Brain function after cryopreservation

[Music] Welcome to Science Sessions, the podcast of the proceedings of the National Academy of Sciences, where we connect you with Academy members, researchers, and policymakers. Join us as we explore the stories behind the science. I'm Paul Gabrielson. Storing biological samples at very low temperatures can preserve them from decay. It's called cryopreservation. But the process of freezing samples, including formation of ice in cells, can damage sensitive structures within those tissues. That's particularly true for brain tissue. In a recent PNAS study, Alexander German of Universitat Erlangeneurinburg and colleagues developed a different approach to brain tissue cryopreservation. It's called vitrification. And instead of freezing the tissue, the process turns the tissue into a glass-like state. The authors vitrified thin sections of mouse brain for cryopreservation. They introduced cryoprotectant chemical solvents that inhibited ice formation as the tissue was cooled. After rewarming, tests showed that key brain structures had been preserved. Alexander, let's start with some background. What are the problems with traditional cryopreservation methods? So the core problem with traditional cryopreservation methods is ice formation. Ice crystals solidify the tissue water into a regular lattice that displaces the fine structure of complex 3D tissues like brain tissue and preclude the recovery of function thereafter. So while there are examples from nature, including freeze tolerant amphibians like the Canadian Wood Frog and the Siberian salamander, that tolerate near-whole body freezing with crystallization down to minus 50 degrees Celsius. This has not been successfully translated to the mammalian organism. In the 1960s, 70s, there have been attempts to apply traditional freezing methods from cryopreservation to organs. And without much success, for instance, in the brain freezing methods result in the over-gloss of synaptic connections. Why do we cryopreserve tissue? Cryopreservation is a method to stabilize tissues, to distribute them across time and space. So for instance, in nature, it is used as a method to endure adversarial environmental conditions like winter and in research, it is a method to permanently preserve cell lines and utilize them on demand and also more complex products like cell constructs, organoids, tissues or even one vision as organ banking to have cryopreserved human organs on demand to simplify or improve a transplantation logistics and improve donor recipient matching to make sure that the immunological fingerprint of the organ and size and other properties is really the right match for the recipient and make the procedure planable for the surgeons to improve the safety of the surgical procedure. Tell us about vitrification and why it's different than traditional cryopreservation. Vitrification is different from traditional cryopreservation because it is ice-free. This has been introduced to cryobiology in the 1980s by Greg Fahey and colleagues in mouse embryos. So by using cryprotectants and rapid cooling and re-warming, the tissue solidifies into a non-chrysaline glass like state and this is leaving the nanostructure of the tissue intact so there are not much effects of the cooling and re-warming procedure. However, the avoidance of ice crystal formation is traded against higher cryoprotectant toxicity and osmotic stress for the tissue. What is osmotic stress? And why is that a concern in cryopreservation? Cellos and tissues are filled with water and the water is flowing across gradients of its chemical potential. So it will diffuse to places where its chemical potential is lowest and the addition of cryoprotectants is reducing the chemical potential of water. So when the cell is exposed to less permeable than water cryoprotectants, the cell will shrink and when the cryoprotectants to be washed out, the reverse will happen and the cell can swell or even burst which would be a verse case. How did your protocol minimize damage to brain structures and tissues? The protocol is really about balancing several forms of damage at once. Not just preventing ice as in traditional freezing. It involves step-wise loading of chemicals at defined temperatures and concentrations for defined durations. Then it involves directional cooling to avoid cracking from thermal mechanical stress after the glass transition has occurred and also rapid cooling to avoid ice formation and rapid re-warming to avoid ice growth during re-warming and then reverse. Unloading washout of these cryoprotectants at defined concentration, temperature and durations using an osmotic buffer to reduce the excess cellular chemical potential of water to avoid self-swelling beyond what the cytoskeleton can tolerate. We are mainly working with 350 micro meter brain sections because there it is easy to access the excess cellular space from all sides via diffusion. This works very well. We also developing whole brain vitrification protocols, however this is complicated by the blood brain barrier which is exacerbating the mismatch between cryoprotectant and water conductivity and therefore resulting in more profound shifts, risking brain dehydration and brain edema. So here substantial optimization is still required. Tell us about the tests you perform to evaluate the preservation and function of the vitrified tissues. We looked at structure, metabolism, single cell, excitability and circuit level plasticity. Recovery showed up on all of these levels. My cross-coupie showed intact synopsis and dendroids. Oxygen consumption showed mitochondrial activity. Electrophysiology showed neurons firing and synopsis communicating. We also induced long-term potentiation which is the cellular mechanism for learning and memory. What's the usefulness of this method? What does it enable researchers to do that they couldn't before? Vitrification turns brain tissue preservation from a purely structural possibility to a functional possibility. So the immediate research implication of preserving viable tissue is that experiments are unrestricted by a time or location. This improves reproducibility, could reduce animal use and make many areas of the human neocortex available for electrophysiological and other types of experimentation for the first time. So it could advance basic neuroscience, also neuropharmacological drug testing and also other types of research that heavily depend on realistic human brain tissue that has been exposed to environmental factors and mature within the skull for decades. This is something that you cannot easily model in a tissue if you compare it for instance to a cancer cell line, a cancer cell line will grow easily in a cell culture incubator. But growing realistic 3D human brain tissue is still out of reach and Vitrification could transform this research on brain tissue slices from a geographically and temporarily constrained opportunity to a more on demand, reliable, widely distributed resource. We're currently launching a spin-off company. It's called Hyber to make this technique available to many more labs and make human brain tissue available to many more labs to translate the insights from our research. What are the caveats or limitations of the study? What we showed is a short-term recovery of brain slices. So we did not reanimate the whole brain. We crop reserve the whole brain with low yield protocol and showed recovery in slices. So we did not revive a conscious brain. Just preserving the brain structure does not allow for functional recovery with our present techniques. However, there are some concepts that the preservation of brain structure might eventually enable recovery of brain function in the distant future. However, this cannot currently be foreseen. So this is a proof of principle. This is not a ready-made route to preserve whole human brains. The acute slices degrade naturally after 10 to 15 hours. So our observation window is limited. This is mouse tissue, not human tissue and it's not a whole conscious brain. And scanning this up to larger organs or even human organs is currently beyond the limits of heat transfer and so a crappy technique in toxicity. for the methods from our study. 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