X-Message-Number: 7715
From: Brian Wowk <>
Date: Thu, 20 Feb 1997 23:05:05 -0600 (CST)
Subject: Progress in Human Vitrification

     MAJOR PROGRESS TOWARD VITRIFICATION OF HUMAN CRYOPATIENTS
 
     by Brian Wowk
 
 
     For more than a decade, cryobiology has tantalized us 
with the hope of eliminating freezing damage via 
vitrification.  While a variety of cell types and small
tissue samples can be successfully vitrified, there have
been daunting technical obstacles to the vitrification of
large organs or whole human beings.  

     Now, however, a fresh approach has opened up exciting 
new possibilities. 

 
     How Ice Damage Occurs 
 
     At room temperature, water molecules are arranged 
randomly and can mingle with other molecules. When the water 
freezes, however, its molecules align themselves in a rigid 
structure that has no room for other substances. This means 
that when ice forms inside human tissue, the ice squeezes 
vital ions and proteins out of the tissue, forcing them 
into shrinking pockets of residual unfrozen water. Even 
the fabric of cells themselves is crushed into these tiny 
spaces among the ice crystals. 

     As cooling continues, more than eighty percent of tissue 
volume can become converted to ice, and the cells are crushed 
beyond recovery. 

     Vitrification eliminates the formation of ice during 
cooling. It is a way of stopping biological time without 
disturbing the natural order inside living cells. 

     During vitrification, ice formation is completely 
inhibited by cryoprotectants (chemicals that prevent water 
from freezing). As a result, liquid water molecules maintain 
their natural random arrangements during deep cooling. There 
is no disturbance of other chemicals or cell components.  
Everything stays exactly where it belongs while cooling 
proceeds. At a temperature below about -100 degrees Celsius, 
molecules cease to move relative to each other, and 
biological time is stopped. This is called the "glass 
transition" temperature. 
 
 
     Partial Vitrification: the Story so Far 
 
     *Partial* vitrification has been the basis of every 
successful cryopreservation experiment during the past fifty 
years. When modest concentrations of a cryoprotectant such as 
glycerol are used to protect cells during freezing, this 
reduces the amount of ice that forms, because the 
cryoprotectant becomes progressively concentrated, lowering 
the freezing point of the remaining unfrozen liquid.  
Eventually the cryoprotectant becomes so concentrated (around 
70% of solution) that no more ice can form. With further 
cooling, the remaining solution vitrifies. Cells that can 
survive being squeezed into these narrow vitrified spaces 
with 70% cryoprotectant concentration will survive the 
freezing process. 

     A problem still exists, however. Collections of 
individual cells can move in response to growing ice, but 
cells in organs cannot move without disrupting the normal 
cell-to-cell relationships that organs require for integrated 
function. This is a primary reason why succesful organ 
preservation by freezing has eluded cryobiologists for 
decades. 
 
 
     Complete Vitrification 
 
     There are obvious advantages to vitrifying an entire 
mass of tissue instead of just narrow spaces between ice 
crystals. If ice is eliminated completely, there is zero 
physical displacement or physical damage to cells, and no 
progressive concentration of toxic chemicals in residual 
solution during ice growth. Instead, everything stays "in 
place" like a movie slowing down and then stopping. 

     The problem is that completely stopping ice growth 
requires cryoprotectant concentrations near 70%. All known 
cryoprotectants are intensely toxic at this concentration, 
often dissolving cell membranes and obliterating the very 
biological structures we seek to preserve. (As we have seen, 
pockets of 70% concentrated cryoprotectant form during 
ordinary freezing with glycerol. However, these pockets do 
not form until deep sub-zero temperatures, where toxic 
effects are diminished.) 

     In 1981 cryobiologist Gregory Fahy working at the 
American Red Cross suggested a way around this problem. Fahy 
noted that highly concentrated (but non-lethal) solutions of 
cryoprotectant were capable of *supercooling* (cooling below 
their freezing point without freezing) if cooled rapidly. In 
particular, Fahy showed that cryoprotectant concentrations 
near 50% could supercool all the way to the glass transition 
temperature without any ice formation (achieving complete 
vitrification) if cooled at a rate near 10 degrees Celsius 
per minute. 
 
 
     Applying Vitrification to Cryonics 
 
     Bearing all this in mind, there are two primary 
obstacles to successful tissue vitrification. The first 
obstacle is the design of cryoprotectant mixtures that are 
sufficiently penetrating and non-toxic to replace 50% of 
water in cells without injuring them. Dr. Fahy has introduced 
cryoprotectants at progressively lower temperatures to reduce 
toxicity, so that the final "vitrifiable" concentration is 
not reached until the temperature falls below -20 degrees 
Celsius. Unfortunately, glycerol is too viscous and non-
penetrating to be usable at such low temperatures in cryonics 
patients. Consequently, this cryoprotectant is unsuitable for 
human vitrification. 

     Researchers at 21st Century Medicine, in collaboration 
with BioPreservation (CryoCare's cryopreservation service 
provider) have been developing new cryoprotectant mixtures that  
can be used at low temperatures. Concurrently, they have 
completed construction of a unique sub-zero operating room in 
which patients can be perfused with the new cryoprotectants. 
Full results of this work will be presented in a future issue 
of CryoCare Report. 

     The second obstacle to vitrification of large organs is 
more formidable, and for years has appeared insurmountable. 
The cooling rate of 10 degrees Celsius per minute, which is 
required for vitrification by supercooling, is 100 times 
faster than the fastest cooling rates ever achieved for human 
cryopatients. Even direct immersion of patients in ultra-cold 
silicone oil only results in cooling at 0.1 degrees Celsius 
per minute, due to the large volume-to-surface-area ratio of 
the human body. Cryoprotectant concentrations that will 
vitrify at such slow cooling rates are devastatingly toxic
(with toxicity further increased by the long cooling time!).
 
 
     A Breakthrough in Cooling Technology 
 
     The cooling-rate obstacle to human vitrification has now 
been overcome. Researchers at 21st Century Medicine have 
developed a radical new method of cooling organs and 
cryopatients by replacing the blood with a fluorocarbon 
substitute that remains a freely flowing liquid to below -100 
degrees Celsius. The compound is completely non-toxic and 
biologically inert, and functions as a heat-exchange fluid 
that allows cooling hundreds of times faster than could be 
achieved previously. Initial results indicate that cooling  
rates near 10 degrees Celsius per minute can be achieved for 
whole bodies, and 50 degrees Celsius per minute for brains. This
new (proprietary) technology opens for the first time the
possibility of vitrifying humans with survivable concentrations
of cryoprotectant. 
 
 
     The Road Ahead 
 
     Work at 21st Century Medicine over the next year will focus
on optimizing the hardware and software necessary for sub-
zero cryoprotective perfusion and rapid fluorocarbon cooling. 
Development of cryoprotective solutions suitable for human 
vitrification will proceed in parallel. If this work goes 
well, a cryopatient vitrification capability may be in place 
as soon as 1998, heralding the end of ice in cryonics. 

***************************************************************************
Brian Wowk          CryoCare Foundation               1-800-TOP-CARE
President           Human Cryopreservation Services   
   http://www.cryocare.org/cryocare/

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