X-Message-Number: 7658
Date: 06 Feb 97 16:47:15 EST
From: Michael Darwin <>
Subject: Fractures as a problem?

Doug Skrecky writes:

>I would like to suggest a possible solution to the problems that have
 >been occurring with replication of the Visser technique. It seems that
 >good viability requires that the rat hearts be retrieved from liquid
 >nitrogen promptly, while prolonged immersion is lethal. I strongly
 >suspect the problem is cracking and believe that an examination of the
 >hearts used in the experiments will back me up in this. Switching to a
 >two step fast then slow cooling rate should eliminate this problem. 

This is a thoughtful suggestion, but, unfortunately fracturing did not appear to
be the problem in 3 of 4 of the Visser rat heart experiments.  I make the
following observations:

1) The 21st Century Medicine (21CM)staff (including myself)  were granted
extraordinary latitude in observing the entire procedure and in insuring that
controls were done which were rigorous.  We carefully observed the hearts after

cryopreservation and did not note any fractures except during the last run where
there was poor perfusion of the cryoprotective agent prior to freezing.  In
particular, I was able to examine the heart at distance of a few centimeters
while it was being reperfused and the surface of the myocardium was crack free.

2) Sandra Russell, a researcher at 21CM (and a former EKG technician) was quite

persistent in asking that the hearts be dissected and examined following thawing
and reperfusion.  She encountered considerable reluctance from Mrs. Visser, but
Sandra persisted and was successful.  The hearts were sectioned sagittally

through the left ventricle.  Two of the hearts revealed uniform blanching of the

myocardium with accompanying edema.  There were no signs of fractures visible to
the naked eye, or with a loupe, in either the atria,  the septum or the
ventricular walls.  One heart had epicardial blanching with evidence of failed
endocardial reperfusion (see below).

3)  Three of the four hearts reperfused to some degree following
cryopreservation.  Their appearance following freezing was the fairly normal
"kidney bean" color typical or blood free hearts.  Shortly after the start of
reperfusion the myocardium began to blanch adjacent to the coronary vessels and
the effluent perfusate from the right heart was noted to be pink-tinged.  This
is a _very_ typical finding after severe cryoinjury to the heart.  The
pink-tinge in the effluent is myoglobin being washed out of ruptured heart
cells.  This is a cell-level injury which has nothing to do with cracking.  The

same sort of injury is seen in red blood cells similarly treated except that the
colored molecule released is hemoglobin, not myoglobin.

4) Similarly, in other studies, kidney and liver slices evaluated for viability

after cryopreservation with 25% DMF exhibit protein loss from cell lysis and the
bathing media takes on a typical "root beer float" appearance.

5) Other investigators, using no cryoprotectants or other cryoprotectants such
as DMSO, have reported similar findings upon reperfusion following freezing of
rat hearts to very low temperatures.  Karow and Fahy in particular have
extensively documented this effect.  Further, they achieved recovery of

contractility in rat hearts subjected to 20 minutes of freezing at -20 degrees C
following cryoprotection with DMSO.

6) The injury I observed in the hearts cryopreserved with the Visser method was

massive.  The myocardium was globally compromised with lysis or major alteration
in membrane permeability of the majority of the cardiac myocytes occuring as
evidenced by onset of thaw rigor, blanching of the myocardium, and the
appearance of myoglobin in the effluent following thawing.  NOTE: the effluent
was crystal clear long before the end of, and during the entire course of
cryoprotective perfusion.

7) Variables between runs were so poorly quantified and so poorly controlled it
is impossible to draw meaningful conclusions about those hearts that recovered
following 60 seconds of immersion in LN2 on the Langendorff column (as reported
by Zawacki/Ettinger).  That was NOT the experiment I observed.  There was no
good measurement or control over "shroud" volume/composition, intracardiac
temperature descent/rewarming, EKG acquisition, etc.  In short, what I saw was
not good science.  Artifact was reported as evidence of electrophysiological
recovery, and no paper strips were available for evaluation.  This is
unacceptable for an event that can occur in a fraction of a second.

In conclusion, I don't think cracking was a limiting factor in this model.  I
think the most likely explanation for failure of the hearts to recover
contractility was massive cryoinjury as a result of inadequate cryoprotection.
Everything I observed in terms of outcome was consistent with the results seen
by other investigators in hearts subjected to cooling to -196 degrees C in the
absence of adequate cryoprotection.

Lastly, I want to take this opportunity to congradulate and to thank Fred and
Linda Chamberlain for what they did that weekend.  Fred was passionately and
unflinchingly insistent that the protocol be followed in terms of time,
temperatures, and rate of cryoprotectant introduction.  He and Linda also
allowed the other investigators present (the 21CM staff and Paul Segall of
Biotime) to observe the experiments first hand.  This was _invaluable_.  Such

accountabilty and unflinching committment to getting at the truth took something
rarely seen anywhere: REAL COURAGE.  Whatever our differences have been, I

congradulate Fred and Linda, and Alcor for this courage.  I also thank them, and
so should the rest of us.

Mike Darwin

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