Every effort is made to limit the ischemic time (the time between the initial interruption of coronary flow by means of aortic cross-clamping in the donor and the removal of the clamp in the recipient) to less than 4 hours. Although laboratory data and isolated clinical reports suggest that good results may be expected with storage times of 8 hours or longer with the use of the newer preservation solutions, [8] increased ischemic time remains a strong and important independent risk factor for poor recipient outcome.

The maximal safe interval for the lung to remain ischemic, even when cooled, has not been defined. Based on empiric observation, 6 hours is the selected limit. [105] This constraint limits the distance that can be traveled to recover donor lungs. The limits of donor-lung ischemia have been expanded because of efforts to develop bilateral, sequential lung replacement. The second lung to be implanted is ischemic for longer than the first because the lungs are not implanted simultaneously. The longest cold ischemic time was 9-10 hours, and the lung functioned well within 24 hours after implantation. Although donor-lung dysfunction occurs in 5-10% of cases, it is usually reversible. This problem does not correlate with prolonged donor-lung ischemic time.

Whether one type of preservation solution for lung allografts is better than another remains to be determined. The Celsior solution may be better for early lung function than the standard Euro-Collins solution. [106] One clinical study demonstrated less reperfusion injury, better immediate and intermediate function, and better early and long-term survival for donor lungs preserved with Celsior than with Euro-Collins solution. [105] Core cooling of the donor with an extracorporeal circuit has been used extensively in the United Kingdom for cardiopulmonary transplantation. Others have used an immersion technique without flushing the pulmonary artery. Hypothermia is applied, along with intracellular-type solutions. The Kyoto solution also appears promising in lung transplantation. [107] The optimal preservation solution for the lungs remains unknown, and deficiencies in the quality and duration of preservation still limit the results of pulmonary transplantation.

Small bowel

The transplantation of segments of small bowel with or without concomitant liver transplantation is being performed with increased regularity as the treatment of choice for short-gut syndrome. The bowel is usually preserved with UW solution, though experimental evidence does not indicate that this solution offers any advantage over simple hypothermia. [108] Experimental studies have also reported that ex vivo application of carbon monoxide in UW solution may also prevent reperfusion injury. [109]

The use of anti-oxidative agents has also shown promise in laboratory studies as an intervention to minimize preservation injury. [110] The same investigators reported that this may occur due to down-regulation of pre-apoptotic and up-regulation of cytoprotective signals in the presence of a nutrient-rich preservation solution. [111] A study in a rat model reported better small bowel integrity and function compared with UW solution. [112]

Most procurements of small bowel are from donors who have many useful organs. Therefore, standard intra-aortic preservation techniques are used. The current limit of cold ischemia time for small bowel is approximately 12 hours. [113] Reports suggest that the use of luminal flush solutions, both UW and amino acid–enriched solutions, improve mucosal barrier function (as measured by mannitol permeability) and decrease villous morphologic injury. [114]

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Future Considerations

Advances in the understanding of the mechanistic processes of ischemic organ damage have enabled the development of a number of preservation strategies to extend the storage limits of organs for transplants.  Whereas conventional static cold storage enabled preservation times measured in hours, advances in normothermic perfusion and high subzero preservation promise to extend this time to days. Finally, the promise of cryopreservation, or true organ banking, is finally entering the realm of practicality. Each of these advances improves on those that came before and the hopes for avoiding ischemic organ damage altogether are becoming closer to achievable.

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