Introduction to Aldehyde-Stabilized Cryopreservation

Table of Contents


Written by:

Robert McIntyre

Aldehyde-stabilized cryopreservation (ASC) is a technique which preserves brains for use in connectomics research. I developed ASC while working at 21st Century Medicine during 2015, with the goal of winning the Brain Preservation Prize. ASC can preserve every single delicate synaptic connection in a whole brain for centuries without any decay. Our best understanding of neuroscience suggests that synaptic connections are the physical basis of long-term memory storage. By preserving all of a brain's synaptic connections, ASC raises the question of human brain preservation from mere academic speculation to serious scientific inquiry.

High-level overview of ASC

ASC consists of multiple steps which lock the brain's molecules into place and prevent them from decaying over time. First, ASC uses glutaraldehyde to almost instantly stabilize the brain against natural decay processes. By itself, glutaraldehyde is able to stop decay for at least several weeks, but we need more powerful stabilization to resist decay for centuries. In the second step, we use ethylene glycol to make it impossible for dangerous ice crystals to form in the brain no matter how cold it becomes. Then the brain can be safely cooled to -135°C (a temperature colder than anything which naturally occurs on Earth!) for long term storage. At -135°C, the molecules in the brain stop moving, forming a solid, "glassy" structure in a process called vitrification. Vitrification stops all decay processes and allows the brain to be stored for centuries with no degradation.

ASC quickly arrests the natural decay process with glutaraldehyde


Figure 1: Chemical structure of glutaraldehyde. Glutaraldehyde acts like molecular "handcuffs", linking proteins together into a solid gel.

Glutaraldehyde is part of a family of aldehyde fixatives including formaldehyde, acrolein and glyoxal, all of which have been used in neuroscience to preserve brain tissue. All aldehyde fixatives have reactive aldehyde groups which can bind to proteins and create molecular crosslink bridges between them. Glutaraldehyde is a dialdehyde, which means it has two aldehyde groups, each of which is able to bind to proteins. Glutaraldehyde can also bind to itself to make chains. You can think of glutaraldehyde as a pair of molecular "handcuffs" — each aldehyde end is a "cuff", and the connecting carbon molecule is the "chain". When you expose brain tissue to glutaraldehyde, the glutaraldehyde rapidly binds to everything it can, transforming the brain from a soft, watery consistency to that of soft rubber. This process is called "fixation" and has been used for decades to preserve biological samples including brains, various organs, and even entire animals.

One of the reasons that brains deteriorate after death is that without a steady supply of energy and oxygen, the brain's metabolic processes go out of control, and the brain essentially digests itself. Glutaraldehyde stops all metabolic processes within seconds of contact and prevents runaway metabolism from damaging the brain's structure. The process of fixation also confers immense strength to the brain and makes it extremely resistant to changes in temperature, pH, and osmolarity. I've personally found that fixed brains can be stored for weeks at 4°C in a refrigerator with no structural degradation.

Glutaraldehyde and other chemicals are delivered via vascular perfusion

While glutaraldehyde is generally recognized as the "best" fixative for fixing biological tissue, it has one problem – it doesn't penetrate tissue very quickly. Glutaraldehyde penetrates tissues at around 0.75 millimeters / hour, which is far too slow to preserve a whole brain effectively simply by placing the brain in fixative solution. For example, it would take glutaraldehyde around 1 day to fully penetrate a whole rabbit brain. By that time, the core of the brain would have suffered severe degradation.

To get around glutaraldehyde's penetration problems, in ASC we deliver glutaraldehyde through the animal's blood vessels in a process called perfusion fixation. Every cell in a brain needs a steady supply of nutrients and oxygen, which are delivered by the bloodstream. Neurons are therefore never very far away from the nearest blood vessel; the average distance between a neuron and its closest capillary is less than 50 microns, meaning that you can fully deliver glutaraldehyde to every cell in the brain via perfusion fixation in less than 4 minutes.


Figure 2: This is a diagram showing the fixation stage of rabbit brain perfusion fixation. Glutaraldehyde is pumped into the rabbit's carotid arteries, where it circulates through the rabbit's brain and head, rapidly fixing the brain's structures and protecting them from decay.

ASC uses vitrification to guarantee centuries of storage time

Once the brain is fully fixed with glutaraldehyde, it is protected from decay for weeks, months, or perhaps even a year or two depending on how carefully it is stored. However, glutaraldehyde only binds proteins together and does not interact with lipids (a fatty substance which serves as the major structural component of cell membranes and myelin). Without further stabilization, these lipids will degrade over time. Fixed brains that have been stored for years often form a thick "film" at the top of their containers, made of lipids that have slowly migrated from their original positions in the brain due to the slow thermal action of water molecules.

In order to store the brain for longer than a century, we need a way to comprehensively stabilize all of the brain's structures against the random movement of water molecules. Extreme cold is one excellent way to do this, because for every 10°C decrease in temperature, the brain will last for twice as long without decay. This exponential increase in storage time adds up fast! For example, one week of cold storage storage at 4°C, a standard refrigerator temperature, is equal to 314 years at -135°C! Since glutaraldehyde allows brains to survive for much longer than a week at 4°C, if we could get the brain to -135°C, then the immense cold would allow us to store the brain for centuries without damage.

Unfortunately, a fixed brain is still mostly composed of water, and if you cool it down to -135°C, ice crystals will crush the brain's delicate neurons and synaptic connections, causing massive structural damage.

ASC avoids ice crystals in brains the same way you avoid them in your car during winter — with antifreeze. In ASC, we use a mixture of phosphate salts, ethylene glycol, and glutaraldehyde as a powerful cryoprotectant (antifreeze) solution. Ethylene glycol is the same chemical used in automotive antifreeze solutions and works by disrupting the hydrogen bonds between water molecules so that they can't link together to form ice crystals. The more ethylene glycol there is in a solution, the colder it can get before ice crystals form. However, once the concentration of ethylene glycol is high enough (around 55% weight / volume), ice crystals will never form, regardless of how cold the solution gets. Instead, as the solution gets colder it becomes more and more viscous until it becomes a vitreous (glass-like) solid, a process called vitrification.


Figure 3: Chemical structure of ethylene glycol. At high enough concentrations, this cryoprotectant can completely suppress ice formation regardless of temperature. ASC uses 65% weight/volume ethylene glycol, guaranteeing that preserved brains will vitrify instead of freeze.

There's no exact dividing line between a "solid" glass and an extremely viscous solution. Solutions that can vitrify become exponentially more viscous the colder they get. Eventually it might take years or even centuries for a very cold solution to flow at all! The glass in your windows is actually a vitrified solution of silicates, and if given enough time it would eventually "melt" into a puddle. (However, you would have to wait many trillions of years for this to happen in practice.) ASC's 65% ethylene glycol-based antifreeze solution becomes solid for all intents and purposes at -122°C (13°C warmer than the storage temperature of -135°C).

Ethylene glycol is normally very damaging to living brain tissue, causing immense dehydration and structural damage and generally "melting" the brain. Brain tissue fixed with glutaraldehyde is a different story; the glutaraldehyde fully protects the tissue from the destructive effects of ethylene glycol and allows us to completely cryoprotect the brain. Glutaraldehyde also buys us enough time to slowly add ethylene glycol to avoid mechanical disruption of the brain as water is gradually replaced with ethylene glycol.

In ASC, we add cryoprotectant slowly using a gradient former (Figure 4), a simple mechanical device which ensures that cryoprotectant is added slowly and uniformly over a course of 4 hours.


Figure 4: This recirculating cryoprotectant circuit is used to introduce cryoprotectant slowly into the fixed brain. Fluid flows from the recirculating reservoir (labeled "FIX" here), through a filter, into the rabbit's brain, and then drips down from the jugular veins back into the recirculating reservoir. Connected to the recirculating reservoir is another reservoir containing full-strength CPA solution. Both containers hold the same level of fluid and are joined with a short piece of tubing. As the fluid flows through the circuit, the WASTE pump very slowly discards fluid at a rate of around 12.5 mL/min. Removing fluid causes CPA solution to flow from the CPA reservoir into the recirculating reservoir so that the levels of the two reservoirs are always equal. Slowly, the concentration of CPA rises from 0% to full strength. Each reservoir contains 1.5 liters of solution, and at a rate of 12.5 milliliters per minute discard it takes around 4 hours to complete the cryoprotectant ramp.

This rabbit brain was preserved with ASC and stored overnight at -135°C in liquid nitrogen vapor, colder than any temperature that naturally occurs on earth!

Over the next 10-15 minutes this brain warmed to room temperature. Then Dr. Hayworth and I processed it for submission to the Brain Preservation Prize. Images from this brain are available at


Packing delicate glassware

I like to think of ASC as a "brain-packing" technology. If you want to ship an expensive tea set across the country, you carefully wrap each class piece securely with bubble wrap and scotch tape. Then you take each wrapped piece of glass, lay them carefully in a box, and fill everything with packing peanuts. Likewise, if you want to send the information in a brain to the future, you need to bind the delicate synapses with glutaraldehyde and immobilize everything with ethylene glycol and vitrification. Glutaraldehyde and ethylene glycol do for synapses what bubble wrap and packing peanuts do for delicate glassware.

Preserving books

If brains are like books, ASC is like soaking a book in crystal-clear epoxy resin and hardening it into a solid block of plastic. You're never going to open the book again, but if you can prove that the epoxy doesn't dissolve the ink the book is written with, you can demonstrate that all the words in the book must still be there, preserved in the epoxy block.

If you really cared about the contents of the epoxy-embedded book, you might be able to carefully slice it apart, scan in all the pages, and print/bind a new book with the same words. But you can only do that if the data is there to be scanned and hasn't been destroyed by the preservation process.

So in the analogy, brain viability is like the ability for you to open a book, leaf through the pages, and read the book's story. The brain's connectome is like the words on the pages of the book. You can preserve the words while making the book impossible to open, and you can prove that the words are preserved by doing your preservation procedure on test books, cutting them open, and seeing that the words haven't been altered.

This is what I'm trying to do with brains — the glutaraldehyde glues all the proteins together while maintaining the structural connections between all the brain's cells. Storage at -135°C completely locks everything in place and guarantees long-term storage. I can demonstrate that all the brain's connections are still intact by preserving rabbit brains with ASC, rewarming them, cutting them into slices, and looking at those slices with electron microscopy. The electron micrographs from these preserved brains look the same as micrographs generally accepted as "the baseline" for connectomics research.

The ancient Greeks used to perform the libation ritual, where they would pour out a small amount of wine to the gods as an offering of thanks. It would have been wonderful if the Greeks had had a similar concept for libraries. They might have made copies of the most important scrolls, soaked them in wax, put them in a lead box, and thrown them into the ocean as part of a grand ceremonial "library libation". If they had done this, then today we would be able to recover those scrolls from the bottom of the ocean and read them again. We wouldn't need to cut through the scrolls or even touch them to reveal the text; modern x-ray technology can easily penetrate a preserved scroll and read all the text within without disrupting even a single ink molecule. Of course, the Greeks never did anything like this and most of the priceless knowledge of western civilization burned away forever with the great library in 48 BCE.


"Every neuron and synapse looks beautifully preserved across the entire brain. Simply amazing given that I held in my hand this very same brain when it was frozen solid… This is not your father’s cryonics." — Dr. Kenneth Hayworth, BPF President

"I witnessed the infusion of a rabbit brain through its carotid arteries with a fixative agent called glutaraldehyde, which binds proteins together into a solid gel. The brain was then removed and saturated in ethylene glycol, a cryoprotective agent eliminating ice formation and allowing safe storage at −130 degrees C as a glasslike, inert solid. At that temperature, chemical reactions are so attenuated that it could be stored for millennia. If successful, would it be proof of concept?" — Michael Shermer, president of the American Skeptics Society

Prospects for the future and further reading

ASC is the first step towards a reliable nanoscale structural preservation protocol that can preserve all the information stored in a person's brain and body for hundreds of years. I've created a neuroscience research company called Nectome to do further research into ASC. I intend to create a robust preservation protocol that could be applied to humans and to clearly establish exactly what information is being preserved with ASC. If you're interested, please persue the following links to learn more!

ASC Frequently Asked Questions
Answers to common questions about neuroscience and cryobiology as related to ASC.
Cryobiology Paper
This is a peer-reviewed scientific account of the ASC protocol and early results obtained on rabbit and pig brains. The paper has been released open-access under a Creative Commons license.
Neuroscience company dedicated to advanced structural brain preservation. I am one of the founders. Sign up for our mailing list if you want to stay informed about our progress.

Author: Robert McIntyre

Created: 2016-11-13 Sun 11:22

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