CI Research Report: 2006

by Yuri Pichugin, PhD, Staff Cryobiologist, Cryonics Institute

I was able to carry out 140 experiments for this year. The experiments were devoted to several research projects.

1. I continued to study the possibility of preservation of patients’ brains during 12-24 hour transportation from regions which are far from CI.

1.1 I finished the study of warm and cold ischemia of rat brains. The results were published in the Immortalist magazine (2006, vol. 38, No. 1-2 and 5-6). The main conclusion is that no present organ preservation solutions and additives were useful for long-term cold storage of the brain. Cryonics is very different from today cryobiology and medicine because it relies on future perfect technology, but today imperfect cryobiology and medicine can use only natural, spontaneous resources of biological systems to recover them from ischemic damage.

1.2 I continued to study Dr. Suda’s experiments using whole rat brains and the K/Na ratio assay. The brains were perfused with glycerol solutions (5, 10, and 15% v/v) at 0ºC, slowly cooled to −20ºC, kept at this temperature for 12-24 hours, and washed out from glycerol. Survival of the rat cerebral tissues was evaluated by the hippocampal slice method I usually used. The tissues were dead.

1.3 I continued to study Dr. Seki’s experiments using rat cerebral tissues. Dr. Seki dehydrated rat hearts in a certain extent and kept them in a non-aqueous medium. He briefly reported in the Cryobiology journal in 1999 that he was able to preserve and resuscitate the rat hearts after 10-26 day cold storage! However, there was none of his articles or patent applications on this subject for 2000 – 2006.

I tried to use 2.5-20% glucose solutions in order to dehydrate rat brains in various extents for an improvement of cold storage of the brains. However, that osmotic dehydration was harmful for the rat brains.

I also tried to use Dr. Seki procedure for rat hippocampuses. The hippocampuses were stored in inert fluid with silica gel at 2-4ºC for 24 hours. There was no positive effect.

Rat hippocampus were dried with silica gel in 20 ml vials without inert fluid at 2-4ºC for various time and subsequent cold storage of the hippocampuses for 24 hours. The experiments were performed to find the influence of various degrees of dehydration of rat hippocampuses by drying on slice survival. There was no positive effect again.

I would like to publish the results of my experiments on Dr. Suda’s and Dr. Seki’s subjects in the Immortalist magazine.

2. I continued to study the quality of vitrification mixture (VM) perfusion for sheep brains that had 24 hour cold ischemia.

It is an important research for CI because most CI patients had long term (12-24 hour) cold ischemia. My experiments showed that the sheep heads were successfully perfused with VM-1 solutions even after their storage at 2-4ºC for 24 hours! The sheep brains had the stable vitrification. It is a very important positive result for us. I also did not observe any breakage of the cerebral blood vessels.

3. I studied some problems of VM perfusion of the body. Saturation of the human body with any vitrification mixtures requires a very large amount of VMs to get its uniform and stable vitrification.

A main problem was an accumulation of a large amount of perfusates in the alimentary tract and abdominal cavity. An exact cause of this phenomenon is not known. Most likely, there was a leakage of blood capillaries in those regions. For now, it is impossible to saturate a patient’s body with vitrification mixtures to get its uniform and stable vitrification.

In the past CI had a little swelling of patients’ abdomens because CI used a very small amount of glycerol solutions for body perfusion. Sometimes CI had a bigger swelling even with the small amount of glycerol solutions because those CI patients had a cancer of inner organs.

I elaborated a method of perfusion of a patient’s body with ethylene glycol (EG) solutions for freezing procedure but not for vitrification one. Concentrated EG solutions are much less viscous than glycerol ones. EG better penetrates in tissues than glycerol.

4. I have written a patent application for the CI method of cryopresevation of cerebral tissues by vitrification.

I needed to perform several additional experiments for the application. Cryonics Institute’s vitrification mixture (CI-VM-1) is very simple and cheap. This is one of most important advantages of CI-VM-1 in comparison with other known VMs.

5. I continued to study the possibility of an improvement of the present CI vitrification method for potential CI patients without long term ischemia.

12-24 hour cold ischemia decreased survival of cerebral tissues to 40% of the control. We should propose CI members an improved CI vitrification method with maximally possible cryopreservation of cerebral tissues. For this, first of all, potential CI patients should avoid the long term ischemia.

I think usual compounds that were used against short term ischemia (i. e. anti-oxidants) cannot improve long term (12-36 hours) cold storage of the brain. I decided to test some of unusual compounds. But I have just a little hope to improve the results of the long term ischemia.

One of harmful factors is excessive dehydration of the brain during VM perfusion. The cause of this problem is a very low permeability of the blood-brain barrier (BBB) for cryoprotective agents. As I reported previously, I was able to saturate rat and sheep brains with VM-1 completely without dehydration using Substance X to open BBB. However, I need to continue the study of the influence of brain dehydration on cerebral tissue survival in order to find an optimal procedure for opening BBB. The procedure could be material for a new patent application.

Saturation of the human brain with 70% VM-1 at -20ºC instead of 0ºC could significantly increase cerebral tissue survival. The main problem is very high viscosity of 70% VM-1 at −20ºC, although CI-VM-1 is less viscous than other VMs. I need to perform more experiments to try overcoming the problem.

I also performed experiments studying the possibility of faster cooling a patient’s head through its blood vessels using inert fluids after saturation of the head with VMs. The models for the human head were rat and sheep heads.

Ben Best proposed me to use trehalose and sucrose in order to try decreasing CPA concentrations in VM-1. I was and now I am against decreasing CPA concentrations in VM-1 because this will decrease the stability of brain vitirfication and so can result in devitrification and ice formation. My experiments with trehalose and sucrose showed the decreasing of the stability of cerebral slice vitirfication. Ice crystallization is a very powerful process and so we should not decrease VM-1 concentration. I propose another way to increase cerebral tissue survival, namely to increase the resistance of cerebral tissues to toxic effects of VM-1.

CI-VM-1 Cryoprotectant and CI-Carrier Solution

The Cryonics Institute (CI) has been using a mixture for vitrification of the brains of cryopreserved pets and humans since August 2004, when it first did an experimental perfusion of the dog of a CI Member. The vitrification mixture goes by the name CI−VM−1 (CI Vitrification Mixture one) and was developed by CI Staff Cryobiologist Yuri Pichugin, PhD. The first published use of the vitrification mixture was in February 2005 with the vitrification of the dog Thor. The first human use was in August 2005 with the the vitrification of the 69th CI Patient.

In August 2006 the Cryonics Institute filed a preliminary patent application for CI−VM−1 in anticipation of filing a complete patent application. Although a patent application was prepared, legal counsel advised that the chances of getting a patent were very slim because of commercial use more than one year prior to filing the preliminary patent application. We were advised to publish the CI vitrification and carrier solutions as a defensive measure so that others would not be able to prevent CI from using them.

The final vitrification perfusion of Cryonics Institute cryonics patients is done using a 70% solution of CI−VM−1 in a carrier solution developed by Dr. Pichugin which he calls m−RPS−2 (modified Renal Preservation Solution two). About 8.3 liters of 70% CI−VM−1 is made with:

          2.83 liters Ethylene glycol (3.15 kilograms)
       + 2.83 liters DMSO (3.14 kilograms)
       + 1.0 liter of (9X concentrated) carrier solution (about one kilogram)
       + 1.70 liters of water (about 1.7 kilograms)

The exact volume does not matter so much because 70% (w/w) is based on a weight/weight calculation:

       About 6.3 kilograms cryoprotectant divided by 9 kilograms total gives about 70% (w/w).

The carrier solution is composed of 28 mM/L potassium chloride, 230 mM/L glucose and 10 mM/L organic TRIS − HCl buffer. One liter of 9 times (9X) concentrated carrier solution is made with 19 grams of KCl (potassium chloride), 372 grams of glucose and 11 grams of Tris [2-Amino-2-(hydroxymethyl)aminomethane] in 72 milliliters of 1.0 Normal HCl (hydrochloric acid) filtered in a 2 micron filter. VM-1 is not filtered.

A determination of the glass transition temperature (Tg) of CI−VM−1 made by the cryobiological research company 21st Century Medicine gave the following results:

60% (w/w) CI−VM−1 ==> Tg = −123ºC
70% (w/w) CI−VM−1 ==> Tg = −121ºC

Dr. Pichugin believes that the combination of his vitrification solution and carrier solution are well optimized for both low viscosity and minimal expense, while providing powerful vitrification capability. He does not believe in the value of high molecular mass agents such as proteins, dextrans, HES, PVP, etc, to support oncotic pressure in brain perfusion in CI’s protocol because he believes these agents increase viscosity and are not necessary due to the dehydrating effect of cryoprotectants. In practice the Cryonics Institute has not seen much brain edema or the need for oncotic support in perfusions of brains with CI−VM−1 and m−RPS−2.

Dr. Pichugin has observed that carrier solution additives such as Ca2+, Mg2+, phosphate ion and inorganic buffers result in additive precipitation in CI−VM−1 plus carrier at low temperature — which can block blood vessels during perfusion. For this reason these agents are not included in his carrier solution.

Dr. Pichugin has assessed the ice blockers (Supercool X-1000 and Supercool Z-1000) from 21st Century Medicine to determine the possible benefit of adding these agents to CI−VM−1.

Dr. Pichugin first determined that in cooling to −130ºC (and rewarming) at 0.3ºC/minute that the minimal (critical) concentration of CI−VM−1 required to vitrify (prevent ice formation) without ice blockers is 55% (52% CI−VM−1 without ice blockers results in ice crystals).

Dr. Pichugin then determined that in cooling to −130ºC (and rewarming) at 0.3ºC/minute that the minimal (critical) concentration of CI−VM−1 required to vitrify (prevent ice formation) with ice blockers is 52% (50% CI−VM−1 with ice blockers results in ice crystals).

If by using ice blockers a lesser amount of CI−VM−1 can be used to achieve an equivalent vitrification, then the result would be increased viability due to the reduced cryoprotectant toxicity associated with the reduced cryoprotectant concentration. Unlike cryoprotectants, ice blockers are not believed to be toxic.

To test the toxic effects of CI−VM−1 (with or without ice blockers) hippocampal slices were saturated with increasing concentrations of ethylene glycol at 0ºC and −7ºC before cooling to −20ºC for ten minutes of saturation with CI−VM−1 (with or without ice blockers). The DMSO in CI−VM−1 is less toxic at lower temperatures, and is least toxic when introduced at −20ºC. Adding the ethylene glycol first and cooling at 0.3ºC/minute ensured that the solution would not be frozen at −20ºC when the CI−VM−1 (with or without ice blockers) is introduced.

The results of the toxicity test were as follows:

86.1% viability +/- 5.8% for 55% concentration CI-VM-1 without ice blockers

89.6% viability +/- 6.2% for 52% concentration CI-VM-1 with ice blockers

Toxicity assay was made using potassium/sodium ratios. Full viability (no toxicity) would be 100%. Note again that this is not a test of the ability of CI-VM-1 (with or without ice blockers) to prevent freezing.

Dr. Pichugin does not believe that these increments of increased viability with ice blockers justify the increase in viscosity or cost if they were added to the CI−VM−1 formula, especially in light of the fact that ice blockers cannot cross cell membranes or the blood-brain barrier. If brain areas are very poorly perfused, then even ice blockers won’t help. If the viscosity of ice blockers reduces perfusion, that reduces the benefit of ice blockers. Freezing within cells is not such a great problem because there are few nucleators inside cells. But there may be as many nucleators the interstitial fluid (between cells) as in the bloodstream. Therefore, the fact that ice blockers do not cross the blood-brain barrier may mean that the proposed benefit for poorly perfused areas is not so great.

The Cryonics Institute uses industrial grade cryoprotectants, which helps keep the cost very low. But, although industrial grade, the cryoprotectants are still of high purity. The major impurity is water, which may be unsuitable for laboratories needing chemicals uncontaminated with water, but CI adds water to its cryoprotectant mixtures, anyway, so water as a “contaminant” is not a matter of concern. The DMSO is 99.7% pure with the only impurities listed being water, “color” and titratable acid (0.001 milliequivalents/gram). The ethylene glycol is 99.94% pure with the major impurity again being water: nearly 0.06%. The next largest impurities in the ethylene glycol are acetic acid (<0.001%) and ash (0.0005%). There are part-per-million amounts of chloride, “color”, diethylene glycol, and iron in the ethylene glycol.

The Cryonics Institute protocol for perfusing the heads (brains) of cryonics patients is a 4-stage stepped open circuit perfusion:

(1) blood washout with carrier solution (4ºC)
(2) 10% Ethylene Glycol (4ºC)
(3) 30% Ethylene Glycol (4ºC)
(4) 70% CI−VM−1 (−7ºC)