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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)
After step (3) a burr hole is drilled into each side of the skull. These holes are about an eighth of an inch in diameter and only go deep enough to reveal the brain, without injuring it. The cryoprotectant causes the brain to shrink away from the hole. Classically burr holes have been valuable in cryonics to monitor for edema and blood washout in the brain. The Cryonics Institute also uses burr holes and effluent from the jugular vein as a means of accessing vitrification mixture concentration in the brain. As the perfusion proceeded samples are extracted from the burr holes and jugular veins for assay with a refractometer.
The objective is to perfuse the brain until the refractive index of the vein effluent and the burr hole samples matches at least matches the refractive index of 60% VM−1. The refractive index of 65% VM−1 is 1.422 and the refractive index of 60% VM−1 is 1.416. A 60% VM−1 solution is deemed adequate for stable vitrification. (As a reference, note that the refractive index of water is 1.333.)
Body perfusion was done with ethylene glycol, and now with glycerol only. A complete description of the Cryonics Institute perfusion and cooling protocol can be found at Outline of CI Cryopreservation Procedures and in the case reports, such as The Cryonics Institute's 77th Patient.
Detailed descriptions of the preparation of CI's carrier and cryoprotectant solutions can be found in the WORD document: Solution Preparation Notes.
Below are Electron Micrographs of cerebral cortex tissue of rats that
were perfused with a stepwise protocol of 10% Ethylene Glycol,
30% Ethylene Glycol, and 70% VM−1, cooled to −130ºC
(below the vitrification temperature), rewarmed, and unloaded by stepwise
immersion. No agent was used to open the blood brain barrier during
cryoprotectant perfusion. All samples are 6,000 times magnification.
The white dots in the images are presumed to be processing
artifacts or lipid droplets (white dots are seen in all
control slides as well as the experimental ones).