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Freezing for the Future
Life-Saving Cryobiology

Photo of frozen cellLondon, England, 1949. Audrey Smith and Charles Polge put a few drops of glycerin into a petri dish containing chicken sperm. The glycerin, which was intended to slow down the sperm so the scientists could study its movement, had an unexpected side effect—when the scientists accidentally froze these sperm in liquid nitrogen, they survived. This "chance observation," as the pair described it in their article in the scientific journal Nature, was a milestone—the first time in history anyone had successfully cryopreserved living mammalian cells or tissue.

The science of cryobiology—the study of living things at low temperatures—has progressed to the point where researchers today routinely freeze red blood cells, bone marrow, sperm, eggs and embryos, corneas, and skin for basic research in biology and medicine, and for clinical applications. But no one has ever successfully revived an entire human organ, and the science fiction stories of thawing out a frozen man after years in suspended animation are just that—science fiction—says UK mechanical engineer Dayong Gao. That is, he says, until scientists can overcome some serious challenges.

How did Gao, who's original dream was to design jet engines, become a cryobiologist? He answers very simply: "It's the challenge."

"In your very first undergraduate biology class you can question the professor, and the professor will often tell you, "I don't know" or "It's not clear now.' But in undergraduate physics and mathematics class, you can always get an answer," Gao says. "In biology there is an enormous amount we know nothing about. It's very challenging. There are a lot of things to be discovered, and that really motivates me."

A native of China, Gao received his bachelor's degree in mechanical engineering at the University of Science and Technology of China then moved to Canada to study bioengineering at Concordia University in Montreal. He earned his Ph.D. in 1991 and was a postdoctoral fellow at Methodist Hospital of Indiana in Indianapolis. Gao ultimately became director of the cryobiology and bioengineering division at Methodist Hospital. In 1998 he left that position to come to UK.

In his two years at UK, Gao has received external funding totaling approximately $1.2 million from the National Science Foundation, National Institutes of Health, American Cancer Society, American Heart Institute, Department of Defense, A/F Protein Co., Analytical Control System Inc., Baxter Healthcare Corporation, and Whitaker Foundation. With internal support from the University of Kentucky, he has established four research labs, and supervises 14 graduate students and visiting postdoctoral scientists, as well as two students from Dunbar High School's Math, Science & Technology Center.

The grant support makes possible his fundamental research on cryoinjury—injury to cells at low temperatures. "Contrary to popular belief," says Gao, "the biggest challenge cells face during cryopreservation is not their ability to endure storage at deep low temperatures (lower than –100°C). Rather, it is the lethality of the cooling and warming processes associated with the intermediate zone of temperature (-10°C to -60°C) that cells must traverse twice—once during cooling and once during warming.

"Cells will be kept dormant but potentially alive in liquid nitrogen (-196°C) for centuries as long as you can escort the cells safely from room temperature to -196°C and bring them safely back to room temperature," he says. To protect cells during the cooling and warming in this temperature "danger zone," scientists use chemicals called cryoprotectants, or cryoprotective agents (CPAs), that safeguard the cell from damage as ice crystals form and melt. "Scientists have developed more than 100 glycerin-like substances that have a similar function in preventing cell injury," says Gao.

Photo of Dayong GaoGao holds a primary appointment as an associate professor in the Department of Mechanical Engineering in the UK College of Engineering, and joint appointments in the Graduate Center for Biomedical Engineering and the College of Medicine's Department of Physiology. He is also an associate faculty member of the Center of Membrane Sciences.

Cryopreservation is a four-step process: (1) adding CPAs to cells before cooling, (2) cooling the cells to the low temperature at which the cells are stored, (3) warming the cells, and (4) removing the CPAs from the cells after thawing.

CPAs protect by modifying the freezing behavior of cells, specifically affecting the rates of water transport across cell membranes and ice crystal growth.

The use of certain CPAs is not enough to ensure cell cryosurvival. If cells cool too slowly, they lose too much water and experience severe volume shrinkage, which can cause the cells to collapse and membranes to fuse. But the opposite is just as bad. If cells are cooled too rapidly, water trapped inside the cell freezes as ice crystals—scientists call this intracellular ice formation (IIF). IIF kills cells. If a scientist tried to preserve an entire human organ by plunging it into liquid nitrogen, the expanding ice crystals would rupture thousands of blood vessels and crush billions of cells, causing irreparable damage.

"For any given cell type, there are specific optimal cooling and warming conditions. If you put all different cell types together to make a tissue, what's the average optimal condition for all of them? This is much more complicated on the tissue level," Gao says.

"Tissue has a structure, and even if you can preserve the individual cells, if the communication between the cells is destroyed, the structure has been destroyed. Tissues can also fracture by non-uniform cooling or warming, just like if you suddenly heat one side of a glass, the glass will fracture."

Identifying the optimal conditions for cooling and warming the compilation of cell types that make up tissues and organs is the key to the future of cryopreservation, says Gao, and nature has given scientists some clues.

"The North American wood frog can survive winter with over 60 percent of its body water frozen. Professor Boris Rubinsky at the University of California–Berkeley demonstrated a few years ago with MRI (magnetic resonance imaging) that when the frozen frog starts to thaw, the first part of fluid that starts to melt is in the heart." One of the keys to the wood frog's survival is slow cooling. A gradual drop in temperature allows the frog time to produce 100 times the normal amount of glucose in its organs. The glucose acts as a cryoprotectant.

"This is not fiction—it's real. We need to learn from the wood frog, just like we learned the basics of flight from studying birds," Gao says. "God created all of these amazing creatures for us to learn from."

Cord Blood on Ice
Gao is focusing his attention on umbilical cord blood (with exciting transplantation applications), platelets and arteries (two things scientists have yet to freeze successfully) in an effort to treat some of the most devastating diseases known to man."

Cord blood remains in a baby's umbilical cord after the cord is cut, and is usually thrown away. Like bone marrow, cord blood is a rich source of stem cells—the building blocks of the blood and the immune system. Stem cells differentiate, or reproduce, into three types of cells: red blood cells (carry oxygen to tissues and organs), white blood cells (fight infections), and platelets (help clot the blood). Researchers have discovered that umbilical cord stem cells are particularly effective in treating a variety of diseases including several types of leukemia and anemia, breast cancer, and brain tumors.

"There are two types of bone marrow transplantation," says Gao. "In one, a patient has a tumor. We need to use radiation or chemotherapy to kill the cancer cells, but chemotherapy also kills stem cells, so we cryopreserve the patient's own bone marrow stem cells before treatment. After chemotherapy, we transfuse those preserved cells back into the patient to re-establish the blood system.

"The second type is for leukemia—which means the blood cells have cancer. In this case we cannot collect that patient's bone marrow for re-transfusion because it's infected. Instead, we have to use other people's bone marrow stem cells," he says. "Cord blood can be used in either case, but in leukemia patients we find a key advantage—umbilical cord-derived stem cells are less likely to be rejected by the recipient's immune system." Why this is the case (whether there is a special antigen or something else) is a very active research area, Gao says.

In 1993, the National Institutes of Health funded the first public cord blood bank in New York City for non-family transplantation. In 1996, NIH granted more than $25 million for three additional public banks to collect and store 15,000 samples over five years for non-family studies. The Cord Blood Registry, the largest for-profit blood bank in the world, estimates that in a five-year period close to 20 million cord blood samples will be thrown out with the placenta as biological waste. Families can pay a number of private blood banks several thousand dollars in a lump sum or in monthly installments to freeze their baby's cord blood in the event that the child develops a disease or a family member needs transplant stem cells. The cord blood stem cells are cryopreserved right after birth to prevent aging or contamination.

Gao and Gary Van Zant, professor of internal medicine and director of bone marrow transplantation research labs in the UK Markey Cancer Center, received NSF funding to study the best conditions under which to grow and preserve cord blood stem cells.

"One single sample of umbilical cord blood may not be enough for adult transplantation," Gao explains. They use a device called the Cellfoam system, made by the Cytomatrix Company, which Gao describes as a "bioreactor." "This device, or engineered tissue, looks like a sponge. Cells can get into this porous medium easily and grow inside it. This allows us to multiply the number of stem cells in umbilical cord blood for transplantation." Gao is working to predict the optimal cooling and warming temperatures for these cells and create novel devices for adding and removing cryoprotectants to preserve the cells.

Saving Platelets
Platelets, the cells in blood that help it clot, have a five-day shelf life at room temperature. With funds from the Whitaker Foundation and the American Cancer Society, Gao and Van Zant are studying fundamental methods to freeze platelets, as well as practical approaches to meet the needs of doctors in the operating room. The researchers are finding ways to extend the life of platelets. "In the blood, platelets live nine days, but you can only bank them at room temperature for five; after that you get bacterial contamination and aging. On day five, the Central Kentucky Blood Bank and banks all over the world have to throw out all the platelets," says Gao.

"In bone marrow transplantation, preserving human platelets is an important step. A few days after chemotherapy treatment, the blood does not have many platelets left, and the patient is at high risk for internal bleeding. There is an urgent need for long-term freezing and banking of human platelets. Our results may save as much as hundreds of millions of dollars each year in cancer treatment," Gao says.

Frozen Arteries
Through funding from the American Heart Association, an agency that seldom supports work by mechanical engineers, Gao is also developing technology for cryopreservation of human arteries. "We need conduit for bypass surgery. Right now there are only two types of blood vessels that can be used: the patient's own veins or artificial (plastic) tubes, both of which don't last long," Gao says. "Using a vein from the patient is not as good as using an artery. The thickness, structure, and biological function are different between a vein and an artery." And sometimes patients who need bypass surgery don't have a spare good artery anywhere else in the body because the harmful plaque that"s built up near the heart is often in other blood vessels too.

"Cryopreserved arteries could provide a virtually limitless supply of arterial conduit for use as long-lasting bypass grafts. Cryopreservation would allow banking of a large quantity of arteries for typing, matching and proper donor selection, facilitate the transport of arteries, and allow sufficient time for disease screening," Gao says. "The major barrier to cryopreservation of arteries is structural failure (fractures) caused by thermal stress during cooling and warming. Studies up to this point have failed to prevent fractures largely because of a lack of understanding of thermal and mechanical properties of the artery at low temperatures. We are using novel experimental and computational techniques to discover optimal freezing conditions."

If Gao can discover why thermal-stress-induced fractures occur, he may open the door to cryopreservation of organs like the heart, liver, kidney, pancreas and ovaries. "This work will provide a scientific foundation and database of thermal and mechanical properties of frozen biomaterials to develop novel technology for cryopreserving many other organs and tissues in the future," says Gao.

"I believe one of the most important purposes of why we study science, why we do research, is to improve the environment and our health. Science generates knowledge to the benefit of the human being. If we can preserve living cells, tissues and organs, we can save lives. The idea of preserving the human body now—living forever—that is just a joke. That's a selfish idea," he says. "That's not my purpose, but eventually maybe we'll find a way."

Tommorrow's Artificial Kidney
Photo of several artificial kidneysEach year 260,000 Americans suffer from chronic kidney failure, requiring hemodialysis or a kidney transplant to live. "Many other countries have the same rates, but they don't have this kind of care—the people just die," says Dayong Gao, who this summer was invited to help establish a research center in China. Gao was awarded the Chang-Jiang Chair Professorship from the Ministry of Education, People's Republic of China, and received major funding to establish a biomedical engineering institute at his alma mater, the University of Science and Technology of China. "One of my goals in going to China to establish the biomedical center is to open China's first artificial kidney research center," he says.

Gao has obtained a large amount of funding from the Baxter Healthcare Corporation in Chicago, an international biomedical company with research and manufacturing facilities in the United States, Europe and Asia. Professor Charles Knapp, director of the Graduate Center for Biomedical Engineering (CBME), and Gao established a new artificial kidney research lab at CBME a year ago. Four graduate students and one postdoctoral researcher are currently working in this area.

Patients in end-stage kidney failure must be hooked up to this dialysis machine several days a week, four hours each day. The machine removes toxic solutes (known as uremic solutes) that build up in the patient's blood because the kidneys are no longer filtering them out.

"A dialyzer is a tube with 10,000 porous, hollow fibers inside," Gao explains. "The blood enters, works its way into the hollow fibers, and then exits to the veins. And meanwhile, a cleaning solution (dialysate) enters and runs through the gaps in the fibers, washing away the uremic solutes diffusing out from the blood through the hollow fiber walls (made up of porous membranes).

"We use a clean medium to wash dirty blood," Gao says, "but this clean medium might still contain bacteria. The blood is dirty not because of bacteria, but because sometimes the solution to clean the blood actually sends bacteria into the patient's body. With current dialyzers we may give patients high doses of anti-bacterial medicine after treatment. People can be infected or killed by the bacteria." Researchers call this problem "back filtration," and Gao is developing a way to combat this by creating a new generation of dialyzer with internal filtration capabilities.

But there are other problems with current artificial kidneys. "The pores in the fiber wall easily filter out the toxic substance that is small; but for the larger size molecules that are still toxic, it's more difficult," Gao says. "If we try to remove them, we will probably also remove the nice, useful proteins at the same time because the molecules are similar in size. This is a big challenge for us."

To reveal what is really happening on the membrane level, Gao's colleague Peter Hardy, an assistant professor at the CBME and Magnetic Resonance Imaging and Spectroscopy Center at UK, is developing a novel MRI method to study flow patterns inside the dialyzer. The researchers are using this MRI data to calculate how effectively toxic solutes are removed under different operating conditions.

"Forty years ago, final-stage renal failure patients' lives were counted by days. Later by weeks, now by years or tens of years, due to advances in the new artificial kidney," Gao says. His first goal is to make a model patients can take home with them, so they're not tied to a treatment center. The next step would be a portable model. But before a portable model is possible, Gao must devise a way to purify the solution that takes the toxic solutes from the blood so it can be reused over and over again.

"These patients live a rather miserable life," Gao says. "They can't travel more than one or two days at a time because they have to go back to the dialysis center. It would be ideal to have a portable dialyzer patients could carry with them."

Alicia P. Gregory