X-Message-Number: 6417
Date: 30 Jun 96 00:16:43 EDT
From: "Kent, Saul" <>
Subject: Progress In Kidney Vitrification Research

	Saul Kent asked me to respond to notes posted recently by Mike
Darwin and Brian Wowk concerning the present state of my research on
vitrification of rabbit kidneys.  I am going to limit myself to the
technical issues involved, not to some of the political issues raised by
Brian, to avoid endless discussions of unresolvable issues.
	Mike predicts major problems with electromagnetic warming.  He
may, of course, be absolutely correct.  I personally would rather do the
experiments than speculate on their outcome in advance, particularly since
the results should become available, as Brian notes, well before the end
of 1996.  Whatever happens, we will take the next logical step.	
	I have to gently correct some of Brian's statements.  It is
improper use of terminology to say that we can "cryopreserve" kidneys at
around -45 deg. C, because the word "cryopreserve" implies the ability to
store for indefinite, or at least for significant, periods of time. 
Kidneys stored at -45 deg. C, unfortunately, will freeze in about 30 min
to an hour, presumably precluding recovery on warming.  Therefore, while
it is accurate to say we have recovered a kidney from around -45 C, we
still cannot be said to have cryopreserved a kidney even at that
temperature.  Slices stored at similar temperatures in the past, even when
they did not freeze, died after 24 hours of storage.
	Second, the chief obstacle to cooling to -130 C is not only the rf
heating technology but also the fact that 50% survival is not 100%
survival.  My feeling is that cooling to -130 and warming therefrom may
induce (likely will induce) additional injury, of presently undefinable
nature.  If we begin cooling kidneys that have only a 50% probability of
survival even without this additional stress, then the imposition of the
additional stress of actual vitrification and rapid warming, even if this
additional stress is only very minor, could tip the fine balance between
life and death.  In other words, if we wait until we can buck up survival
to nearly 100% before we attempt actual cryopreservation, we will have a
greater margin for error and therefore a greater probability of
recovering viable kidneys.  (Of course, if we can find no way to improve
upon a 50% survival rate, we will vitrify anyway, having run out of other
options.  Fortunately, our current results suggest that we will not have
to settle for this state of affairs.)
	Another correction has to do with the endpoints of our studies. 
Brian says that 50% of the time, our protocol provides "essentially
perfect ultrastructural preservation."  But our endpoint is not
ultrastructure, it is life support.  When I say 50% of the kidneys
survive, I mean 50% of the kidneys support the life of the recipient
indefinitely as the sole kidney.  We do not routinely examine each kidney
for ultrastructure for a variety of good reasons.  In general,
histological examination will continue to be sufficient for us until we
arrive at our blue ribbon "final" group, which will be examined
ultrastructurally to document in more detail the acceptability of the
results.  In the meantime, survival is good enough for me, as long as
function is equivalent to a control kidney.  This can happen even if
there is minor nephron loss, which need not be a serious concern.  More
important perhaps is the fact that our standard of excellence is more
stringent than that used in human kidney transplantation today: survival
with no dialysis.
	Brian says, speaking of kidneys cooled to "high sub-zero
temperatures", that "these kidneys are vitrified" but -- of course -- they
are not!  The glass transition temperature for our cryoprotectant mix is
around -125 C.  Until we reach that temperature, no vitrification takes
place.  Between -39 C and around -125 C, any kidney is simply supercooled.
	We still have our work cut out for us, even at the current highly
advanced state of progress.  It takes a lot to vitrify a kidney.  But we
are getting closer and closer, the evidence that this can eventually be
done has never been better, and we know of no clear evidence that we
cannot accomplish our goal.  So onward we go.
     -- Greg Fahy


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