Measuring phase change one cell at a time
Standing in front of the MizzouCentral stage at the Missouri State Fair this summer, microphone in hand, Gary Solbrekken worked the crowd like a pro. His demonstration of scientific principles was one of several sponsored by the University of Missouri College of Engineering to accentuate its informational booth.
Holding a grape over a canister of liquid nitrogen, the mechanical engineering assistant professor asked a group of rapt children in the front row, “What do you think will happen?”
Guesses flew as the grape dropped.
After being fished out with a tongs by Solbrekken’s graduate assistant, William Zhao, a volunteer from the audience was called upon to verify the outcome. As some in the audience had predicted, the grape shattered under a hammer blow, fragments skittering along the cement floor.
At minus 321 degrees, the liquid nitrogen froze the grape within seconds, a process with direct ties to the National Science Foundation-funded research project that Solbrekken and Zhao are doing with cryopreservation of biological tissues.
The research project, a collaboration with John Critser, a professor of comparative medicine with MU’s School of Veterinary Medicine, is aimed at developing more effective ways to store biological tissues at low temperatures, while still preserving their mechanical functionality.
“The cell type we are working on with Gary is the oocyte—mouse, bovine and swine—for agricultural research,” said Critser.
Challenges involved with the rapid freezing of tissue occur at a cellular level when water molecules form ice crystals, and increased concentrations of salt result in cell dehydration—both potentially lethal to cells. To further compound the difficulty, each type of cell responds differently.
“We’re relatively good at preserving sperm cells, but the larger egg cells are harder to cryopreserve, ” Solbrekken said. “The probability of cell survival depends on the cooling rate. Each cell type has a cooling rate for which its survival probability is optimum.”
“We are building a tool—a micro-scale differential scanning calorimeter (micro-DSC)—to evaluate the intracellular phase change process within a single cell,” said Solbrekken. Similar tools exist on a large scale, but none are sensitive enough to measure phase change at such a minute level.
Scientists follow a series of steps in cryopreservation during which a cryoprotectant such as glycerol—think antifreeze—is added to biological samples to prevent the formation of ice crystals during rapid freezing. During this ice crystal-less freezing process, known as vitrification, molecules move increasingly slower as they are cooled, eventually reaching a solid, glassy state without structural damage to the cell. When thawing, the protocol must be reversed.
“Our goal is to understand the fundamental biology and each of the steps we subject cells to during the process,” said Critser. “We want to be able to measure basic biophysical and biomechanical criteria and use the data to improve tissue survivability.”
Engineers Solbrekken and Zhao are utilizing the micro-DSC with its thermoelectric heating and cooling capabilities that operate based on the Peltier effect to do just that. The device will measure and closely characterize phase changes within a single cell as it is cooled. Solbrekken explained that it is the same technology used in “coolers” that plug into a car’s cigarette lighter and offer the option of keeping its contents cool or warm.
“With the micro-DSC, we can control the amount and direction of the electric current and therefore control the amount of cooling and heating within a cell in a very precise way,” said Solbrekken. “This will allow for accurate measurement of ice formation inside the cell during the cooling process.”
“Some ice crystals may be tolerated,” he said, speculating the results. “Just how much cryoprotectant, toxic to a cell at high concentrations, is necessary to suppress the formation of ice crystals? Is there an optimum time to add it? Maybe there are other options entirely.”
“Developing a more efficient and effective protocol for cryopreservation of sperm and oocyte cells is just the first step,” said Solbrekken. “Then we can move on to other things, like skin tissue.”
“In my mind, “he added, “cryopreservation of organs is the holy grail.”