11 replies to this topic

[Not A Naked Ape]

Thanks for the detailed answer. This reminds me of another question I have: How long can a patient be kept at the temperature of dry ice without major damage? This is especially important if you arrive from another country to the US.

[bgwowk]

Thanks for the detailed answer. This reminds me of another question I have: How long can a patient be kept at the temperature of dry ice without major damage? This is especially important if you arrive from another country to the US.

If you are frozen without cryoprotectant (so-called "straight freeze"), the answer is probably years because whatever tissue debris remains between ice crystals will be solidified in concentrated salt. If you are perfused with a concentrated vitrifcation solution prior to cooling to dry ice temperature, tissue will be in a viscous liquid state at dry ice temperature. Limited experimental data suggests that days might be okay in this state, but risk of ice growth increases with time. Conventional wisdom says that once perfused with vitrification solution, cooling below -100 degC should occur as fast as possible to minimize risk of ice formation and growth.

By the way, some laypeople have called perfusing with vitrification solution and cooling to dry temperature "vitrification at dry ice temperature" (-79 degC). However this is not correct because vitrification (solidification) doesn't occur until temperatures below -120 degC (the glass transition temperature). There is no name for the unfrozen state above the glass transition temperature because nobody but cryonicists ever pauses there.

Perfusing with a freezable concentration of cryoprotectant, such as glycerol as was used in the 20th century, and pausing for long periods at dry ice temperature is less hazardous than for a vitrification solution because any ice that was going to form will have already formed by that temperature. The only residual damage mechanism would be slow cryoprotectant toxicity in the viscous liquid between ice crystals. This is probably okay for up to several months. Some of the best micrographs of brain tissue ever obtained were from a brain frozen at -90 degC for a year

http://www.alcor.org...servation1.html

To summarize, stability at dry ice temperature is a complicated question that strongly depends upon what concentration of cryoprotect is present in tissue. This is something often not known very well under field perfusion conditions.

[benbest]

Perfusing with a freezable concentration of cryoprotectant, such as glycerol as was used in the 20th century, and pausing for long periods at dry ice temperature is less hazardous than for a vitrification solution because any ice that was going to form will have already formed by that temperature.

I don't understand this point.

When ice forms in a vitrification mixture
above glass transition temperature, I understand
that it forms as ice balls (as seen in a pure
solution in a flask) -- so why is that any
different from glycerol? Why would a glycerol
solution have formed ice when cooling to dry ice
temperature, but not form any more ice after
reaching dry ice temperature? Why would this
be true of glycerol, but not of a vitrification
mixture? Doesn't cooling rate matter?

I am not challenging your statement, I just want to know.

-- Ben Best

[bgwowk]

If a solution is far enough below the concentration needed for vitrification that it doesn't supercool very much during cooling, then ice will form and grow at relatively high subzero temperatures. An equilibrium amount of ice will form so that the melting point of the solution between ice crystals is approximately equal to the solution temperature as cooling progresses. That means that by the time you get down to -79 degC with such a solution, all the ice that can form at that temperature will have already formed because the solution was in approximate thermodynamic equilibrium during the whole slow cooling process. In fact, cooling with glycerol solutions in the 20th century was deliberately slow to maintain equilibrium and avoid supercooling. Only if a solution is supercooled when -79 degC is reached (i.e. solution melting point higher than -79 degC) will a solution continue to grow ice during holding at -79 degC.

[benbest]

If a solution is far enough below the concentration needed for vitrification that it doesn't supercool very much during cooling, then ice will form and grow at relatively high subzero temperatures. An equilibrium amount of ice will form so that the melting point of the solution between ice crystals is approximately equal to the solution temperature as cooling progresses. That means that by the time you get down to -79 degC with such a solution, all the ice that can form at that temperature will have already formed because the solution was in approximate thermodynamic equilibrium during the whole slow cooling process.

You seem to be relying on the idea that perfusion is perfect. But the dry ice storage is primarily to be used for overseas cases, is it not, and ischemia may prevent perfect perfusion. (Germans, for example, cannot begin vitrification perfusion much before an hour or two after cardiac arrest, due to the laws in that country.) With imperfect perfusion, many areas of the brain are likely to have less than a concentration needed to vitrify of vitrification solution.

In fact, cooling with glycerol solutions in the 20th century was deliberately slow to maintain equilibrium and avoid supercooling. Only if a solution is supercooled when -79 degC is reached (i.e. solution melting point higher than -79 degC) will a solution continue to grow ice during holding at -79 degC.

What danger would there have been to supercool?

[bgwowk]

What danger would there have been to supercool?

In conventional freezing with dilute cryoprotectant solutions, excessive supercooling leads to damaging intracellular ice formation if ice forms in the supercooled state. So one of the worries with vitrificaton protocols that cool rapidly to deliberately to avoid ice formation is that if you stop and hold at -79 degC in the supercooled state then ice that does form may form inside cells, which is more damaging than ice forming outside cells as would normally occur with freezing.

You are right to be concerned about sub-optimal perfusion with vitrification solution, which would make the vitrification solution become a defacto freezing solution in areas where perfusion is poor. Unfortunately I can't tell you that cooling rapidly directly to the glass transition temperature completely solves the problem either, because freezing in poorly-perfused areas may still occur.

It may also be that freezing in supercooled sub-vitrifiable concentrations of cryoprotectant doesn't result in the kind of intracellular freezing that is seen with the very dilute cryoprotectant concentrations used by cryobiologists for cell freezing. Cryoprotectant concentrations between those of classical freezing solutions and vitrification solutions is a netherworld that hasn't been studied in cryobiology as far as I know. The only practical setting in which it occurs, beginning with utilization of high molarity glycerol in the 1990s, is cryonics.

[JJN]

This is a very important basic issue, not only for people overseas, but also for people who are remote from cryonics facilities in the US, and from KrioRus. Ben brought up a good point about differing regulations regarding the 'deceased' in Germany.

It is good to see that Ben and Brian are having a cordial dialogue. Obviously, both are prominent point-people for CI and Alcor. The issue raised is paramount for prescribing protocols for various situations.

Here's to hoping you continue to have a healthy dialogue. It would be reactionary to make a call for having a set of regulations for various situations at this time, and in this place, but hopefully progress will be made, as I am sure it is. So much is still not known, and needs to be studied.

Kind regards,

Jeff

[bgwowk]

To be clear on this, I think based on available evidence that shipment on dry ice from overseas locations after perfusion with a concentrated vitrification solution is a reasonable thing to do. I had more concerns about this strategy several years ago than I do now. I've never had concerns about shipping on dry ice after perfusion with freezable cryoprotectant solutions, such as glycerol-based solutions, or after straight freezing with no cryoprotectants.

I'm not sure why any cryonicist would consider regulation in this context. The biological effects of transporting tissue at temperatures above the glass transition temperature subject to various cryoprotection scenarios is a good example of an issue that is so complex, understood by so few experts (absolutely none in medicine), that it would be a farce for government to regulate it based on a pretense of cryonics quality assurance.

Edited by bgwowk, 11 December 2010 - 01:32 AM.

[benbest]

What danger would there have been to supercool?

In conventional freezing with dilute cryoprotectant solutions, excessive supercooling leads to damaging intracellular ice formation if ice forms in the supercooled state. So one of the worries with vitrificaton protocols that cool rapidly to deliberately to avoid ice formation is that if you stop and hold at -79 degC in the supercooled state then ice that does form may form inside cells, which is more damaging than ice forming outside cells as would normally occur with freezing.

To be clear on this, I think based on available evidence that shipment on dry ice from overseas locations after perfusion with a concentrated vitrification solution is a reasonable thing to do. I had more concerns about this strategy several years ago than I do now. I've never had concerns about shipping on dry ice after perfusion with freezable cryoprotectant solutions, such as glycerol-based solutions, or after straight freezing with no cryoprotectants.

This brings us back to the original question which started this thread:

How long can a patient be kept at the temperature of dry ice without major damage?

I take that to mean: "How long can a patient [who has been perfused with vitrification solution] be kept at the temperature of dry ice without major damage?" In addition to the "how long" question, is the question: if a patient is to be held for an indefinite period of time on either glycerol or a vitrification solution at dry ice temperature, which would be more damaging, and why?

[bgwowk]

I take that to mean: "How long can a patient [who has been perfused with vitrification solution] be kept at the temperature of dry ice without major damage?"

Without getting back into the all the technical aspects of that question, you must surely realize that it depends on too many different variables to have one answer. Just minimize the time as much as you can.

In addition to the "how long" question, is the question: if a patient is to be held for an indefinite period of time on either glycerol or a vitrification solution at dry ice temperature, which would be more damaging, and why?

Conventional wisdom would be that it's safer to store tissue that followed an equilibrium freezing process down to dry temperature than to store tissue in a metastable supercooled state at dry ice temperature. Ice formation in supercooled solutions can lead to intracellular freezing because the speed of ice growth when it occurs may not permit cells to dehydrate in response. So I believe a glycerol solution that resulted in equilibrium freezing during cooling would be "safer" for indefinite storage at dry ice temperature than a vitrification solution. This does not address the issue that the glycerol solution may result in more total damage than the vitrification solution. It's just that with glycerol all the freezing damage is over and done with by the time you reach dry ice temperature. Only slow chemical effects would remain.

[benbest]

In addition to the "how long" question, is the question: if a patient is to be held for an indefinite period of time on either glycerol or a vitrification solution at dry ice temperature, which would be more damaging, and why?

Conventional wisdom would be that it's safer to store tissue that followed an equilibrium freezing process down to dry temperature than to store tissue in a metastable supercooled state at dry ice temperature. Ice formation in supercooled solutions can lead to intracellular freezing because the speed of ice growth when it occurs may not permit cells to dehydrate in response. So I believe a glycerol solution that resulted in equilibrium freezing during cooling would be "safer" for indefinite storage at dry ice temperature than a vitrification solution. This does not address the issue that the glycerol solution may result in more total damage than the vitrification solution. It's just that with glycerol all the freezing damage is over and done with by the time you reach dry ice temperature. Only slow chemical effects would remain.

When you say "safer" you seem to be referring to
where the damage occurs (extracellular for glycerol,
intracellular for vitification solution), but you
seem to be ignoring this point when referring to
"more total damage" if "total" means quantity
rather than quality of damage.

How slowly would a glycerol-perfused patient
need to be cooled in order to avoid supercooling?
What methods are readily available for doing this?
I should think that most of those needing to store
a patient in dry ice would simply pack the patient
in dry ice. I should think that most of the resultant
freezing would be extracellular, given that fact that
it is not possible to cool large tissues very quickly.

So the question becomes, under conditions in which
a perfused patient is to be stored for an extended
period (two weeks or more), and the patient is
simply packed in dry ice after perfusion, would
glycerol perfusion result in more or less damage
than vitrification perfusion?

[bgwowk]

So the question becomes, under conditions in which
a perfused patient is to be stored for an extended
period (two weeks or more), and the patient is
simply packed in dry ice after perfusion, would
glycerol perfusion result in more or less damage
than vitrification perfusion?

I don't know.