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 Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=6417