X-Message-Number: 8415 Date: 22 Jul 97 04:18:07 EDT From: Paul Wakfer <> Subject: Prometheus Pilot Project Plans The $100,000 offer of research funds by a pathology professor and research scientist at a research/teaching hospital associated with a major university which I announced a couple of months ago has led to a series of planning discussions which have resulted in the plans detailed here. There is a highly skilled cryobiologist who is very eager to see this project happen and to be part of it. He has already formulated a research proposal with as much detail as possible at this stage. This proposal will be modified, detailed, solidified, and costed during the next few weeks. It is expected that a finished contract proposal ready for submission to the review board of the institution involved will be completed by the end of September at the latest. The scientist who made the offer, the cryobiologist, and I have been conversing regularly concerning the design, implementation and execution of all aspects of this project. However, before planning can proceed much further, we need to know how much money we can expect will be added to the $100,000 from other sources. As I stated in my first announcment of this possibility, since the idea of a pilot project has been discussed among the Prometheus pledgers before, it is my hope and expectation that substantial funds will be forthcoming either from current pledgers or newly attracted cryonicists and life-extensionists. Frankly, I would like to see at least *another* $100,000 added to this pilot project fund not only for its execution, but to finance the full-time participation of the cryobiologist in the Prometheus Project and the initiation of detailed planning efforts for it. The rigorous, conservative, institutional review process for this research contract means that strict confidentiality must be maintained. However, I am ready and willing to disclose the location of the institution, and the names of the principal scientists involved to those who phone me to discuss making a funding decision. No written disclosure (or verbal disclosure to "outsiders") of this information will be made (or should be made by others) in order that this connection with a major academic institution and the scientists involved is not jeopardized. Once the research is underway, has started generating results and successes, and more money is available, more complete disclosure should be possible. Timing, Staff, Organization Plans: 1. The pilot project is planned to be a year long, beginning some time before the end of 1997. As you will see, as you read on, this is actually the beginning of the Prometheus Project research itself. 2. If funding and plans proceed as expected, the cryobiologist will receive a faculty appointment at the institution involved and could have an established working relationship by January 1998. With this appointment, he would be in a position to take part in several research activities in which he is interested at the institution involved and would be eligible to apply for and receive grants from various granting agencies. 3. A research technologist will be hired to work in the lab full-time on this project. For this position, we will attempt to find someone who is sympathetic to the goals of the Prometheus project. This person needs to have a science/lab education/background, but will receive on-the-job training in the specific work to be done. We will also need the full-time services of an EM (electron microscope) technologist. 4. A for-profit corporation will be formed to finance the portion of this project beyond the offered $100,000. Those who put money into the project will do so either by purchase of shares in that corporation, or by donations to a 501(c)3 organization which are 100% "passed on" to that corporation (either by donation or purchase of stock). Some of the money that the corporation furnishes to the project will be donated to the research institute earmarked for the project. This money plus the original $100,000 will be spent on salaries and other consumables. The bulk of the money from the (our) corporation will be spent initially to purchase the specialized equipment needed for the pilot project and the continuing Prometheus research. This equipment will be made available to the research institute for use on the pilot project and whatever research continues from it, either at the institution or in a private lab. 5. The facilities at the research/teaching hospital while offering good electron microscopy resources are somewhat limited physically with respect to carrying out the full Prometheus Project research, even during its first ten years. Our present plans call for establishing a private lab nearby to do the larger portions of the research sometime after the pilot project begins and when the full Prometheus Project research is ready to begin. The reason why we want to retain ownership of all equipment purchased is so that it can be located to best advantage (as well to maintain some coporate assets for the shareholders). The lab at the research institute will continue to do work related to the ongoing effort even after the pilot project is complete and the private lab is in operation. In order to maintain the academic/establishment connection, new, or ongoing contract research proposals (sub-projects of the main Prometheus goal) will continually be made to the research institute. THIS IS IT GUYS AND GALS. WE CAN BE UNDERWAY AND ON THE ROAD TO PERFECTED SUSPENDED ANIMATION AND AN OPEN-ENDED LIFESPAN, IF WE *CHOOSE* TO BE. IT IS ALL UP TO US NOW. Partial Science plans: The complete current science plans are available to those who are seriously considering making a monetary contribution. Such potential contributors will also be given detailed updates as modifications to these plans and their costing occurs. The background and summary of the research proposal are given below. Please understand that this research proposal is a conservative, but essential first step toward the broader goal of the Prometheus Project. Research Concept for Initial Prometheus Project Pilot Study: "Feasibility of Brain Cryopreservation by Vitrification" Informal Draft July 6, 1997 Background It was reported in 1984 that rabbit brains perfused with 6 M glycerol appeared unaltered by light microscopy after prior freezing and thawing, but that severe disruption of neuropil was visible when only 3 M glycerol was employed. Unpublished informal follow-up studies using electron microscopy have shown that ultrastructure is not adequately preserved at 6 M glycerol, but is often excellently preserved at 7 M glycerol. These results imply that for consistently excellent ultrastructural preservation, concentrations are required that approach those needed for vitrification. Vitrification is cryopreservation in the absence of freezing. This is possible because very concentrated cryoprotectant solutions allow water to remain liquid until the temperature becomes so low that the solution as a whole reverts from the liquid state to the glassy state. The glassy state is an arrested liquid state, wherein translational molecular motions are largely precluded. Because molecular motions are effectively terminated, molecular change and diffusion do not take place over relevant time scales. This allows indefinite preservation without the mechanical damage to tissue architecture that accompanies crystallization (freezing). The process of going from the liquid state to the glassy state (vitrification) and its reverse (vitromelting) are not in themselves biologically damaging. For this reason, vitrification has in other contexts been proposed as a means of preserving the viability of whole organs at cryogenic temperatures, and has been used for the successful cryopreservation of many simple living systems. This shows that the addition of high concentrations of glass-forming cryoprotectants as needed for ultrastructural preservation need not result in major biochemical lesions. Enzymes appear not to be denatured by either glycerol or dimethyl sulfoxide. An unpublished attempt was made in 1981 to vitrify a perfused, sectioned rabbit brain using 1,2-propanediol as the cryoprotectant. Visually, it appeared that both the grey matter and the white matter vitrified, whereas a similar experiment on a cryoprotected, sectioned canine brain using a vitrification solution containing acetamide led to vitrification of the cortex but not of the white matter. It therefore seems that the choice of cryoprotectant is important for vitrification particularly of the white matter. Conceptually, vitrification of white matter is problematic due to the barrier function of the myelin sheath, but appears possible. Brightman has shown that horseradish peroxidase can travel through the nodes of Ranvier and along the axolemma for large distances, implying that cryoprotectants can gain access to axoplasm if permitted sufficient time and if they are sufficiently membrane permeable. Research Goal The primary goal of the proposed research is to determine whether vitrification of all parts of the brain, with retention of good brain ultrastructure following rewarming, is feasible. The verification of vitrification can only be accomplished using freeze fracture techniques which allow the tissue to be observed in the cryopreserved state. We will use this technique alone and in combination with freeze substitution. To verify that ultrastructure is preserved after warming, brains will be fixed both before and after elution of the vitrification solutes and processed for routine transmission electron microscopy. Should the proposed studies be successful, the most promising methods will be studied in a subsequent proposal using a variety of pertinent biochemical end points. Paul Wakfer Voice/Fax:909-481-9620 Page:800-805-2870 HELP TO ACHIEVE - PERFECTED SUSPENDED ANIMATION WITHIN 20 YEARS! Check out the Prometheus Project web site at URL: http://www.prometheus-project.org/prometheus/ Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=8415