Principles Of Organ Preservation

±CaCl2 was omitted when the solution was used for the initial flush.

Simple Cold Storage

This method involves flushout of the organ with a suitable solution to remove blood elements and to cool the organ. The organ is then stored in the preservative solution at a temperature of 0° to 4°C.

The success of this method is dependent upon the use of hypothermia. Cooling the organ from 37°C to 0° to 4°C slows enzymatic reactions by 10-fold or more.18 The low temperature is one of the most critical components of successful preservation. This is evident by considering the differences in organ viability when exposed to cold ischemia versus warm ischemia. In warm ischemia, organs lose viability in a couple of hours or less, depending upon the organ. Under the condition of cold ischemia, organ viability is extended many fold. The kidney tolerates up to about 12 hours of cold ischemia when flushed out and stored in cold blood,1 but will tolerate only about 60-90 minutes when stored warm.19

Why hypothermia is effective is deduced from mechanisms of ischemic injury proposed to cause organ death at normothermia.20,21 In warm ischemia the lack of oxygen and perfusion leads to a very rapid decrease in the availability of energy (ATP) derived from mitochondrial and glycolytic catalyzed reactions. Without ATP, the tissue loses control of its intracellular environment, resulting in changes in cytosolic ionic composition (protons, Ca2+, K+, Na+), activation of hydrolytic enzymes (phospholipases, proteases, endonucleases), and destruction in the stability of the intracellular structural components (microtubules, cytoskeleton membranes). It is readily apparent that the loss of ATP is the critical factor in induction of ischemic injury. For instance, the tolerance of the liver to an ischemic or hypoxic insult is extended significantly by stimulating anaerobic energy production through glycolysis.22,23 Similarly, inhibition of anaerobic energy production in the heart24 leads to a more rapid onset of irreversible ischemic injury. Preservation of the cold stored kidney can be extended by persufflation of oxygen into the or-gan,25 which appears to affect viability only by stimulating the rate of ATP turnover. The preservation of the pancreas has also been extended by exposing the organ to high concentrations of oxygen without continuous perfusion.26 This method also appears to improve preservation by a mechanism related to ATP generation.27 Although hypothermia slows down the rate of ATP loss in tissues and organs, this does not explain the beneficial effect of hypothermia. Most organs that are cold stored lose 90% or more of their ATP within 2 to 4 hours, yet when stored appropriately remain viable for one or more days.

Hypothermia may be effective, therefore, by blocking the activities of various hydrolytic enzymes. This has been shown to increase the tolerance of organs, tissues and cells to ischemia.28 Various phospholipase and protease inhibitors have been shown to improve the viability of cells and tissues exposed to warm and cold ischemia.29 Hypothermia is the simplest method to block the activity of these enzymes. Clearly, enzymatic reactions continue at 0°-4°C, as is evident by the accumulation of end products of metabolism (lactate, glucose, purine nucleotides, etc.) in cold-stored tissues. It is likely that continued enzymatic activity leads to the ultimate destruction of the organ by altering its capability to re-establish metabolic control when rewarmed and reperfused, i.e., transplanted. The combination of hypothermia and chemical inhibition of enzymes is an attractive approach to suppressing cold ischemic injury. However, it is unclear which of the many intracellular enzyme systems is responsible for ischemic injury. Most likely a wide range of inhibitors would be needed to suppress the activity of multiple enzyme activities.

Even with metabolic suppression by hypothermia, the longer an organ is subjected to ischemia, the greater the extent of reperfusion injury. At some point in time, the reperfusion injury becomes irreversible.

Although hypothermia is the single most important factor in successful storage of organs for transplantation, there are other important considerations as well; lack of attention to them can negate the beneficial effect of hypothermia. One important factor appears to be the presence of impermeant molecules that remain outside the cells of the organ, helping them resist the tendency to swell during cold storage. Cell swelling is caused by the metabolic inhibition associated with hypothermia and lack of energy production due to hypoxia. The intracellular milieu, which contains a large number of impermeants (proteins, phosphory-lated compounds, etc.), exerts osmotic and oncotic forces that tend to draw water into the cell. To resist this, the cell under normal conditions uses membrane-bound ion pumps (such as the Na+-K+ ATPase) to maintain a high concentration of K+ inside the cell and a high concentration of Na+ outside the cell. In this way, the large concentrations of Na+ act as an impermeant and energy is needed to maintain an equilibrium between intra- and extracellular water activities. This equilibrium helps the cell resist the tendency to gain water and swell, preventing the concomitant disruption of the cellular concentrations of reactants, activities of enzymes, and cell ultrastructure. In cold storage of organs, impermeants, such as saccharides (glucose, mannitol, sucrose, raffinose), colloids (hydroxyethyl starch, dextran, polyethylene glycols), and anions (phosphate, sulfate, lactobionate, gluconate) counteract the tendency for hypothermia-induced cell swelling and appear to provide stability to the ultrastructure of the cell during cold storage.

The presence of the appropriate impermeant appears to be essential for successful long-term preservation of organs.30,31 Many of the earlier cold storage solutions, such as Collins, EuroCollins, Marshall's hypertonic citrate, and others used saccharides as agents to counteract hypothermia-induced cell swelling. However, many of these agents were not completely impermeable and would enter the cell with time, thus losing their efficacy. Lactobionate and gluconate, the two impermeants in the UW solutions, have so far been the most successful in suppressing hypothermia-induced cell swelling for long periods of time and in every organ tested. These agents appear to be the most important ones in the UW solutions for successful preservation of organs for 24 to 30 hours.

Other agents in preservation solutions may be less important than the impermeants, but were chosen for their potential usefulness in suppressing preservation or reperfusion injury.31 There is evidence that some of these agents do, in fact, assist recovery of function in cold stored organs; however, controversy exists about these issues.

The use of antioxidants in organ preservation is a common approach to improving results. There are numerous studies32-34 showing that warm and cold is-chemia/reperfusion leads to the rapid formation of oxygen free radicals (OFR). Furthermore, the generation of OFR during reperfusion of numerous organs leads to tissue injury. Numerous types of antioxidants have been shown to ameliorate reperfusion injury in many model systems.35-37 Also, during cold ischemia, there is

a loss of the naturally occurring antioxidants vitamin E and glutathione.38,39 Thus, cold-stored organs are sensitive to OFR-induced injury because of the lower concentration of endogenous antioxidants. In the development of the UW solutions we used glutathione to enhance the antioxidant capacity of the solution and al-lopurinol to block the activity of xanthine oxidase. Xanthine oxidase has been proposed to be a major source of superoxide anions during reperfusion of ischemic organs.40 Studies have now shown that glutathione is an important component of the UW solution and can increase the viability of preserved dog livers,41 rat livers,42 kidney tubules,43 isolated hepatocytes,44 and hearts.45 The mode of action of glutathione is not clear but may be related to inhibition of proteases46 or reduction in lipid peroxidation stimulated by OFR generation.47 Furthermore, mitochondria are highly dependent upon glutathione for suppression of oxida-tive injury; preserving the liver in UW solution maintains the mitochondrial GSH level.48 GSH, however, is unstable in solution and is oxidized during storage.49 For this reason, our center adds fresh GSH (3 mmole per liter) to the UW solution prior to use for organ preservation.

A controversy also exists over the need for a colloid (hydroxyethyl starch) in cold-storage solutions.50 The presence of a colloid in a preservation solution was based upon the need for an agent to counteract the hydrostatic pressure in continuous machine perfusion of organs. In cold storage, however, the organ is not exposed to an hydrostatic perfusion pressure except during the initial flushout of blood. Therefore, theoretically, there should be no need for a colloid in cold storage solutions. This is in fact the case and many preservation solutions, such as Collins or Marshall's hypertonic citrate, have been used successfully for short periods of time without the presence of a colloid. Also, over the past few years, many investigators have attempted to develop their own preservation solution by using the basic components of the UW solution and subtracting various agents, such as the colloid, hydroxyethyl starch.51,52 Some studies suggest that there may be a role for the colloid, even in cold storage of organs. For instance, better preservation of the dog pancreas was obtained in the UW solution with starch versus that with-out.53 Preservation of the rat heart also seems improved when hydroxyethyl starch remained in the UW solution.54 Finally, hydroxyethyl starch has recently been shown to suppress proteolysis in cold-stored rat livers. Proteolysis is thought to be a contributing factor to liver injury during cold storage55 and its suppression may be a key factor in prolonging the viability and quality of preserved livers. For kidney preservation, however, the presence of a colloid has never been shown to be essential.

Because the UW solution is used for the preservation of all transplantable organs, and because some organs are preserved better with the colloid, it would appear prudent to use the UW solution that contains hydroxyethyl starch for all cold stored organs.

Another controversy in cold-storage solution composition is related to the solutions' content of sodium and potassium. The clinically most popular and successful cold storage solutions were the so-called 'intracellular-type' solutions. These contained high concentrations of K+ relative to Na+, making them similar

to the intracellular environment of most cells. Solutions such as Collins, EuroCollins, Marshall's hypertonic citrate, and the UW solution all contain high concentrations of K+. The benefit of a high K+ concentration was thought to relate to the suppression of hypothermic-induced efflux of K+ during cold ischemia. Thus, the cell would retain a near-normal K+ concentration and would not need to expend a great deal of energy during reperfusion to re-establish a normal intracellular K+ concentration had the K+ been replaced with another cation, such as Na+. This has not been proven, but the concept may be valid. Conserving energy during the first minutes of reperfusion may be essential to providing the necessary energy for cellular repair and other critically important cellular functions.

Many investigators,57 as well as our own studies,58 have shown that the electrolyte content of preservation solutions may not be critical and can be reversed to contain a high Na+ content relative to K+. Some investigators have attempted to show that a high K+ concentration is detrimental to organ storage,59 but others have found no such effect.60 Therefore, it appears clear that a preservative containing a high concentration of Na+ or K+ is probably equally effective for short-term (24-30 hours) organ preservation.

A key factor in successful organ preservation may be the rate of regeneration of ATP upon reperfusion. This requires leaving the energy-generating machinery (mitochondria) intact during preservation and reperfusion, as well as supplying the cell with the appropriate precursors for ATP regeneration. During cold storage there is a loss of the precursors for ATP synthesis. ATP is degraded to phosphate and its purine constituents. The purines are permeable across the cell membrane and rapidly flushed out of the tissue upon reperfusion.61 Thus, in developing the UW solution, adenosine was added to elevate the concentrations of this ATP precursor and provide substrate of ATP regeneration.62 Although there is not an apparent critical dependency of successful short-term organ preservation on adenosine, some studies have attested to its value in stimulating regeneration of ATP62 in the kidney, liver, heart and pancreas.

The suitability of the UW solution for liver preservation has been recently questioned by some investigators63 because of the presence of microcrystalline materials. These investigators showed that in some bags of the UW solution there were crystals made up of salts of naturally occurring fatty acids (palmitate and sterate). Palmitate and sterate are components of the packing material and necessary as plasticizers for the manufacture of the containers. The fatty acid crystals form because of the relatively high pH of the UW solutions (pH 7.4) compared to other parenteral solution. The higher pH causes the fatty acids to slowly leach out of the container and form salts of Ca2+ or Mg2+. The study that showed that these crystals could be injurious in liver preservation came from measuring microcirculation of rat livers after about 16 hours cold storage.63 In livers stored in the presence of crystals, there was poorer blood reperfusion after cold storage than in livers stored in UW solution without crystals. Because of the presence of crystals in UW solution, the manufacturer has contacted the United States Food and Drug Administration, and with their guidance advised users of the UW solution to 1) discard bags if visible crystals are present, and 2) to use a blood filter (Pall) in

the administrations of the UW solutions. It was shown in studies submitted to the FDA that the Pall filter effectively removed crystals from bags of UW solutions containing known loads of crystals. This demonstrates that there are certainly reasonable solutions to this problem.

Our center has had little concern for the presence of these crystals in the UW solution for a number of reasons. First, on a macroscopic level, we have never observed crystalline materials in commercially produced bags of UW solution. Second, studies have shown a problem in rat livers but not yet in larger animals. Third, if the crystals are injurious, their effect should be seen in short-term preserved livers (1 to 2 hours) as well as in longer-term preserved livers, and this has not been shown. In fact, the incidence of liver problems, including primary nonfunction and initial poor function, increases slightly, but significantly, with preservation times above 24 hours,64,65 not including the early stages of preservation. Fourth, we have used commercially prepared UW solution for the past 10 years in clinical preservation of kidneys, livers, pancreases, hearts, and lungs, and have reported outstanding results. In fact, the results have been superior to those obtained with other solutions, presumably not containing crystalline materials. Fifth, other centers in the USA and Europe have also used commercially prepared UW solution for over 10 years and have also reported excellent results in organ preservation. Although it is clear that the presence of crystals of any type is not desirable in preservative solutions, it is also clear that they have caused no apparent problems in organ storage in the UW solution. Work is underway to find a suitable method to prepare the UW solution without crystalline materials without decreasing its efficacy and importance in organ preservation.

In conclusion, simple cold storage is the most popular method for organ preservation because of its simplicity. The primary reasons cold storage is successful are because of the hypothermic-induced inhibition of metabolism, use of impermeants to suppress cell swelling, the presence of agents to stimulate metabolism on reperfusion, and the short periods of preservation used. This method is very suitable for preservation times of 24-30 hours, and most organs can be transplanted within this period of time. However, the simplicity of cold storage is also one of its drawbacks. First, this method is not well suited for preservation of nonideal donor organs, such as those exposed to hypoxia or ischemia during or prior to the harvest. This fact may become more important in the future because there is an increasing need for organs and great interest in procuring organs from non-heart-beating cadavers. These organs are exposed to varying periods of low flow and ischemia (20 to 60 minutes) and will require better methods of preservation (such as machine perfusion) than ideally harvested organs. Furthermore, many organs come from donors that are not ideal due to age, weight, cause of death, nutritional factors. Preservation of these organs may require more sophisticated means than simple cold storage to assure that good organ function is restored immediately after transplantation. Preservation/reperfusion injury may be a factor in acute and chronic rejection and may complicate immunosuppressive therapy after transplantation.66,67

Finally, improving organ preservation by cold storage may be very difficult and we may have reached the clinically practical limits of successful cold storage (24 to 48 hours). In the laboratory, cold storage allows successful 48- to 72-hour preservation of the liver, kidney, and pancreas, but this is under relatively ideal conditions. The donor (a dog, pig, or rat) is usually ideal, healthy, well fed, and not traumatized by accidental death. Furthermore, the recipient is not ill with serious complications due to organ failure. Thus, clinically safe preservation by cold storage is probably more likely to be shorter than shown in the laboratory. Also, during cold storage there is a nearly complete loss of metabolic control due to the lack of a continuous input of energy and removal of metabolic end products. This leads slowly even at hypothermia to disruption of the functional and structural components of the cell, which are only able to remain stable with a constant supply of energy. It is sometimes argued that if we knew precisely the mechanisms of cold storage injury, we could use appropriate drugs to block the injurious reactions, either during cold storage or during reperfusion. There have been numerous attempts to accomplish this, but there has been very limited laboratory success. Most of the results have not found serious clinical utility. The reasons for this may be that the mechanism of cold ischemic injury is multifactorial, and blocking one or two reactions that lead to organ injury is not sufficient to prevent organ failure or injury. The agents that are effective may be those that improve the reperfusion of the organ, allowing a better opportunity for repair of damaged tissues and cells. Thus, agents such as nitric oxide donors, calcium channel blockers, antioxidants, and phospholipase or protease inhibitors all may function by increasing reperfusion of the microvascular of the organ. The future of organ preservation, therefore, may lie in developing or utilizing new methods, such as continuous machine perfusion.

Continuous Machine Perfusion

Continuous machine perfusion, developed by Belzer et al,2 is used in about 6 to 8 centers in the US. Kidneys are perfused by a pulsatile perfusion pump (about 60 beats per minute) at a pressure of about 40-50 mm Hg, resulting in a flow rate of 0.5-1.0 mL/min/g per kidney. The perfusate is a UW-gluconate-based solution (Trans Med, Belzers MPS) as described above. The temperature is maintained at about 4°-6°C. A retrospective analysis of kidneys preserved by machine perfusion shows a one-year graft survival rate similar to those cold stored.67 However, there is significantly less delayed graft function (DGF) (need for dialysis) in machine-perfused kidneys versus cold-stored kidneys. Centers that machine perfuse kidneys have reported DGF rates of less than 5 to 10%, compared to DGF rates for cold stored kidneys of, on average, greater than 20%.68-70 The cost of DGF nearly doubles the cost of a renal transplant71 and therefore, the possibility of reducing DGF should be an important consideration in choosing a method of preservation. Furthermore, the possibility of late graft failure due to chronic rejection (progressive injury to the renal vascular system) may be greater in kidneys showing initial poor function than in those with good initial function,72 as discussed below.

These results suggest that machine perfusion is a better method for preserving organs than simple cold storage. This appears true for the kidney, liver, heart, lung, and pancreas. The lung and pancreas can be successfully preserved without continuous perfusion, but the continuous delivery of oxygen to the organ (one of the important functions of perfusion) maintains the ATP content and provides better protection than in the ischemic state. Machine perfusion not only maintains a high ATP content, but also removes end products of metabolism that could accumulate to toxic concentrations in the tissue. Machine perfusion also allows control of cellular pH and can continuously deliver substrates and other cytoprotective agents to the tissue. For instance, antioxidants, enzyme inhibitors, and precursors for cytoprotective agents could be delivered to the preserved organs. Thus, upon reperfusion (transplantation) the organ will be better suited to resist injury and regenerate more normal cellular concentrations of metabolites.

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