The goal is to make human eggs, ovarian tissue, blood vessels, even whole organs available when needed.
To get there, researchers are directly comparing slow-freezing techniques, used successfully for decades to preserve sperm and embryos, to a more rapid method of cryopreservation that transforms tissues into durable glass-like structures.
Phase I trials under way at the Medical College of Georgia are comparing the two approaches in human ovarian tissue and eggs, or oocytes, as well as human-like cow ovarian tissue and eggs.
They start with reproductive tissues because young women with cancer produce a compelling need and are a good model for other tissues and organs.
"What we tell patients is that right now the standard of care for people who are going through cancer therapy is to use egg donors later on," says Dr. Adelina M. Emmi, reproductive endocrinologist and medical director of MCG Reproductive Laboratories of Augusta.
Treatment for leukemia and cervical, ovarian, breast or other cancers often leaves women infertile because systemic chemotherapy and more focused radiation therapy, designed to kill rapidly spreading cancer cells, also can destroy dynamic reproductive tissue.
"I don't think when you are faced with the reality that you may die, your fertility is the most important thing you are thinking or talking about, but there are a lot of women interested in talking about it," says Dr. Emmi. She hopes her work with Dr. Ying C. Song, cryobiologist, will one day give her more to say.
They are collecting ovarian tissue from volunteers age 16 to 37 who need the tissue taken for some reason other than cancer, such as a hysterectomy for benign disease, says Dr. Song, MCG clinical associate professor at MCG and director of research for Augusta-based Xytex Research/Xytex International. Collaborators at the University of Texas Health Science Center and M.D. Anderson Cancer Center are doing the same.
With some of the tissue, they are using conventional cryopreservation. Chemicals to protect cells from the hazards of freezing are added before taking tissue from the refrigerator temperature of 4 degrees Celsius to minus 80 degrees Celsius over two- and one-half hours. Later, liquid nitrogen takes it to minus 196 degrees
"You put it in a control-rate freezer that takes down the temperature one degree centigrade per minute so it drops the temperature very, very slowly," says Dr. Song.
Slow cooling works well for simple tissue, such as sperm or even embryos, and for blood. "In blood, for example, conventional cryopreservation freezes the liquid part but not the cells inside. Liquid freezes and the water inside the cells moves out gradually so they dehydrate," Dr. Song explains.
But, for more complex structures, such as a human egg or ovarian tissue, resulting ice formation can be destructive. "Ice crystals break up your inside organelles. That is what hurts eggs, which are very delicate," he says.
"When you trigger ovulation with a hormone or naturally, you get the last separation of the chromosomes, from 46 to 23," says Dr. Emmi. That separation enables a future baby to get half his chromosomes from mom and half from dad. Fragile spindles, which line up chromosomes for division, are easily broken during freezing so chromosomes can't properly divide. Typically the resulting embryo dies. Plus, fertilization is unlikely since freezing often hardens the egg's outer shell that sperm must penetrate.
"That is why we have tried to develop technology without freezing," says Dr. Song, who has pioneered use of vitrification in blood vessels, cartilage and heart valves.
Vitrification, which takes tissue from room temperature to minus-100 degrees Celsius in 20 minutes, solidifies tissue into a clear, glass-like structure minus the opacity of ice cubes and frozen meats, a tell-tale sign of ice crystals within.
Dr. Song, whose research lab is in MCG's biotech incubator, has developed cryoprotectants that can be used safely in higher doses as well as agents to help protect tissue during the ultra-rapid process of de-vitrification.
"People use low concentrations of cryoprotection because they are toxic," he says. "The problem is, if you use lower concentrations, you cannot get true vitrification." The agents are needed to intercept water so it won't form ice. Interestingly if small ice crystals form during cooling, they can get larger during de-vitrification, which takes place in seconds.
"We developed a solution where we can warm up tissue in under five minutes and still get no ice formation," says Dr. Song, adding that ice formation aside, it is difficult to thaw rock-solid tissue at room temperature in a matter of seconds, meaning the current approach could have extremely limited use.
A study he published in March 2000 in Nature Biotechnology showed the approach he uses works well, at least in blood vessels. "Now we want to try this on eggs and ovarian tissue and see if we can develop a robust technology and improve outcomes," Dr. Song says.
Later, researchers will put ovarian tissue preserved both ways into mice to see if it survives and starts making proper connections.