Should you ever need a new kidney, a replacement heart or another vital organ, you won’t exactly be spoilt for choice. That’s because when it comes to healthy human organs for life-saving transplants, there is a vast chasm between supply and demand.
In the United States alone, 26,517 organs were transplanted in 2013, yet over 120,000 patients are stuck on the waiting list. Quite simply, there are not enough donations to go around. To make matters worse, even the organs that are made available sometimes go to waste because they don’t have much of a shelf life once they’ve been removed from a donor. At the moment the best we can do is to preserve them in a special solution just above 0C for a day or two, which doesn’t leave much time to find well-matched patients to receive them.
But there is a possible answer. If scientists could find a way to deep-freeze organs and bring them back without incurring damage, we could potentially bank them for weeks or months. The same could be done for lab-engineered organs, if we can create them. With that in mind, the Organ Preservation Alliance, a charity incubated by Singularity University Labs at Nasa’s Research Park in California, is planning a multi-million dollar prize to incentivise breakthroughs. So could we see a time when transplant surgeons can dip into freezers, and take their pick of kidneys, livers or hearts to carry out life-saving operations?
Scientists have been successfully freezing, or cryopreserving, small collections of human cells for 40 years. They preserve eggs and embryos by flooding cells with solutions of so-called cryoprotectant compounds – which prevent the formation of ice crystals that can rip cells apart, and also guard against lethal shrinkage. Unfortunately, they hit major hurdles when trying to scale this process up, as the architecture within more complex tissues and organs is much more vulnerable to ice-crystal-related damage.
Nevertheless, a small cadre of researchers has not given up, warming to the challenge in part by taking cues from nature. Antarctic icefish, for example, survive in waters as cold as -2C thanks to antifreeze proteins (AFPs), which lower the freezing point of their bodily fluids and bind to ice crystals to stop them spreading. Researchers have used solutions containing icefish AFPs to preserve rat hearts for up to 24 hours at a few degrees below zero. Any colder, however, and icefish AFPs backfire: they force budding ice crystals to form sharp spikes that pierce cell membranes. Another anti-freeze compound, recently discovered in an Alaskan beetle that can tolerate -60C, might prove more useful.
But anti-freeze ingredients alone won’t do the job. That’s because freezing also wrecks cells by affecting the flow of fluids into and out of them. Ice first forms in the spaces between cells, reducing the volume of liquid and increasing the concentration of dissolved salts and other ions. Water rushes from cells out to compensate, causing them to shrivel and die.
For eggs and embryos that’s where cryoprotectant compounds such as glycerol come in handy: they not only displace water to prevent ice formation within cells, but also help to prevent cell shrinkage and death. The problem is these compounds can’t work the same magic in organs. For one, cells in tissue are much more susceptible to ice penetration. And even if cells are protected, ice crystals forming in the spaces between cells shred the extracellular structures that hold the organ together and facilitate its function.
One way to overcome the dangers of ice formation is to stop it from happening in the first place. That’s why some scientists are betting on a technique called vitrification, in which tissues are cooled in such a way that they’re transformed into an ice-free glass. The approach is already used by some fertility clinics, and it has produced some of the most encouraging results to date in terms of preserving complex tissues.
In 2000, for example, Mike Taylor and colleagues at Cell and Tissue Systems in Charleston, South Carolina, vitrified 5cm-long segments of rabbit vein, which falls somewhere between cells and organs in terms of complexity, and demonstrated that they retain most of their function after warming. Two years later, Greg Fahy and colleagues at 21st-Century Medicine, a California-based cryopreservation research company, made a significant breakthrough: they vitrified a rabbit kidney, keeping it below the glass transition temperature of -122C for 10 minutes, before thawing and transplanting it into a rabbit that lived for 48 days before it was killed for examination.
“It was the first time a vital organ has been cryopreserved and transplanted with life support afterwards,” says Fahy. “It was proof that this is a realistic proposition.” But the kidney didn’t function as well as a healthy version, largely because one particular part, the medulla, was slower to soak up the cryoprotectant solution, which meant that some ice formed there during thawing. “Even though we had tremendous encouragement, we also knew we needed to do a better job,” Fahy adds.
“That’s the nearest we’ve come,” says Taylor, adding a note of caution. “That was over 10 years ago, and if the technique was sufficiently robust then there should have been follow-up studies and reports substantiating the finding, which there has not been.” Further progress has been slow, in part, Fahy says, because a chemical that was key part of his method went out of production. Nevertheless, his group has made up the ground and taken a step further: at the annual meeting for the Society of Cryobiology in 2013, Fahy presented a method that allows them to more quickly load the medulla with cryoprotectants.
Despite Fahy’s optimism, it’s clear that when it comes to preserving large organs, vitrification poses some formidable challenges. For a start, you need high concentrations of cryoprotectants – at least five times higher than in conventional slow cooling – which can poison the cells and tissues they’re supposed to protect. The problem gets worse with larger tissues because it takes longer to load the compounds, meaning slower cooling times and more opportunity for toxic exposure. In addition, if cooling is too rapid, or it reaches temperatures that are too low, cracks can appear.
The exceedingly delicate warming process presents more hurdles. If the vitrified specimen is not heated quickly or evenly enough, glassiness gives way to crystallisation – a process known as devitrification – and, again, cracking can occur. “[This] is a challenge we’ve not yet met,” says John Bischof, a cryobiologist and engineer at the University of Minnesota. “The limiting factor is how quickly and uniformly we can thaw it.” And that’s because warming is usually done from the outside in.
Last year, Bischof and graduate student Michael Etheridge proposed a way around the problem: add magnetic nanoparticles to the cryoprotectant solution. The idea is that the particles disperse through the tissue and, once excited by magnetic fields, heat the whole thing evenly and rapidly. The duo is currently working with Taylor and his colleagues to test the method on rabbit arteries
Ice in action
For the most part, advances in the field have arrived by trial and error: testing combinations of solutions and freezing/thawing methods. But researchers have also begun to take advantage of new technologies to get a closer look at how ice behaves in cells and tissues. If you understand the processes in detail, the hope is that you can design new and more effective approaches to control them.
The last 12 months has seen significant advances in this area. Taylor, working with Yoed Rabin, a mechanical engineer at Carnegie Mellon University in Pittsburgh, introduced a new device that enables high-resolution full-colour thermal imaging in large-volume tissues. Meanwhile, Jens Karlsson at Villanova University in Pennsylvania has recently captured ultra-slow-motion microscopic video footage of ice penetrating tiny pockets between two close-knit cells and then triggering crystallisation within them.
Insights from these methods could bring new ideas about how to manipulate the freezing process, says Karlsson, who is trying to figure out ways to cryopreserve tissues by carefully controlling the freezing and thawing process, rather than via vitrification. One possibility is to genetically engineer cells to coax them to make cell-cell junctions capable of withstanding cryopreservation. The next task would be to find a way to direct the formation of extracellular ice so that it doesn’t affect an organs’ function.
Karlsson is also eager to use computer simulations of the freezing process to efficiently test millions of possible protocols. “We need these sorts of tools to accelerate progress,” says Karlsson, who likens the task to “trying to get to the moon with a fraction of the funding that went into that endeavour”.
Even with limited resources, the field has demonstrated that ice-free cryopreservation is practical for small tissues such as a segment of blood vessel. “The remaining barrier, and it’s significant one,” says Taylor, “is scaling it up to a human organ.” For Karlsson, who suspects that such efforts “may hit a brick wall” before vitrification will ever work for human organs, freezing – or what he calls ice-assisted – approaches represent an equally, if not more, viable route to success.
But there is one final sobering thought. “No cryopreservation technique ever offers 100% survival of the component cells,” says Taylor. “In many applications this can be tolerated but for a single organ this may be a significant amount of injury to repair post-storage or transplantation.” Ultimately, that means no matter how well cryopreserved specimens are, they are likely to be sub-standard compared with freshly procured organs.
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