Cold facts

Oregon team refines cryopreservation technique

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CORVALLIS, Ore.—A new approach to “vitrification,” or ice-free cryopreservation, could ultimately allow a much wider use of extreme cold to preserve tissues and even organs for later use, according to researchers in the College of Engineering at Oregon State University. If less-toxic cryoprotectants could be discovered, many more applications of vitrification could be feasible, they said. The researchers, whose work was supported by the National Science Foundation, recently announced their findings in PLOS ONE.
Describing the work as “an important step toward the preservation of more complex tissues and structures,” Dr. Adam Higgins, an associate professor in the OSU School of Chemical, Biological and Environmental Engineering and expert on medical bioprocessing, explains, “Vitrification as a strategy for cryopreservation has been available for decades and some of the first reports of successful vitrification of mammalian cells were reported about 30 years ago. Since that time, the toxicity of cryoprotective agents has been recognized as one of the major hurdles to achieving successful vitrification, particularly for large samples like tissues and organs.”
While cryopreservation is widely used to preserve semen, blood, embryos, plant seeds and other biological samples, it is limited by crystallization when water freezes and sometimes damages or destroys tissues and cells, according to Higgins. As he explained, “The major challenge with cryopreservation using vitrification methods is that the cryoprotective agents (CPAs) used to suppress ice formation are toxic. Successful vitrification requires selection of CPAs and equilibration of the sample with CPAs in a way that minimizes toxicity. Our mathematical modeling approach helps to identify minimally toxic methods for CPA equilibration.”
The mathematical model developed by OSU engineers is designed to simulate the freezing process in the presence of cryoprotectants and thereby minimize damage. The engineers determined that cells that are initially exposed to a low concentration of cryoprotectant and given time to swell can be vitrified after rapidly adding a high concentration of cryoprotectants. The end result is diminished overall toxicity—healthy cell survival after vitrification rose from about 10 percent with a conventional approach to more than 80 percent with the new optimized procedure.
Higgins says that vitrification refers to “solidification of a liquid without crystallization,” adding that the resulting vitrified solid is typically referred to as a glass. Vitrification “avoids the damaging effects of ice formation so it is a particularly attractive approach for cultured cells, tissues and organs—systems that are particularly susceptible to damage by ice,” he said.
To achieve vitrification, it is necessary to cool and warm quickly enough to “outrace the ice crystallization process.” The crystallization process is slowed and the freezing point is depressed by addition of CPAs. Therefore, as Higgins says, “the typical strategy is to load the sample with sufficient CPA to enable the sample to be vitrified at the cooling and warming rates that are achievable experimentally.” For large samples, these cooling and warming rates are relatively low, so high CPA concentrations are required.
“Our long-term goal is to apply our mathematical optimization strategy to 3D tissues and organs,” he adds. “This will require refinement of the mass transfer model, among other modifications to the optimization algorithm. While there is still substantial work to do, our recent work shows that our general optimization strategy is effective and lays the groundwork for these future studies.”
Higgins believes that there may be commercial potential as the method is applied to more valuable biological materials, including tissues and organs on a chip. While he believes that the model can help to identify less-toxic cryoprotectants, and ultimately open the door to vitrification of more complex tissues and perhaps complete organs, he noted that nothing the OSU engineers have done to date has intellectual property protection “and our methods and mathematical optimization strategy are publicly available.”
He concludes: “There is still a lot of work to do to realize the full potential of our mathematical optimization approach for designing cryopreservation procedures. With continued funding, I expect we could successfully apply this approach to 3D tissues in a few years, and possibly to organs as well.”

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