Nanoparticles Key to Tissue Preservation

A multidisciplinary research team has discovered a groundbreaking process to cool and rewarm large-scale animal heart valves and blood vessels preserved at very low temperatures. The discovery could result in a major step forward in saving millions of human lives by increasing the availability of organs and tissues for transplantation, according to John Bischof, professor of both mechanical and biomedical engineering at the University of Minnesota, a past president of the Society for Cryobiology and senior author of the group’s study, published in Science Translational Medicine [1].

Schematic illustrating tissue vitrification, convective warming, and nanowarming. Image: Manuchehrabadi et al., Science Translational Medicine (2017)

Schematic illustrating tissue vitrification, convective warming, and nanowarming. Image: Manuchehrabadi et al., Science Translational Medicine (2017)

More than 60 percent of the hearts and lungs donated for transplantation must be discarded each year, for example, because the tissues cannot be kept cool for longer than four hours. But recent estimates suggest that if only half of unused organs were successfully transplanted, transplant waiting lists could be eliminated within two years.

The new method uses silica-coated iron oxide nanoparticles dispersed throughout tissue samples within a cryoprotectant solution applied during the vitrification process. Long-term preservation methods, like vitrification, that cool biological samples to an ice-free glassy state using temperatures between -160 and -196°C, have been around for decades. The biggest problem for researchers has been with the rewarming process, where tissues often suffer major damage that makes them unusable, especially at larger scales.

“This is the first time that anyone has been able to scale up to a larger biological system and demonstrate successful, fast and uniform warming of hundreds of degrees Celsius per minute of preserved tissue without damaging the tissue,” says Bischof. “These results are very exciting and could have a huge societal benefit if we could someday bank organs for transplant.”

Bischof says that previous research has only shown success at about one milliliter of tissue and solution. The new study, however, scales up to 50 milliliters, and indicates there is a strong possibility the team could scale up to even larger systems, like organs, according to Bischof.

The researchers used a vitrification solution known as VS55, initially developed as VS41 by Patrick Mehl in 1993 [2]. Both Bischof and Kelvin Brockbank, a team member from Clemson University and Tissue Testing Technologies LLC, were familiar with VS55 prior to the study, having used it in other experiments in their respective labs.

“We stuck with VS55 and found ways to make it work,” says Brockbank. ‘So, in other words, rather than change the formulation of the solution, we changed how we deployed it: How many steps did you use? What temperature was that step performed at? All of those things can make quite a significant difference during both the cooling phase, the introduction of cryoprotectant and the removal. And hopefully, if you get it right, there’s no freezing involved at all. In fact, usually I have to go through everybody’s manuscript crossing out the word freezing, because it’s not a freezing method, it’s a vitrification method.”

The researchers precooled the VS55 solution to -10°C and began introducing it to harvested tissue at ice temperature. The sample and solution mix was then cooled to the low temperatures necessary for vitrification. The team used traditional refrigeration equipment available at both labs and medical institutions for biomedical storage, cooling the samples about 10°C per minute. The vitrification process essentially takes a liquid to a point of infinite viscosity. A properly vitrified sample becomes glassy, brittle in a way but not cracked or crystallized.

Conventional convective warming puts cryogenically preserved tissues in danger of becoming cracked or crystallized. Image: Manuchehrabadi et al., Science Translational Medicine (2017)

Conventional convective warming puts cryogenically preserved tissues in danger of becoming cracked or crystallized. Image: Manuchehrabadi et al., Science Translational Medicine (2017)

“With everything we’re doing here, we’re right on the edge of killing the tissue,” says Bischof. “You’re racing against time and racing against toxicity with each one of these steps. And that’s what makes it difficult, challenging.”

The iron oxide nanoparticles then act as tiny heaters around the tissue when they are activated using noninvasive electromagnetic waves to rapidly and uniformly warm tissue at rates 10 to 100 times faster than previous methods. With convective warming, for example, the researchers found that samples warmed unevenly, with temperatures rising much slower in the center of a sample compared to its edge. “And that leads to two problems,” says Bischof. “One is that the rate will be too slow and you will crystallize and the second is that the difference in the temperature between the middle of the system and the edge of the system will increasingly get larger and larger until you build up a thermal stress in the system that’s like dropping an ice cube into a glass of water. It’s just gonna crack.”

After rewarming the samples and washing away the iron oxide nanoparticles, the team tested for viability and found that none of the tissues displayed signs of harm, unlike control samples rewarmed slowly over ice or those using convection warming.

Bischof says the idea for the experiment grew from his team’s research in many different fields to preserve or destroy cells and tissue at either ultrahigh or ultralow temperatures, but that its success ultimately resulted from contributions from an interdisciplinary group of established and capable researchers from all walks of complementary science. “This isn’t just coming out of my lab,” he stresses, “it’s a very collaborative project.” In addition to Bischof, several experts from the University of Minnesota contributed to the project, including postdoctoral researchers Navid Manuchehrabadi, Zhe Gao, Jin Jin Zhang, Hattie Ring and Qi Shao; graduate student Feng Liu; undergraduate student Michael McDermott; dentistry professor Alex Fok; radiology professor Michael Garwood, and chemistry professor Christy Haynes. Other team members included Brockbank and mechanical engineering professor Yoed Rabin at Carnegie Mellon University.

Moving forward, the team plans to expand the experiment to rodent organs (such as rat and rabbit) and then scale up to pig organs and then, hopefully, human organs. The technology might also be applied beyond cryogenics, including delivering lethal pulses of heat to cancer cells. “We’ve gone to the limits of what we can do at very high temperatures and very low temperatures in these different areas,” Bischof says. “Usually when you go to the limits, you end up finding out something new and interesting.”

[1] Manuchehrabadi et al., “Improved tissue cryopreservation using inductive heating of magnetic nanoparticles,” Science Translational Medicine.
[2] Editor’s Note: The print version of this article contained an error, attributing the development of VS55 to Dr. Greg Fahy, who only used the solution for study. Dr. Patrick Mehl discovered VS55, and discusses it in “Nucleation and Crystal Growth in a Vitrification Solution Tested for Organ Cryopreservation by Vitrification,” Cryobiology. 1993 Oct;30(5):509-518. ■