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After 13 years of unsuccessful attempts to improve his own best cryoprotectant formula, a cryobiologist has found a way to develop a whole new family of cryoprotectant solutions that should enable organs to be vitrified successfully in the near future. "Vitrification" means changing a liquid to a glasslike solid as temperature falls, without forming ice crystals that damage cells. For twenty years, cryobiologists have questioned whether vitrification of human organs will ever be practical. The investigators at 21st Century Medicine should get the first glimpse of the answer in 1999.
Concurrently, biophysicist Brian Wowk, a former President of CryoCare Foundation, has discovered a different family of cryoprotectant compounds which enable vitrification at lower concentrations and higher temperatures. Wowk has also developed synthetic "ice blockers" that enhance many other cryoprotectants and eliminate problems associated with rewarming vitrified organs.
Finally, Mike Darwin, founder of BioPreservation, has led a highly successful initiative to minimize ischemic injury--the damage that is caused by insufficient blood flow, typically when the heart stops beating. Darwin's team now holds the unofficial world record for resuscitating dogs after up to 17 minutes of "death" at normal body temperature.
These multiple breakthroughs should enable preservation of human brains with minimal or even zero ice damage, and may lead to reversible brain cryopreservation within ten years. If this goal is achieved, cryonics will not have to rely on future technology to repair damage caused by freezing or toxicity, and will take a major step toward credibility in conventional science.
Long before that, however, the research will have applications outside cryonics that should be highly profitable for 21st Century Medicine and its stockholders.
Biologist Christopher Rasch, hematologist Nooshin Mesbah-Karimi, and surgeon Yasumitsu Okouchi collaborated with Brian Wowk on their work, while Steven B. Harris, MD, Sandra Russell, Joan O'Farrell, and Carlotta Pengelley participated with Mike Darwin.
21st Century Medicine was founded in 1993 by Saul Kent and Bill Faloon, long-time cryonics activists who run a lucrative vitamin mail-order business and offer information on dietary supplements via their Life Extension Foundation. In 1997, after Kent and Faloon won a long legal battle with the FDA, they purchased a second building for 21st Century Medicine, hired additional personnel, and are spending currently almost $2 million a year on research.
At a seminar on November 8th, 1998 in Ontario, California, the principal researchers from 21st Century Medicine described some amazing payoffs that have resulted from the investment by Kent and Faloon, far sooner than anyone expected.
The presentations were tantalizing, because key information is being withheld while patents are being filed. Still, a huge amount of information was communicated, and I can provide only a partial summary here. 21st Century Medicine is selling videotapes to anyone who wants the complete version.
Brian Wowk began the presentations by describing his search for cryoprotectant molecules that would bind less readily with each other, and more readily with water molecules, thus reducing viscosity and enabling faster perfusion. "The idea that we came up with was to replace hydroxyl groups on cryoprotectant molecules with methoxyl groups," he said.
For example, propylene glycol consists of a chain of carbon atoms, with two OH (hydroxyl) atomic groups attached to the first two atoms in the chain. Wowk proposed replacing one of the hydroxyl groups with an OCH3 (methoxyl) group, creating a methoxylated version of propylene glycol. "We can make similar modifications to a variety of other standard cryoprotectants," he said. "If you do this, you get some rather dramatic results."
In the case of propylene glycol, the methoxylated version is almost 100 times less viscous than the regular version. Ethylene glycol and glycerol can be modified in the same way, though the improvements are less extreme.
The modified compounds penetrate cells much faster than conventional cryoprotectants. Ethylene glycol is one of the most penetrating cryoprotectants known, but the methoxylated version gets into red blood cells about four times faster.
Better still, the methoxylated compounds inhibit ice formation and enable vitrification far more effectively. Wowk showed a cooling curve for a 45 percent glycerol solution, and another curve for methoxylated glycerol. The former indicated significant ice formation; the latter showed virtually none.
Moreover, methoxylated compounds vitrify at higher temperatures. Wowk predicted that in the future, we won't need to use liquid nitrogen for long-term storage because a suitable cocktail of methoxylated compounds should vitrify above -79 degrees Celsius (dry-ice temperature), which will reduce storage costs and the risk of structural cracking.
One problem with the new compounds is that they are more toxic to cells. However, Wowk has found that toxicity can be mitigated by mixing appropriate compounds. In the lab, viability of cells has been measured in terms of their ability to pump potassium and sodium ions across their membranes after exposure to and removal of cryoprotective agents. Ultimately Wowk found that if he replaced propylene glycol with methoxylated glycerol in VS4-1A (the previous state-of-the-art cryoprotectant developed more than ten years, it produced no more injury than VS4-1A itself. Given that VS4-1A formerly was the least toxic vitrifying agent known, Wowk felt that this was "a pretty impressive result." However, he went on, "... completely destroyed these results with new results that surpassed them by almost an order of magnitude."
Initially he suspected that solutions which are more liable to denature proteins would be more toxic--but found that just the opposite is true, which "makes no sense." He also thought that a less-concentrated solution would be less likely to disrupt biological systems, but found inconsistent correlation between concentration of cryoprotectants and viability of cells.
In 1998, a cryobiologist came up with a novel idea to make sense of the data. This led him to a new way to measure concentration of cryoprotectants, which does correlate properly with viability of cells.
"Suddenly all the data points fall on a straight line," He told his audience at the 21st Century Medicine seminar.
He would not reveal the exact nature of his insight, but claimed it enabled him to understand how to reduce toxicity in cryoprotectants more effectively than has ever been achieved before. He came up with a solution which he calls VX. For thirteen years he had been trying to find something less toxic than his previous achievement, VS4-1A, a 55 percent solution of DMSO, formamide, and propylene glycol. VX turned out to be the answer.
Using it as a starting point he developed four new vitrification solutions, "each of which is statistically significantly superior to the previous world champion solution, VS4-1A." One of the new VX mixes should enable 100- percent survival of perfused rabbit kidneys, according to him. Still, this did not solve the problem posed by larger organs that cannot be cooled as rapidly as rabbit kidneys, and tend to suffer from increased ice damage as a result. The cryobiologist said he considered using "some tricks from nature" to inhibit the ice crystal growth.
The trick he tried was an antifreeze protein found in Antarctic fish. When he added it to conventional cryoprotectants in a standard salt solution (the solution that carries the cryoprotectant into and out of organs during perfusion), it achieved barely measurable results. However, when he used a new solution to "carry" the cryoprotectant, and then added the antifreeze protein, he reduced the amount of ice formed in a dilute version of VS4-1A known as VS4 by a factor of 1,000.
He also tried a third "vehicle solution" designed to enhance a different antifreeze protein found in a species of beetles. This reduced ice formation even more effectively, by an additional factor of 10 when no protein was present, and by an additional factor of 1,000 when beetle antifreeze protein was present. The practical bottom-line result was that he could achieve vitrification with a slow cooling rate of 1 degree Celsius per minute--which is practical for human kidneys--even using a version of VS4-1A that was diluted to the point of virtual nontoxicity.
Also he found that the beetle protein would eliminate another intractable problem: ice crystals forming when a vitrified sample is rewarmed. Typically, a sample has to be rewarmed extremely fast to get it from its deep subzero temperature to above freezing point without ice crystals causing catastrophic damage along the way. Since raising the temperature of large organs rapidly is quite difficult, zero- damage rewarming has always been a formidable challenge. But with his new vehicle and 1 percent beetle protein, he found he could avoid ice formation at a warming rate of just 1 degree per minute, even with a solution so dilute as to be essentially nontoxic.
"This is wonderful," he told the audience at his presentation, "but beetle protein is hard to come by, and is expensive. We wanted to come up with our own solution, our own ice-blocking agent, which is dirt cheap. Why not? Let's ask for the moon, maybe we'll get it. And luckily Brian found the moon for us, and now Brian will deliver it."
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