X-Message-Number: 7762
Date: 26 Feb 97 07:16:05 EST
From: Mike Darwin <>
Subject: Copy of: Advances in cryopreservation

Recently Brian Wowk posted some disclosures about the  (FC) cooling approach
being developed at 21st Century Medicine.  This prompted a number of questions
(all good ones) which I had hoped to have time to answer sooner.  As it is, we
have all been busy doing ICU care on one of our asanguineous (blood washed out)
deep hypothermic circulatory arrest dogs.  Since we are using a lot of new
technology and pharmacology on this run and it has been sometime since we last
pumped a survival dog, we have had our hands full! This post will be messy; I
have had little sleep since Saturday.  I make no apologies.

I will try to address the questions asked from memory, and I will also expand a
bit on FC cooling and elucidate some problems and pitfalls which will hopefully
put things in a clearer perspective.

1) Thomas Donaldson asks how sure we are this will work.  The answer is, pretty
sure.  Others have perfused organs with pure fluorocarbons (i.e., non
emulsified)  for reasons not related to heat exchange and have been able to
achieve good flows and normal histology and acute metabolism following 24, 48,
and 72 hours of storage at near 0 degrees C.  I can provide the cite if anyone
is interested.  We have done several dog runs and we have found a number of
significant pitfalls.  FCs are totally insoluable in water or
cryoprotectant-water mixtures.  Most are approximately 2X the density of water
and most have kinematic viscosities of far less than that of water and

dramatically less than that of blood or cryoprotective perfusate (CPA) mixtures.
These three factors mean that getting FCs in and evenly distributed requires
clever techniques.

The best way to visualize the problem is to imagine trying to deal with air in a
complex plumbing system.  Air bubbles will float to the top of horizontal pipes
and form long, linear bubbles which will be broken up and displaced slowly as
fluid is pumped through the system.  In this case the "air equivalent" is
perfusate and it does not all come out of the system easily.  On the low
pressure venous side you are left with a long, linear bubble of perfusate
running the length of the vena cava.  If you tip the animal the perfusate will
behave just like air would in such a system since it is less dense; it will
quickly flow to the highest point.  If you are moving the animal into a cold
bath and you stop flow of FC and tip the animal during the move the CPA
perfusate will displace FC in the cannulae.  If the cannulae are in a bath at
below the freezing point of the perfusate you will get a frozen plug of

perfusate in the cannulae and/or the connecting lines.  This little failure mode
cost us a $5,000 experiment!

Another problem is the complexity of flow through a multi-pathed circuit which

is filled with a high viscosity fluid (CPA perfusate) and which is being flushed
with a low viscosity fluid (FC).  What will tend to happen is that the shortest
connecting paths or shunts between the input and output sides of the plumbing
array will be flushed out with FC first.  In other words, the shortest paths
between the arterial and venous elements of the circulatory system will be
flushed clear of CPA perfusate and filled with FC.  The problem is that once
this happens, these shorter, lower resistance pathways become "short circuits"
through which most of the FC flow occurs through.  The FC in effect takes the
path of least resistance.  In a mammal the brain, kidneys and liver are high

flow and will tend to get most of the flow of FC.  But, even within these organs
there are short circuits in the capillary bed.

Consider this fact: in a normal, healthy person only about 1/3rd of the
capillaries are open to flow at any one time.  Most of the capillaries are cut
off from flow by the precapillary sphincters which open and close in a pattern

called vasomotion.  Once one area of tissue gets well perfused it closes to flow
and another area ges flow.  Then, the previously perfused area opens up again
when it needs more nutrients and oxygen.  This vasomotion or autoregulation of
capillary flow is mediated by induceable nitric oxide production (and other
factors).  In multisystem organ failure normal vasomotion is disrupted and
autoregulation is lost.  Cardiac output goes from a normal of 4.5 liters per
minute (LPM) to 30 or 40 LPM!  This is called hyperdynamic shock and is a
devastating medical problem which has occupied a great deal of my research time
and attention over the past few years. Most cryonics patient experience loss of
normal vasomotion during the agonal period in slow death.

The importance of understanding vasomotion is to realize that much of the
capillary bed is _not_ being perfused at any one time.  Further, low viscosity
FCs will find the shortest pathways and preferentially flow at tremendous rates
through these pathways.  To open up more of the capillary bed would require
increasing the pressure (and flow).  In small models we've used just this
approach.  For larger animals the FC viscosity must be carefully modulated
during washout of CPA and low viscosity mixtures (with lower pour points) must
be ramped in.

However, even with this approach we see "perfusate embolization" during FC
perfusion up until the point that the CPA perfusate solidifies (by freezing or
vitrifying).  Perfusate embolization consists of little streams or bubbles of
perfusate entering the main venous FC outflow as more and more of the capillary
bed is opened up.  This presents a tremendous engineering problem because if

this water-CPA perfusate is not _completely_ removed from the venous flow before
it is pumped back into the arterial circulation it will act exactly like an air
embolism or a blood clot.  I have solved this problem with a series of
separators, traps and filters but it has been costly and difficult.

Seeing this phenomenon has helped to explain why when we wash blood out of dogs
or people we see a slow accumulation of red cells in the formerly RBC free
perfusate once we close the circuit and start recirculating.  We have always
wondered how we could wash out a human with 20 liters of perfusate (4X blood
volume!) and still see the RBC count rise once we closed the circuit.  Now we
know why: there is a lot of capillary bed which is still blood filled and the
lower viscosity cell free perfusate is "channeling" through the paths of least
resistance.  This also explain why we see a steady stream of RBCs emerge from

the patient as cryoprotective perfusion with glycerol proceeds: as the perfusate
viscosity rises and the perfusion pressure rises we open up more and more
formerly un-perfused capillary bed.

We've done several large animal runs with FC perfusion to and from deep subzero
temperatures and we're reasonable confident it is workable. It is certainly
workable for heads (brains) or other single organs.

We are, however, a LONG way from having all the bugs worked out for whole
animals such that revival would be possible.  This will remain a problem until
we can access _most or all_ the capillary beds with FC and displace all or

nearly all the cryoprotective perfusate. What we DO have now is a technique that
will allow fairly even and controlled rate cooling of cryopatients.  We do

perfuse most of the vasculature with FC and the ultrastructure looks much better

in dogs so treated following cryopreservation.  Small animal runs are easier and

less costly and we will probably concentrate on these for the foreseeable future
because of:

2) Costs.  The FC mixture we are using is astronomically expensive.  The FC for
one dog alone is nearly $3,000.  Yes, we can recover some of it and reuse it.
But it is still very expensive stuff.  This expense has lead us to develop  an
alternative to the traditional immersion bath which has been used to cool
cryopatients for years.  Instead of using a tank full of astronomically costly
FC the patient will probably be  placed on a conductive metal lattice-work

elevated above the bottom of a tank and a continuous, high flow spray of FC will
be directed over the the surface of the patient.  Why use FC for external
cooling at all, you may ask?  Because some areas may not perfuse well with FC
due to clotting, injury, short-circuiting, etc., and we will be relying on
surface cooling.  Secondly, the FCs we are using have viscosities of 0.5 cs.
(about half that of water) at room temperature and the viscosity climbs only to
2-3 cs. at -90 C!  FCs also have very low surface tensions and excellent
spreading coefficients which means that they will flow through ANY hole or tear

in the system.  A needle stick becomes a geyser of FC!  These agents are used as
leak-check agents in industrial applications for just this reason.  If you will
recall a recent technical brief from me on the use of Super Glue to achieve
"hemostasis" in cryopatients you should now understand why this became so

critical an issue:  the smallest incision will _massively_ leak FC.  In patients
with median sternotomies the FC will leak out very rapidly and will flow into
the tank holding the patient.  This leakage will then be recycled through a
"clean-up loop" which will remove all particles and fluid (CPA, ice, solid

debris, etc).  The same FC used to cool the patient externally will be tapped to
provide intravascular cooling as well.  Which brings us to:

3) Thomas' question about whether FC will wash out cryoprotective and Jan
Cotzee's question about FC's penetrability into cells or their components:  FCs

are best thought of as liquid teflon.  Almost nothing will dissolve in them with
the exception of some gases (oxygen, CO2 and nitric oxide being the important

ones to us).  Virtually all cryoprotectants (old and new) are insoluble in fully

perfluoronated chemicals.  So, cryoprotectant is perfused first and then this is

followed by FC perfusion which displaces the perfusate from the vasculature, but
of course does not displace the cryoprotectant or water from the tissues.

Jan Cotzee's question about FC solubility in biological systems can be most
simply answered by stating that FCs do not cross cell membranes or capillary
membranes.  They thus remain exclusively in the circulatory system.  However,
and this is an important _however_, fentogram quantities of FCs appear to
dissolve into cell membranes that are in contact with FC.  For once, we get a
lucky break: in living animals this appears to be the mechanism by which FCs
radically down-regulate ICAM activity; an important mediator of the

immune-inflammatory response.  What this means in practice is that in the living
mammal FCs greatly reduce adhesion and de-granulation (release of toxic
hypohalous acids like chlorine bleach) of white blood cells.  They do not
inhibit white cell chemotaxis, but do greatly inhibit WBC adhesion and release
of destructive chemicals.  This is of tremendous benefit to the lung during
liquid ventilation with FCs since the WBCs are the primary mediators of
pulmonary injury in shock, multisystem organ failure, pneumonia, etc.

4) There were questions about FC toxicity.  We have ventilated _many_ dogs with
FCs.  Some have been ventilated for days with no ill effects.  Right now of the
8 or nine dogs in our colony only 1 has not had FC ventilation.  Aside from the
dog now in the ICU who has other problems (related to being at 0.6 degrees C
with no flow for hours  ;-)), all are fine with no residual effects from FC
toxicity.  Perhaps more to the point, the FCs used for liquid ventilation and
the others used for intravascular subzero cooling have high vapor pressures and

high spreading coefficients which means that they get all over the place.  Thus,
the staff of 21CM has been breathing FCs almost continually for years.  For
those of you concerned about the ozone layer; the FCs we use are not
contributors to ozone depletion; it is the chlorinated and brominated
fluorochemicals that do this.  

5) What about the Prometheus Project?  Frankly, it is needed more than ever.
The FC mix we'd most like to usefor vitrification is simply not available to us
and will not be until after 2003.  Costs for implementing FC perfusion in a
reversible fashion will be well over 21CM's anticipated ability to bear the
costs.  In fact, the major reason FC perfusion is not on-line for humans now is
the incredibly demanding technology required to deliver subzero fluorochemical
perfusion and the high cost of the materials.  BioPreservation is making
incremental purchases of the system components, but the pace is glacially slow
(pun intended).  We have bough the heat exchanger and the heat exchanger
cabinet, but are a LONG way from a working system for humans.  As it is, I have
enough FC to allow me to perfuse one human under poorly controlled conditions
(not good enough to allow for vitrification).

6) Why do we want to FC cool people now, even though we can't yet vitrify them?
The answer is that with the current immersion method of cooling used to freeze
people now, freezing takes place at relatively high subzero temperatures and
this results in a large increase in the concentration of cryoprotectant agent;
ice freezes out as pure water.  Right now, that means nearly a doubling of the
concentration of cryoprotectant that patients are exposed to.  The reason thin
tissues (skin, corneas) or cells in culture can survive freezing is that a

cooling rate of between 0.5 and 1.0 degrees C per minute can be achieved.  _This
means that once freezing occurs the cells are exposed to hyper-concentrated CPA
at high temperatures only briefly._

When we freeze humans this NOT the case.  Freezing occurs and then the patient
sits exposed to massive concentrations of agent for _hours_ during subsequent
cooling to -90 degrees C.  Exposure to these concentrations of agents over a
time course of many hours appears to be dissolving cell membranes; thus I am no
longer at all sanguine that people frozen with today's techniques are coming
back as who they were.  Somebody may come back, but the question is, WHO?  Mary

may have a cloned little lamb who's fleece is white as snow, but the question is
does Mary the_ lamb_ still know? 

It is thus critically important that we start achieving cooling rates in humans
that are consistent with cellular survival (membrane integrity) during
cryopreservation.  This is a staggeringly complex and difficult task and it has
been rough sledding going it alone; doing this is BPI's job, not 21CM's and BPI

doesn't have that kind of money. (If you want to know where every spare nickel I
have has been going, now you know).

7) I believe Prometheus quite understandably underestimated the cost to to
deliver reversible brain cryopreservation rather than overestimated it.  No one

should feel bad about this if this turns out to be the case.   We have dozens of
cryoprotective compounds to evaluate and many, many more times that number of
CPA mixtures.  This will take time and money.  The most promising agent we've
found so far costs nearly $2.00 per gram and is no longer being manufactured.
Tooling to make this molecule and related ones of interest will be very costly.

We already have two organic chemists on retainer, having pretty much hit the end
of the road with most of the big and small chemical houses including
Sigma-Fluka-Aldrich, 3M, BASF, Dow, and just about everybody else you can think

Finally, I want to point out that moderating reperfusion injury and dealing with
the intracellular cascade of events which occurs following stress to cells is
another area we are working on and which I believe we need to continue to work
on.  Greg Fahy cannot recover _any_ kidneys unless he moderates reperfusion
injury with aspirin, prostaglandin and other drugs upon blood reperfusion.  His
technology for doing this is dated and crude compared to what we now have
available.  I believe that controlling the intracellular PARS cascade and

moderating inducible nitric oxide synthase and other mediators of injury will be

critical to achieving suspended animation.  I _know_ that this work will pay big
dividends in the treatment of dying cryonics patients by allowing us to stop
edema and reperfusion injury and allow us to better perfuse people dying now.  

These technologies will take time and money.  Lots of BOTH. Prometheus was never
more needed.

I guess that just about covers it.

Mike Darwin

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