Attending with Argentine biogerontologist and Cryonics Institute Member Rudy Goya (who was profiled on page 6 of the Nov/Dec 2011 issue of LONG LIFE magazine) may have reduced the interaction I had with the cryobiologists. There were fewer sessions than usual, and thus more free time. The welcome reception was not held until the evening of the first day of the sessions.
The first session dealt with an aspect of Argentine cultural heritage, the Llullaillaco children — three Inca children who had been mummified by dehydration high on a volcano and preserved for over 500 years. Two of children were selected by the Incas because they were “perfect” (beautiful and pure) at 6 or 7 years of age. It was believe to be an honor to go directly to heaven, not really death or sacrifice. The children were given an intoxicant and buried alive atop the Llullaillaco volcano. Much of the session focused on the conditions that caused the children to be so well-preserved, and the conditions the curators should use to preserve the children for the future — involving careful regulation of temperature, atmosphere, humidity, and an environment inhospitable to most microbes.
If reanimated cryonicists receive anything like the care these children are receiving, there should be no concerns about being welcome in the future. In a sense, the Incas had it right when thinking they were sending the children to heaven. Of course the Inca children were deprived of life and are unable to experience or enjoy their treatment by modern curators — and cryonicists should not encourage hastening death based on reliance on unproven future technologies.
At this conference there were special “How to do it?” sessions overlapping part of the lunch hour that focused on practical techniques unrelated to the experimental results and theoretical considerations covered in the regular sessions. Sunday’s topic was proteomic analysis, which covered removal, isolation, and identification of proteins from cells. The presenter (from the Institute of Molecular Cell Biology in Rosario) claimed that instrumentation allowing high throughput and resolution had given proteomics a maturity comparable to genomics.
The afternoon sessions were concerned with cell and tissue preservation. Elza Cabrita reported on improved cryopreservation of fish sperm through a combination of cryoprotectants and antioxidants. Locksley McGann reported on experiments sequentially exposing human articular cartilage to four CPAs (DMSO, glycerol, propylene glycol, and ethylene glycol) at lowering temperature (0ºC, −10ºC, −15ºC). Vitrified samples were cooled to liquid nitrogen temperature, and demonstrated 75% cell recovery when rewarmed.
Adam Higgens reported on an improved procedure for washing glycerol from red blood cells. Currently about 99% of banked blood is stored at refrigerator temperature (2-4ºC), with a shelf life of 42 days. Only 1% of blood (mostly rare blood types) is cryopreserved with glycerol and stored at −80ºC, with a shelf life of ten years. A major deterrent preventing more blood from being banked at −80ºC is the 30-60 minute glycerol washout procedure. Adam’s group developed a procedure that can wash the glycerol out in 30 seconds, but 5 seconds longer or shorter results in too much hemolysis. A three minute washout procedure is less time sensitive (one minute longer or shorter is tolerable), but the method needs to be scaled-up from the 0.5 milliliter test volumes being used.
On Monday, Peter Mazur reported that in vitrifying mouse oocytes, it is the warming rate and not the cooling rate that is most critical for success. He spoke of microwave warming and the problem of thermal runaway (uneven warming). Ice blockers would not cross cell membranes, and thus would not be of use against intracellular ice formation. Pier Morin reported on miRNA microarray assessment of miRNA expression of the freeze-tolerant insect goldenrod gall fly at control (+5ºC) and freezing (−15ºC) temperatures. mIR-210 was down-regulated and mIR-1 was up-regulated at freezing temperature (the latter is involved in cell cycle regulation).
Ali Eroglu reported on epigenetic perturbation resulting from human oocyte cryopreservation techniques. Both the slow freezing and vitrification methods he used resulted in down-regulated expression of H19 and Ube3a genes. Igf2r was down-regulated by vitrification, but not by slow freezing.
Monday’s “How to do it?” session described a combination of nanotechnology and stem cells for tissue engineering. Specifically, electrospinning can be used to create a nanometer scale web of biodegradable fibers that can be populated with mesenchymal stem cells by electrospraying. The main challenge is vascularization of the tissue. Vascular Endothelial Growth Factor (VEGF) increases cell adhesion, but not necessarily vascularization.
Barry Fuller reported on successful hypothermic perfusion of liver. A kidney hypothermic perfusion machine has been in operation for ten years, but liver has been more challenging, because of its large size and the fact that two vessels supply the organ (hepatic artery and portal vein). The liver hypothermia perfusion machine uses two pumps.
PhD student Na Guan described her study of gene expression changes associated with chilling injury of rat liver slices. Cryoprotectant solutions supplied by 21CM (Greg Fahy) were used to ensure no ice formation interfered with the process. ATP levels indicated that the cryoprotectant solutions used were causing no damage, although the composition of those solutions was not disclosed. 1108 genes were observed, of which 251 were up-regulated and 77 were down-regulated by chilling at −15ºC. Focusing on the top ten up- and down-regulated genes: inflammatory and DNA repair genes were considerably up-regulated, and genes associated with biosynthesis of cholesterol and polyunsaturated fatty acids were down-regulated. The latter seems paradoxical in light of the up-regulation of cell surface-linked signaling pathways, which indicate cell membrane injury.
During the question period, both Andreas Sputtek and Arthur Rowe were sharply critical of the undisclosed composition of the 21CM cryoprotectant solutions being used. Sputtek said that because science is about disclosure of methods and materials, Guan’s work was not science. Guan said she had begged 21CM for disclosure, but said she was told that anyone wanting to replicate the experiments could buy the solutions from 21CM. Tiantian Zhang said that gene analysis only done 30 minutes after chilling injury does not give the whole picture. She said that in her own work doing gene analysis of fish oocytes or embryoes after chilling injury, gene expression changes dramatically with time — that it is a mistake to only analyze the expression 30 minutes after exposure as Guan had done. After the presentation, Arthur Rowe spoke with Guan telling her how much trouble he has had over the years with her collaborator (Dr. Fahy) in connection with the non-disclosure issue. I spoke with Guan myself after her presentation. She told me that the greatest chilling injury occurs at −90ºC. She also said that she would be getting her PhD in July and did not know who would be continuing her work. When I spoke to Dr. Fahy about the presentation, he told me that the composition of the vitrification solution had been disclosed and that Guan was mistaken in believing that she could not disclose the composition.
Tuesday morning had been scheduled to begin with a lecture by Ken Storey. Storey typically has no interest in what other cryobiologists have to say, is fairly ignorant of areas of cryobiology outside of hibernation and effects of low temperature on animals in nature, and only comes for his own presentation before leaving. His ignorance is on display when journalists get him to do cryonics-bashing, which he does with relish, but the general public only sees the comments of a respected cryobiologist, not the ignorant misunderstandings of cryobiology. I would not have expected Storey to come all the way to Rosario, Argentina only for his own presentation, but this is what he attempted to do — and he missed one of his flight connections. Ironically, this year Storey was honored by being made a Fellow in the Society for Cryobiology. Storey does, admittedly, have a fabulous knowledge of molecular biology, and is an outstanding scientist in connection with his own work.
To compensate for Storey’s absence the conference organizers arranged a makeshift follow-up session on the Llullaillaco children. This wasn’t entirely a waste, because many issues had not been addressed in the first round. I was going to question using a 2% oxygen and 98% nitrogen atmosphere for the children rather than pure nitrogen, but Barry Fuller raised this objection before I was called upon. I did, nonetheless, suggest that the goal should be to perfect the preservation environment rather than try to recreate the conditions of the mountain. Even this had not been done because the relative humidity had been raised to 70% on the bad advice of an expert rather than held to the 40% present on the volcano. The children were reportedly gaining 300 grams per year, probably from the humidity. There is a lower humidity limit below which no microorganisms can grow, but 0% relative humidity in the −20ºC preservation chambers would run the risk of freeze-drying.
For the second session on Tuesday, John Crowe had been scheduled to lead a symposium composed of 3 other speakers besides himself, but all of the other 3 speakers cancelled-out. John, nonetheless, did an excellent job of speaking for the whole session on the basis of his own work. John is an expert in dehydration and freeze-drying of organisms as well as on tardigrades and trehalose. Drying DNA with trehalose prevents fragmentation, and drying proteins with trehalose prevents denaturation. John discovered that drying liposomes with trehalose prevents membrane fusion — although he lost most of the patent rights on commercially valuable processes by publishing too soon. Dehydration of samples containing sucrose drives the glass transition temperature (Tg) from 20ºC to 60ºC, but dehydration of samples containing trehalose raises the Tg from 20ºC to 120ºC. More recently, however, it has been found that LEA proteins can be as protective as trehalose, but in a way that is distinctive and complementary to trehalose — stopping liposome fusion, preventing protein aggregation, and changing sample Tg. Yeast cells are protected against dehydration damage not only by trehalose, but by the trehalose transporter protein which exports the trehalose to the exterior membrane surface and imports the trehalose to the internal membranes of organelles such as mitochondria. But although the genome of tardigrades has been sequenced, the tardigrade trehalose transporter has not yet been identified.
Barbara Reed is probably the world’s foremost expert on plant cryopreservation, and she has spoken a lot about the benefits of antioxidants for cryoprotection. But the presentation Barbara gave on Wednesday gave me the strongest indication that oxidative stress could be a significant mechanism of cryoprotectant toxicity. Not only because a variety of cryopreserved patients show improved viability with Vitamin E, Vitamin C (if iron is removed), lipoic acid, glutathione, and melatonin — but because oxidative damage was shown to increase significantly associated with cryoprotectant loading.
Roland Fleck works with the UK Stem Cell Bank. The Bank conducted studies indicating that a 2-step freezing protocol results in better viability than vitrification. But examining the results of 8 technicians showed that in the hands of the most experienced technician vitrification was as effective as the 2-step freezing protocol. Protocols should not be so highly dependent upon technician expertise. After his presentation, Roland told me he was concerned that he was only able to assay viability by the use of trypan blue, which only indicates membrane integrity and does not provide a very fine measure of cell function. He said that the requirement to use the trypan blue viability assay was imposed by bureaucrats or scientists who do not have much knowledge of cryobiology.
Igor Katkov said that he believes any sperm cell can be vitrified simply by choosing the right cooling and warming rate. He said he was advised by his patient attorney to drop seven slides from his PowerPoint presentation.
At the business meeting the Society membership was reported to be down to 186. The journal CRYOBIOLOGY continues to be profitable. CRYOBIOLOGY has a 33% rejection rate, a 1.83 impact factor, and 33 Members on the Editorial Board. The Society has $300,000, which the IRS thinks is too much for a charitable organization, but the IRS is allowing the society to retain tax-exempt status. Increasing travel awards is the preferred use of money, but there is a problem that on the one hand travel awards are a taxable benefit, and on the other hand it is illegal to pay the taxes on travel awards. The 2013 conference (the 50th annual conference) is to be held in Washington, DC, where the first conferences were held. The 2014 conference might be Istanbul, Turkey and the 2015 conference could be in Isreal, but definite decisions have not been made.
Last year’s new Society for Cryobiology Fellows Barbara Reed and John Crowe each gave presentations reviewing their careers. Barbara Reed began as a plant biologist in 1985, but was brought into the field of cryobiology by a need to preserve germ tissue. John Crowe said that after the Sputnik shock of 1957 the US government sought to encourage more young people to go into science, including him. As a teenager, John was sent to a number of different science laboratories on his summer vacations. John considers himself more of a “dryobiologist” than a cryobiologist. He entertained us with photos taken in the many exotic countries he and and his wife have visited since his retirement.
The two new Fellows for 2013 are Ken Storey and Mehmet Toner.
This conference was attended by not more than about 80 people, at least half of whom were South America. There were maybe 30 or so hard-core Society for Cryobiology Members. This was my 9th annual meeting in a row, but for the most part I made little effort to relate to the cryobiologists, although one of my intentions in attending these meetings has been to soften the hostility of cryobiologists to cryonicists. I sat near the front of the meetings with Rudy who told me that he learned a great deal about the cryobiology behind cryonics practices by attending this conference. Very many of the cryobiologists were reporting on using vitrification at this conference, and including articular cartilage and plant tissue as well as single cells. I was fairly active in my questioning and comments — about which a few of the cryobiologists complimented me.
I lost my sense of urgency about talking to Peter Mazur. Peter recently told a journalist that although it is not possible to prove that the chance of cryonics patients being reanimated are zero, “you can, I think demonstrated that the probability of its being done is so extremely low that effectively it is zero” [CANADIAN MEDICAL ASSOCIATION JOURNAL; Monette, M; The Church of Cryopreservation; 184(7):749 (2012)] I am curious about the demonstration Peter has in mind, but I am also committed to learning from cryobiologists rather than arguing with them about cryonics. Peter walked away a few years ago when I asked him when solution effects rather than mechanical damage cause injury to cells due to slow cooling, so that may be a touchy subject with Peter as well.
I did, however, pepper John Crowe with questions — finding him to be friendly and informative. John confirmed what Peter Mazur had told me about cells being able to tolerate the loss of all osmotic water (freezable water, which constitutes at least 80% of cell water) without injury — a matter of great relevance in the vitrification of cryonics patients (assuming inter-cellular effects are not of great significance).
I sought-out Ali Eroglu, with whom I have had little interaction in the past, calling his attention to an article in the most recent issue of CRYOBIOLOGY about transfection of mammalian ovary cells with trehalose [CRYOBIOLOGY; Chakraborty,N; 64(2):91-96 (2012)]. Ali has microinjected oocytes with trehalose (along with low concentrations of DMSO to protect the mitochondria) [BIOLOGY OF REPRODUCTION; Eroglu,A; 80(1):70-78 (2009)]. Ali had not seen the CRYOBIOLOGY article, but he told me that ovarian tissue is easier to work with than oocytes.
At the final banquet I sat next to one of the conference organizers. He told me that John G Baust had been supposed to conduct a symposium, but had cancelled the whole thing a month before the conference without giving any explanation. He agreed with the comments I had made about the Llullaillaco children, and told me that a committee of cryobiologists was going to supplement the questionable advice that the Argentine government has been getting from a single advisor in New York. He told me that National Geographic had discovered the children and attempted to remove them from Argentina on a midnight flight, but the Argentine government got wind of the plan and intervened. Nonetheless, the children were simply kept in −20ºC freezers for several years while planning and building better preservation chambers.
The return bus trip to BA on Thursday took the entire afternoon — much longer than I would have expected. I sat next to Adam Higgens on the bus, and spoke with him much of the time, mostly about his life and work, as well as about our experiences in Argentina. Adam knew Spanish fairly well because he has spent four months of language immersion living in Equador (and visiting the Galapagos Islands). If he gets a patent for deglycerolizing blood, the University would get half the royalties and he would split his half with his collaborators. The advantages of his method would be the ten year rather than 42-day shelf life for banked blood, and the greatly reduced washout time. The latter is a significant savings in labor costs, but would have to be weighed against greater electrical costs for a −80ºC freezer as opposed to refrigeration. Even if he is successful in perfecting his methods, he thinks that the blood banking industry is too conservative to be captivated by superior storage methods. Adam has attended most of the annual conferences since I began attending in 2004, and told me that he would like to become a Governor of the Society. Not once did Adam ask me what work I do, and he evidently does not know because he was surprised when I told him I am not a Member of the Society for Cryobiology. Whether or not I am formally accepted as a Member, my attendance at these conferences is implanting me into the consciousness of the cryobiologists as being a member of their community.