From: kqb@cbnewsl.cb.att.com (kevin.q.brown)
Subject: Organ Perfusion Fluids & Brain Cryopreservation
Organization: AT&T Bell Laboratories
Date: Thu, 7 Jan 1993 16:27:03 GMT
X-Crossposted-To: cryonics mailing list

    ------------------------ Forwarded Message ------------------------

> Date: 05 Jan 93 03:27:26 EST
> From: Mike Darwin <70023.3041@CompuServe.COM>
> Message-Subject: CRYONICS Organ Perfusion Fluids & Brain Cryopreservation

> To: Edgar Swank
> From: Mike Darwin
> Re: Organ Perfusion Fluids
> Date: 4 January, 1992

     I have been away on holiday and thus have been unable to reply to net 
queries and communications sooner.

     Neither DCS 200 or FC-75 are water soluble or water miscible. This is 
an  *advantage* because toxicity is minimized and the  desirable  physical 
properties of the material are not likely to be affected by dilution  with 
water.

     I  do not know offhand what the vapor pressure of these  agents  are, 
however, the point I wish to make is that these fluids are not supposed to 
be used as intermediaries for helium or other gas perfusion but rather are 
designed to *replace* said gas perfusion.  The principal objective of  gas 
perfusion is to clear the circulatory system of fluid that will turn  into 
ice.   These  agents  will not freeze or vitrify until  very  low  subzero 
temperatures and will not EXPAND when they do.  Thus, they would serve  to 
substitute for gas perfusion.

     The  problem  of  large biomasses  fracturing  during  cooling  after 
reaching  the  glass  transition point is not  one  of  "uneven"  cooling.  
Rather it is a complex property of weak glasses interacting with  stresses 
which  occur during the cooling of inhomogenous materials below the  glass 
transition point of the solution which they are embedded in/comprised of.

     To  translate:  Let's use a simple analogy that's easy to grasp.   If 
you have two dissimilar materials such as a sheet of steel and a sheet  of 
glass  and you cool both of these materials very evenly and  homogeneously 
the  fact  still  remains that steel contracts at a  different  rate  than 
glass.  Indeed, the fact remains that almost ANY material will contract as 
it  is cooled.  This is why we have expansion joints in bridges and  other 
large  structures.  If the steel and the glass cool they will contract  at 
different rates.  This is not a problem providing they are not attached to 
each  other  or  are in some kind of frame  or  restraint  which  prevents 
contraction  during cooling.  However, if we glue the glass to  the  steel 
(leaving  aside the extra set of problems introduced by the glue!) we  now 
have  a  situation  wherein  materials  with  different  coefficients   of 
contraction  and  expansion are *bonded to each other*.  As  they  try  to 
contract at different rates they will be unable to slide over each  other.  
Forces  will  begin to build up and at some point the  material  with  the 
lower  tensile strength will break -- or fracture.    Thus, the  *eveness* 
of cooling has little to do with the ultimate occurrence (or lack thereof) 
of fractures.

     Similarly,  large masses of structurally weak glasses may  accumulate 
internal stresses during (even very slow) cooling which will  subsequently 
cause them to shatter.  I have cooled 100 cc volumes of 60% (v/v) DMSO  or 
75%  (v/v)  glycerol  very slowly to -190xC.   Just  lightly  tapping  the 
container  or  warming it slightly by lifting it up out of cold  vapor  is 
enough  to cause "instantaneous" and massive fracturing of  the  solution.  
With  rapid  cooling  below  the  glass  transition  point  (TG)  you  see 
infrequent  large  fractures that occur at relatively  high  temperatures.  
With  very  slow  "differential free" cooling of bulk  solutions  you  see 
literally  hundreds or thousands of smaller fractures.  The same seems  to 
be  true  of  biological material such as cats  which  are  cooled  either 
rapidly or slowly.

     As  to the possibility of "voids" of water based perfusate  remaining 
in  the circulatory system after perfusion with oils or  fluorocarbons;  I 
can't really say.  I have inadvertently administered DCS 200 intravenously 
to  a  rabbit during intraperitoneal toxicity testing and I can  tell  you 
that  a  small volume of this solution (ca. 5 cc)  resulted  in  immediate 
death of the animal from massive pulmonary embolism.  Of course, that  was 
with  the  material  being  given  into  circulating  blood  rather   than 
introduced  as  the sole circulating fluid (where one  would  expect  less 
creation of blebs of oil that would act as emboli).

     As  to  the issue of my remarks about using these agents to  cool  to 
below -80xC I have the following to say:

     From  a cryobiological standpoint most of the events  causing  injury 
which  you  are trying to avoid by using gas,  silicone,  or  fluorocarbon 
perfusion will have already occurred or been avoided.  Here I refer to ice 
formation  and  its subsequent effects.  If you are going  to  freeze  the 
system  it  will  almost certainly be frozen by -80xC and  thus  you  will 
either have reduced or eliminated ice mediated damage by gas perfusion  or 
you will have failed.

     Gas  or "inert" liquid perfusion below -80xC is not going  to  effect 
this  kind  of damage one bit, and increasing the eveness  of  cooling  to 
below  these  temperatures  isn't  going to  help  you  avoid  fracturing.  
Indeed, delaying fracturing until very low temperatures seems to result in 
more  numerous fractures which would seem to present a greater barrier  to 
subsequent repair.

     As to the issue of rates of reaction, storage temperature and so  on, 
it  is important to realize that the Arrhenius equation is predictive  and 
useful  only in aqueous or gaseous systems where the two products can  get 
at  each other and react.  If an enzyme cannot reach its target  substrate 
because  it  is  embedded in glass, then no reaction  will  occur.   Also, 
enzymes,  unlike  inorganic chemicals, are very complex  and  depend  upon 
their  shape (among other things) i.e., stereospecificity, in order to  be 
able to engage in a reaction.  Many enzymes have "energies of  activation" 
below  which  there is a sharp drop-off in their activity,  if  not  their 
complete inactivation.

     What  is  the  relevance of this to  cryopreservation  of  biological 
systems?   The  answer is simple:  Below the glass transition  point   the 
Arrhenius  equation  breaks  down.   When  enzyme  and  substrate   become 
immobilized in glass, chemistry is, for all intents and purposes,  brought 
to  a  halt.  This "theoretical" observation is confirmed in  fact.   Most 
cells  and  tissues  are stored not *in* liquid nitrogen,  but  rather  in 
liquid  nitrogen *vapor* at about -135xC to -150xC.  Higher  temperatures, 
such  as  -80xC  have been tried, but do not work  probably  because  many 
enzymes  are  still  somewhat active at this temperature  and  *much  more 
importantly, because a substantial fraction of the cell volume is still in 
the  liquid state.*  It is very important to realize that biochemistry  is 
largely  *diffusion  driven* and if you stop the diffusion  you  stop  the 
chemistry.  Several companies manufacture *mechanical* refrigerators which 
take  advantage of this fact and deliver a temperature of  -135xC.   These 
freezers  are  advertised and successfully sold and used to  store  viable 
biological  systems such as sperm, embryos, heart valves, and so on  at  -
135xC.  Queue Systems is one such company and a great deal of thought  has 
been  given  to using this kind of technology for  storage  of  suspension 
patients.

     The  crux  of all this is that if you do not cool much below  TG  you 
should  not  experience  fractures.  Furthermore, a  large  body  of  both 
theoretical and practical evidence indicates that storage at  temperatures 
not far below TG should be biologically acceptable.  TG for glycerol-water 
systems is around -100xC.  For DMSO water systems it is probably closer to 
-130xC  (I'd have to check to be sure).  Based on my  limited  experience, 
solutions tend to fracture about 10xC to 20xC below TG.

     I  have  no ideas on the cost of a vacuum chamber to  carry  out  the 
experiment you outline.

     I  have  no comment on the DMSO-trehalose idea because I  don't know 
what  the result would be.    I know that a noted cryobiologist  did some
experiments  in this  area  and found the results disappointing, but I do
not recall the specifics.

     I  have  tried trehalose alone with my own RBCs and  found  a  modest 
increase  in survival with slow cooling.  This work and the work of  Crowe 
and  Anchordougy  was  responsible for Alcor switching  from  mannitol  to 
sucrose   as  the  impermanent  osmotic  agent  in  the  base   perfusate.  
Conversations  with membrane cryobiologist Tom Anchordougy indicated  that 
sucrose  was  almost as effective as trehalose, but it  costs  many  times 
less.

     Keep  in  mind however that trehalose and these other  agents  cannot 
protect  against  mechanical damage from ice.  THAT is the major  kind  of 
injury  we  appear  to  dealing with right now, and in  any  event  it  is 
certainly  a major factor.  Trehalose and related sugars and  amino  acids 
such  as  glycine  provide membrane cryoprotection: they  do  not  provide 
colligative  cryoprotection by significantly reducing ice formation.   For 
that  we  need a penetrating agent which alters the total  amount  of  ice 
formed and/or its location.

>To: Ben Best
>From: Mike Darwin 
>Re: Brain cryoprotection
>Date: 4 January, 1992

     We are in complete agreement: theorizing is THE BEST way to have your 
research ask the right questions.  Indeed, it is the essential first  step 
in any research: create a hypothesis.  I did not mean my remarks to be  in 
any  way  disparaging of your comments or suggestions.  I just  wished  to 
point  out that we need to have laboratory verification before we  go  out 
and change human protocol.
     
     I will ask our cryobiologist to comment on a nondehydrating  protocol
for introducing  6M glycerol.  I seem to recall that what was required was
to perfuse at 20xC or above.

     Per  the paragraph above I have the following information  to  relay:  
the  nondehydrating protocol consisted of starting glycerol  perfusion  at 
normothermia  and then cooling down to room temperature.  He  wasn't  sure 
how  long was spent during cooldown or if the whole procedure was  carried 
out at room temperature or whether further cooling during  glycerolization 
was employed.

     Alas, things are not as simple as they seem.  Molecular weight is not 
everything  in terms of cryoprotectant penetrability.  Charge,  and  other 
properties  are  also critical.  There also exist  substantial  variations 
between  species.  Rat brains are almost completely dehydrated  by  agents 
that appear to freely penetrate rabbit brains.

     In  fact,  formamide is a significant candidate for causing  the  CNS 
dehydration that our cryobiologist  observed  with  VS. solutions.   Also,
formamide  makes a lousy CPA since it does not depress the freezing  point
very well and it is poor at forming glasses (ie, supporting vitrification).
He includes formamide in the mix largely to counteract the toxicity of the
DMSO.   Given a choice it would  be  far  better to replace the  formamide
mole per mole with DMSO in terms of reducing mechanical  damage  from  ice
formation.

     '[He]  sounds so awesomely confident in CRYOMSG 486 when he  says  "I 
may  or may not be able to work out a technique for vitrifying the  brain, 
but  I  can  certainly  reduce  ice  crystal  growth  enough  to  preclude 
structural  damage  which  would be good enough."'   I  concur  completely.  
Furthermore, I think that that is an objective that we can achieve in this 
laboratory  as  well.   However, I would like to  aim  higher  (i.e.,  for 
substantial or complete preservation of viability) and for that I  believe 
he is essential.   Our cryobiologist has  made a major  commitment to work
with  us,  giving  us as a  minimum of at least  1/3rd  of his time.    We
definitely  have  the physical  plant  and  personnel  to support  such an
operation.

     Why  isn't  implementing such a program the number  ONE  priority  of 
cryonics  research?   I don't know?  You tell me!  A major reason  for  my 
dissatisfaction with Alcor was lack of attention to this area.  I spoke up 
more  and more vociferously and was ignored.  Indeed, several people  have 
actually accused me of being the reason the work hasn't been done.
     
     Well, we now have an independent capability to do this kind of  work, 
indeed  we have the ONLY capability that I know of.  Now the question  is, 
will the cryonics community support it both in terms of dollars and  other 
efforts?

     Keith  Henson's  inappropriate  remarks  discouraging  involvement  in 
our cryobiologist's efforts, my efforts and the efforts of a growing number
of  others to achieve this end is not the kind of response we need.   I had
hoped that despite  division in other areas, this whole community could get
together on the need to do this kind of work.   To this end, I  have  tried
to minimize my participation in the many areas of contention.

     A   comprehensive   approach   to   improved   or   perfected   brain 
cryopreservation  will  demand  enormous amounts of  financial  and  other 
support.  Fragmentation in this area will lead to FAILURE.

     Our cryobiologist has been working  on  the  master research proposal
for brain  cryopreservation  for  several  months now.   A careful program
is being mounted to present this proposal to the entire cryonics community
in a thoughtful and orderly way.

     If  you  (Ben) or others are interested in knowing  more  about  this 
and/or  helping,  please feel free to call me at (909)824-2468  M-F  after 
3:00 P.M. PST.