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Cryonics Institute Research Report for 2002

by Yuri Pichugin, PhD, Staff Cryobiologist, Cryonics Institute

"The broad purposes of our research plans are (1) to improve our standard procedure in order to maximize our patients' probability of revival, within constraints of technical and financial feasibility; and (2) to investigate alternative procedures, whether more or less expensive, so as eventually to offer the best possible range of options, depending on the circumstances of the members or patients. The earliest plans include the following.

1. Further evaluation of the current standard CI procedure. This will include use of the rat brain slice model and the K/Na ratio assay.
2. Modification of the blood washout phase and the solution used. This will probably involve the RPS−2 solution (described in existing literature, but modified according to my requirements and decisions) at a temperature of 4ºC.
3. Modification of the perfusate, according to conclusions I have reached, which will be further tested, including changing the buffer and some of the electrolytes, and the use of some new additives.
4. Testing of the above and of cooling rates at different stages of the procedure, separately and in combination. Also trials and evaluations of a few parameters of equilibration and washout of cryoprotectant. Trials and evaluations of different rates of rewarming.
5. If things go as I hope, and some of my new ideas prove correct, I may be able to create a new cryopreservation method somewhat like vitrification but easier and more stable, and not requiring very fast cooling and warming rates for the best results. But in any case we will make advances."

I started my research work since November 1st, 2001. I performed 215 experiments in the CI research laboratory and 50 experiments in the other three labs, namely in the Harvard Medical School, Boston, in the Kharkov Institute of Cryobiology, the Ukraine, and in the Moscow Institute of Neurophysiology, Russia.

Please see the points 1 and 4 of the last year research agenda.

I created a method for determination of general glycerol concentrations in a patient’s body and head without taking biopsies. I’ll start to use the method for a first future CI patient in order to get more information about the current standard CI perfusion procedure for its better improvement.

The CI suspension method based on glycerol was carefully investigated using the rat brain slice model and K/Na ratio assay. I studied brain slice survival using various glycerol concentrations (from 15% to 80%), various glycerol exposure conditions, various cooling and warming rates (from very slow to very fast), and various final freezing temperatures ( from −20ºC to −196ºC, LN2 temperature). Dependence of brain slice survival upon the cryopreservation parameters was found . In general, the survival was maximal (about 60%) when about 70% glycerol, very fast cooling and warming rates, and the final cooling temperature −130ºC were used.

Please see the points 2 and 3 of the last year research agenda.

I proposed the new blood washout and vehicle perfusion solutions based on modified RPS−2 (Renal Preservation Solution Two). CI will use the solutions.

I also recommended to saturate a patient’s body with glycerol up to 70% and to use faster cooling rates from 0ºC to −100ºC but then to use very slow cooling rates from about −120ºC to −196ºC to avoid body cracking.

I think I investigated the glycerol cryopreservation method for rat hippocampal slices completely. After that, I studied toxicity of seven cryoprotective agents for brain slices. I am naming the agents in the order of the increasing their toxicity: ethylene glycol, glycerol, 1,2 propanediol, dimethylformamide, dimethyl sulfoxide, monomethyl ether of ethylene glycol, and formamide. The five first compounds were individually used to try finding a cryopreservation method that could be better than the glycerol method. Only ethylene glycol can be used for that.

I have recently started to carry out experiments to try creating a new cryopreservation method somewhat like vitrification but easier and more stable, and not requiring very fast cooling and warming rates for the best results. It was the fifth point of my research plan.

Before I present my plans in outline, I would like to express my paradigm. My present scientific paradigm for cryonics is that, most likely, a complete recovery of even frozen animal brains will not be achievable without the use of nanotechnology. So, I will try to find a cryoprotectant mixture which would be least toxic for brain tissue in concentrations up to 70% or, maybe, even up to 85% in order to use slow cooling rates for human bodies to vitrify them with a good practical cryotechnology. My paradigm is based on my great experience in my experimental work with vitrification of brain tissue. Brain tissue is the most delicate tissue of all the biological tissues.

I will try to find a complex, many component cryoprotective solution with minimal toxicity and sufficiently high vitrification activity. The vitrification solution will have the following constitutive parts:

1. a mixture of penetrating cryoprotectants;
2. a non-penetrating cryoprotectant for facilitating vitrification and inhibiting devitrification;
3. better components for vehicle medium than RPS−2;
4. possibly effective ice growth inhibitors;
5. possibly effective compounds prepared brain tissue against toxic and/or freezing stresses.

After the use of the brain slice model, I will test the findings using a fresh dead animal model. Also, I agree to work with human cadavers in order to adapt direct the future CI vitrification technology for real patient’s cases. Of course, if the work will be possible.