How Is Cryopreservation Currently Used?

What if you could choose how long you lived? The goal of human cryopreservation is to preserve patients for the future and give them a chance(!) to be revived once medical technology has advanced enough to treat them. For a deeper understanding of the process involved, this article will define what cryopreservation is and how it’s currently used. 

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What is Cryopreservation? 

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Cryopreservation is the process of preserving cells, tissues, and other biological materials by lowering core temperatures to sub-freezing levels (usually at -196°C) without ice formation. This reduces metabolic rate to a point where biological activity is completely paused. The most common coolant for cryopreservation is liquid nitrogen, which has a natural boil-off temperature of -196°C. Cryoprotective agents and vitrification allow cells to be preserved virtually indefinitely. 

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Cryoprotective agents (CPAs) are a type of medical-grade antifreeze that helps reduce the formation of ice crystals. Vitrification is the transformation of a substance into a glass-like amorphous state. This occurs once cells pass the glass-transition temperature of e.g. -125°C, where they are solid but not frozen. This is one of the final steps in the cryopreservation procedure. It’s the point where all biological processes stop, allowing cells to be preserved indefinitely without decay or damage. 

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Current Applications of Cryopreservation

While medical research is constantly progressing, it’s not yet possible to revive a human after they’ve been cryopreserved. However, there are several cells and tissues that have been successfully cryopreserved and rewarmed. These include early human embryos, sperm, eggs, skin, bone, red and white blood cells, bone marrow, and more. An entire rabbit kidney was even vitrified, cryopreserved, re-warmed, and transplanted back into a rabbit where it demonstrated a full functional recovery. Let’s explore more of the current applications of cryopreservation below. 

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Sperm Cryopreservation 

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The first successful preservation of mammalian cells occurred by chance in 1949 when Christopher Polge and his colleagues (Smith and Parks) were studying the effect of glycerol on rooster sperm mobility. Polge was using liquid nitrogen to freeze the control samples in his lab, but one day accidentally added the liquid nitrogen to a sperm sample that contained glycerol. When he rewarmed the sample, he noticed that the rooster sperm regained mobility, thus discovering that sperm could be cryopreserved without cellular death. 

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This sparked several comprehensive reviews of sperm cryopreservation, which eventually led to the techniques used in sperm banking or sperm freezing today. Semen can be cryogenically stored indefinitely and used for sperm donation. Once warmed, sperm regain viability and motility. 

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Sperm cryopreservation has a wide range of applications but is most commonly used to help an individual (or couple) conceive a child. This includes applications for women without a male partner or couples experiencing male infertility. 

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Red Blood Cell Cryopreservation 

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The following year, in 1950, Smith extended their findings on sperm cryopreservation to examine the effect of vitrification on red blood cells in glycerol. He found that in rabbit and human blood diluted with a glycerol solution, the red cells did not undergo the rupture usually caused by freezing and thawing. This research led him to successfully cryopreserve human red blood cells. 

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This study was extended in 1951 when Sloviter found that rabbit red cells recovered after extended storage survived and were successfully reintroduced into circulation. After further research, Sloviter found that human red cells that were cryopreserved and rewarmed using glycerol were viable for transfusion. 

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The cryopreservation of red blood cells continued to evolve over time and is now used for many purposes throughout the medical community. Red blood cell cryopreservation is a common practice that allows rare blood types to be stored from donors for patients who need it in the future. Currently, units of red blood cells that have been cryopreserved can last 30 years or more. 

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Oocyte Cryopreservation 

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In 1952, researchers and scientists made a breakthrough in the cryopreservation of oocyte germ (egg) cells. Chang and his colleagues began exploring the effect of low temperature on the survival of a mammalian oocyte (rabbit). Similar studies continued to test the viability of oocytes after being warmed and found that suspending the germ cells in a glycerol mixture and cryopreserving them resulted in successful pregnancies. 

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Research slowed for oocyte cryopreservation until the late 1970s when the first mice were produced from cryopreserved and rewarmed oocytes. While mammalian oocytes are more susceptible to damage during cryopreservation, Steponkus and Mazur eventually found success with extremely rapid cooling rates [3]. The first successful human pregnancy following oocyte cryopreservation occurred in 1986.

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Now, oocyte cryopreservation is commonly used for in vitro fertilization (IVF). Women can freeze their eggs to postpone pregnancy for several reasons. If they choose not to use them in the future, they have the option of donating them to others. 

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Embryo Cryopreservation 

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Following the cryopreservation of individual sperm and oocytes, embryo research began. The first mammalian embryos to be successfully cryopreserved were that of a mouse in 1972. During their research, Leibo and Mazur studied the effect of slow cooling rates on ice formation within embryonic cells. They found that "in order to successfully cryopreserve embryos to lower temperatures, the embryos must be frozen at rates slow enough for water to move out of the cell before it crystallizes into ice" [1].

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A year later, cryopreserved embryos were used to produce Frostie. Frostie was a calf born in 1973 from a cryopreserved embryo that was warmed and implanted into a surrogate cow. This breakthrough accelerated the development of techniques used for in vitro fertilization today.

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Later, in 1983, the first successful human embryo was cryopreserved. While this embryo did not survive until birth, it occurred quickly after. In March 1984, Zoe Leyland was born in Australia from an embryo that had been cryopreserved, warmed, and implanted. 

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Since the mid-1980s, embryo cryopreservation has been an important component in human-assisted reproduction techniques. The cryopreservation of embryos involves preserving an embryo (usually at the stage corresponding to pre-implantation) at sub-zero temperatures. This is a common practice in fertility treatments such as in-vitro fertilization. If a couple becomes pregnant, leftover embryos that have been cryopreserved can be donated to others. Cryopreservation has not been shown to increase the rate of birth defects or lead to developmental abnormalities.

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Another interesting use of embryo cryopreservation occurs in the livestock industry. It’s used to improve conservation efforts. In 2005 alone, over 370,000 frozen-thawed bovine embryos were transferred worldwide. However, live offspring has only been produced in a few species, notably in rabbits, cattle, felids (cats), primates, and ungulates (hoofed mammals like deer).

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Stem Cell Cryopreservation 

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Stem cells are unspecialized cells that have yet to develop their specific function. They can become brain cells, bone cells, blood cells, heart cells, etc. Due to this diversification, stem cells can be used to treat a wide range of diseases. The cryopreservation of stem cells involves harvesting donor cells, adding cryoprotective agents, rapidly cooling the cells, assessing viability after 72 hours, rewarming cells, then washing and conditioning for transplant. They can be used to treat both malignant and non-malignant diseases. 

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There are several cryopreservation methods used for stem cells, many of which vary in freezing temperature, rate, CPAs, and rewarming factors. However, stem cells have been undergoing cryopreservation for years as it’s a key element in the successful delivery of cell-based therapies. 

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Organ Cryopreservation 

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Organ cryopreservation has been continually studied since its inception in the 1960s. One of the first notable studies examining cryopreserving organs was done in Japan when Isamu Suda and colleagues perfused a cat brain with a glycerol solution, chilled it to -20°C for over six months, and then rewarmed it. After examination, they found recognizable brain waves—an exciting development for cryonics!

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More systematic studies relating to organ cryopreservation began in the 1980s and 1990s, when cryobiologist Gregory M. Fahy and his colleagues began exploring the process of solidification without ice formation for organ preservation. His findings, published in 2003 and 2006, deemed him the world’s foremost expert in organ cryopreservation by vitrification. 

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From the early days of vitrification research, Dr. Fahy and his colleagues set out to learn more about cryoprotectant toxicity and how to mitigate it using vitrification solutions. In 2005, his research culminated in the "vitrification (to -135°C), rewarming, and transplantation of a rabbit kidney with good viability and functionality" [2]. 

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This research has sparked several other studies in the field of cryonics. Most notably were the findings of Robert McIntyre and his team in 2016. Their research resulted in the first entire mammalian brain being cryonically preserved and recovered in “near perfect” condition. After gently rewarming it and flushing out the CPAs, it was discovered that cell membranes, synapses, and intracellular structures remained intact [5]. 

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The process of cryopreserving organs could have a substantial impact on the transplant community. For example, as stated in PNAS, “current cryopreservation tools are already being applied to help revive damaged organs… and stretch the amount of time a transplant organ can be preserved“ [4]. Discovering a method for long-term organ banking through cryopreservation could further extend the time organs can be maintained before transplant or surgery and save thousands of lives. 

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While there have been successful cases of cryopreserving and rewarming animal organs, there are still challenges (like toxicity) that need to be addressed before human use. 

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Pet Cryopreservation 

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Currently, there are over 85 pets cryopreserved, some of which include dogs, cats, turtles, hamsters, parrots, a turtle, a chinchilla, and even a monkey. The process is similar to the cryopreservation of cells, just on a larger scale. Using vitrification, the animal’s body is cooled until it reaches a glass-like amorphous state. They can then be put into a dewar and preserved indefinitely.

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The main difference between pet (and human) cryopreservation, when compared to the applications above, is that the technology to rewarm them does not yet exist. Researchers don’t guarantee anything and they don’t know if it will work, but cryopreserving pets is not something that lacks foundation. There’s also no scientific evidence that proves technology and treatment that can rewarm pets, address their illnesses, or reverse aging will never exist.

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Human Cryopreservation 

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The same mentality applies to human cryopreservation. Cryoprotectant agents are introduced into the body by perfusion via the circulatory system. These CPAs replace the water in the body and reduce the freezing point of the remaining liquid while minimizing the formation of ice crystals. Once vitrification occurs and the patient is in a glass-like amorphous state, all biological processes are put on pause, allowing for preservation without decay or damage. 

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Like cells, the combination of cryoprotective agents and vitrification allows patients to be preserved indefinitely. There are some limitations to human cryopreservation, but - as one example - several cryopreservation researchers (including us) are currently working on a system that would allow patients to be stored at higher temperatures (less thermal stress). 

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Scientists can currently cryopreserve patients, but there’s no technology to rewarm them. Still, James Bedford was the first person to be cryopreserved in 1967 and has been stored at Alcor since 1991. Today, there are roughly 500 patients cryopreserved with ages ranging from 2 to 101. While revival is not yet possible, the preservation process allows them to be stored indefinitely. 

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Today, cryopreservation is predominately used for the preservation of cells, but scientific, technological, and medical research continues to advance. While the technology to revive a human after being cryopreserved has not been developed yet, there’s no fundamental biological reason why revival would not eventually be possible.

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Limitations of Cryopreservation

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Since cryopreservation pauses biological activity, there will need to be an external force to reactivate it and revive patients. One of the biggest limitations in cryopreservation today is the fact that this type of technology does not yet exist. The technology to rewarm tissues homogeneously or treat aging and disease also does not yet exist. That’s not to say it never will though. Advancements in nanotechnologies and nanowarming could help us with this. 

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The world has come a long way in 100 years and current cryopreservation applications in science and medicine demonstrate that technology is still advancing. Think about it this way: there’s currently no evidence that suggests cryonics won’t work in the future. Just because someone can’t be saved today, doesn’t mean they can’t ever be saved. So, consider reframing your mindset—some diseases are just not treatable… yet. 

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Another challenge is that vitrification can result in toxicity for complex tissues, especially in regard to organs. Cryoprotective agents used are often a combination of penetrating and non-penetrating solutions. Penetrating CPAs prevent the formation of ice crystals inside the cells, while non-penetrating CPAs prevent the formation of ice crystals outside of cells. The challenge is that the higher the concentration of CPAs, the more toxic the solution is. Alternatively, if the concentration of CPAs is too low, complete organ vitrification can’t be achieved. 

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Overcoming this challenge is an important factor in the ongoing advancements of cryopreservation and successful revival.

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The Future of Cryopreservation

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For decades, scientists have been successfully cryopreserving and rewarming parts of the body, but patient revival is still not currently possible. While we’re unable to predict exactly when revival could be feasible, patients will remain cryopreserved for however long it takes. Whether it’s 10, 50, 100 years, or longer, there’s no time limit for how long a person can be cryopreserved.

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Conclusion

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Nobody knows what will happen in the future, but what we do know is that you can be cryopreserved today. Cryonics is currently the only technology that could bring you to that future, so what have you got to lose?

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 References

[1] Schnebly, R. A. (2020, October 1). The Embryo Project Encyclopedia. "Survival of Mouse Embryos Frozen to -196 ° and -269 °C" (1972), by David Whittingham, Stanley Leibo, and Peter Mazur | The Embryo Project Encyclopedia. Retrieved July 20, 2022, from https://embryo.asu.edu/pages/survival-mouse-embryos-frozen-196-deg-and-269-degc-1972-david-whittingham-stanley-leibo-and  

[2] de Wolf, A., & Platt, C. (2022, May 23). Human Cryopreservation Procedures. | Alcor. Retrieved July 20, 2022, from https://www.alcor.org/docs/cryopreservation-procedures-book.pdf 

[3] Borini, A., & Coticchio, G. (2010). Preservation of human oocytes. CRC Press. Retrieved July 20, 2022, from https://thaisrm.com/docs/Ri%20Chian%20Book/Preservation%20of%20Human%20Oocytes%20-%20From%20Cryobiology%20Science%20to%20Clinical%20Applications%20copy.pdf 

[4] Scudellari, M. (2017, December 12). Cryopreservation aims to engineer novel ways to freeze, store, and thaw organs. PNAS. Retrieved July 20, 2022, from https://www.pnas.org/doi/full/10.1073/pnas.1717588114 

[5] Crew, B. (2016, February 10). A Mammal’s Brain Has Been Cryonically Frozen And Recovered For The First Time. ScienceAlert. Retrieved July 20, 2022, from https://www.sciencealert.com/a-mammal-s-brain-has-been-cryonically-frozen-and-recovered-for-the-first-time