Cryopreservation

Aliyyah Noel

August 9, 2012

Cryopreservation

Over the past three weeks we have covered many topics involving neuroscience, and many more were touched on in Sebastian Seung’s book Connectome. Although I thought many of these topics were engaging, I found cryopreservation to be of particular interest. I was interested in this topic because I find the idea of freezing tissue for use later on fascinating. The possibilities that could sprout from successful reversible cryopreservation are endless. If reversible cryopreservation can be done with nearly 100% success it will open the door for countless applications. Not only will people who want to be reanimated have a better guarantee that a future generation will be able to revive them without much damage having been done to their brain, but organs in the current time could be saved. Cryopreservation could be used to store organs from the deceased for transplantation later on. At this point there is no way of saving harvested organs for extended periods of time, so if an appropriate donor is not immediately available, the organ goes to waste. If these organs could be saved instead of simply wasted, many lives could potentially be saved. In addition to the human organ aspect of cryopreservation are the germplasm and sperm storage aspects. There has been debate about the formation of a Genome Resource Bank to help with the preservation of biodiversity. This would do wonders for the repopulation of endangered species. It would allow scientists to have access to a much larger gene pool. The genes of deceased species would still be available, as well as those from animals in the wild, allowing the repopulated species to not be confined to the genes of a few individuals. Not only would the sperm and germplasm of endangered species be saved, but also those of rats and mice that have been genetically altered for scientific purposes., There are countless new strains of rats and mice that have been created, and more are always in the process of being created, and it is important that these not be lost. They are important for scientific research and advancement, so having a way to save their genetic codes is extremely important. There are many important and world changing applications, but problems still remain related to the best and most efficient way of successful freezing. At this point in time there are two main ways of cryopreserving samples. The favored way is vitrification, but the other way simply involves cooling the sample down extremely slowly. Using this method allows intracellular water to flow out of the cell before turning into extracellular ice. This concentrates the intracellular solution to a point where it does not freeze. The intracellular water osmosis out of the cell due to a difference in chemical potentials. Through the process of cooling the extracellular water ends up with a lower chemical potential than the intracellular water, thereby forcing the water to osmos from high to low concentration. Although this works well to concentrate the intracellular solution, it can damage the cells. In place of intracellular ice is a higher salt concentration, which in itself can be damaging. On top of that the cells shrink in size and gets lodged between the growing extracellular ice crystals. Due to these problems, vitrification is sometimes viewed as the better way to cryopreserve samples. It involves the use of cryoprotective solutes that induce the formation of glass. Vitrification occurs while cooling and heating samples extremely fast. Cooling quickly does not give the water a chance to form the typical crystalline structure of ice, instead forming intracellular glass (with the assistance of the cryoprotective solutes). This glass does not harm the cells like ice does. As Seung said in his book Connectome, “It’s not easy to sort out the complex phenomena happening inside cells during cooling”. Rewarming vitrified samples must be done very quickly as well though, because if it is not devitrification occurs and harmful intracellular crystalline structures form. In the figures below the differences between slow freezing and vitrification on the cellular level can be seen.

Figures from Alcor Life Extension Foundation

As can be seen from the images on the right, simple freezing shrinks and misshapes the cells, while vitrification leaves them whole. What is happening on the molecular level can be seen on the left. Freezing the samples allows the water to form the ice crystals, but during vitrification the water remains locked in place (not in crystals). Although it appears to be a relatively simple procedure, it is not. The cryoprotective solutes are needed in order for vitrification, however they are often times toxic. This then requires knowledge about what concentration of the toxicity cells can handle before being damaged. It is a fine line between a low level of toxicity and not forming intracellular ice. Current research is being done on trying to find better cryoprotective solutes and figuring out the proper concentrations in order for reversible cryopreservation to be successful. In 2005 Alcor created a new cryoprotective solution called M22 that inhibited intracellular ice formation, had very low toxicity, and had a reasonable cooling rate. Further research also includes finding a way to preserve entire organs for use later on, more reliable ways of successfully storing germplasm and sperm of endangered species, and using hypothermic preconditioning. There are still many technical questions that remain unanswered in the study of cryonics, more than are answered at this point. However, the biggest and most all-encompassing of these questions is how to be 100% successful at reversible cryopreservation. This is only one simple question, but it has many different parts. It requires answering more difficult questions such as what the optimal rate of cooling for every cell type for every species is. It also requires scientist to know the proper concentrations of cryopreservation solutes to ensure that the toxicity level is low, but that the cells do not have intracellular ice formation. On top of these difficult to answer questions are even more dynamic problems, such as the best type of cryopreservatives to use. This is very important because it is what allows the cells to be frozen without being harmed. Not only are there technical questions that remain unanswered about the actual process of freezing, thawing, and successfully reusing, but there are also economical questions that remain. Storing all the bodies, tissue, samples, etc. that would be saved using cryonics would take up a lot of space and require a vast amount of money. For example, for Genetic Reserve Banks there is debate about where the money to support them would come from. There is also the question of where the samples would be stored. This then begins to run into political strains between countries and what would be best for the science in general. Aside from these more straightforward questions are ethical questions, especially related to the discipline of freezing human bodies for reanimation in the future. People argue about what the true definition of ‘death’ is. If the person is believed to be brain dead, but are being cryogenically saved for the future, are they dead or should they still be considered living? Some say they are undoubtedly dead, while others argue that because the information in their brain is being saved, they are still living. This debate will probably never be answered, but some of the other questions could be. The problems with the simplest answers will most likely be the ones related to the technical part of reversible cryopreservation, however these also might take the longest to answer. The uncertainty of which cryopreservation solute will work best will take a lot of research. It may be helpful to study, in depth, organisms such as water bears that have the ability to freeze and remain living. If we are able to fully understand how it is that they do this it could be applied to cryobiology. Perhaps they contain some molecule in their cells that allows them to inhibit ice formation. If that is the case it would be a matter of synthetically creating that molecule and seeing if it is compatible with other cell types. The economical and ethical questions are dynamic and have many different possible solutions. As long as people are disagreeing about the definition of death there will be room for debate. Because people love to argue and are often set in their beliefs, and rightly so, there will probably never be a time when everyone has the same definition of what it means to be dead. However the economical questions will hopefully have concrete answers. The money could come from government sources and institution based programs. Because of its possible application with transplants, it could also come from medical research money. As long as there is interest in this field, there will be people willing to fund it. The study of cryopreservation is relatively new, but very important. If the ability to freeze full organs, sperm, and maybe even bodies for future use becomes a possibility, not only will many lives be saved with transplants, but many endangered species could be saved, among other bonuses. There is still a long way to go, lots of research to be done, and things to be discovered about the different aspects of cryopreservation. In time the questions will be answered, the technology will be improved, and the goal of successful reversible cryopreservation will be attained.

Resources

Critser, J.K, and L.E. Mobraaten. "Cryopreservation of Murine Spermatozoa." ILAR Journal. 2001. Web. 2 Aug 2012. . De Wolf, Aschwin. "Neural Cryobiology and the legal Recognition of Cryonics." Institute for Evidence Based Cryonics. (2011): Web. 2 Aug. 2012. . What is Vitrification?. Graphic. Alcor Life Extension Foundation. Web. 2 Aug 2012. . Wildt, David E.. "Genome Resource Banking for Wildlife Research, Management, and	 		 conservation." ILAR Journal. 2001. Web. 2 Aug 2012. http://dels-old.nas.edu/ilar_n/ilarjournal/41_4/Genome.shtml