X-Message-Number: 33241 From: Date: Sat, 15 Jan 2011 23:49:41 EST Subject: Response to Metzger Part I My attempted post to Cryonet early this AM bounced because it exceeded the allowable file length. So, I've broken the file into two parts, pretty much arbitrarily, and am now re-posting them. Mike Darwin My responses are interleaved with the two posts below and start with my initials (MD) and end with >> Message #33230 Date: Fri, 14 Jan 2011 16:51:49 -0500 From: "Perry E. Metzger" <> Subject: Glass vs. Crystal transitions References: <> It is rare that a topic comes up here that is actually of direct interest to improving cryopreservation quality. However, as Mike Darwin has had the temerity to interrupt the usual chatter around here by discussing actual experiment, I thought I'd chime in: > From: > Date: Thu, 13 Jan 2011 21:56:09 EST > Subject: Intracellular Freezing & Vitrification [...] > vitrify. A potential problem arises, especially when ice growth > inhibiting molecules are also present, in that such tissues will > tend to supercool far below their true freezing point, One side note here. (I'm sure Mike is aware of this, but others may not be.) A uniform idealized substance (and sometimes a uniform idealized mixture) can have a single definable freezing point, but non-uniform substances do not. Biological tissues and individual cells are nearly the definition of a non-uniform substance -- if they were uniform, they would not function. Furthermore, not every substance has a well defined freezing point even when it is perfectly uniform. A good everyday example (just for purposes of illustration) is paraffin. Why mention this? Because even in idealized conditions, getting an entire cell or block of tissue to undergo a phase transition at the same time may be a bit of a challenge, and because things one learns from uniform systems may warp when applied to non-uniform systems like biological tissues. MD: First, before I get into specifics, I'd like to thank Perry for asking some very good questions - so good in fact, that I'm sure I will not be able to answer all of them. My hope is that Brian Wowk will step up to the plate, especially where I don't know, or where I err. Second, meaning no disrespect, Cryonet is not really the proper place for these, or any discussions that involve complex scientific concepts that, by their very nature, cry out for visual aids and examples. I was hoping that a blog or interactive web 2.0 space would be available by now, but so far that hasn't happened. Now, to the matters at hand. Yes, as you point out, biological systems are inhomogeneous and this is greatly amplified when a whole organ or organism is considered. In fact, one of the primary obstacles to successful renal vitrification at this point is that the renal medulla is comparatively poorly perfused compared to the cortex and may, in addition, have structural features that make cryoprotectant agent (CPA) equilibration more problematic. Having said that, cryoprotective perfusion in a healthy animal probably does a fair bit to decrease the inhomogeneity in various organs and tissues because, if successful, it swamps the tissue with massive amounts of colligative agents AND it dehydrates the tissues to just about the maximum degree possible. A cryoprotected brain is massively dehydrated at the end of CPA loading, and basically consists of CPA in a concentrated protein gel. All but the vascular extracellular space is abolished; and the tissue ground substance is extremely dense - in fact it makes it very hard to see fine intracellular structure with TEM. So, what you have is sort of a CPA loaded 'brain-jerky.' Still, there will be regional variations of some kinds, and if freezing is going to take place, these may be material. But the point here is that the severe dehydration, coupled with the massive freezing point depression, will probably tend to collapse a lot of the normally present regional variation in things like water, protein and lipid content.>> > What Brian pointed out to me is that a > 7.5 M solution of glycerol has a melting point of ~ -50 deg C.Thus, > even if tissues containing 7.5 M glycerol solution nucleated > perfectly upon passing below their melting point during cooling, > all the ice growth would take place at deep subzero temperatures > where the mobility of water (and cell membrane permeability to > water) is putatively low. An added complicating factor is that 7.5 > M glycerol solutions are virtually certain to supercool well below > their thermodynamically ideal freezing point - and indeed to do so > by tens of degrees. Supercooling is common across most substances. Extremely pure water with no nucleation sites can be cooled to below -40C before it will undergo spontaneous "homogeneous nucleation". Crystal formation itself is a fairly complicated process, and without a nucleation center the transition can be disfavored even if the final crystal would be perfectly stable. MD: Yes, water has two freezing points: 0 deg C, which is the heterogenous nucleating temperature, and -40 deg C, which is the homogenous nucleating temperature. In theory, if you could purge a solution of all exogenous nucleators, like bacterial proteins, gas bubbles, etc., then you could cool water to -40 deg C and it would remain in a liquid state. In fact, this works well for small volumes of water, and some living organisms survive by a combination of purging nucleators and using a modest amount of a colligative agent. But, for large volumes of water, thermodynamics and chance dictate that you will get freezing, and sooner, rather than later.>> The specific issue, as I understand it, is this. (If someone understands the process better than I do, please chime up -- I know a bunch of physical chemistry but it is not my area of expertise.) Forming an interface between the two phases requires energy, which is proportional to the size of the surface area at the interface. Now, there is a source of energy available, to whit, the energy liberated by the formation of the crystal itself (the crystal necessarily has lower free energy than the liquid), which is proportional to the volume of the forming crystal. However, the smaller an object is, the larger the ratio of its surface area to its volume. Thus, a very small spontaneously formed crystal will not liberate enough energy to account for the energy of the interface. Assuming a spherical nucleus, one can calculate a critical radius below which a spontaneously formed crystal is unstable. (A very similar process happens in water droplet formation in clouds, where below a critical radius the droplets are disfavored.) However, as the temperature becomes lower and lower, the critical radius itself lowers (one can model this mathematically though it has been some years since I knew the details), until finally you hit a point where the critical radius is small enough that spontaneously formed crystals are larger than it and you get homogeneous nucleation followed by a spontaneous phase transition throughout the liquid. MD: Yes, I believe this is a succinct and accurate description, and it applies not just to freezing water, but to boiling water as well. With the advent of microwave ovens, many people are now familiar with superheating of water, as when a cup of water in the oven fails to boil at 100 deg C. As soon as it is nucleated, either by moving it or by adding a powder or a tea bag, explosive boiling occurs as all that 'stored' heat that should have been disposed of by steam escaping during boiling, is rapidly dissipated. It is the same phenomenon in reverse when freezing occurs after deep supercooling. And, as you point out, crystal formation yields free energy - in this case in the form of heat. If a LOT of ice forms quickly, the very rapid release of this latent heat of fusion can, in theory, re-warm the system and cause damage by inducing localized CPA toxicity, melting and refreezing, and by driving recrystallization in already frozen areas.>> > This will be even more true of glycerol > solutions at these temperatures because of their very high > viscosity and the resultant decreased mobility of water in the > solution. I know little about the physical chemistry of glycerol undergoing this transition. Are the crystals formed pure water? If so, the concentration of glycerol of the surrounding liquid would rise during crystal formation, inhibiting further growth if diffusion was slow enough. That would tend to explain the phenomenon you describe below: MD: Yes, ice freezes out as pure water under these conditions, and with these molecules. Some putative cryoprotective agents such as the gases Xenon and Argon can form non-ice structures called clathrates. However, to my knowledge, the CPAs currently in use leave water either hydrogen bonded to the CPA and present as a CPA-water liquid - or as ice, frozen out as pure water. In fact, the definition of ice is a crystal of water, and a crystal of water is made up only of H2O.>> > As Brian points out, if you carefully examine a vial of > such a solution being slowly cooled to well below its freezing > point, what you notice first is the presence of a few scattered > large ice balls in the solution. These are the points in the > solution where nucleation and subsequent ice growth first began, > and they will typically have formed and begun growing at between > -50 deg C and -60 deg C; at the warmest temperatures that ice can > form in a 7.5 M glycerol solution. The "sentinel" ice balls you describe may fail to grow faster precisely because their formation alters nearby concentration of glycerol, a self limiting process especially since the formation of pure water ice requires energy since it is entropically disfavored. It would be interesting to learn if this is true. However, with time, as the liquids diffuse, they should grow until the overall concentration is sufficient to impede further change. The phenomenon, as you describe it, is self limiting even in a slowly cooled solution, which is slightly puzzling. MD: Brian can better answer the second part of your question. But yes, as the ice front grows it is largely excluding not only glycerol (or other CPAs) but also the salts and colloids present in the solution. The latter can amount to as much as 6-10% of the volume, and more importantly, concentrated colloids at subzero temperatures are VERY viscous, and probably comprise a huge barrier to the already slowed kinetic diffusion of water. Additionally, the concentration of CPAs and other dissolved solids will be highest right at the crystal-solution interface. This will mean that the freezing point of the microenvironment surrounding the ice crystals will be the lowest it is anywhere, with the possible exception of the intracellular spaces. And diffusion is greatly impeded at temperatures like -50 deg C not only because of cooling, per se, but because of the tremendous increase in viscosity most CPAs undergo when they are cooled to these temperatures. So, you can't simply do an Arrhenius-based calculation of the diffusion kinetics based on water alone - you have to account for the high viscosity of the CPAs/carrier solution.>> > These sentinel ice balls almost certainly occur as a result of the > presence of bacterial ice nucleating proteins that are present just > about everywhere in the environment. That may or may not be the case. It should be straightforward to determine which, however, if it proved to be important. MD: Well, what started Brian off on his criticisms of my previous post was that, in fact, the experiment HAS been done - and most embarrassingly, by me! When we loaded our experimental dogs with 7.5 M glycerol we dropped the freezing point of the tissues to ~50 deg C and we virtually guaranteed both supercooling and inhomogenous freezing. And yet, we saw stunningly good histological and ultrastructural preservation - with the exception of 'random' areas of neuropil that had a 'blasted' appearance - rather like what you'd see in an aerial photograph after a hurricane of Tsunami hits an urbanized area. These were probably areas of primary nucleating with large ice masses. Also, there large holes or tears around many of the brain capillaries. At the time we did that work we were very pressed for money - or more accurately, I was, since BioPreservation (my company) paid for that work. I cut corners in what seemed a perfectly reasonable way at the time, and that was to do just one control that was glycerol perfused and fixed, but NOT frozen. I knew what I was going to see, so I only submitted one sample for TEM and only paid for a few high magnification 'survey shots' to confirm that everything looked as expected. When we got the EMs back on the frozen dogs,they looked spectacular, except for two things - the small 'blasted areas' of neuropil, and much more disturbing, peri-capillary holes. There were these huge gaping holes that looked like tears from ice around many (but not all) of the brain capillaries. Woebetide the scientist who knows what he will see BEFORE he sees it. As it turns out, these peri-capillary holes are NOT from ice formation, but rather are a result of the cerebral dehydration due to glycerolization. This was discovered by Greg Fahy when they began vitrifying brains at 21CM, because they saw the same exact holes in both vitrified and CPA perfused but not vitrified brains! I mention this because, shockingly (to me) as I look back over the micrographs of the 7.5M frozen brains, I find that if I exclude the peri-capillary ice holes, which weren't really ice holes, high molarity glycerol yields structural preservation that is arguably not that much worse than you get with vitrification. Furthermore, Hugh Hixon told me some years ago that it is possible to perfuse 8M glycerol in humans. If you can add ice inhibiting molecules and cool at 0.5 deg C/min it may well be possible to vitrify. AND, if you can perfuse the brain vasculature with ultra-cold gas, preferably helium (I originally thought nitrogen would do, but there is now evidence that it is undesirable) it should be easily possible to cool the brain homogeneously at somewhere between 1-3 deg C min, depending upon the condition of the vasculature. That should handily allow for 'vitrification.' Indeed, just extending blast cooling with cold gas to naso-oropharynx should double or triple the currently achievable cooling rate of 0.3 deg C/min for human heads. The is material because glycerol is far less membrane toxic than current vitrification solutions and in places where tight control of temperature is not possible, it may be a much safer alternative. It is also vastly cheaper, easier to prepare and handle, and perhaps most importantly, results in much less edema in ischemic patients.>> > Brian further notes that these initial ice balls (several > millimeters in diameter) grow as large as they do because they have > lots of time to do so during slow cooling. That would seem on its surface to be correct, but what limits them to a few millimeters? MD: I think this is definitely a Brian question, but my untutored answer would be viscosity and decreasing temperature. It's true that if you HOLD at a favorable temperature, ice will grow to its maximum possible volume. In fact, it was because I did these same experiments as a kid with dry ice that I failed to understand what was happening. In order to prevent freezing of a glycerol water solution at -77 deg C you need ~ 70% v/v glycerol! Since I didn't have sustained access to LN2 as a teenager, all my 'work' with glycerol-water solutions had to stop at dry ice temp. However, if you CONTINUE to cool, and do so with some rapidity, you can stay ahead of the ability of ice to grow. Indeed, that is what vitrification as it is currently practiced depends upon, because no vitrification solution that is biologically innocuous has a critical cooling or re-warming rate of infinity. You always have to cool or warm rapidly enough to avoid devitrification. The ice inhibiting molecules greatly relax those cooling rates and time constraints, but they don't abolish them.>> > However, what is easy to miss, or at least not to > understand, is that as the rest of the rest of the 7.5 M glycerol > solution is further cooled, it will grow cloudy. This cloudiness is > due to the formation of millions of microscopic tiny ice balls > that refract the light. Those microscopic ice balls were areas in > the solution that nucleated LONG after those few big ice balls > formed, in fact, tens of degrees C later, when the solution was > much more viscous. As a result, those tiny ice balls couldn't grow > much before the glass transition temperature (Tg) of the solution > was reached, which in the case of 7.5M glycerol, is ~ -100 deg C. Again, that seems reasonable, but are you sure of the details? One would tend to believe that an abrupt change in optical characteristics has to be associated with a partial phase change, but the exact nature of the phase change is of interest. In particular, can you be sure these are not formed of some sort of organized glycerol-h2o crystals or small particles of glassy solid and are instead pure water? I would tend to agree that pure water seems most plausible, but mere plausibility isn't enough, and this actually may be important. Also, can you be sure the crystals grow no larger because they have no time to do so, or could it be because the overall concentration of glycerol has gone up enough to impede growth, or for some other reason? It seems to me that this would require some experimentation. MD: I'll leave this question to Brian, because he can discourse at length on the magic that is differential scanning calorimetery (DSC). DSC can detect the minutest of phase changes in a sample, and if your numbers all sum out right, then you can be pretty sure "what's what" after cooling to any given temperature. I was still at 21CM when Brian began this work, and it was horrible - the kind of thing that would drive me barking mad. Basically you crimp a tiny volume of solution 'just so' into sealed metal pans, and put them in the device and cool them. Of course, the catch is, that you must do this thousands and thousands of times to build a picture of how different solutions behave under different regimens of cooling and re-warming. It is boring, repetitive and truly dull work.>> Content-Type: text/html; charset="US-ASCII" [ AUTOMATICALLY SKIPPING HTML ENCODING! ] Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=33241