Ultrastructural and Histological Cryopreservation of Mammalian Brains by Vitrification

1. Problem Statement

The cryopreservation of whole mammalian brains has historically been hindered by two major obstacles:

• Ice Damage: Traditional freezing methods cause ice crystal formation, leading to severe structural disruption, cavities, and loss of connectomic detail (as seen in previous studies using glycerol and slow freezing).
• Osmotic Injury and Shrinkage: Vitrification (ice-free cryopreservation) requires high concentrations of cryoprotective agents (CPAs), such as the M22 solution (~9.45M). The blood-brain barrier (BBB) is more permeable to water than to CPAs. This creates a massive osmotic gradient, causing rapid water efflux from brain cells and severe tissue dehydration (shrinkage).
• The Knowledge Gap: While previous studies showed that aldehyde-fixed brains could be vitrified with preserved ultrastructure, it was unknown if unfixed brains could survive vitrification without prior fixation. Specifically, it was unclear if the extreme osmotic shrinkage caused by M22 perfusion was reversible or if it caused irreversible structural damage that would erase biological information (connectome) or prevent future medical resuscitation.

2. Methodology

The study employed a combination of animal (rabbit) and human brain models to evaluate vitrification without prior aldehyde fixation.

• Subjects:
  • Rabbits: New Zealand White rabbits were perfused with M22 vitrification solution, vitrified, and rewarmed.
  • Human: A 73-year-old male terminal cancer patient donated his brain. The brain underwent M22 perfusion approximately 2 days after agonal hypotensive hypoxia and 3 hours post-cardiac arrest, followed by vitrification.
• Vitrification Protocol:
  • Perfusion: Brains were perfused with M22 (a solution containing ethylene glycol, 1,2-propanediol, dimethyl sulfoxide, N-methylformamide, and polymers) to achieve a concentration of ~9.45M.
  • Cooling: Samples were cooled to below the glass transition temperature (~-123°C) using forced-convection liquid nitrogen vapor to prevent ice nucleation.
  • Warming: Rapid rewarming was performed to prevent devitrification (ice formation during warming).
• Post-Processing Variations:
  • Group A (Fixed in M22): Brains were fixed with aldehydes while still saturated with full-concentration M22.
  • Group B (Partial Rehydration): Brains were perfused with M22, vitrified, rewarmed, and then subjected to graded washout (dilution) to 5M, 3M, or 1M M22 concentrations before fixation. This tested the reversibility of shrinkage.
  • Human Biopsies: Human cortical biopsies were taken after perfusion, stored in liquid nitrogen for 4 years, then warmed and processed for histology and electron microscopy.
• Analytical Techniques:
  • Differential Scanning Calorimetry (DSC): Verified the absence of ice formation during cooling and warming.
  • High-Performance Liquid Chromatography (HPLC): Measured actual CPA concentrations within the tissue.
  • Microscopy: Extensive use of Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and Scanning Transmission Electron Microscopy (STEM) to assess ultrastructure at high magnification.

3. Key Contributions

• First Direct Evidence in Unfixed Brains: This is the first study to demonstrate that whole mammalian brains (both rabbit and human) can be vitrified and rewarmed without prior aldehyde fixation while retaining predominant ultrastructural integrity.
• Human Brain Viability: It provides the first direct inspection of human brain ultrastructure after practical cryopreservation conditions (including post-mortem ischemia), showing that histological detail is preserved.
• Reversibility of Shrinkage: The study demonstrates that the severe morphological distortion caused by osmotic dehydration is largely reversible upon partial rehydration, restoring normal cell shapes (e.g., pyramidal cells) and revealing intact synapses.
• Identification of Specific Injury Mechanisms: The authors identified four specific types of injury to be mitigated in future protocols:
  1. BBB-limited dehydration.
  2. Arteriolar separation/delamination.
  3. Myelin sheath distortion.
  4. Osmotic swelling damage during CPA washout.

4. Key Results

• Absence of Ice Damage:
  • DSC confirmed that human biopsies did not form ice during cooling or warming.
  • Microscopy revealed no ice crystals, no cavities, and no loss of ground substance in either rabbit or human brains, contrasting sharply with the severe distortion seen in slow-frozen controls (Fig. 1).
• Ultrastructural Integrity in Dehydrated State:
  • In brains fixed at full M22 concentration, cells were extremely shrunken and electron-dense.
  • Despite this, synapses, mitochondria, and neuronal membranes remained intact.
  • Capillaries remained intact and attached to the neuropil, though some arterioles showed separation from the surrounding tissue or delamination of the tunica adventitia (likely due to osmotic vasodilation followed by contraction).
  • Myelinated fibers showed shrunken axoplasm and partial separation of myelin layers, but the axonal paths remained denoted.
• Reversibility of Morphology:
  • Partial Rehydration (5M - 3M M22): When brains were diluted to 3M–5M M22 before fixation, the tissue re-expanded. Pyramidal cells regained their normal spindle shape, and apical dendrites became clearly visible.
  • Synaptic Preservation: High-magnification imaging at 3M M22 revealed abundant, intact synapses with visible presynaptic vesicles.
  • Limit of Rehydration: Dilution to 1M M22 caused "exploded" or ruptured neuronal compartments, indicating that rapid removal of too much CPA without counteracting osmolytes causes osmotic swelling injury.
• Human Brain Specifics:
  • Human cortical biopsies stored for 4 years showed identical preservation qualities to the rabbit brains.
  • The preservation was achieved despite the donor having significant agonal hypotension and hypoxia prior to perfusion, suggesting the method is robust against ischemic injury.

5. Significance

• Feasibility of Human Cryopreservation: The results provide the first concrete scientific evidence that human brains can be cryopreserved with the retention of the connectome (structural information) without prior fixation. This supports the theoretical feasibility of "medical time travel" (cryonics), where future technology might repair the specific, non-lethal injuries identified (e.g., BBB permeability, osmotic swelling).
• Brain Banking and Research: This technique allows for the banking of rare or endangered species' brains, or human brains with rare neurological conditions, for future ultrastructural and molecular analysis without the artifacts introduced by aldehyde fixation.
• Roadmap for Optimization: By identifying specific failure modes (arteriolar separation, myelin distortion, and osmotic swelling during washout), the paper provides a clear roadmap for refining vitrification protocols. Future work involving BBB permeabilization (e.g., using SDS) and the use of extracellular osmolytes during washout could potentially eliminate these remaining injuries.
• Connectome Preservation: The study confirms that the "wiring diagram" of the brain (synapses and axonal paths) is preserved even under extreme dehydration, a prerequisite for any future attempt to revive or reconstruct a cryopreserved mind.

In conclusion, the paper establishes that vitrification is a superior method to freezing for whole-brain preservation and that the structural damage observed is primarily osmotic and reversible, rather than catastrophic ice damage, marking a significant milestone in cryobiology.