Frozen
How cryofreezing technologies work
Sergey Amstislavsky, Post-science
Cryocosmononauts who have slept for decades on their way to a distant star system are a frequent topic in science fiction books. Cryopreservation technologies are developing very rapidly today – is humanity really going to fly to the stars? We talk about cryonics, cryobiology and other "cryo" in our article.
"The elderly professor was ready for any outcome of events and lay down to rest from his labors, reveling in the colossal, unprecedented results that he would be able to achieve. His body will never be destroyed; and his bones will never turn white to return to the dust of the earth, from which all people originally came and to which they must return. His body will be perfectly preserved for millions of years, untouched by the gray hand of that time, which only geologists and astronomers can imagine."
The novel "Jamison's Companion", by Neil Jones.
What is this quote about?
Freezing people is a favorite topic of science fiction writers. Special chambers for long-term storage of human bodies are regularly mentioned in literature and cinema. Sometimes freezing occurs accidentally, as a result of severe frostbite of a person. This image is found both in serious films, such as "The Flight of Mr. McKinley", "Avatar" by James Cameron, new films of the "Alien" franchise by Ridley Scott, and in satirical works: the film "Idiocracy", the TV series "South Park" and "Futurama".
A special role in the series of works about cryopreservation was destined to be occupied by Neil Jones' story "Satellite Jamison". The main character of this story, Professor Jamison, sends his body into Earth orbit in order to freeze himself in space and live to the distant future. He succeeds, and millions of years later, a race of mechanized people picks up Jamison and brings him back to life.
At the age of 12, Rob Ettinger, who later became the main popularizer of cryonics, got acquainted with this story.
Cryonics is a teaching according to which the bodies of dead people can be frozen and brought back to life in the distant future, when technology will allow to correct any defects of the body. The scientific community considers cryonics a pseudoscientific teaching, and the provision of services for freezing dead people is quackery. Organizations practicing cryonics require a lot of money for their services. For example, in Russia, freezing the whole body will cost about $ 36,000, the brain – $ 15,000. Save DNA (for subsequent hypothetical cloning) – $900.
He is also, in a sense, a science fiction writer: in 1964, his book "Prospects for Immortality" was published, where he tried to present his vision of how cryopreservation technology, combined with other inventions of the future, could forever change human society. Despite its many artistic merits, it has little in common with real science. It will take a long time to get close to the horizons of cryobiology indicated by Ettinger.
Is it possible?
Cryopreservation is a method that really exists in cryobiology for storing biological objects at a temperature of liquid nitrogen –196C. Cryopreservation is considered successful if the object has fully preserved its viability after defrosting. About why scientists actually freeze living cells and organisms, post-science asked cryobiologist Sergei Amstislavsky.
Experiments with lowering the storage temperature of biological samples began in the XX century and never aimed at freezing a person or other animals entirely. One of the pioneers in this field was the Russian scientist Ilya Ivanov, a specialist in the field of artificial insemination. In the course of his work in the 1910s and 1920s, he repeatedly resorted to cooling the seed of horses to temperatures of the order of minus 15C.
Full cryopreservation became possible after the discovery of cryoprotectors – substances that protect cells from the damaging effect of freezing. They lower the crystallization temperature and protect cell membranes. The first known cryoprotector was glycerin, whose protective properties were discovered by British scientists Christopher Polge, Audrey Smith and Alan Parks in 1949. They managed to cool the spermatozoa of several animal species to the temperature of liquid nitrogen, and then defrost and successfully carry out artificial insemination. Thanks to this discovery, since the middle of the XX century, the freezing of the seed of farm animals has spread widely in many countries.
Scientific base
There are two approaches to cryopreservation of biological objects. The first of them is software freezing, based on the discoveries and developments of Peter Mazur and his students. They found that with a properly selected cooling rate of a cryoprotectant-saturated biological object, intracellular ice crystals, inevitably formed during cooling, will be small enough not to tear cell membranes.
Another approach is vitrification. It was first developed by a team of scientists led by Basil Leweth in the 1930s. According to their mathematical calculations, at very high concentrations of cryoprotectors and a sharp decrease in temperature, the object is immediately vitrified – it turns into a vitreous state, bypassing the crystallization phase.
Before freezing, it is necessary to saturate the biological sample with a cryoprotector. Vitrification, however, requires a higher concentration of these substances. Then the sample is placed in a container, where it is gradually impregnated with a cryoprotector in several stages.
After that, the cooling process begins. During program freezing, a freezer is used, which gradually lowers the temperature of a biological sample according to a pre–selected program. First, cooling proceeds at a rate of 1-2 degrees per minute, then, after the so-called siding, that is, the beginning of induced crystallization, cooling continues, but more slowly: the temperature is reduced by tenths of a degree per minute. After bringing the sample to the desired point (usually up to about –30-45C), it is immersed in a cryopreservation filled with liquid nitrogen, where it is on demand.
Vitrification does not require a freezer – a cryoprotector is sufficient, that is, a container for storing biological samples and a source of liquid nitrogen. The samples are immediately immersed in nitrogen, which is why the liquids in them do not turn into ice, but instantly "glaze over". However, despite its apparent simplicity, this type of cryopreservation requires high skill when performing: you need to manually quickly immerse the object in liquid nitrogen without harming the object being frozen.
Sergey Amstislavsky, Doctor of Biological Sciences, Embryologist, Head of the Cryopreservation and Reproductive Technologies Sector of the Institute of Cytology and Genetics SB RAS:
– A modern study of physicists from the Institute of Automation and Electrometry of the Russian Academy of Sciences, conducted jointly with us, showed that under certain conditions of program freezing and when using a French straw as a cryocontainer, not all liquid turns into ice: part of it glasses and remains in this amorphous state. This suggests the prospects of combining program freezing and vitrification to reduce the negative effects of each of the methods.
Technically, the procedure for decommissioning cryopreservation is similar for programmatically frozen and vitrified objects. The only difference is the speed: the vitrified object needs to be defrosted abruptly, and the one that has been programmatically frozen – more smoothly. To do this, the sample container simply needs to be heated: placed in warm water, held in the air at room temperature, or even simply blown with warm air – depending on how the cooling took place and on which carrier or in which container cryopreservation was carried out. The work of the founder of modern cryobiology Peter Mazur has shown that the heating rate of the sample is no less important component of the success of cryopreservation than the cooling rate. During vitrification, heating should occur very quickly. If this is not done, ice crystals form, and cells can die.
In 2017, a group of researchers from the laboratory of John Bischoff managed to successfully cryopreservate the embryos of danio-rerio fish, which could not be done before due to the peculiarities of the structure of their shells. They were cooled to the temperature of liquid nitrogen at a rate of 90 thousand degrees per minute, and a laser beam was used to warm them up, which hit the embryo at the very moment of its extraction from liquid nitrogen. Gold nanoparticles in the cryoprotector helped to make the heating "volumetric" and almost instantaneous – 14 million degrees per minute.
After defrosting, it is necessary to get rid of cryoprotectors. Almost all of them are more or less toxic substances that, at positive temperatures, can lead to the death of a biological sample. For example, glycerin itself in doses used in cosmetics and as a food emulsifier practically does not harm a person. However, in high concentrations, glycerin can cause serious damage to human health. The biological object that has been frozen is literally impregnated with this substance or other cryoprotectors penetrating into the cells (ethylene glycol, propylene glycol or dimethyl sulfoxide). In addition, the defrosted object, as a rule, is very dehydrated after exposure to the cryoprotector, so it must be "washed", at the same time allowing it to be saturated with moisture.
Experiments
The main field of activity of modern cryobiology is the freezing of germ cells of living organisms, as well as their embryos. These objects are simple enough to cope with a sharp and strong temperature drop without consequences. But even with them there are difficulties: for example, oocytes (female germ cells) are much more difficult to preserve than spermatozoa, and embryos are already full-fledged multicellular organisms, to which it is not so easy to find an approach in a number of vertebrates.
There is a great demand for this technology among specialists engaged in working with animals. The key task solved with the help of cryopreservation is the preservation of biological diversity. Many species of wild mammals are on the verge of extinction today, and not all of them have an environment where they could survive on their own. In order to preserve the genetic resources of these animal species, cryobanks are being created all over the world – complexes similar to storage barrels filled with liquid nitrogen. They house racks in which containers with biomaterial are stored. Cryobanks are needed for storing germ cells and animal embryos, as well as for some other types of cells.
Cryopreservation is also used to optimize the work of vivariums with laboratory animals. Currently, there are already thousands of different lines of mice and rats, in the future there will be even more of them, and even now it is impossible to do without cryoarchiving. In large genetic centers, many lines of mice are preserved in the form of spermatozoa and embryos. Only the lines most frequently used by experimenters are kept in the form of live collections.
Cryopreservation was useful for breeding. By freezing the genetic material, it is possible to preserve and use large stocks of germ cells of individuals with advantageous characteristics: greater fertility, greater weight, increased hair growth rate, and so on.
Cryobanks that provide services for the storage of human germ cells are also popular today. However, the goals of these banks are purely reproductive.
There are many unsolved problems in cryobiology. For example, effective methods of freezing embryos and larvae of amphibians and fish have not yet been found. That is why in 2017 the work of American scientists, which showed the possibility of vitrification of the embryos of the danio-rerio fish, became a breakthrough. Difficulties may also arise when working with mammals, although reliable methods of cryopreservation of gametes and embryos are already available for many of them. For example, embryos and oocytes of wild cats or domestic pigs are problematic objects due to the high content of lipid granules. Lipids play a key role in maintaining the functioning of the cell nutrition system, while lowering the temperature affects the aggregate state of lipid granules, which can disrupt metabolism and lead to cell death.
A lot of research is being conducted, the purpose of which is to learn how to cool and in the future freeze larger biological objects – organs and tissues. We have already managed to cool the rabbit kidney to –45C, after which it retained its functionality and was successfully transplanted.
The results of another impressive experiment were published in 2006. The authors of the study demonstrated that the cells of the hippocampus of the rat brain are able to maintain viability after vitrification. The results of the experiment, however, were ambiguous. First, very thin slices of the brain, only 500 microns thick, were subjected to cooling. Secondly, the ability to maintain a high level of intracellular potassium was chosen as a criterion of viability, which does not necessarily imply the normal functioning of nerve cells and the maintenance of connections between neurons.
These successes are impressive, which once again raises the question: is Rob Ettinger's dream of cryopreservation of a person or at least his brain feasible in the foreseeable future?
What discoveries are missing to make this a reality?
It will not work to freeze a person. In fact, cryopreservation is not applicable today not only to humans, but also to the overwhelming number of multicellular organisms. So far, only the roundworm Caenorhabditis elegans (and some other small parasitic worms), as well as free-floating larvae of several coral species, small in size and not distinguished by a complex structure of the organism, have been successfully frozen.
The main obstacle to freezing large and complex creatures is the variety of cell types and tissues in the body. Intercellular contacts are very sensitive to any kind of influences, so it is necessary to spend a lot of time and effort to learn not to damage the interaction of cells in organs and tissues during cryopreservation. Each cell type needs its own approach, and sometimes the optimal cryopreservation regimes for them differ. If all this is not taken into account, successful freezing of individual organs and organisms will be impossible.
It is difficult to say when this task will become feasible, but the key to solving the problem may be to unravel the phenomenon of suspended animation. Many living creatures – insect larvae, mollusks, amphibians and reptiles – survive the cold period in a frozen state and "come to life" when more favorable conditions occur. Of course, none of them independently reached a body temperature of –196C, but their abilities are still impressive. The forest frog, when it goes into hibernation, is able to turn 35% of the fluid in its body into ice, while ceasing to breathe, eat, and even "turning off" the heartbeat.
At the same time, it is known that lowering a person's body temperature also significantly slows down his metabolism and gives a chance to survive in the most difficult situations. The most impressive story happened to 35-year-old Japanese Mitsutaka Uchikoshi in 2006. After getting lost during a hike in the mountains with friends, he suffered a fractured pelvis and lost consciousness. He lay at the scene for 24 days. But the ambient temperature around was quite low, which is why Uchikoshi's own body temperature dropped to 22 degrees. After receiving medical care, the victim was able to fully recover. The doctors who treated him attribute this to the fact that for some reason Uchikoshi fell into a state close to hibernation, which allowed him to last so long without food and water.
Given these facts, there is reason to hope that the disclosure of the secrets of suspended animation will allow us to get closer, albeit very soon, to the cryopreservation of people described in science fiction.
It's worth thinking about
However, even a complete restoration of the vital activity of the body does not guarantee the return of a person to life after thawing. At the moment, we do not have enough information about how the brain forms a person's personality. Proponents of cryonics insist that with the cessation of electronic activity of the brain, its formal death does not occur.
Therefore, if activity is restored after cryopreservation, then the person will return to life as if nothing had happened. Nevertheless, there is no convincing evidence, and it is impossible to conduct an experiment confirming or refuting this. In addition to a technological breakthrough, the realization of the cryonics concept requires the work of neuroscientists and philosophers of consciousness: they will have to develop criteria for the identity of a person so that his full return to life can be guaranteed. Otherwise, we risk getting either a living body without the presence of certain cognitive processes, or some new personality with fragmentary memories of a past life.
About the author: Sergey Amstislavsky – Doctor of Biological Sciences, Head of the Cryopreservation and Reproductive Technologies Sector of the Institute of Cytology and Genetics SB RAS.
Portal "Eternal youth" http://vechnayamolodost.ru