Avoiding the formation of ice crystals at the extreme temperatures required for cryopreservation is vital. Ice crystals can destroy cell membranes and damage the integrity of the body. The best way of deterring ice is by using cryoprotectants.
Cryoprotectants, cryoprotective agents or CPAs are a type of medical-grade antifreeze that is introduced to the body through a process called perfusion. This allows for vitrification of the body after further cooling - a key process for high quality cryopreservation.
Finding the right balance of CPAs is important because of their varying levels of toxicity at certain concentrations. Cryonicists are constantly fine-tuning the mixture to enable the best cryopreservation with as little toxicity as possible.
This article will help you better understand what cryoprotectants are and how they work.
Water freezing within the body causes two types of damage: mechanical and chemical. Cryonics researchers aren’t fans of either. Mechanical damage is the distortion of cell structure and shape caused by the formation of ice crystals. These ice crystals are extremely sharp and can cut through cell membranes and other tissue around it. We want to prevent this from happening during cryopreservation at all cost as it would otherwise contribute to cellular death.
Chemical damage, on the other hand, is caused by the exclusion of molecules when water freezes. Usually, water inside a living organism is part of a solution that consists of many different molecule types. As the water molecules freeze, they seek eachother out and form a pure substance, pushing all other molecules away. This leads to a high concentration of damaging solutes in the remaining unfrozen water.
To avoid this, cryoprotectants are deployed which in turn allow for vitrification in cryopreservation. Vitrification is the transformation of a substance into a glass-like amorphous state; this happens at around -130°C, the so-called glass transition temperature. Cryoprotectants hold molecules in place until vitrification can occur, which increases cell survival significantly compared to freezing.
Cryoprotectants behave similarly to antifreeze chemicals like the ones you add to your car when the outside temperature drops below zero. They dissolve in water and help lower its freezing point. However, CPAs are a specialised medical-grade variety of these chemicals.
Widely used CPAs are glycerol, ethylene glycol, propylene glycol, and dimethylsulfoxide (DMSO). Of these CPAs, there are two main classes of cryoprotectants:
Penetrating CPAs are regularly used together with non-penetrating CPAs because ice forms more readily extracellularly than intracellularly. With the presence of non-penetrating CPAs, penetrating CPAs do not need to be so concentrated. This is crucial for high quality cryopreservation since the higher the concentration of penetrating CPAs, the more toxic the solution is.
Water molecules inside the body need to be replaced with cryoprotectant molecules for successful vitrification. However, at higher concentrations, CPAs become increasingly toxic.
The level of CPA toxicity changes at different temperatures. To reduce toxicity during perfusion, CPAs concentrations are increased at a slow rate, while temperatures in the body are lowered. This is done because CPAs administered at near-freezing have a much lower level of toxicity than those same chemicals at warmer temperatures.
Cryoprotectants are not toxic at cryogenic temperatures. They only become toxic upon rewarming. This means that upon eventual revival, CPAs would need to be removed extremely quickly to avoid cell death. This poses an issue that future medical technology will need to find a solution for. Looking at current medical progress, we are optimistic that future technology could find a way to overcome these challenges and allow for revival from biostasis.
Scientists are aware of the limitations of cryoprotective agents, and are actively working towards counteracting, minimising and eventually removing their toxicity. Currently, there are a few things that can be done to lower CPA toxicity:
Research has found that different CPAs have differing levels of toxicity - depending on the situation they’re used in. We know that there are certain combinations of CPAs that do reduce overall toxicity.
Scientists are currently investigating the optimal combinations of CPAs.
Scientists are trying to find alternative cooling methods for cryopreservation. One such example is the deployment of cold gas to cool down animal organs for cryopreservation without vitrification. The results showed that cold helium cooled a pig kidney down to −180°C without fractures and suggested that using 20 atmospheres of pressure could allow for significantly faster cooling rates.
Cooling rates as rapid as this could reduce the exposure time of CPAs. This would therefore reduce the toxicity - as long as damage such as cold shock can be avoided.
Certain CPAs release chemicals that can cause cellular death. By introducing inhibitors into warmed cells, this can be avoided. In addition, certain CPAs’ toxicity can be reversed with gene expression changes (achieved through Nanotechnology).
The hope is that as our understanding of the toxicity of cryoprotectants improves, we will be able to counteract their effects more effectively.
Cryoprotectants, although toxic at certain concentrations, play a crucial role in cryopreservation of all biological materials, from tissue, sperm, eggs, organs and human beings. Cryonics organisations understand that too. Therefore, scientists involved in biostasis research are constantly investigating ways to improve current methods. Discovering improved mixes of CPAs with lower toxicity is one of our R&D objectives at Tomorrow Bio. We are the fastest-growing cryonics organisations in Europe, and advancing biostasis research is one of our main goals.
As our knowledge of the behaviour of cryoprotectants increases, we will be able to understand and remove the detrimental effects they have on the body. Once researchers grasp this, revival after cryopreservation will become far more likely.
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