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/

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