08. July 2008 · Comments Off on Vitrification agents in cryonics: M22 · Categories: Cryonics · Tags: , , , , , ,

M22 represents the culmination of decades of work in applied cryobiology by researchers Gregory Fahy , Brian Wowk, and others to develop a vitrification agent that can recover complex organs (such as the kidney) from cryogenic temperatures without ice formation and minimal toxicity. In 2005, M22 was licensed by the patent holder 21st Century Medicine (21CM) to the Alcor Life Extension Foundation to replace their previous vitrification agent B2C. As a result, the least toxic vitrification agent for complex organs that has been documented in peer review journals is currently being used for cryonics patients at Alcor.

M22 incorporates a number of important discoveries in cryobiology:

1. High concentrations of a cryoprotective agent (or a mixture of different cryoprotective agents) can prevent ice formation during cooldown and warming.

2. The toxicity of some cryoprotectants can be neutralized by combining them with other cryoprotective agents.

3. The general toxicity of a vitrification agent can be predicted by using a measure called qv*, allowing for the rational formulation of less toxic vitrification agents.

4. Within limits, non-penetrating agents can reduce the exposure of cells to toxic amounts of cryoprotectants without reducing vitrification ability.

5. Synthetic “ice blockers” can be included in a vitrification mixture to reduce the concentration of toxic cryoprotective agents necessary to achieve vitrification.

6. Substituting methoxyl (-OCH3) for hydroxyl groups (-OH) in conventional cryoprotective agents can decrease viscosity, increase permeability, and reduce the critical cooling rate necessary to avoid ice formation.

7 Chilling injury can be eliminated by introducing the vitrification agent with a hypertonic concentration of non-penetrating solutes.

8. In cryonics, with a minor proprietary modification, M22 can be used for whole body perfusion without causing severe edema that has been a problem for some other solutions.

Vitrification is the solidification of a liquid without crystallization. When a solution is cooled down to the glass transition point (-123.3°C for M22) the extreme elevation in viscosity will produce a glass in which all translational molecular motions are arrested. Although water vitrifies at cooling rates exceeding a million of degrees Celsius per second, such cooling rates are relaxed when other solutes are substituted for water. In cryobiology solutions with high concentrations of cryoprotective agents can be used to vitrify complex organs such as the kidney or the brain.

Vitrification has a number of clear advantages over conventional cryopreservation. The most important advantage is the elimination of ice formation. Although the adverse effects of ice formation can be mitigated by the use of cryoprotective agents (glycerol, DMSO) and optimization of cooling rates, massive ice formation does not permit recovery of complex organs with full viability. Another advantage is that vitrification eliminates the need to strike a balance between the risk of intracellular freezing induced by fast cooling on the one hand, and cell dehydration and solution concentration induced by slow cooling on the other hand.

The challenge in formulating successful cryoprotective agents is to design vitrification solutions that are non-toxic but allow for vitrification at realistic cooling and warming rates. For more than a decade the least toxic vitrification agent was Greg Fahy’s VS41A, which is an 55% weight/volume equimolar mixture of DMSO and formamide plus propylene glycol. The “1A” in VS41A reflects the solution’s ability to vitrify at normal atmosphere pressure (as opposed to an older, more dilute solution, VS4, which requires 1000 atmospheres of pressures to vitrify). The equimolar concentrations of DMSO and formamide reflect Baxter and Lathe’s research who concluded that amides can neutralize the toxicity of DMSO, a finding that Greg Fahy later revised in favor of the theory that it is actually DMSO that neutralizes the toxicity of formamide. The ability of DMSO to neutralize the toxicity of formamide (up to certain concentrations) allows for the formulation of vitrification agents with reduced toxicity. This finding has been so fundamental that an equimolar concentration of DMSO and formamide remains the core of M22.

Another major step was made when the researchers at 21CM found that high concentration of (penetrating) cryoprotectant agents do not necessarily increase toxicity. Contrary to conventional cryobiology expectations, Fahy et al. found that weaker glass formers favor higher viability. They proposed a new compositional variable called qv* to predict the general toxicity of vitrification solutions. Using qv* they made the “counter-intuitive” decision to substitute a higher concentration of the weaker glass former ethylene glycol for propylene glycol to create a solution called Veg, which produced a substantial improvement in terms of viability as measured by K+/Na+ ratios.

Because cells contain higher concentrations of protein, the intracellular space is more favorable to vitrification than the extracellular space. As a consequence, the concentration of penetrating (toxic) cryoprotectants can be reduced in favor of non-penetrating polymers like polyvinylpyrrolidone (PVP). Variations of Veg in which the concentration of DMSO and formamide was reduced in favor of PVP increased viability without decreasing its ability to suppress ice formation. The concentration of penetrating cryoprotectants can be further reduced by inclusion of non-penetrating “ice-blocking” polymers. These ice-blockers also reduce the critical cooling and warming rates necessary to avoid ice formation, which is an important requirement for solutions that are used to vitrify complex organs such as the human brain.

Because concentrated vitrification solutions depress the homogeneous nucleation temperature (Th) below the glass transition temperature (Tg), a major obstacle to successful vitrification is the presence of heterogenous nucleators. Some organisms have antifreeze proteins (AFPs) and anti-freeze glycoproteins (AFGPs) that mitigate heterogenous nucleation by binding to nucleators. Because adding such anti-nucleating proteins to vitrification solutions would be prohibitively expensive and less effective, Greg Fahy proposed the creation of synthetic ice-nucleation inhibiting polymers. In 2000 Wowk et al. published work that showed the effectiveness of a co-polymer of polyvinyl alcohol (PVA) and vinyl acetate in inhibiting heterogenous ice-nucleation. This co-polymer is now being sold by 21CM under the name “X-1000”. X-1000 is particularly effective in glycerol solutions, presumably because glycerol itself is a poor anti-nucleation agent. Increasing the concentration of X-1000 in vitrification solutions decreases ice formation and relaxes minimum cooling rates. Although X-1000 is presumed to be non-toxic, the maximum concentration in vitrification solutions does not exceed 1% w/v because no further benefits were observed beyond this concentration. In 2002, 21CM announced the discovery of another synthetic “ice-blocker” called Z-1000. Z-1000 is the polymer polyglycerol (PGL), which specifically inhibits ice nucleating activity caused by the bacterium Pseudomonas syringae. Mixtures of PVA and PGL are more effective in inhibiting ice formation than either agent alone, suggesting the PVA and PGL complement each other by inhibiting different sources (bacterial and non-bacterial) of ice nucleation.

A variant of Veg that includes the low molecular weight polymer polyvinylpyrrolidone K12, X-1000, and Z-1000 named VM3 improved viability in renal cortical slices and decreased the critical cooling and warming rates necessary to avoid ice formation and de-vitrification (ice formation during rewarming) while maintaining the same molar concentration as VS41A. The transition from Veg to VM3 reflects the two breakthroughs mentioned above: reduction of cryoprotectant toxicity by inclusion of non-penetrating polymers and ice blocking agents. VM3 also was the least toxic agent in vitrification of rat hippocampal brain slices, which is of particular importance for cryonics. The first vitrification agent ever to be introduced to cryonics was a hyperstable variant of VM3 called B2C. B2C was used until late 2005, when it was replaced by M22.

M22 takes advantage of two other discoveries: the ability to design better glass formers by methoxylation of conventional polyols, and inhibition of chilling injury by delivering the vitrification agent as a hypertonic solution. Because hydroxyl groups can bind either to water or hydroxyl groups on other cryoprotective agents, substituting methoxyl groups for hydroxyl groups should decrease interaction between cryoprotectants and increase interaction between the cryoprotectant and water. As a result, methoxylated compounds have stronger ice inhibiting ability, thus reducing the critical cooling rate for vitrification or reduce the concentration of (toxic) cryoprotective agents in a solution. Methoxylated cryoprotectants also decrease viscosity and increase cell permeability, allowing for shorter perfusion times, and thus reduced cryoprotectant exposure at higher temperatures. For example, the methoxylated glycerol derivative 3-methoxy-1,2-propanediol has a higher glass transition point and vitrifies at ~ 5% lower concentration than the corresponding conventional cryoprotective agent. Complete exploitation of these advantages is limited by the fact that they are more toxic than their non-methoxylated compound, as predicted by qv*. As can be seen in the table, the major difference between VM3 and M22 is the reduction of PVP K12 in favor of the penetrating cryoprotectants 3-methoxy-1,2-propanediol and n-methyl-formamide, and increased concentration of the ice-blocker Z-1000. The final molar concentration of 9.345 M demonstrates that more concentrated vitrification agents do not necessarily have to be more toxic.

VS41A

Veg

VM3

M22

Dimethyl sulfoxide

3.10 M

3.10 M

2.855 M

2.855 M

Formamide

3.10 M

3.10 M

2.855 M

2.855 M

Propylene glycol

2.21 M

Ethylene glycol

2.71 M

2.713 M

2.713 M

N-methylformamide

0.508 M

3-methoxy-1,2-propanediol

0.377 M

Polyvinyl pyrrolidone K12*

7% w/v

2.8% w/v

X-1000 ice blocker*

1% w/v

1% w/v

Z-1000 ice blocker*

1% w/v

2% w/v

Total Molarity

8.41 M

8.91 M

8.41 M

9.345 M

* Non-penetrating polymers are in w/v

M22, so called because it was intended to introduced at -22 degrees Celsius, constitutes a major landmark in vitrification of complex organs. In 2005 Fahy, Wowk et al. announced routine recovery of rabbit kidney slices from temperatures around -45 degrees Celsius. Although consistent recovery of vitrified organs is not yet feasible, continued progress in solution composition and perfusion techniques inspire optimism that this may be possible in the future. In 2007, Greg Fahy of 21CM reported recovery of electrical activity in vitrified brain slices and induction of long-term potentiation (LTP), which indicates that the structures for processing memory are maintained after vitrification, storage and rewarming of brain tissue. Visual evidence that M22 can preserve the ultrastructure of the brain better than B2C was published on the Alcor website in 2005.

M22 also needs to be used in a suitable carrier solution to support cell metabolism at low temperatures and decrease oxidative injury and edema. The carrier solution for M22 is called LM5 to reflect the 50% reduction of glucose (as compared to the older carrier solution RPS-2) in favor of equimolar concentrations of mannitol and lactose, to address compatibility problems with the ice blockers. The combination of the isotonic LM5 plus the non-penetrating polymers in M22 creates a hypertonic solution, which has been shown to eliminate chilling injury, which is the injury that is caused by exposure to low temperatures as such. For cryonics, the composition of M22 is further enhanced by including a proprietary components that allows perfusion of whole body patients without edema.

The research breakthroughs discussed above allow for a global reconstruction of the composition of M22 using the table. Maintained is the equimolar combination of DMSO and formamide from Fahy’s older vitrification solutions to reconcile strong glass formation ability and minimal toxicity. The discovery of the  compositional variable qv* allows for substitution of higher concentrations of the weaker glass former ethylene glycol for propylene glycol. Substitution of a non-penetrating polymer, PVP K12, and the ice-blockers X-1000 and Z-100 allow for further reduction of DMSO and formamide, reduction of critical cooling rates, and increased stability against ice formation. In M22, PVP K12 is reduced to optimize hypertonicity of the non-penetrating agents for suppression of chilling injury. Added are the methoxylated cryoprotectant 3-methoxy-1,2-propanediol and the highly permeable amide n-methyl-formamide, producing the least toxic but most concentrated vitrification solution to date.

The most striking differences between Alcor’s old perfusate and the newer vitrification agents licensed from 21CM are complexity and cost. Until 2002, Alcor patients were perfused with high molar glycerol in an MHP-2 based carrier solution. M22 itself consists of 8 (!) different components, putting the total number of components of M22 in carrier solution above 15. Such perfusates makes great demands on preparation skills and quality controls. Components such as the ice blockers and 3-methoxy-1,2-propanediol have put the cost of Alcor’s whole body perfusate alone close to the cost of complete cryopreservation arrangements at the Cryonics Institute (CI). This raises obvious questions about costs and benefits. As evidenced by CI’s VM-1, potent protection against ice formation can be achieved with a vitrification agent that solely consists of DMSO and ethylene glycol. It is plausible to assume that vitrification lessens demand on future repair technologies, but it speculative to assume that minor differences in toxicity between different vitrification agents will translate in earlier resuscitation and less expensive repair protocols. However, more toxic vitrification solutions, such as CI’s VM-1, may cause acute injury to endothelial cells. As Brian Wowk notes, “good cryoprotection depends on good perfusion, which depends on preservation of vascular integrity during perfusion. The ability to perfuse M22 into whole bodies with tolerable edema is likely to be intimately related to its low toxicity to vascular endothelium.” And of course, there are also PR advantages to the fact that a cryonics organization uses a vitrification agent that is also the state of the art in conventional cryopreservation of organs.

M22 produces substantial brain shrinking during perfusion of (non-ischemic) patients. As a matter of fact, cerebral dehydration may be a major contributing factor to vitrification of the brain and even allow for reduced concentrations of M22 for brain preservation. This does not mean that the (expensive) non-penetrating polymers could be replaced for any high molecular weight polymer because the ice blockers and non-penetrating cryoprotective agents also protect the extracellular space against ice formation and are effective in ischemic patients with a compromised blood brain barrier (BBB). The limited ability of some components of M22 to cross the BBB and, and differences in permeability of the various components of M22, does raise questions about the exact composition of M22 beyond the BBB and within brain cells after completion of cryoprotective perfusion.

Patients outside of the US may not fully benefit from cryopreservation with M22 because of the of long cold ischemic times during transport. This raises the question if cryonics patients can be perfused outside of the US and shipped in dry ice. Experiments with VM-1 in bulk solution indicate that this solution is very stable against ice formation, even during long storage periods. M22 in bulk solution seems to form ice crystals overnight if stored in dry ice. This does not necessarily mean that M22 cannot be used in combination with dry ice for overseas patients because human tissue perfused with M22 (or any cryoprotective agent) is not the same as M22 in pure solution. But regardless of M22’s compatibility with dry ice shipping, cryonics organizations may benefit from formulating a highly concentrated inexpensive vitrification solution that is extremely robust against formation of ice, which can be used for simple perfusion of non-US patients in combination with dry ice shipping. The decreased cold ischemic times of such a solution may outweigh the increased toxicity of such solutions.

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