Cryonics technologies classified by phases of their application

1.    Preliminary preparation of patients for the cryopreservation

1.    Signing up of a contract

Client need to choose the type of the preservation: full-body or the brain/head only (the so-called neuropreservation). Approximately half of the cryonics patients choose the full-body preservation, the other prefer the neuropreservation.
In addition, you need to sign various wills depending on the requirements of the country of residence and the country where the cryopreservation will be perform. In general, all these issues are easily solved with the help of employees of the cryonics company.

2.    Preliminary period

It is very important, to initiate surface cooling as soon as possible, especially in relation to the head of the cryonics patient after his or her legal biological death declaration. Therefore, everything required for the next phase of the cryopreservation must be prepared in advance.

3.    Premedication and primary cooling

Immediately after the legal declaration of the patient's death the primary cooling, the introduction of the necessary medicines (premedication) and perfusion begin. Also, during the initial phase (up to 4 hours after the moment of the legal death), it is necessary to inject heparin into the body to prevent the formation of thrombi prior to the perfusion.
In addition, scientists believe, that it makes sense to inject various medications that will help to stop the negative processes in the cells and improve the efficiency of the perfusion.
The perfusion of the head and body with a special cold wash solution can very quickly cool the head to 0 ° C, thereby protecting the brain cells from the destructive effect of thermal ischemia.
An experienced surgeon-perfusionist connects the perfusion system to the arteries and veins of the cryonics patient depending on the particularities of the circulatory system
In addition, to the perfusion pre-cooling, external cooling of the body using cold packs or even a special cooling bath is also use during the whole cryopreservation process.

2.    Methods of Perfusion

In order to obtain the maximum survival rate of the brain cells, it is necessary to stick to the correct procedure of the perfusion. Parameters of brain perfusion with vitrifying mixtures:

1.     The formula of the vitrification mixture;
2.    Volumes and concentrations of perfusion solutions;
3.    Head and perfusion solutions temperatures;
4.    Perfusion pressure and rate. Viscosity of the solutions at different temperatures;
5.    Methods for monitoring the degree of brain saturation with a vitrification mixture.

Let us study these parameters and methods in the description of the vitrification perfusion procedure, which is currently used in the whole world.

2.1.    The formula of the vitrification mixture
There are several vitrification mixtures used in cryonics.
American cryonics organization “Alcor” uses the M22 mixture (from the laboratory 21th Century Medicine). The cost of M22 for perfusion of one is $ 16,000.
According to thorough experiments on the heads of rats and sheep, it was founded out that the vitrification mixture consisting only of ethylene glycol and DMSO (1: 1) demonstrated the results similar to M22. The cost of this vitrification mixture for perfusion of one head is only about $ 100. A license is not required, because this mixture is not patented. This mixture and its modifications are used by the Cryonics Institute.
Yuri Pichugin, a well-known scientist-cryobiologist, has developed an alternative, equally qualitative vitrification mixture for KrioRus.

2.2.    Volumes and concentrations of the perfusion solutions
The volume of the using perfusion solutions depends on the degree of brain saturation with cryoprotectants. In order to avoid possible damage to cells from osmotic shock, the concentration of cryoprotectants should be increased gradually from 0% to 70% with a use of the special apparatus. In practice, perfusion solutions are administered on different stages with discrete increasing concentrations.
At the initial stage, one injects ethylene glycol without DMSO from 0% to 35% and simultaneously decreases the temperature of the perfusion solutions from 0 ° C to -10 ° C. At this stage, hemoencephalic barrier modifiers are also injected. Ethylene glycol is less toxic than DMSO. As the toxicity of cryoprotectants decreases with decreasing temperature, a more toxic DMSO should be introduced into the head at a lower temperature. DMSO is gradually introduced from 0% to 35% in a 35% solution of ethylene glycol with a simultaneous synchronous decrease in the temperature of the perfusion solutions from -10 ° C to -50 ° C.

2.3.    Head and perfusion solutions temperatures
For the perfect perfusion technology, it is necessary to maintain the temperature of the head/body and perfusion solutions equal to the freezing temperature of the corresponding perfusion solutions. It is necessary in order to minimize the toxic effects of cryoprotectants. A special cryogenic chamber in which the patient's head will be fixed and perfused and which will lower the temperature of the chamber synchronously with the perfusion process is required. Some excess amount of perfusion solutions should be used for the fastest, most effective cooling of the head. The introduction of a final 70% vitrification mixture should occur at the lowest possible temperature from -30 ° C to -50 ° C.

2.4.    Perfusion pressure and rate. Viscosity of solutions at different temperatures
The rate of the perfusion depends on the perfusion pressure, which depends on the viscosity of perfusion solutions. Viscosity of solutions dramatically increases with the decrease of the temperature of solutions. If the perfusion pressure is maintained equal to the physiological pressure (120 mm Hg), then the perfusion rate must be dramatically reduced, which drastically reduces the efficiency of the perfusion.
However, the rule P = 120 mm Hg. Is correct only for the temperature of 36.6 ° C. However, even at T = + 10-0 ° C, the vessels become rigid and more resistant to the increased perfusion pressure.

2.5.    Methods for monitoring the degree of brain saturation with a vitrification mixture.
Modern, yet imperfect control of the degree of brain saturation with vitrification mixture is carried out according to the index of refraction of solutions flowing out of the veins and trephine holes in a skull. For stable vitrification of the brain, it is necessary to saturate it evenly with a 60-65% vitrification mixture. For faster saturation, a 70% vitrification mixture is used. The introduction of this solution is terminated when the refractive index of the solutions emanating from the trephine holes in the skull corresponds to the one of a 65% vitrification mixture. This method was firstly verified by direct testing of the vitrification of the sheep brain after the perfusion. The results were good.

One of the aims of the CryoGen project is to further develop and implement a wide range of tests to improve the definition of perfusion quality during the cryopreservation process and upon its completion.

3.    The final stages of the cryopreservation

After the brain is saturated with the vitrification mixture, the brain must be cooled as soon as possible to -130 ° C in order to minimize the toxic effect of the cryoprotective mixture. This temperature is advisable to store the vitrified. However, it is technically more difficult than the storage in liquid nitrogen, which is currently used. The vitrified brain storage at -130 ° C would have allowed to avoid its cracking during cooling from -130 ° C to -196 ° C. To minimize or even completely avoid the cracking of the vitrified brain and the body, it is necessary to apply ultra-slow cooling rates (1-0.2 ° C per hour) in the temperature range from -130 ° C to -196 ° C. For all this, modern cryogenic technology is required i.e. programmable freezers.

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