Humans have long been intrigued by the possibility of suspended animation by freezing to low temperatures. Only relatively recently has this interest been channelled into practical developments, such as the frozen preservation of foods or the storage of viable cells and tissues.
But freezing is not new for many species of plants, microorganisms, insects, and reptiles. These species survive winter in Alberta using a variety of strategies to avoid or tolerate freezing within their tissues. There remains a lot to be learnt from these natural systems.
There are many centres around the world, including Edmonton and Calgary, where concerted efforts are being made to understand the responses of living systems to low temperatures, and to apply these concepts to the preservation of cells, tissues, and organism at low temperatures for various purposes.
The cryopreservation of most cells in suspension is now routine, with some notable exceptions that we will discuss later. Bone marrow and other progenitor cells from cancer patients are routinely stored for transplantation after ablative therapy in the treatment of leukemia and other malignancies. Sperm and embryos from many different species (plants, insects, mammals, other animals) have been stored for several reasons, including preservation of biological diversity in endangered species. The Islets of Lanagerhans, the structures in the pancreas that produces insulin in response to blood glucose levels, are being banked using cryopreservation for treatment of leukemic patients. Transplantation of human tissues is becoming increasingly routine as human tissue banks are being developed around the world, and tissues are more readily available.
The understanding of low-temperature responses in tissue systems also allows the development of strategies to maximize cryoinjury in localized areas. Cryosurgery uses freezing to destroy tissues, particularly malignant tissues, while minimizing injury to the surrounding normal tissues. Cryosurgery is commonly used for treating dermatological malignancies, but increasingly being used on cancers of the liver, prostate and breast.
2. Approach to this course
In course would like to stress the understanding of:
It seems that the more one understands the changes which occur on freezing, the more one is amazed that a living thing can survive these changes.
At the end of this course, you should have an even better understanding of cryobiology than some of the scientists working in this area. You should be able to predict and interpret outcomes of different procedures for cryopreservation or cryosurgery, and you should have a better understanding of the manner in which animals, insects, and plants survive the winters in Alberta.
3. Historical Perspective
The word cryobiology is relatively new, apparently first used in the early 1950’s to describe the newly-developing field in low-temperature biology. The word "low temperature" is relative. To a cryogenic physicist, low temperatures are within a few millidegrees of absolute zero. To a cryobiologist, low temperatures often mean temperatures below the freezing point of water, but is sometimes used as any temperature below the normal operating temperature of the organism in question.
The cosmic scale
The average temperature of the universe has been estimated to be 2.735 degrees above absolute zero. Of course there are heat sources such as our sun where the temperature varies between 6,000°C at the surface to several million degrees at the corona. Within our solar system, the planets are cooler. The average temperature of the earth close to 0°C, with a range of ~-80 to +50°C.
Human encounters with cold
For most of human development, cold has likely been a significant factor, particularly in Canada. It is surprising that our major efforts have been to avoid the cold rather than using it!
Apparently during the glacial periods, it was much colder on the earth, so early humans certainly perceived cold, and had to insulate their bodies against it. They perhaps used cold for food preservation, and it is likely that cold was a motivating force for the use of fire.
The struggle of humans with cold slackened as the polar ice caps receded and milder temperatures prevailed.
The advent of research
In last few centuries, humans have been looking more closely at the world and how it is put together. Early Greek thinkers asked "What is cold?", but the answer had to wait for 19th century physics. The advent of modern research (defined as the use of experimentation as a tool in thinking) in the 19th century has led to extensive now information in many fields. It is from this time forward that Cryobiology gradually emerges.
What is cold?
Physicists developed a kinetic theory to address the old question What is cold? Hot and cold are statements on the relative kinetic energies of molecules. The statement "the surface feels cold" implies that molecules in the tips of my fingers have greater kinetic energy than molecules on the touched surface. An immediate consequence of the kinetic theory was the prediction of a lower limit of temperature, a point of zero kinetic energy where molecules stopped moving – absolute zero (the zero point on a temperature scale of ideal gases, denoted by 0 K on the Kelvin scale or 0ºR on the Rakine scale –273.16ºC on the Celsius scale, or –459.60ºF on the Fahrenheit scale).
The kinetic theory spurred many advances in low temperature physics, including the use of a phenomena called electronic paramagnetism to produce temperatures calculated to be within a millionth of a degree of the absolute zero.
Early publications in low-temperature biology
In 1683, a monograph New Experiments and Observations Touching Cold was published by Robert Boyle (of Boyle's law fame in physics). This monograph described experiments on the effects of freezing on living organisms.
In 18th and 19th centuries, Leenwenhoek in Holland, Needham in England, Spallanzini in Italy, Reamur and Claude-Bernard in France, all prominent scientists of their time, engaged in experimental work related to Cryobiology.
Scientists were generally attracted by prospects of suspended animation by cooling. This is still the motivation, if you consider cryopreservation of organs for transplantation as an expression of suspended animation.
Early experiments with microorganisms and seeds were encouraging, sufficient to sustain interest despite failure of experiments with frogs, fish, and rabbits.
About this time it was observed that some insects and aquatic animals could survive freezing of water in winter.
With the development of refrigeration and its use for food storage, Cryo went home into the kitchen. Refrigeration revolutionized the preparation and distribution of food in most of the countries in the world, and it is now common in Canada to have as many as 4 different storage temperatures in the home (room temperature, +5°C in the refrigerator, -5°C in a freezing compartment, and -20°C in a deep freezer). The appropriate storage temperature depends on the material to be stored and the storage time required. A decision on storage temperature and storage time utilizes a considerable amount of information on low temperature preservation, indicating that this information has now become common knowledge in Canada.
The modern era of Cryobiology
In 1940, modern era in Cryobiology began with publication of the book Life and Death at Low Temperatures by Luyet. Luyet was remarkable man (I had opportunity to meet him many years ago), a Jesuit priest with overwhelming curiosity, looking in awe at the wonders of the universe through his microscope. He held a Ph.D. degree in Biology and, in 1920, studied life by observing living and dead organisms. He used freezing as a method to kill yeast, and attempted to observe and measure differences between live and dead yeast. While most of the yeast were killed by freezing, some survived. This triggered his interest in understanding the responses of yeast at low temperatures, which is the science of cryobiology. He observed that the major biological effects occurred with the phase change from water to ice, and realized that the phase change was important. Being a humble biologist with little knowledge of phase changes, he continued his studies and earned a Ph.D. degree in Physics. Later in his life he went to the U.S.A. in Madison, Wisconsin, where he founded a Research Institute. Two sentences from his book capture the essence of this remarkable character. Both are on the page after the title page. The first is the dedication "The authors dedicate this book to all those straining in the pursuit of Truth". The second is the copyright notice "The honesty of the readers being beyond all suspicion, no author’s right is reserved in any country."
A famous trio: Polge, Smith & Parkes
In 1949 Polge, Smith, Parkes in England discovered of the protective action of glycerol, and there are several variants of the story, all with the same theme. While working in the area of reproductive biology, Audrey Smith and Chris Polge were attempting to cryopreserve spermatozoa from roosters. The media commonly used at that time for handling sperm cells was a saline solution supplemented with skim milk powder, egg yolk, sugars, and other compounds. The researchers were modifying the composition of the cryopreservation solution, and testing the motility of sperm after cryopreservation. Normal results were about 5% motility. They also performed morphological examinations of the sperm cells to assess the nature of cryoinjury sustained, and then design experiments to avoid or repair that injury. Glycerol was used as an agent to immobolize the sperm for microscopic examination. It was another routine day in the series of experiments when the investigators were surprised by the high (>50%) recovery of motility after cryopreservation. They repeated the experiment several times with the same remarkable result. Convinced that they had solved the problem, they went out to celebrate. The next day, while demonstrating the remarkable development to their colleagues, the post-thaw was back the normal 5%. The only difference was that they had mixed a new batch of the cryopreservation solution. Retrieving the empty bottle from the previous day, and extracting the last few drops of solution remaining, chemical analyses showed that the solution contained a high concentration of glycerol. On retracing the events, the discovered that the labels had falling off several bottles in the refrigerator, and someone had replaced the labels on the wrong bottles, so what they thought to be their cryopreservation solution was, in fact, the solution used to immobilize the sperm for morphological assessment.
It is interesting to note that glycerol is still widely used in cryopreservation solutions, and the cooling and warming conditions are still similar to those used in that serendipitous experiment.
I had opportunity to meet Audrey Smith, Chris Polge, and Sir Allan Parkes during my postdoctoral fellowship in England, before coming to Alberta.
Chris Polge pioneered the development of techniques for embryo preservation in domestic animals, and was involved in the recent cloning of sheep.
Audrey Smith wrote several books on Cryobiology, most of which were dedicated to Katie. Few people realize that Katie was Dr. Smith’s dog.
The discipline of Cryobiology continues to grow as demands for cryopreserved tissues for transplantation increases,
The need to store reproductive tissues for important plants, crustaceans, microorganisms, and animals is becoming increasingly urgent as the world struggles to feed an ever-growing population. The demand for cryopreserved tissues increases as transplantation has become an effective and cost-effective intervention for a range of conditions. The use of cryosurgery is growing in many countries as an effective approach to treat malignancies. More recently, the development of an ever-increasing range of biosynthetic tissues for transplantation has added an urgency to the development of procedures for the cryopreservation of complex tissue systems. The discipline of Cryobiology continues to grow to meet these challenges.
Please review the paper Cryobiology: the freezing of biological systems, Science 168:939-949, 1970 for an overview of cryobiology.