Cool it down
If you’ve ever made the mistake of putting certain fresh fruits or vegetables in the freezer, then you’re already familiar with the effects of freezing on biological tissue. Banana skins turn black and slimy. Whole oranges leak and deflate. Lettuce comes out limp and soggy. The cause of all this spoilage? Ice crystallization.
Fruits and vegetables, like all biological systems, are mostly water, and when that water freezes, the resultant ice crystals pierce and shred cell membranes like tiny knives. This same thing happens with most of the biological materials that we’d like to preserve — from genetic samples to transplant organs.
“If you want to keep something forever, you need to store it at cryogenic temperatures, but ice kills biological tissues. So, how do we get cryogenic temperatures without the ice?” asks Boris Rubinsky, Professor of the Graduate School at the Department of Mechanical Engineering and professor emeritus of bioengineering.
“If you want to keep something forever, you need to store it at cryogenic temperatures, but ice kills biological tissues. So, how do we get cryogenic temperatures without the ice?”
The answer, he has found, lies in a peculiar property of water. Most matter condenses when it solidifies, but water expands, which had Rubinsky and his team wondering: could crystallization be prevented by restricting the expansion of water as it cools? Now, the process that they have devised and refined — known as isochoric preservation — is being used to save corals from extinction, preserve foods and even extend the viability of organ transplants.
Rubinsky first theorized isochoric freezing in 2005, when he proposed a system designed to cool liquid far beyond its freezing point while preventing crystallization. There are chemicals used to prevent crystallization called cryoprotectants, but they’re extremely toxic and limit the use cases in biological preservation. Rubinsky aimed to achieve the same outcome with thermodynamics alone. The key was confinement. If enclosed in a robust, air-tight container, water has no room to expand as it freezes.
Tony Consiglio, Alan Maida and Boris Rubinsky in their Etcheverry Hall lab. (Photo by Adam Lau/Berkeley Engineering)
But achieving isochoric freezing in practice proved more difficult. Matt Powell-Palm (Ph.D.’20 ME), now an assistant professor of mechanical engineering at Texas A&M University, was a Berkeley graduate student in 2019 when he began working with Rubinsky.
Powell-Palm recalls that when he and Rubinsky designed the first isochoric chambers, they struggled to achieve isochoric freezing. In one early experiment, Powell-Palm dipped an isochoric chamber into a cooling bath over and over, but the sample inside just wouldn’t freeze. In a moment of frustration, he hit the chamber with a hammer, hoping the vibration might initiate a state change. An idea struck him.
“I thought, what if this bug is actually a feature of some other technique?” Powell-Palm says.
That other technique turned out to be isochoric supercooling, a whole new type of isochoric preservation. Over years of research, Rubinsky and Powell-Palm eventually identified three different varieties of isochoric preservation: isochoric vitrification, isochoric freezing and isochoric supercooling.
As Powell-Palm describes it, these types of isochoric preservation are all low-tech, high-science endeavors. The devices the team developed are little more than fancy metal jars. The only thing that’s special about them is that they’re designed to anticipate thermodynamic scenarios that might emerge inside. Each is dependent on temperature and cooling speed, and each has different applications, all of which are now being pursued by researchers at Rubinsky’s Bio-Thermal Laboratory.
Isochoric vitrification: Preserving threatened species
Vitrification refers to the transformation of a substance into glass. Biological samples can be vitrified when cooled very quickly to temperatures around -200 degrees Celsius, suspending all biomolecular processes. According to Powell-Palm, so long as temperature is maintained, there is virtually no limit to the duration for which a vitrified sample could be stored. This makes it particularly appealing for preserving genetic samples from threatened species.
Brooke Chang (B.S.’22, M.S.’23 MSE), a visiting scholar in the Bio-Thermal Laboratory and Director of Vitrification Research at BioChoric Inc., joined Rubinsky’s coral preservation research team in 2021. Currently studying best practices for preserving coral fragments, Chang was drawn to the project by the challenge of solving basic science questions in the service of conservation, like the design of the isochoric chambers.
Brooke Chang holding an an isochoric chamber.
On a recent morning, Chang visited the Bio-Thermal Laboratory to demonstrate the design of the chamber used to preserve coral samples. It’s just a rectangular jar with two small holes at the top that cause water to be squeezed out of the chamber when a threaded lid is screwed in — all air must be removed because ice forms easily where air and water meet. The jars are composed of an aluminum alloy (aluminum 7075) that combines strength with high thermal conductivity.
Chang currently works with the Smithsonian’s National Zoo and Conservation Biology Institute to vitrify coral samples for preservation and test their revivability. Because of the rapid cooling, the samples are infused with cryoprotectant chemicals, which must be flushed and replaced with water if the coral is revived. Chang recently visited the Hawaii Institute of Marine Biology and worked with marine biologists there to find gentler ways of rehydrating the samples. They haven’t yet seen any growth in revived corals, but the samples appear healthy.
“It’s great to see things through the eyes of the marine biologists, because they have a completely different perspective. We see the project as engineers, and so we would never notice the kinds of things that they notice,” says Chang.
Isochoric freezing: Better food storage
While coral can survive inundation with cryoprotectants, those chemicals are completely toxic to humans and can’t be used in food preservation. But the relatively slower cooling speeds and warmer temperatures of isochoric freezing make the process ideal for preserving food.
In isochoric freezing, water begins to crystallize around the edges of the chamber, but the expanding ice causes increased pressure that inhibits its own growth. A state of equilibrium is reached in which part of the water is ice, but most is still liquid, even when it reaches temperatures well below the freezing point — around -20 degrees Celsius. If the complicating factor in isochoric vitrification is cooling speed, in isochoric freezing, it’s pressure. Alan Lenon Maida, a graduate student researcher in the Bio-Thermal Laboratory, says that pressure can deliver unexpected benefits in food preservation.
The pressure generated by isochoric freezing can range anywhere from 0 to 220 megapascals — double the pressure of the deepest part of the ocean. The team discovered that high pressures can kill bacteria. Though freezing temperatures alone will suspend the growth of bacteria, once thawed the bacteria can bounce back, causing foodborne illness and faster spoilage of the food.
“We’re looking for the sweet spot where pressure is high enough to kill bacteria but low enough to preserve quality,” said Maida.
A recent study conducted by the lab found that higher pressures were associated with limited preservation time. When frozen at pressures of 75 megapascals, milk could only be stored for five weeks before some of the proteins were altered. Maida said that the ideal pressure would likely turn out to be somewhere between 65 and 75 megapascals.
Commercial applications may not be far off. Rubinsky has spun off a private enterprise, BioChoric Inc., that has licensed Berkeley’s patent of isochoric freezing technology to sell commercial units. Some of these were recently sold to the government of Iceland for the purpose of exploring the long-term preservation of fish. The company also has received a $25 million dollar grant from NASA to develop new methods of preserving food for space travel.
The benefits of utilizing isochoric freezing in food storage could have an even greater impact here on earth. A study conducted by Rubinsky’s lab in 2021 found that freezing foods under isochoric conditions might reduce global energy use by as much as 6.49 billion kilowatt hours per year — the resultant decrease in carbon emissions would be equivalent to removing a million cars from the road.
Isochoric supercooling: Extending organ transplant viability
In 2021, some 9,000 liver transplants were performed in the United States, and there are more people who need liver transplants than donor livers. According to the Health Resources and Services Administration, of the over 100,000 people waiting for donor livers, 17 die each day for want of one. Liver transplants are further complicated by the need to perform the surgery within 9 to 12 hours of the donor’s death.
Through isochoric supercooling, the Bio-Thermal Laboratory hopes to double that transplantation window. Tony Consiglio (M.S.’20, Ph.D.’23 ME), a postdoctoral scholar at the lab, recently demonstrated the isochoric liver preservation chamber. At the heart of the device is a cylindrical aluminum vessel surrounded by cooling units. The device cools the material inside the chamber to -5 degrees Celsius.
A conventional liver transplant is packed in ice and transported at temperatures between 0 and 5 degrees Celsius. Consiglio explains that the colder the storage temperature, the longer an organ can be preserved. If cryoprotectants could be added to the saline mixture that is currently used in the isochoric chamber, they might be able to preserve organs at colder temperatures for longer periods.
“One of the major challenges that people are trying to solve is how to make these cryoprotectant chemicals less toxic to the organs. If you could do that, you could reduce the temperature even further,” says Consiglio.
In a previous study, the team found that they could preserve pig livers at -2 degrees Celsius for as much as 48 hours before tissue changes became apparent. Moving forward, they’re partnering with the Department of Surgery at UCSF to test the viability of the technology in human livers. They’ll preserve livers donated for scientific research for a period of 24 hours. Once removed from storage, the livers will be evaluated for signs of tissue damage by UCSF gastrointestinal surgeon Tammy T. Chang.
Isochoric supercooling will be more expensive than current organ preservation methods, which involve packing the organs with ice in coolers. But it will likely be much less expensive than other advanced alternatives like normothermic perfusion — machines designed to keep transplant organs alive by flushing them with blood-like solutions and mimicking in-body conditions. The team believes that their isochoric supercooling method will provide a good middle-ground between cost and storage time.
Rubinsky says that such discoveries are possible because of the interdisciplinary nature of his lab. Though isochoric preservation may be low-tech, achieving it requires a thorough understanding of thermodynamics, mechanics and bioengineering. The pursuit of basic science, often yielding simple, elegant solutions, is to Rubinsky’s mind, a beautiful process — and he gives a great deal of credit to the undergraduates who’ve come to his lab and helped advance his research.
“The quality of undergraduates at Berkeley makes all the difference,” says Rubinsky. “They’re driven by science, they’re willing to pursue new ideas, and they’re very good.”
