February 12, 2016

Rabbit brain defrosted from cryopreservation without damage

A mammal brain has been defrosted from cryogenic storage in an almost perfect state for the first time. This breakthrough, accomplished using a rabbit brain, brings us one – albeit tiny – step closer to the prospect of reanimating a human brain that has been cryogenically preserved.

After death, organs begin to decay, but we can delay this by cooling these tissues, just like freezing food. But in the same way that a frozen strawberry becomes soggy when defrosted, it is difficult to perfectly preserve mammals at cold temperatures. We, and strawberries, contain large amounts of water, which freezes into ice crystals that damage cells.

Cryoprotectants can prevent this ice damage, working like medical-grade antifreezes and preventing organs from freezing. This works in small worms and rabbit kidneys, but it needs to be administered quickly, which usually causes brains to dehydrate and shrink.

The company 21st Century Medicine in Fontana, California, have developed a technique that appears to prevent dehydration and preserves the brain in a near-perfect state. By draining the blood immediately and replacing it with a chemical fixative called glutaraldehyde, they can instantly stop decay, allowing them to add cryoprotectants more slowly to prevent dehydration.

The brain is then cooled to -135 °C, which turns it into a glass-like state that can be stored for centuries without decay. When they tried this technique on rabbit brains, thawing them up to a week later, Fahy and McIntyre say the preservation appeared “uniformly excellent” when examined using electron microscopy. They have been awarded a US$26,735 prize by the Brain Preservation Foundation for the technique.

Mastering cryopreservation of the brain and other organs can also improve organ transplantation.

Cryopreservation of the brain could be used for cryogenic suspension of those who are critically ill for possible revival in the future after medical advances.

Although cryopreservation techniques have not yet been perfected, more than 100 people worldwide have already been cryogenically frozen after death by companies like Alcor.

Freezing normally damages cells, but this defrosted rabbit brain was in a near-perfect state. Kenneth Hayworth, Brain Preservation Foundation

Cryobiology - Aldehyde-stabilized cryopreservation


• We introduce aldehyde-stabilized cryopreservation (ASC), a brain-banking technique for preserving detailed brain ultrastructure over long time scales.
• ASC uses glutaraldehyde to rapidly stabilize brain ultrastructure.
• ASC uses vitrification to preserve brains over indefinite time scales.
• Fixative/cryoprotectants are delivered via perfusion, enabling preservation of brains of any size.
• Cryoprotectants can be removed either by diffusion from slices or via perfusion, yielding a fixed brain compatible with many connectomics assays.

Abstract - Aldehyde-stabilized cryopreservation

We describe here a new cryobiological and neurobiological technique, aldehyde-stabilized cryopreservation (ASC), which demonstrates the relevance and utility of advanced cryopreservation science for the neurobiological research community. ASC is a new brain-banking technique designed to facilitate neuroanatomic research such as connectomics research, and has the unique ability to combine stable long term ice-free sample storage with excellent anatomical resolution. To demonstrate the feasibility of ASC, we perfuse-fixed rabbit and pig brains with a glutaraldehyde-based fixative, then slowly perfused increasing concentrations of ethylene glycol over several hours in a manner similar to techniques used for whole organ cryopreservation. Once 65% w/v ethylene glycol was reached, we vitrified brains at −135 °C for indefinite long-term storage. Vitrified brains were rewarmed and the cryoprotectant removed either by perfusion or gradual diffusion from brain slices. We evaluated ASC-processed brains by electron microscopy of multiple regions across the whole brain and by Focused Ion Beam Milling and Scanning Electron Microscopy (FIB-SEM) imaging of selected brain volumes. Preservation was uniformly excellent: processes were easily traceable and synapses were crisp in both species. Aldehyde-stabilized cryopreservation has many advantages over other brain-banking techniques: chemicals are delivered via perfusion, which enables easy scaling to brains of any size; vitrification ensures that the ultrastructure of the brain will not degrade even over very long storage times; and the cryoprotectant can be removed, yielding a perfusable aldehyde-preserved brain which is suitable for a wide variety of brain assays

SOURCES - Cryobiology, New Scientist, 21st Century medicine

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