Embryos of Fruit Flies Are Frozen and Revived
By MALCOLM W. BROWNE
Published: December 22, 1992
The art of preserving live organisms in frozen storage has taken an important step with a report that scientists have successfully frozen and revived the embryos of fruit flies.
The achievement will greatly simplify the work of the many laboratories that use fruit flies in experiments and is also expected to improve techniques of cryogenic preservation generally.
Dr. Peter Mazur of Oak Ridge National Laboratory, a principal scientist in the achievement, predicted in an interview that the long-term frozen storage of human hearts, kidneys and other organs for use in transplant operations was no more than a decade away. Weapon Against Disease
Success with frozen fruit fly embryos will also enable scientists to preserve the living embryos of mosquitoes and house flies, both of which are disease carriers and therefore targets of laboratory investigations.
Dr. Mazur said, however, that although the embryos of mice, cattle and sheep were routinely frozen for use by breeders, the freezing of entire animals for subsequent resuscitation -- a goal of cultists who advocate the freezing of human beings -- was out of the question.
Although fish and frogs sometimes survive brief immobilization in relatively high-temperature ice, they have never been kept alive in long-term storage, which requires temperatures low enough to kill them.
The fruit fly Drosophila melanogaster, whose embryos were the subject of Dr. Mazur's project, is one of the most widely used of all animals in the study of genetic mutation, because Drosophila breeds prolifically, undergoes rapid mutations and can produce many generations in a very short time.
Until now there was no way to freeze Drosophila embryos for future use, so laboratories had to maintain large breeding stocks of pure strains of live flies, a costly system with many disadvantages.
Laboratory colonies of flies are easily contaminated by outside strains, and even without genetic contamination, they can undergo spontaneous changes in their genes that spoil mutation experiments.
Although the bodies of fruit flies are much simpler than those of mammals, their embryos have proved much harder to keep alive in frozen storage than those of mice or even of human beings, said Dr. Mazur and his colleagues at Oak Ridge National Laboratory in Tennessee and the University of Chicago.
In a report in the current issue of the journal Science, they say the reason is that fruit fly embryos, unlike those of mammals, nematode worms and other organisms, are naturally protected by waxy membranes that are impermeable to water. These membranes prevent fruit fly embryos from being dehydrated as they are frozen. That step is essential in preventing ice crystals from forming inside the cells of embryos, destroying their functions and killing them.
"We finally succeeded by preparing the Drosophila embryos with a series of treatment steps, essentially using household bleach, gasoline and radiator antifreeze," Dr. Mazur said.
The bleach attacks the embryo membrane, the gasoline (heptane) removes the water-resistant wax and the ethylene glycol antifreeze displaces most of the water from the embryo cells and impedes the formation of crystalline ice.
Some water remains, but because the cryogenic process freezes it almost instantly, it has no time to form ordinary crystalline ice -- the kind that destroys living cells. Instead, it takes the form of "glassy" ice, which like window glass lacks the regular lattice structure of atoms assembled into crystals and is relatively benign to cell structures.
But the scientists learned after many trial-and-error experiments that other conditions were essential to keeping the fruit fly embryos alive in frozen storage. Discovery by 2 Teams
Two independent research teams, one headed by Dr. Mazur and the other by Dr. Peter L. Steponikus at Cornell University, discovered that fruit fly embryos, unlike those of mammals, were less sensitive to the destructive effects of freezing if they were first allowed to grow to a stage where the gut, nervous system and head had developed. By contrast, mammalian embryos survive best when they are frozen at the earliest possible stage of development.
Even under optimum conditions, some mammal embryos, including those of pigs, have never been successfully stored.
"Coming 20 years after our laboratory first learned how to store live mouse embryos cryogenically," Dr. Mazur said, "the Drosophila success gives us cause to celebrate."