001 Milestones in cryobiology

Cryobiology is a discipline that has its origins in experiments that were published exactly 350 years ago. In 1663, Henry Power, a British physician, reported that he had used a mixture of snow and salt to freeze nematodes. When he thawed the samples three hours later, he stated that “all my little Animals made their re-appearance and danced and frisked about as lively as ever.” Over the centuries, this discipline has evolved and grown into a formal science that ranges from theoretical analysis of the behavior of solutions and the response of biological cells at subzero temperatures to application of very practical procedures to cryopreserve 250 million doses of cattle sperm annually as well as sperm of more than 60 species of mammals, fish and birds. Cryobiology now plays a critically important role in human and animal medicine, agriculture, research and as a fundamental discipline itself. Its applications are so diverse that it is not possible to even mention all of the milestones that have been achieved. Throughout the 18th century, scientists studied the phenomenon of cryptobiosis, the interruption of normal metabolism of cells and simple organisms by cooling them to low temperatures. By the 19th century, such studies had developed into formal experimental biology in which systematic measurements of the effects of freezing were made. By the 1920s and 30s, investigators were attempting to preserve cells of many animal species by cooling them to temperatures as low as that of liquid nitrogen and even liquid helium. In some isolated cases, cells of various types survived; for example, Jahnel in Germany observed motile human spermatozoa that had been cooled to −269 °C. In the 1940s and 1950s, several observations significant enough to be called milestones had been made. These included the demonstration by Polge and his colleagues that glycerol protected rooster spermatozoa against the damaging effects of freezing. Within a short time, this finding had been extended to cattle and human spermatozoa and then to erythrocytes. Freezing of sperm resulted in live births of calves and children. To search for possible causes of injury, Luyet and Meryman independently studied formation and re-crystallization of ice crystals in aqueous solutions. Lovelock began to apply formal physical–chemical analysis to the subject. Other biologists measured effects of freezing and thawing on a wide variety of microorganisms and animal cells. In the 1950s, there was sufficient interest in these observations of freezing to justify convening several symposia. In the 1960s, Mazur formulated his mathematical analysis of the response of cells when they are frozen. Shortly thereafter, it was demonstrated that cells of higher animals subjected to freezing and thawing exhibited responses very similar to those of microorganisms. This, in turn, led to the successful cryopreservation of numerous types of animal and plant cells. In the 1970s, application of fundamental principles resulted in the successful cryopreservation of mouse embryos and then to those of more than 25 other species. Then, procedures were devised to cryopreserve organized tissues of many types and from diverse species. Now, in the 21st century, an incredible variety of cells and tissues of plants and animals are routinely preserved not only by freezing but also by vitrification and even by drying. Many of these achievements were first attempted by the earliest investigators of cryptobiosis. Source of funding: None declared. Conflict of interest: None declared. sleibo@uno.edu