Michael J. Taylor Ph.D F.Soc Cryobiol.
Vice President R&D, Cell and Tissues Systems, N. Charleston, SC, USA and Adjunct Professor, Mech Engineering. Carnegie Mellon University. Pittsburgh, PA.
It is universally recognized that hypothermia has provided the bed-rock upon which clinical organ preservation has served the transplantation community since the advent of clinical organ transplantation in the 1960s. Hypothermia became widely used clinically after the introduction of cardiopulmonary bypass in the early 1950s.1,2 Lowering the temperature of a euthermic subject to a temperature below that normally maintained by homeostasis reduces the metabolic rate, and thus the demand for oxygen and substrates by the tissues. On this basis, many modern day surgical procedures, particularly in the areas of cardiovascular surgery, neurosurgery, and sometimes trauma, rely on some degree of imposed or regulated hypothermia as a relatively safe modality for effecting biologic protection during circulatory and/or cardiac arrest.3,4 Similarly, the ultimate application of hypothermia to organ preservation is based upon sound scientific principles for the protective effect of reduced temperatures against the deleterious effects of ischemia and hypoxia that ensues when an organ is removed from its normal physiological environment during procurement. However, low temperatures have a multitude of effects, not all of which are desirable or beneficial. As such, special conditions have been developed to harness the protective properties of cold for effective organ preservation. The development of specialized storage solutions is the primary factor in the evolution of hypothermic organ preservation technology as this article sets out to summarize.
Basics of cellular hypothermia
At the cellular level, the fundamental basis of hypothermic protection is the effect of temperature on reaction rates which, according to Arrhenius’ theory, are generally slowed by a reduction in temperature (typically 50% reduction for each 10 degree drop in temperature for most reactions). Since the processes of deterioration associated with ischemia and anoxia are mediated by chemical reactions, it has proven well founded to attempt to prevent or attenuate these changes by applying hypothermia. These deleterious changes, often referred to as the ischemic cascade, begin with early onset biochemical events arising from the immediate depletion of high energy reserves (adenosine triphosphate and creatine phosphate) and membrane depolarization, culminating in structural changes and eventual cell death.5 While hypothermia is known to influence reaction rates; energy metabolism; active ion transport and ion homeostasis; membrane fluidity; and function and secretion of hormones and neurotoxins, the effects are not exclusively beneficial and harmful effects of hypothermia have to be “weighed in the balance”.5 Nevertheless, cooling has proven to be the first line of defense against hypoxic injury and is necessary to reduce cellular metabolism and the requirements for oxygen to prevent tissue injury. A great deal of information has been gathered over the centuries on the effects of cold on physiology and biochemistry and documented in recent reviews pertaining to organ preservation.5–8
Interventions to maximize the benefits of hypothermia and approaches to organ preservation
The application of cold per se by simply cooling an organ is not sufficient to provide good preservation. It is imperative that the benefits of hypothermia are enhanced by careful control of the extracellular environment of the component cells by vascular flush or perfusion, with specialized solutions designed to counteract the detrimental effects of cooling. Many solutions have been formulated over the decades for this purpose following early reports of acellular solutions designed to mimic, in simple terms, the intracellular electrolyte balance of mammalian cells (see reviews:5,7–9). Early attempts to use blood-based solutions, even hemodiluted to reduce viscosity, proved futile for two principle reasons:
1. Dissociation of oxygen from oxyhemoglobin ceases at temperatures below 12°C, and
2. Cold-induced changes in erythrocyte membranes causes hemodynamic problems during hypothermic vascular perfusion and microvascular blockage during reperfusion.3,4,10
Synthetic crystalloid/colloid solutions have proved to be the only way to go for effective hypothermic preservation, with certain standards emerging to support the time-honored practice of storing and shipping donor organs on ice. The gold standard developed by Belzer and Southard at the University of Wisconsin (UW Solution [Organ Recovery System SPS-1]),11,12 embodies many of the key components now known to be critical for hypothermic preservation by static cold storage on ice after an initial vascular flush.5,7,8 The common elements of all solutions developed for organ preservation include a biophysical component to counteract the passive diffusional processes that ensue when membrane pumps are switched off, and biochemical and pharmacological components to restrict the deleterious effects of ischemia and reperfusion.5 This approach has served the transplantation community well for several decades by providing a logistic supply-line of viable organs. However, the technique suffers serious shortcomings when applied to less than ideal donor organs such as those that suffer significant warm ischemia at the time of procurement, or less than optimal organs from expanded criteria donors. The static cold storage method is also constrained by restrictive cold ischemia times that vary according to the type of organ (e.g. 24 hours for kidney, 12–15 hours for liver, a max of 8 hours for lung and <6 hours for heart). In recent times, these constraints have been eased significantly by the alternative approach to hypothermic preservation involving machine perfusion. Here the technology provides a system for continuous perfusion of a synthetic acellular preservation solution under controlled conditions of temperature and pressure, offering a variety of advantages over the classical static cold storage method (Table 1).13,14 These attributes of hypothermic machine perfusion have emerged from the technological development of both standardized, portable, user-friendly machines coupled with an adequate, but not necessarily optimized, hypothermic perfusion solution.7,8,13,14 This approach has been shown to offer significant clinical benefit for renal transplantation.15–17 A third approach that may emerge in the future involves hypothermic perfusion of gaseous oxygen (persufflation) as a modality that is showing great promise in experimental transplantation studies of a variety of organs.18
The modern era of organ transplantation, which is making increasing use of organs from marginal donors (including those compromised by prior warm ischemia), is becoming increasingly reliant on preservation technology to provide the means to stabilize and even resuscitate the donor organ. Ischemic insults (warm and cold) are incurred by the donor organ during each stage of the transplantation process from procurement, through preservation for storage and shipping, to re-implantation and reperfusion. Each stage demands optimized conditions for the best outcome. Hypothermia enhanced by appropriately designed preservation solutions is not only the basis for current standard practices, but offers the path towards improved techniques in the future. The most promising approach for further improvement of hypothermic preservation technology is the careful selection of cytoprotective and resuscitative agents as supplements to the baseline perfusates to counteract ischemia/reperfusion injury.7,13 Specific targets include reactive oxygen species, pro-inflammatory cytokines and other cell stress mediators that lead to death by apoptosis or necrosis. This approach has recently been applied successfully to hypothermic perfusion preservation of clinical livers.19,20
Table 1. Benefits of hypothermic perfusion preservation13
- Maintains patency of the vascular bed
- Provides nutrients and low demand O2 to support reduced energy demands
- Removes metabolic by-products and toxins
- Provides access for administration of cytoprotective agents and/or immunomodulatory drugs
- Increases available assays for organ viability checks
- Facilitates change from emergency to elective surgery with reduced costs and improved outcomes
- Maintains endothelial viability: endothelial response to pulsatile perfusion leads to enhanced nitric oxide release and vasoprotective gene expression, resulting in a reduced pro-inflammatory response (cytokines, adhesion molecules) and eventually to reduced immunogenicity21–24
- Improves outcomes (as demonstrated by reduced primary non-function and delayed-graft function)
- Permits use of expanded-criteria kidneys, or organs from non-heart beating donors to increase donor pool
- Economic benefit for transplant centers via reduced health care costs
- Provides technology for ex vivo use of non-transplanted organs for pharmaceutical research
1. Bigelow WG et al. Hypothermia: Its possible role in cardiac surgery: An investigation of factors governing survival in dogs at low body temperature. Annals of Surgery 1950;132:849–66.
2. Swan H et al. Hypothermia in Surgery: Analysis of 100 Clinical cases. Annals of Surgery 1955;142(3):382–400.
3. Taylor MJ et al. Hypothermia in Relation to the Acceptable Limits of Ischemia for Bloodless Surgery. In: Steponkus PL, editor. Advances in Low Temperature Biology. London, UK Greenwich,CT: JAI Press 1996;1–64.
4. Taylor MJ. Hypothermic Blood Substitution: Special Considerations for Protection of Cells during ex vivo and in vivo Preservation. Transfusion Medicine and Hemotherapy 2007;34:226–44.
5. Taylor MJ. Biology of cell survival in the cold: The Basis for Biopreservation of Tissues and Organs. In: Baust JG, Baust JM, editors. Advances in Biopreservation. Boca Raton: CRC Press 2007;15–62.
6. Taylor MJ. Hypothermia. In: Fink G, editor. Encyclopedia of Stress. Oxford: Academic Press 2007;428–38.
7. Guibert EE et al. Organ Preservation: Current Concepts and New Strategies for the Next Decade. Transfus Med Hemother 2011;38(2):125–42.
8. Hafez T, Fuller B. Applications: Organ Preservation for Transplantation. In: John G.Baust, John M.Baust, editors. Advances in Biopreservation. Boca Raton: Taylor & Francis 2007;197–270.
9. Brockbank KG, Taylor MJ. Tissue Preservation. In: Baust JG, Baust JM, editors. Advances in Biopreservation. Boca Raton: CRC Press 2007;157–96.
10. Taylor MJ et al. A New Solution for Life Without Blood: Asanguineous Low Flow Perfusion of a Whole-body Perfusate during 3 hours of Cardiac Arrest and Profound Hypothermia. Circulation 1995;91(2):431–44.
11. Southard JH, Belzer FO. Organ Preservation. Annual Review of Medicine 1995;46:235–47.
12. Belzer FO, Southard JH. Principles of Solid-Organ Preservation by Cold Storage. Transplantation 1988;45(4):673–76.
13. Taylor MJ, Baicu SC. Current State of Hypothermic Machine Perfusion Preservation of Organs:The Clinical Perspective. Cryobiology 2010;60(3S):S20–S35.
14. Fuller BJ, Lee CY. Hypothermic perfusion preservation: the future of organ preservation revisited? Cryobiology 2007;54(2):129–45.
15. Moers C et al. Machine perfusion or cold storage in deceased-donor kidney transplantation. N Engl J Med 2009;360(1):7–19.
16. Moers C et al. Machine perfusion or cold storage in deceased-donor kidney transplantation. N Engl J Med 2012;366(8):770–71.
17. Gallinat A et al. Machine perfusion versus static cold storage in expanded criteria donor kidney transplantation: 3-year follow-up data. Transpl Int 2013;26(6):E52–E53.
18. Suszynski TM et al. Persufflation (or gaseous oxygen perfusion) as a method of organ preservation. Cryobiology 2012;64(3):125–43.
19. Henry SD et al. Hypothermic machine preservation reduces molecular markers of ischemia/reperfusion injury in human liver transplantation. Am J Transplant 2012;12(9):2477–86.
20. Henry SD, Guarrera JV. Protective effects of hypothermic ex vivo perfusion on ischemia/reperfusion injury and transplant outcomes. Transplant Rev (Orlando) 2012;26(2):163–75.
21. Gallinat A, et al. Role of pulsatility in hypothermic reconditioning of porcine kidney grafts by machine perfusion after cold storage. Transplantation 2013;96(6):538–42.
22. Minor T and Paul A. Hypothermic reconditioning in organ transplantation. Curr Opin Organ Transplant 2013;18(2):161–7.
23. Tozzi M, et al. Impact of static cold storage VS hypothermic machine preservation on ischemic kidney graft: inflammatory cytokines and adhesion molecules as markers of ischemia/reperfusion tissue damage. Our preliminary results. Int J Surgery 2013;11(Suppl 1):S110–4.
24. Chatauret N. Machine perfusion of warm ischemic kidney grafts improved eNOS phosphorylation during preservation and vasodilation after reoxygenation. (Paper submitted for publication).
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