Peering through the double eyepieces of a $20,000 microscope equipped with a $30,000 high-speed video camera, Assistant Professor Adam Higgins sees more than just microbes, cells and corpuscles freezing and thawing. In his mind’s eye, he sees portable devices for kidney dialysis and new cures for sepsis — a blood infection that kills more people than AIDS, prostate cancer and breast cancer combined.
He sees wounded soldiers getting blood transfusions more quickly thanks to new preservation techniques. He sees infertile couples celebrating the birth of long-awaited babies. He envisions artificial lungs that provide life-giving oxygen to blood flowing through microscopic channels. And when he considers what life will be like in the 22nd century, he foresees freeze-dried bacteria and sugar-encrusted plants and animals transported to distant planets in a state of suspended animation.
The National Science Foundation shares at least one of Higgins’ visions. In 2012, it gave him a prestigious CAREER award of $400,000 to support his research into the use of micro-channels and freezing to preserve cells and tissues. The award, given to faculty in the first five years of their careers, will be used entirely to operate his lab on the second floor of Gleeson Hall. The research that drew the NSF’s attention lies at the intersection of Higgins’ Ph.D. studies in tissue preservation and his application of microfluidics to preserve and handle blood.
Higgins and his Oregon State research team — Allyson Fry, Ratih Lusianti, John Lahmann and Alex Vian — are developing techniques that could revolutionize blood banking and battlefield medicine by allowing frozen blood to be readied for transfusion in minutes. In hospitals and blood banks, most blood is refrigerated rather than frozen. Refrigerated blood can be stored for only six weeks, but frozen blood can be preserved for 10 years, perhaps longer. Nevertheless, only rare blood types are frozen and stockpiled.
“The prospect of thawing blood as needed in an emergency is not practical now,” Higgins explained. “Complicated things happen when you freeze something. To keep red blood cells from shrinking when they freeze, glycerol is added. But it takes about an hour to coax the glycerol out of the cells after the blood is thawed.”
Like explorers of a microscopic arctic, Higgins and his researchers are experimenting with life at the edge of ice. Besides the Leica microscope, which is equipped with a temperature-controlled stage and a slow-motion video camera, the main tool for their research is math. Sophisticated computer models predict how cell membranes will respond to freezing and thawing.The models make predictions. The microscope shows whether cells survived or died. The video camera enables them to share their findings with the world.
“We have done some mathematical modeling of red blood cells, and theoretically it is possible to remove the glycerol in less than one minute,” said Higgins. “We are working on a practical method of implementing this.
That’s where microfluidics comes in. Our short-term goal is to make it more practical to use freezing in the blood banking industry. The long-term goal is bedside warming — hanging a bag of thawed blood above a micro-fluidic device that removes the glycerol as the blood is transfused into the patient. This capability would make it possible to stockpile frozen blood and make the nation better prepared for a national emergency.”
Dry and high
Higgins and his team are pursuing another method for preserving blood: freeze-drying. Technically called lyophilization, freeze-drying makes blood light and easy to transport without refrigeration.
“There is no completely successful freeze-drying procedure,” Higgins said. “You can dry, store and rehydrate blood, but only about half of the blood cells survive. We think we can do better than that.”
In the future, freeze-drying techniques may be the key to transporting large quantities of oxygen-producing bacteria into space. Payloads of protozoans may one day generate a new atmosphere on Mars, turning the red planet green and making it habitable for human colonization.
Although freeze-drying is intriguing, low temperature storage in an ice-free, glassy state — a technique called vitrification — holds more immediate promise. In 2011, Higgins joined others at the university to bring the annual meeting of the Society of Cryobiology to Oregon State. At the conference, Allyson Fry, a Ph.D. researcher in Higgins’ lab, won an award for her work in which she used computer modeling to optimize vitrification of cells. She later applied that optimization approach at Life Technologies Corp. in Eugene to improve cryopreservation of stem cells, which hold great promise in the treatment of illnesses like heart disease and Parkinson’s.
Reliably coaxing frozen egg cells back to life could avoid one of the most vexing moral and political dilemmas facing science, medicine and society — freezing and banking human embryos, the seeds of life.
“If we can freeze and thaw oocytes — the unfertilized egg cells — without losing too many, then we could affect the way in vitro fertilization is done,” said Higgins. “You can collect and freeze sperm cells by the millions, so a 50 percent survival rate is acceptable, but egg cells are much more precious. You want every egg to survive.”
Higgins is reminded of the vast potential of his work by a microscopic creature commonly called the water bear or moss piglet. Formally named the tardigrade, the tiny creature has the ability to replace the water in its body with sugars that put it into a state of suspended animation that is nearly impervious to heat, cold and radiation. It has even survived trips into the vacuum of space.
For Higgins and his team, the tardigrade shows how much remains to be learned. Cryopreservation techniques and microfluidic devices developed in their lab may one day give life, save lives and carry life out into space. Higgins is reminded of those tantalizing possibilities every time he puts his eyes to the microscope.
— Warren Volkmann