Solid organ transplantation is considered one of the great advances in modern medicine, progressing from an experimental treatment to a procedure that is successfully performed around the world. Kidney, liver, heart, lung, pancreas, intestine, and composite tissue allografts are now all possible. This accomplishment has been facilitated by the advent of donor organ cooling, standardized surgical procedures, and a robust understanding of immunology that has aided the discovery and implementation of novel immunosuppressive drugs. Concomitant reductions in infective mortality through coordinated intensivist support and use of antiviral agents have significantly lowered recipient morbidity and mortality. However, the age of such dramatic improvements in patient and allograft survival has plateaued in the past decade(s), demonstrating only marginal gains.
The field of transplantation recognizes unmet clinical needs1 that impede making substantial improvements in short- and long-term outcomes. Glaringly, we lack treatment options for ischemia reperfusion injury (IRI), an inevitable consequence of organ procurement that manifests as delayed graft function (or worse, primary nonfunction). In kidney transplantation, IRI is associated with an increased risk of acute rejection, reduced short-term allograft function, and higher rates of graft failure.2 It is also implicated in primary graft dysfunction following heart3 and lung4 transplantation, with marked effects on early (30-d) mortality. The pathophysiology of IRI is well characterized,5 and the underlying molecular mechanisms involve activation of the innate immune and complement systems, pro-inflammatory cytokine production, and generation of reactive oxygen species (ROS) that culminate in cell death. The inflammatory and vasomotor instability of brain death, cold and warm ischemic times also impact on the severity of IRI and consequent organ dysfunction.6
Good organ preservation is crucial to optimizing graft outcomes by reducing the impact of IRI, and the development of reliable, compatible organ preservation solutions has been key to this process. Despite recent advances in machine perfusion at a range of temperatures, static cold storage—the deceptively simple approach of flushing donor organs with cold preservation solution and storing on ice before implantation—remains the gold standard. Cooling increases the resistance of tissues to a lack of oxygen and is therefore fundamental to decreasing hypoxic injury. Temperature reductions benefit cells by minimizing (but not eliminating) cellular metabolism, ATP, and oxygen requirements. However, ongoing metabolic activity in a hypoxic environment leads to cellular dependence on glycolysis for ATP generation but detrimental lactic acidosis, ROS production as ATP is broken down to purines (which are enzymatically altered by xanthine oxidase) and loss of redox potential (no longer homeostatically maintained by the flow of electrons and H+). This is accompanied by intracellular accumulation of Ca2+ and Na+ (as the Na+/K+ ATPase transporter is disrupted), altering osmotic balance and affecting mitochondria and lysosomal function. These factors all contribute to decreased cell viability.
Preservation solutions were conceived to provide metabolic support, energy supply, and defense mechanisms to counteract hypoxic injuries that exacerbated by revascularization. The development of University of Wisconsin (UW) preservation solution by Folkert et al7 in the 1980s allowed extended cold ischemic times and organ sharing across distances, and permitted (liver) transplantation to be performed as a semielective procedure. The basis of UW solution is a phosphate buffer with high potassium and low sodium (mimicking the intracellular milieu), high molecular weight raffinose and lactobionic acid to prevent transmembranous water shifts, hydroxyethyl starch to prevent expansion of the extracellular space, adenosine (ATP substrate), glutathione (for antioxidant activity), and allopurinol to inhibit xanthine oxidase (and therefore generation of ROS). Several other preservation solutions exist (Euro-Collins, HTK, Celsior, IGL-1) that vary slightly in terms of chemical composition, but the literature certainly demonstrates the superiority of UW solution for multiple abdominal organs (reviewed in the study by Latchana et al8).
Cold storage is traditionally maintained at 4–8°C, because temperatures below this increase the risk of cold injury through protein denaturation, mechanical injury from ice crystal formation, and osmotic stress occurring during the freeze-thaw process. The article by Que et al9 in this issue pushes these underexplored boundaries by using a supercooling technique, where mouse hearts successfully underwent prolonged preservation at subzero temperatures. Cryotherapy is currently only suitable for cells, but supercooling allows storage of organs without ice nucleation. The authors use a novel cooling apparatus, maintaining murine hearts at –8°C in a modified Institut Georges Lopez-1 solution containing polyethylene glycol, glucose, trehalose, and lidocaine. Following warming, syngeneic heterotopic heart transplantation was performed with a standardized warm ischemic time. Hearts were successfully revived when supercooled for up to 144 hours, but long-term survival was only apparent for hearts supercooled up to 96 hours.
In additional experiments, murine hearts preserved for 24 hours with supercooling or in UW solution at 4°C were transplanted. The authors demonstrated a shorter time to spontaneous return of circulation, reduced markers of cardiac injury and histologic damage, as well as lower pro-inflammatory cytokine transcript expression and oxidative stress. There was no clear evidence of structural abnormalities or metabolic deterioration as a consequence of supercooling.
This preservation technique and solution, which circumvents ice-induced cellular injury, potentially represents a significant advance in the field since the introduction of the acellular Collins solution. Supercooling has been demonstrated in rodent livers,10 but the donor heart represents a more fickle organ crucially dependent on short cold ischemic times. Further work will be required to establish whether this technique is as effective in an orthotopic transplant model, and in large animals. It also remains to be seen whether the solution is compatible with other solid organs, and whether components are modifiable for optimal effects based on the organ of interest. However, preservation solutions merely limit ischemic/hypoxic damage and the transplant community is still in desperate needs of a therapeutic agent to robustly prevent cellular injury.
1. O’Connell PJ, Kuypers DR, Mannon RB, et al. Clinical trials for immunosuppression in transplantation: the case for reform and change in direction. Transplantation. 2017; 10171527–1534
2. Siedlecki A, Irish W, Brennan DC. Delayed graft function in the kidney transplant. Am J Transplant. 2011; 11112279–2296
3. Singh SSA, Dalzell JR, Berry C, et al. Primary graft dysfunction after heart transplantation: a thorn amongst the roses. Heart Fail Rev. 2019; 245805–820
4. Lee JC, Christie JD, Keshavjee S. Primary graft dysfunction: definition, risk factors, short- and long-term outcomes. Semin Respir Crit Care Med. 2010; 312161–171
5. Bonventre JV, Yang L. Cellular pathophysiology of ischemic acute kidney injury. J Clin Invest. 2011; 121114210–4221
6. Louvar DW, Li N, Snyder J, et al. “Nature versus nurture” study of deceased-donor pairs in kidney transplantation. J Am Soc Nephrol. 2009; 2061351–1358
7. Belzer FO, Glass NR, Sollinger HW, et al. A new perfusate for kidney preservation. Transplantation. 1982; 333322–323
8. Latchana N, Peck JR, Whitson BA, et al. Preservation solutions used during abdominal transplantation: current status and outcomes. World J Transplant. 2015; 54154–164
9. Que W, Hu X, Fujino M, et al. Prolonged cold ischemia time in mouse heart transplantation using supercooling preservation Transplantation. 2020; 104:1879–1889
10. Berendsen TA, Bruinsma BG, Puts CF, et al. Supercooling enables long-term transplantation survival following 4 days of liver preservation. Nat Med. 2014; 207790–793