The current generation of vitrification agents in cryonics permit elimination of ice formation using realistic cooling rates. But attempts to vitrify the brain require high concentrations of cryoprotective agents to inhibit ice formation. Such high concentrations of cryoprotectants can produce injury to tissues that is distinct from damage caused by ice formation.

Vitrification of complex tissues requires perfusion to substitute the cryoprotective agent for water. Because the cryoprotectant concentration necessary to vitrify (CNV) is higher than than the concentration of solutes in the cells, exposing cells to such high concentrations at once will result in cell injury as a result of osmotic stress. This osmotic effect of cryoprotectants requires that the introduction of the vitrification agent be gradual to allow the cryoprotective agent to be exchanged with cell water without injury.

How important is osmotic shock as a form of injury?

In 1984, Greg Fahy published a paper (Fahy GM, Cryoprotectant Toxicity: Biochemical or Osmotic? Cryo-Letters 5:79-90) to distinguish cryoprotectant-induced osmotic injury from biochemical injury. Fahy reviews the literature and presents his own data obtained in renal cortical slices that indicate that substantial hypertonic osmotic stress does not produce major changes in viability. Conversely, reducing exposure time to higher concentrations of the cryoprotectant can contribute to improved viability. These results suggest that biochemical toxicity, not osmotic stress, is the major factor in cryoprotectant-induced injury.

A number of caveats for cryonics are in order. Osmotic stress as a result of rapid introduction of the cryoprotectant depends on the specific cryoprotective agent(s) and tissue. For example, glycerol, the prevailing cryoprotectant in cryonics until the more recent vitrification agents were introduced, has relatively high viscosity and poor permeability at low temperatures compared to other cryoprotective agents such as DMSO and ethylene glycol. W.M. Bourne et al. found that the highest concentrations of different cryoprotectants that did not cause a loss of human cornea endothelial cells were higher with the ramp method (gradual increase) for glycerol and higher for DMSO, 1,2-propanediol and 2,3-butanediol using a step method. These results indicate that more toxic cryoprotective agents with good penetration may benefit from a stepped approach to reduce cryoprotectant exposure times.

What the optimal introduction rate for specific cryoprotective agents (or mixtures of cryoprotective agents) is in the brain we do not know. The brain is also unique in the sense that an intact blood brain barrier (BBB) limits introduction of vitrification agents to the brain. This is especially important in case the vitrification solution includes non-penetrating agents such as polyvinylpyrrolidone (PVP) and ice blocking polymers. In many cryonics patients, the BBB may be compromised as a result of warm and cold ischemia, which introduces another variable that may affect the optimal introduction rate of the vitrification agent.

Osmotic shock as a result of too rapid diffusion of water from the cells should be distinguished from dehydration injury as such. Vitrification agents like M22 are assumed to confer some of their ice inhibiting effects by dehydration of the brain. Whether such (extreme) dehydration affects (long term) viability in the brain is another area that warrants investigation. Research that would investigate the effects of different introduction and removal protocols for various vitrification agents on the brain would be a step towards finding the right balance between the need for gradual introduction of the vitrification agent on the one hand and minimizing cryoprotectant toxicity on the other.