X-Message-Number: 7439
From: Brian Wowk <>
Date: Mon, 6 Jan 1997 23:04:02 -0600
Subject: Visser Method Improvements

In CryoNet message #7423 Doug Skrecky writes:

>    In cryonet message 7415 Saul Kent revealed
>that the cryoprotectant used in the Visser technique is
>dimethylformamide. Apparently relatively low and nontoxic concentrations
>of this are used in conjunction with fast freezing to cryopreserve rat
>hearts without vitrification. A possible improvement in this technique
>might be achieved by choosing a different cryoprotectant or perhaps
>combination of cryoprotectants.

	Not necessarily, as the "Visser method" (as indicated by
Visser) putatively involves novel physical principles which may be
particular to dimethylformamide (DMF).  (Although work at 21st Century
Medicine, and previously published studies of DMF, fail to show any
unique physical behavior of this agent over a large range of
concentrations.)


>    When one examines the effect of cryoprotectants on subzero
>nonfreezing storage of rat hearts one finds an interesting correlation.
>The order of merit is methanol > ethanol > ethylene glycol > propylene
>glycol > glycerol. (1)  It is interesting that this is also the same 
>order ranking for the membrane permeability of these various
>substances. (2)

I'm familiar with this work by Layne's group (Cryobiology 30:366-375
1993), and it should be realized that this experiment involved
single-step addition of hyperosmolar concentrations of cryoprotectant.
The experiment was thus designed to be specifically sensitive to
osmotic injury, so it's not surprising that the level of injury
depended on the permeance of the agent.  If a more reasonable
cryoprotectant concentration ramp was followed, it's 
likely that the results would have more closely tracked the
traditional toxicity ordering of these compounds (ethanol > methanol >
propylene glycol > ethylene glycol, as I recall). 

       
>Yet more effective cryoprotectants might be found in those with either a
>still greater membrane permeability or a lower toxicity. The later factor
>may be the reason polyethylene glycol is better at preserving function in
>frozen hearts than glycerol and why glucose is in turn better than
>polyethylene glycol at preserving unfrozen hearts at high subzero
>temperatures. (3) (4)

	Polyethylene glycol of course has zero membrane permeance
compared to glycerol, so clearly one cannot conclude that membrane
permeance alone determines cryoprotective efficacy.  High permeance
is valuable for easy loading and unloading of agent, but many other
factors (ice inhibition, membrane toxicity, biochemical toxicity)
also come into play.  High permeance may actually be a drawback for
many cryonics patients, as any patient with more than a couple minutes
of ischemic time will have a compromised capillary endothelium, and will
likely suffer massive cerebral edema if perfused with a high-permeance
cryoprotective solution.  This was certainly the case with DMSO
and propylene glycol in past cryonics experience (although direct
toxicity of these agents may have played a role as well).


>     Possible improvements in the cryoprotectant solution might accrue by
>considering solutes with permeabilities higher than dimethylformamide,
>possibily in combination with glucose.

	There are several thousand organic compounds that penetrate
cell membranes to some degree, and perhaps a hundred compounds that
penetrate better than DMF.  21st Century Medicine is now in patent
on several compounds (actually families of compounds), some of which 
show better permeance, lower toxicity, and stronger cryoprotective
effects than DMF.  The best vitrification results are now being obtained
with mixtures as you suggest, none of which include DMF because of its
hemolytic effects.  More details will be disclosed later this year.
 
***************************************************************************
Brian Wowk          CryoCare Foundation               1-800-TOP-CARE
President           Human Cryopreservation Services   
   http://www.cryocare.org/cryocare/

Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=7439