In a new study, researchers have vitrified mouse brain slices and then a complete brain with encouraging results: upon rewarming, much of the neuronal function was preserved [1].
The bumpy road to cryopreservation
Successful cryopreservation is a coveted prize in medicine, as cryopreserving organs and tissues can make transplantation more accessible. It is also a hot (or rather very cold) topic in the longevity community, where many people see it as the last resort if aging is not completely defeated during our lifetimes. While several companies have operated in this field for years, and the number of cryopreserved humans is now in the hundreds, cryopreservation remains a huge leap of faith, as there is no currently reliable way to freeze and rewarm a human body or even the brain.
Recent years have seen successes with rat kidneys (with subsequent life-sustaining transplantation) [2], livers [3], and hearts [4]. However, the brain had never been shown to recover function after cryopreservation. In a new study from Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU) and Uniklinikum Erlangen in Germany, published in the journal Proceedings of the National Academy of Sciences, the researchers set out to change that.
The central obstacle to cryopreserving any tissue is ice. When water freezes, expanding ice crystals physically rupture cell membranes, shear synaptic connections, and destroy tissue architecture. Traditional freezing of adult brain tissue has repeatedly failed to preserve synaptic function.
“The formation of ice crystals is the reason why extreme cold is usually so harmful to living beings,” explains Dr. Alexander German from the Department of Molecular Neurology at Uniklinikum Erlangen. “This is because the crystals can mechanically damage cells, thereby destroying the sensitive nanostructure of the tissue.”
A technique named vitrification (from the Latin vitro, meaning “glass”) offers a theoretical solution. Instead of letting water freeze into crystals, much of the tissue water is replaced with cryoprotective agents (CPA), such as dimethyl sulfoxide, ethylene glycol, and formamide. At sufficiently high concentrations, with rapid enough cooling, the aqueous phase turns glass-like rather than into ice, preventing mechanical disruption. Vitrification basically stops all molecular processes and preserves the current state of the tissue virtually indefinitely.
Structure and function mostly intact
The team designed a proprietary vitrification protocol and tested it first on mouse brain slices. After vitrification, slices were stored in liquid nitrogen at −150 °C for 10 minutes to 7 days and then rewarmed. Tweaking their vitrification solution recipe, the team was able to completely avoid crystallization.
The question became whether these slices were actually alive and functional. To test whether mitochondria still functioned, the authors measured oxygen consumption rate in the CA1 region of the hippocampus. In the optimal CPA concentration, they found a 22% reduction in basal respiration versus fresh controls – mild but not negligible. However, the entire slump in mitochondrial function came from CPA toxicity rather than from the vitrification/rewarming process, which was demonstrated when the researchers used CPA loading and washing out without vitrification/rewarming. This suggests that safer CPA cocktails could further improve the results.
Mitochondrial respiration is a coarse measure of viability, and the researchers needed to determine whether the fine structure – synapses, dendrites, membranes – survives. Electron microscopy of ultrathin sections from the CA1 region revealed clear membranes and intact neuronal and synaptic structures. Quantitative analysis of dendritic spine density and spine length showed no differences between control and post-vitrification slices.
The synaptic structure seemed intact, but was it working? The team found that basic synaptic transmission was preserved but modestly attenuated. Short-term plasticity (STP) – brief, transient changes in synaptic strength – was attenuated in postvitrification slices but not due to neurotransmitter availability, which was identical in all three groups.
The crucial test was long-term potentiation (LTP), the activity-dependent, persistent strengthening of synaptic connections, widely regarded as the cellular mechanism behind learning and memory. If LTP survives vitrification, it means the molecular machinery for encoding new memories remains operational. Cryopreserved slices reliably produced LTP, and in one particular synapse type it was actually stronger than before vitrification. However, this is not necessarily a good sign, and the authors currently don’t have an explanation for this effect. Single-neuron excitability was also largely preserved, although that differed somewhat across cell subtypes.
Full brain vitrification achieved?
Having established that brain slices survive vitrification, the authors scaled up to the whole mouse brain, a substantially harder problem because CPA must be delivered through the vasculature, which means crossing the blood-brain barrier. This caused a major complication: when the CPA solution was perfused through the vasculature, water exited the brain faster than CPA entered, causing catastrophic dehydration and physically shrinking the brain. The imperfect solution that the team found was partial rehydration between CPA loading pulses.
After craniectomy, brains were vitrified in situ, stored at -140°C for 1 to 8 days, rewarmed, and had CPA washed out. The success rate, however, was less than stellar: one out of three iterations of the final protocol produced tissue suitable for physiological evaluation. The good news is that most functional metrics were preserved after rewarming, although the team only analyzed the neuron subtype that fared best in the slice experiments – granule cells in the dentate gyrus (DG) region of the hippocampus – so we currently don’t know whether other neurons fared just as well.
While the road to successful cryopreservation of a full organism or a large brain is still long, this is an encouraging proof-of-concept study. German has high hopes for their discovery: “This could be an option for space travel, for example, or for people suffering from a currently incurable disease, because at a later date, there may be a treatment option that can help the person affected.”
Literature
[1] German, A., Akdaş, E. Y., Flügel-Koch, C., Erterek, E., Frischknecht, R., Fejtova, A., … & Zheng, F. (2026). Functional recovery of the adult murine hippocampus after cryopreservation by vitrification. Proceedings of the National Academy of Sciences, 123(10), e2516848123.
[2] Han, Z., Rao, J. S., Gangwar, L., Namsrai, B. E., Pasek-Allen, J. L., Etheridge, M. L., … & Finger, E. B. (2023). Vitrification and nanowarming enable long-term organ cryopreservation and life-sustaining kidney transplantation in a rat model. Nature communications, 14(1), 3407.
[3] Sharma, A., Lee, C. Y., Namsrai, B. E., Han, Z., Tobolt, D., Rao, J. S., … & Finger, E. B. (2023). Cryopreservation of whole rat livers by vitrification and nanowarming. Annals of biomedical engineering, 51(3), 566-577.
[4] Chiu-Lam, A., Staples, E., Pepine, C. J., & Rinaldi, C. (2021). Perfusion, cryopreservation, and nanowarming of whole hearts using colloidally stable magnetic cryopreservation agent solutions. Science advances, 7(2), eabe3005.
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