Just Add Water

The first time John Crowe brought “water bears” back from the dead, he got hooked.

By Kathleen Holder

John Crowe was a high school senior in North Carolina in 1961 when he first experimented with water bears, or tardigrades— speck-sized organisms that can dry up and survive for years in suspended animation. He never imagined that his fascination would lead to discoveries that might one day save lives.

“It was sort of a mystical experience,” Crowe says of the first time he watched through a microscope as the Osiris of the invertebrate world came back to life. “Here was this creature that was completely dry—by some definitions dead. Then by adding water, you create life.”

Crowe was so fascinated that he would devote his career to unraveling the metabolism-stopping tricks of tardigrades, as well as “sea monkeys” or brine shrimp, certain nematodes, baker’s yeast, an African desert plant and other extreme-drought survivors. Joining him in this exploration?have been his wife and fellow biophysicist, Lois Crowe, veterinary professor Fern Tablin and other colleagues.

Their discoveries about a natural sugar called trehalose and its cell-protecting abilities would offer new tools for preserving food, medicines, blood components and other cells so they can be freeze-dried, stored at room temperature and later reconstituted with water.

Blood work

Platelets (A), red blood cells (C), white blood cells (D) and plasma (E) at the site of a wound (B).

About 30 people at the UC Davis Center for Biostabilization, directed by Crowe and Tablin at a research park about two miles east of campus, are researching ways to preserve three types of cells: blood platelets, red blood cells and “nucleated cells,” or cells with nuclei, such as kidney cells or bone-marrow stem cells.

So far, they have developed a method for extending the shelf life of blood platelets, which clot blood and promote wound healing, from five days to up to two years. They similarly are exploring ways to freeze-dry and revive red blood cells, which carry oxygen throughout the body, and bone marrow stem cells, which produce blood, muscle, cartilage and bone. Researchers hope that stem cells can be used someday to repair damaged tissues and grow new bone.

In addition, center scientists are working with colleagues at Massachusetts Institute of Technology to develop an off-the-shelf kit for detecting bioterror agents like anthrax
and plague.

Freezing, drying and thawing are hard on cells. Water expands as it freezes, and the ice crystals can tear cells apart. As cells dry out, membranes shrink and stick together. Thawing and rehydrating damage cells as well.

As Crowe told a newspaper reporter two years ago after the remains of baseball star Ted Williams were reportedly frozen: “Trying to revive a body that’s been frozen is like trying to make a cow out of hamburger.”

Red blood cells can be stored frozen, but that requires adding glycerol to protect the cells. Then the syrupy liquid must be washed from the blood cells before transfusions—a process that is difficult and costly. Scientists typically use chemicals, such as dimethyl sulfoxide, or DMSO, to protect stem cells during freezing, but the toxic chemicals must also be rinsed off before the thawed cells can be used.

Platelets, used to treat patients with leukemia, cancer and massive wounds, are damaged by chilling and currently must be stored at room temperature. Federal regulations require blood banks to throw out donated platelets after five days because of risks of bacterial infection.

That’s where trehalose comes in. In nature, tardigrades, brine shrimp or “sea monkeys,” baker’s yeast, mushrooms, the resurrection plant or “rose of Jericho” of African and Asian deserts, and certain other organisms produce this sugar to survive extreme dehydration.

The Crowes and colleagues, following their curiosity, studied the biophysical properties of trehalose in the 1970s and early 1980s. They found that trehalose acts as a water replacement as cells freeze, protecting cells from drying out too fast, stopping ice crystals from forming and preventing cell membranes from falling apart.

That soon caught the attention of food and drug companies. And U.S. military leaders, looking to save wounded soldiers from bleeding to death on the battlefield, recognized the potential of the Crowe’s research findings for preserving blood platelets.

In 1994, the Navy asked Crowe to conduct research on freeze-drying platelets. Crowe said he initially resisted because he knew little about blood. “Then they made me an offer I couldn’t refuse,” he said.

The center is now is supported by about $2.5 million a year in grants, mostly from the Defense Advanced Research Projects Agency, which also funded research that led to creation of the Internet. “They are very willing to take risks and gamble on new technology,” Crowe said.
Crowe contacted Tablin, who had been studying platelets and their progenitor cells since 1985. Tablin recalled: “When John first called me, I said, ‘OK, that’s great, but I don’t know anything about membranes, and I don’t know anything about biophysics. I’m a platelet cell biologist.’ He said, ‘That’s fine.’”

That led to a collaboration unique even at UC Davis, where cross-disciplinary research is common—a merger of labs in the Division of Biological Sciences and the School of Veterinary Medicine.

Blood cell biology and membrane biophysics might seem closely related. But Crowe and Tablin said that in the beginning it was as if researchers from their separate labs spoke different languages.

“We spent the first couple of months having a discussion that looked a lot like a golf game,” Tablin said. “Somebody would hit a ball, and the other person would try to catch up to it. And the next person would hit a ball, and they had no relationship to one another. My lab would propose an experiment, and we’d be corrected about membrane biophysics. John’s lab would propose an experiment, and we’d correct them about the platelets. It took four or five months before we all could sit down at the table and say, ‘OK here’s a reasonable experiment for the right reasons.’
“We learned a humongous amount in the process. It was a tremendously good opportunity for open intellectual exchange that really had no biases because everybody came to it in a naive way. A lot of times in science when you ask naive questions, you get a lot more information because you’re not stuck in the dogma.”

The breakthrough

A main obstacle in their research with trehalose was getting it to work in mammalian cells. Trehalose must be present on both sides of a cell membrane for its preservation magic to succeed, but while that happens naturally with tardigrades and certain other invertebrates like lobsters, trehalose doesn’t pass through mammalian cell membranes.

About six years ago, center researcher Wim Wolpers and colleagues discovered a way to get trehalose inside mammalian cells, a breakthrough that allowed the freeze-drying of blood platelets.

A Maryland company, AdLyfe Inc.—started by one Crowe’s former graduate students, Alan Rudolph, Ph.D. ’85—has bought rights to the platelet-storage technology. Human clinical trials on a “smart” bandage, containing freeze-dried platelets to speed wound healing, could begin within two years, Crowe and Tablin said. “We’re both very, very confident that it would work,” Tablin said.
Blood banks are also interested in the research.

Platelets treated with trehalose are rehydrated with little damage (left). Compare with fresh platelets (right).

The No. 1 clinical problem with transfusion of platelets from blood banks is septic shock, caused by bacterial contamination, Tablin said. It’s a particular problem for U.S. armed forces because the three- to five-day shelf life for platelets is too short to ship them to combat areas. “The only way they can get platelets is in direct, whole-blood transfusions that are done literally on the spot.”

While blood banks refrigerate and store red blood cells up to 42 days, freeze-drying could further extend their shelf life, increase supplies and make them more portable. That would make donated red blood more available in developing countries where there is little refrigeration, as well as in battle zones.

“We hope that one day every soldier will be able to carry his own freeze-dried red blood cells with him on the battlefield,” said Nellie Tsvetkova, who leads the red blood cell research team.

Tsvetkova is a Bulgarian native who did her doctoral research on cell membranes at a Sofia cryobiology institute where other scientists developed food and bandages for the Russian cosmonauts.

A challenge in freeze-drying red blood cells, she said, is preserving their hemoglobin, which carries oxygen to all the tissues in the body and removes carbon dioxide. But the addition of trehalose shows promise here, too.

Preserving stem cells and other nucleated cells presents harder challenges still because the nuclei, which hold the cells’ DNA, must survive intact in order for the cells to divide and grow after they are revived.

Ann Oliver, who directs the center’s nucleated-cell research, envisions a day when bone-marrow stem cells might be used to form new bone or other tissue.

In addition to trehalose, Oliver and her team are studying the cell-protecting qualities of heat-shock proteins from brine shrimp and arbutin, a substance that is found in resurrection plants and used in skin-lightening cosmetics.

“Our goal is to create a dehydrated product that after hydration grows viable cells,” Oliver said.

Other researchers in the field say such work is critical to a new wave of medicine made from cells.

“There’s no question that in the next 10 to 20 years we will be using cells for therapeutic reasons,” said Mehmet Toner, professor of biomedical engineering at Harvard Medical School and Massachusetts General Hospital. “They are more complicated drugs in some ways. The preservation and stabilization of mammalian cells has become a much more important problem in medicine as these applications become reality.”

Crowe is a world leader in cryopreservation, Toner said. “What he really has done, in my opinion, is create a whole new field.” His critical and creative work, joined with Tablin’s expertise, has made the latest advances possible, he added.

A method patented by the Crowes in 1989 for freeze-drying liposomes, cell-like sacs made from fatty substances used in medicines and cosmetics, is already used to preserve a drug for treating a deadly systemic fungal infection that afflicts AIDS and other immune-depressed patients.

“The physician gets a serum bottle with dried liposomes at the bottom,” Crowe said. “He squirts in water, shakes it up, and they’re ready to go. It’s just that simple.”

They are also working on ways to freeze-dry and store a new drug the company is developing to treat cancer patients.

The Crowes and Tablin say such unexpected benefits show the importance of basic, curiosity-driven science.

“There was no thought of any practical purpose to this,” said Lois Crowe, who is now retired. “That was just an offshoot. The experiments were really done to prove a principle: that trehalose and some other sugars could preserve [cell] membranes.”

John Crowe said curiosity still drives the research at the center. “At a fundamental level, we don’t understand all the mechanisms. We feel that we have to understand how it works.”

Kathleen Holder is associate editor of UC Davis Magazine.