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Category Archives: Cryonics

Perfusion & Diffusion in Cryonics Protocol – BEN BEST

Posted: June 6, 2016 at 4:44 pm

by Ben Best CONTENTS: LINKS TO SECTIONS BY TOPIC

Preparing a cryonics patient for cryostorage can involve three distinct stages of alteration of body fluids:

(1) patient cooldown/cardiopulmonary support

(2) blood washout/replacement for patient transport

(3) cryoprotectant perfusion

During patient cooldown/cardiopulmonary support, a cryonics emergency response team or health care personnel may inject a number of medicaments to minimize ischemic injury and facilitate cryopreservation. The first and most important of these medicaments would be heparin, to prevent blood clotting. (For more details on the initial cooldown process, see Emergency Preparedness for a Local Cryonics Group).

Once the patient is cooled, the blood can be washed-out and replaced with a solution intended to keep organs/tissues alive while the patient is being transported to a cryonics facility. At the cryonics facility the organ/tissue preservation solution is replaced with the cryopreservation solution intended to prevent ice formation when the patient is further cooled to temperatures of 120C (glass transition temperature) or 196C (liquid nitrogen temperature) for long-term storage.

For both organ/tissue preservation & cryoprotection it is necessary to replace the fluid contents of blood vessels & tissue cells with other fluids. The process of injecting & circulating fluids through blood vessels is called perfusion. The passive process by which fluids enter & exit both blood vessels & cells is called diffusion.

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Body fluids can be described as solutes dissolved in a solvent, where the solvent is water and the solutes are substances like sodium chloride (NaCl, table salt), glucose or protein. Both water and solute molecules tend to move randomly in fluid with energy and velocity that is directly proportional to temperature. When there is a difference in concentration between water or solute molecules in one area of the fluid compartment compared to the rest of the compartment, random motion of the molecules will eventually result in a uniform distribution of all types of molecules throughout the compartment. In thermodynamics this is termed a decrease in potential energy (Gibbs free energy, not heat energy) due to an increase in entropy at constant temperature leading to equilibrium.

The movement of molecules from an area of high concentration to an area of low concentration is called diffusion. The rate of diffusion (J) can be quantified by Fick's law of diffusion:

dc J = DA---- dx J = rate of diffusion (moles/time) D = Diffusion coefficient A = Area across which diffusion occurs dc/dx = concentration gradient (instantaneous concentration difference divided by instantaneous distance)

Fick's First Law states that the rate of diffusion down a concentration gradient is proportional to the instantaneous magnitude of the concentration gradient (which changes as diffusion proceeds). For movement of molecules from a region of higher concentration to a region of lower concentration dc/dx will be negative, so multiplying by DA gives a positive value to J. Diffusion coefficient is higher for higher temperature and for smaller molecules.

Diffusion can occur not only within a fluid compartment, but across partitions that separate fluid compartments. The relevant partitions for animals are cell membranes and capillary walls. Cell membranes are lipid bilayers that allow for free diffusion of lipid soluble substances like oxygen, nitrogen, carbon dioxide and alcohol, while blocking movement of ions and polar molecules. But cell membranes also contain channels made of protein. Protein channels for water allow for very rapid diffusion of water across the membranes. Protein channels for potassium(K+), sodium(Na+) and other ions allow for more restricted diffusion across cell membranes. There is also facilitated diffusion (active transport) of many types of molecules across membranes.

For a normal 70kilogram (154pound) adult the total body fluid is about 60% of the body weight. Almost all of this fluid can be described as extracellular or intracellular (excluding only cerebrospinal fluid, synovial fluid and a few other small fluid compartments). Extracellular fluid can be further subdivided into plasma (noncellular part of blood) and interstitial fluid (fluid between cells that is not in blood vessels). Cell membranes separate intracellular fluid from extracellular fluid, whereas capillary walls separate plasma from interstitial fluid. The relative percentages of these fluids can be summarized as:

Intracellular fluid 67% Extracellular fluid Interstitial fluid 26% Plasma 7%

Note that blood volume includes both plasma & blood cells such that adding the intracellular fluid volume of blood cells to plasma volume makes blood 12% of total body fluid.

Osmosis refers to diffusion of water (solvent) across a membrane that is semi-permeable, ie, permeable to water, but not to all solutes in the solution. If membrane-impermeable solutes are added to one side of the membrane, but not to the other side, water will be less concentrated on the solute side of the membrane. This concentration gradient will cause water to diffuse across the semi-permeable membrane into the side with the solutes unless pressure is applied to prevent the diffusion of water. The amount of pressure required to prevent any diffusion of water across the semi-permeable membrane is called the osmotic pressure of the solution with respect to the membrane.

Osmotic pressure (like vapor pressure lowering and freezing-point depression) is a colligative property, meaning that the number of particles in solution is more important than the type of particles. One molecule of albumin (molecular weight 70,000) contributes as much to osmotic pressure as one molecule of glucose or one sodium ion. At equilibrium all molecules in a solution have achieved the same average kinetic energy, meaning that molecules with a smaller mass have higher average velocity. Thus, a one molar solution of NaCl will result in twice the osmotic pressure as a one molar solution solution of glucose because Na+ and Cl ions exert osmotic pressure as independent particles.

Solute concentrations are generally expressed in terms of molarity (moles of solute per liter of solution). The osmolarity of a solution is the product of the molarity of the solute and the number of dissolved particles produced by the solute. A one molar (1.0M, one mole per liter) solution of CaCl2 is a three osmolar (three osmoles per liter) solution because of the Ca2+ ion plus the two Cl ions produced when CaCl2 is added to water. Osmolarity, the number of solute particles per liter has been mostly replaced in practice by osmolality, the number of solute particles per kilogram. (For dilute solutions the values of the two are very close.) For describing solute concentrations in body fluids it is more convenient to use thousandths of osmoles, milli-osmoles (mOsm). Total solute osmolality of intracellular fluid, interstitial fluid or plasma is roughly 300mOsm/kgH2O. About half of the osmolality of intracellular fluid is due to potassium ions and associated anions, whereas about 80% of the osmolality of interstitial fluid and plasma is due to sodium and chloride ions.

As stated above, both osmotic pressure and freezing point depresssion are colligative properties. All colligative properties are convertible. One osmole of any solute will lower the freezi
ng point of water by 1.858C. For this reason, a 0.9% NaCl solution is 0.154molar or about 308mOsm/kgH2O, and will lower the freezing point of water by about 0.572C.

The osmolality of a solution is an absolute quantity that can be calculated or measured. The tonicity of a solution is a relative concept that is associated with osmotic pressure and the ability of solutes to cross a semi-permeable membrane. Thus, tonicitiy of a solution is relative to the particular solutes and relative to a particular membrane specifically relative to whether the solutes do or do not cross the membrane. Cell membranes are the membranes of greatest biological significance. Whether a cell shrinks or swells in a solution is determined by the tonicity of the solution, not necessarily the osmolality. Only when all the solutes do not cross the semi-permeable membrane does osmolality provide a quantitative measure of tonicity. It is common to speak as if tonicity and osmolality are equivalent because body fluid solutes are often impermeable. Each mOsm/kgH2O of fluid contributes about 19mmHg to the osmotic pressure.

A solution is said to be isotonic if cells neither shrink nor swell in that solution. Both 0.9%NaCl (physiological saline) and 5%glucose (in the absence of insulin) are isotonic solutions (roughly 300mOsm/kgH2O of impermeable solute). (In the presence of insulin, 5%glucose is a hypotonic solution because insulin causes glucose to cross cell membranes.) Hypertonic solutions cause cells to shrink as water rushes out of cells into the solute, whereas cells placed in hypotonic solutions cause the cells to swell as water from the solution rushes into the cells.

An exact calculation of the osmolality of plasma gives 308mOsm/kgH2O, but the freezing point depression of plasma (0.54C) indicates an osmolality of 286mOsm/kgH2O. Interaction of ions reduces the effective osmolality. Sodium ions (Na+) and accompanying anions (mostly Cl & HCO3) account for all but about 20mOsm/kgH2O of plasma osmolality. Plasma sodium concentration is normally controlled by plasma water content (thirst, etc.)[BMJ; Reynolds,RM; 332:702-705 (2006)]. Normal serum Na+ concentration is in the 135 to 145millimole per liter range, with 135mmol/L being the threshold for hyponatremia. Intracellular sodium concentration is typically about 20mmol/L about one-seventh the extracellular concentration. Glucose and urea account for about 5mOsm/kgH2O. Osmolality of plasma is generally approximated by doubling the sodium ions (to include all associated anions), adding this to glucose & urea molecules, and ignoring all other molecules as being negligible. Protein contributes to less than 1% of the osmolality of plasma. (Cells contain about four times the concentration of proteins as plasma contains.)

Although ethanol increases the osmolality of a solution, it does not increase the tonicity because (like water) ethanol crosses cell membranes. A clinical hyperosmolar state without hypertonicity can occur with an increase in extracellular ethanol (which diffuses into cells)[ MINERVA ANESTESIOLOGICA; Offenstadt,G; 72(6):353-356 (2006)]. Glycerol also readily crosses cell membranes, but it does so thousands of times more slowly than water which means that glycerol is "transiently hypertonic" (only isotonic at equilibrium). Ethylene glycol crosses red blood cell membranes about six times faster than glycerol (and sperm cell membranes four times faster than glycerol). Actually. even for water there is a finite time for hydraulic conductivity across cell membranes.

Cells placed in a "transiently hypertonic" solution (containing solutes that slowly cross a membrane) will initially shrink rapidly as water leaves the cell, and gradually re-swell as the solute slowly enters the cell (the "shrink/swell cycle"). As shown in the diagram for mouse oocytes at 10C, water leaves the cell in the first 100seconds, whereas 1.5Molar ethylene glycol (black squares) or DMSO (DiMethylSulfOxide, white squares) take 1,750seconds to restore the volume to 85% of the original cell volume[CRYOBIOLOGY; Paynter,SJ; 38:169-176 (1999)]. Even if a cell does not burst or collapse due to osmotic imbalance, a sudden change in osmotic balance can injure cells. Nonetheless, cells are somewhat tolerant of hypotonic solutions. Granulocytes are particularly sensitive to osmotic stress, but granulocyte survival is not significantly affected by hypertonic solutions until the osmolality of impermeant solutes approaches twice physiological values (about 600mOsm/kgH2O)[AMERICAN JOURNAL OF PHYSIOLOGY; Armitage WJ; 247(5Pt1):C373-381 (1984)].

PC3 cells show almost no decline of survival upon exposure to 5,000mOsm/kgH2O NaCl for 60minutes at 0C, and show nearly 85% cell survival on rehydration. Nearly 85% survive 9,000mOsm/kgH2O NaCl for 60minutes at 0C, but less than 20% survive rehydration. But although at 23C most cells survive exposure to 5,000mOsm/kgH2O NaCl for 60minutes, only about a third of cells survive rehydration. At 23C and 9,000mOsm/kgH2O NaCl only about half of cells survive 60 minutes and no cells survive rehydration, indicating the protective effect of low temperature against osmotic stress. Water flux at 23C was the same for 9,000mOsm/kgH2O as for 5,000mOsm/kgH2O, and hypertonic cell survival was not affected by the rate of concentration increase[CRYOBIOLOGY; Zawlodzka,S; 50(1):58-70 (2005)].

Hyperosmotic stress damages not only cell membranes, but damages cytoskeleton, inhibits DNA replication & translation, depolarizes mitochondria, and causes damage to DNA & protein. Heat shock proteins and organic osmolytes (like sorbitol & taurine) are synthesized as protection against hyperosmotic stress. Highly proliferative cells (like PC3) suffer from osmotic stress more than less proliferative cells because the latter can mobilize cellular defenses more readily due to fewer cells undergoing mitosis at the time of osmotic stress[PHYSIOLOGICAL REVIEWS; Burg,MB; 87(4):1441-1474 (2007)].

An important distinction to remember in replacing body fluids is the distinction between two kinds of swelling (edema): cell swelling and tissue swelling. Cell swelling occurs when there is a lower concentration of dissolved membrane-impermeable solutes outside cells than inside cells. To prevent either shrinkage or swelling of a cell there must be an osmotic balance of molecules & ions between the liquids outside the cell & inside the cell. Capillary walls are semipermeable membranes that are permeable to most of the small molecules & ions that will not cross cell membranes, but are impermeant to large molecules referred to as colloid (proteins). The colloid osmotic pressure on capillary walls due to proteins is called oncotic pressure. For normal human plasma oncotic pressure is about 28mmHg, 9mmHg of which is due to the Donnan effect which causes small anions to diffuse more readily than small cations because the small cations are attracted-to (but not bound-to) the anionic proteins. About 60% of total plasma protein is albumin (30 to 50 grams per liter), the rest being globulins. But albumin accounts for 75-80% of total intravascular oncotic pressure. Tissue swelling occurs when fluids leak out of blood vessels into the interstitial space (the space between cells in tissues). Injury to blood vessels can result in tissue swelling, but tissue swelling can also result from water leaking out of vessels when there is nothing (like albumin) to prevent the leakage.

Both forms of edema (cell & tissue swelling) can impede perfusion considerably, and is frequently a problem in cryonics patients who have suffered ischemic or other forms of blood vessel damage. Maintain
ing osmotic balance of the fluids outside & inside cells is as important as maintaining oncotic balance, ie, balance of fluids inside & outside of blood vessels.

Much of the isotonicity of the intracellular and extracellular fluids is maintained by the sodium pump in cell membranes, which exports 3sodium ions for every 2potassium ions imported into cells. Proteins in cells are more osmotically active than interstitial fluid proteins. Because of the Donnan effect the sodium pump is required to prevent cell swelling. When ischemia deprives the sodium pump of energy, cells swell from excessive intracellular sodium (because sodium attracts water more than potassium does) resulting in edema. Inflammation can also cause cell swelling due to increased membrane permeability to sodium and other ions. Interstitial edema can occur when ischemia or inflammation increases capillary permeability leading to leakage of larger plasma solutes into the interstitial space.

[For further details on the sodium pump see MEMBRANE POTENTIAL, K/Na-RATIOS AND VIABILITY]

Near the hypothalamus of the brain are osmoreceptors (outside the blood-brain barrier) that monitor blood osmolality, which is normally in the range of 280-295mOsm/kgH2O. A 2% increase in plasma osmotic pressure can provoke thirst. An increase in plasma osmolality can indicate excessive loss of blood volume. To compensate, the posterior pituitary (neurohypophysis) secretes the hormone 8arginine vasopressin (AVP), which is two hormones in one hence the two names vasopressin and anti-diuretic hormone. AVP action on the V1 receptors on blood vessels causes vasoconstriction (vasopressin). AVP action on the V2 receptors of the kidney causes water retention (anti-diuretic hormone). Deficiency in AVP secretion can lead to diabetes incipidus, so called because the excessively excreted urine is tasteless (incipid), in contrast to the sweet (glucose-laden) urine of diabetes mellitus. Cortisol opposes AVP action on excretion, leading to dehydration and excessive urination of fluid. Reduced blood flow to the kidney stimulates release of renin, which catalyzes the production of angiotensin. Like AVP, angiotensin causes vasoconstriction and kidney fluid retention.

Rats subjected to experimental focal ischemia have shown reduced edema when treated with an AVP antagonist[STROKE; Shuaib,A; 33(12):3033-3037 (2002)]. Hypertonic saline(7.5%) has been shown to halve plasma AVP levels in experimental rats, whereas mannitol(20%) had no effect[JOURNAL OF APPLIED PHYSIOLOGY; Chang,Y; 100(5):1445-1451 (2006)]. Increases in plasma osmolality due to urea or glycerol have no effect on plasma AVP levels[JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY; Verbalis,JG; 18(12):3056-3059 (2007)]. The effect of hypertonic saline on osmotic edema due to AVP in a cryonics patient would likely be negligible because of negligible hormone release and transport. So some of the advantage of hypertonic saline over mannitol seen in clinical trials would not occur in cryonics cases.

The net movement of fluid across capillary membranes due to hydrostatic and oncotic forces can be described by the Starling equation. The Starling equation gives net fluid flow across capillary walls as a result of the excess of capillary hydrostatic pressure over interstitial fluid hydrostatic pressure, and capillary oncotic pressure over interstitial fluid oncotic pressure modified by the water permeability of the capillary. For a normal (animate) person, the hydrostatic pressure (blood pressure) at the arterial end of a capillary is about 35mmHg. The hydrostatic pressure drops in a linear fashion across the length of the capillary until it is about 15mmHg at the venule end. The net oncotic pressure within the capillary is about 25mmHg across the entire length of the capillary. Thus, for the first half of the capillary there is a net loss of fluid into the interstitial space until the hydrostatic pressure has dropped to 25mmHg. For the second half of the capillary there is a net gain of fluid into the capillary from the interstitial space. The flow of fluid into the interstitial space in the first half of the capillary is associated with the delivery of oxygen & nutrient to the tissues, whereas the flow of fluid from the interstitial space into the second half of the capillary is associated with the removal of carbon dioxide and other waste products.

Actually, there is a tiny (tiny relative to the total diffusion back and forth across the capillary wall) net flow of fluid from the capillaries to the interstitial fluid which is returned to the blood vessels by the lymphatic system. The lymphatic vessels contain one-way valves and rely on skeletal muscle movement to propel the lymphatic fluid. Infectious blockage of lymph flow can produce edema. A person sitting for long periods (as during a long trip) or standing a long time without moving may experience swollen ankles due to the lack of muscle activity. Swollen ankles is also a frequent symptom of the edema resulting from congestive heart failure. Venous pressure is elevated by the reduced ability of the heart to pull blood from the venous system, whereas vasoconstriction can better compensate to maintain pressure on the arterial side. Reduced albumin production by the liver as a result of cirrhosis or other liver diseases can reduce plasma osmolality such that the reduced oncotic pressure results in edema typically swollen ankles, pulmonary edema and abdomenal edema (ascites).

The Starling forces are different for the blood-brain barrier (BBB) than they are for other capillaries of the body because of the reduced permeability to water (lower hydraulic conductivity) and the greatly reduced permeability to electrolytes. The osmotic pressure of the plasma and interstitial fluid effectively become the oncotic pressures.

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A critical distinction is made in fluid mechanics between laminar flow and turbulent flow in a pipe. For laminar flow elements of a liquid follow straight streamlines, where the velocity of a streamline is highest in the center of the vessel and slowest close to the walls. Turbulent flow is characterized by eddies & chaotic motion which can substantially increase resistance and reduce flow rate. The Reynolds number is an empirically determined dimensionless quantity which is used to predict whether flow will be laminar or turbulent with 2000 being the approximate lower limit for turbulent flow. Transient localized turbulence can be induced at a Reynolds number as low as 1600, but temporally peristant turbulence forms above 2040[SCIENCE; Eckhart,B; 333:165 (2011)].

Turbulent flow could potentially be a problem in cryonics if it reduced perfusion rate or increased the amount of pressure required to maintain a perfusion rate. It is doubtful that turbulent flow ever plays a role in cryonics perfusion, however. Even for a subject at body temperature (37C) Reynolds numbers in excess of 2000 are only seen in the very largest blood vessels: the aorta and the vena cava.

The formula for Reynolds number is: v D Re = ------ = fluid density (rho) v = fluid velocity D = vessel diameter = fluid viscosity

The fact that diameter (D) is in the numerator indicates that only high diameter vessels have high Reynolds number. Velocity (v), also in the numerator, is highest in the aorta & arteries. But the use of cryoprotectants and the increase in viscosity () with declining temperature essentially guarantee that turbulent flow will not occur in a cryonics patient.

More serious for cryonics is the Hagen-Poisseuille Law, which describes the
relationship between flow-rate and driving-pressure: pressure X (radius)4 Flow Rate = ---------------------- length X viscosity

Typically in cryonics the flow rate will be one or two liters per minute when the pressure is around 80mmHg. But because flow rate varies inversely with viscosity and varies directly with pressure, pressure must be increased to maintain flow rates when cryoprotectant viscosity increases with lowering temperature. This poses a serious problem because blood vessels become more fragile with lowering temperature. If blood vessels burst the perfusion can fail.

At 20C glycerol is about 25% more dense (=rho, in the numerator) than water. But the role of viscosity is far more dramatic, with high viscosity in the denominator reducing Reynolds number considerably. The viscosity of water approximately doubles from 37C to 10C, but the viscosity of glycerol increases by a factor of ten (roughly 4Poise to 40Poise). At 37C glycerol is nearly 600 times more viscous than water, but at 10C it is about 2,600 times more viscous.

Although turbulence is not a concern in cryonics, the increase in viscosity of cryoprotectant with lowering temperature certainly is. Fortunately, the newer vitrification mixtures are less viscous than glycerol.

The most common strategy in cryonics has been to cool the patient from 37C to 10C as rapidly as possible and to perfuse with cryoprotectant at 10C. Lowering body temperature reduces metabolism considerably, thereby lessening the amount of oxygen & nutrient required to keep tissues alive. Cryoprotectant toxicity drops as temperature declines. But the very dramatic more-than-exponential increase in cryoprotectant viscosity with lowering temperature poses a significant problem for effective perfusion. When open circuit perfusion is used, a higher temperature may be preferable because the opportunity for diffusion time into cells is so limited (about 2hours 1hour for the head, 1hour for the body) although ischemic damage is difficult to quantify.

With closed circuit perfusion, the perfusion times are longer up to 5hours. If a good carrier solution is used for the cryoprotectant the tissues may receive adequate nutrient. This, along with the oxygen carrying-capacity of water at low temperature, may limit ischemic damage while allowing time for cells to become fully loaded with cryoprotectant. If ischemic damage can be safely prevented in perfusion, the only critical issues for temperature selection are the relative benefits of reduced cryoprotectant toxicity at lower temperatures as against increased chilling injury. The fact that the more-than-exponential increase in viscosity with lowering temperature will increase perfusion time will not be problematic if the risk of ischemia is minimized.

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Typically a cryonics patient deanimates at a considerable distance from a cryonics facility and must be transported before cryoprotectants can be perfused. Blood could be washed-out and replaced with an isotonic (ie, osmotically the same as saline) solution, such as Ringer's solution. The patient is then transported to the cryonics facility at water-ice temperature. Freezing must be avoided because ice crystals would damage cells & blood vessels to such an extent as to prevent effective cryoprotectant perfusion. Water-ice temperature will not freeze tissues because tissues are salty (salt lowers the freezing point below 0C).

As body temperature approaches 10C, metabolic rate has slowed greatly and the oxygen-carrying capacity of blood hemoglobin is no longer required. Cool water, in fact, may carry adequate dissolved oxygen at low temperatures. (Water near freezing temperature can hold nearly three times as much dissolved oxygen as water near boiling temperature. Oxygen is about five times more soluble in water than nitrogen.) The tendency of blood to agglutinate and clog blood vessels becomes a serious problem at low temperature so the blood should be replaced if this does not cause other problems (such as delay and reperfusion injury.)

Replacing blood with a saline-like solution for patient transport, however, does not do a good job of maintaining tissue viability or preventing edema and would likely cause reperfusion injury. For this reason an organ preservation solution such as Viaspan, rather than Ringer's solution, has been used for cryopatient transport. Blood is not simply an isotonic solution carrying blood cells. Blood contains albumin, which attracts water and keeps the water from leaving blood vessels and going into tissues (maintains oncotic balance). Tissues which are swollen by water (edematous tissues) resist cryoprotectant perfusion. One of the most important ingredients in Viaspan preventing edema is HydroxyEthyl Starch (HES), which attracts water in much the way albumin attracts water acting as an oncotic agent by keeping water in the blood vessels. Viaspan contains potassium lactobionate to help maintain osmotic balance. Because HES is difficult to obtain and can cause microcirculatory disturbances, PolyEthylene Glycol (PEG) has been used in organ preservation solutions as a replacement for HES with good results[THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS; Faure,J; 302(3):861-870 (2002) and LIVER TRANSPLANTATION; Bessems,M; 11(11):1379-1388 (2005)].

The same benefit might not apply to cryonics patients, however, because of the prevalence of endothelial damage due to ischemia. Larger "holes" in the vasculature can mean that a larger molecular weight molecule is required for oncotic support. HES molecular weight is about 500,000, whereas the molecular weight for PEG used in organ replacement solutions is more like 20,000. Albumin (which has a molecular weight of about 70,000) provides most of the oncotic support in normal physiology. A PEG with molecular weight of 500,000 would be far too viscous and will form a gel. HES has the benefit of being large enough to always provide oncotic support while being much less viscous than PEG of equivalent molecular weight.

Viaspan (DuPont Merck Pharmaceuticals) contains other ingredients to maintain tissue viability, such as glucose, glutathione, etc. (the full formula can be found on the Viaspan website). Viaspan is FDA approved for preservation of liver, kidney & pancreas, but is used off-label for heart & lung transplants. Viaspan is being challenged in the marketplace for all these applications by the Hypothermosol (Cryomedical Sciences, BioLife Technologies) line of preservation solutions.

Rather than use these expensive commercial products, Alcor and Suspended Animation, Inc. use a preservation solution developed by Jerry Leaf & Mike Darwin called MHP-2. MHP-2 is so-called because it is a Perfusate (P) which contains mannitol (M) as an extracellular osmotic agent and HEPES (H), a buffer to prevent acidosis which is effective at low temperature. MHP-2 also contains ingredients to maintain tissue viability and hydroxyethyl starch as an oncotic agent to prevent edema. Lactobionate permeates cells less than mannitol and can thus maintain osmotic balance for longer periods of time, but mannitol is much less expensive. Mannitol also has an additional effect in the brain. Because of the unique tightness of brain capillary endothelial cell junctions ("blood brain barrier"), little mannitol leaves blood vessels to pass into the brain. This means that mannitol can act like an oncotic agent for the brain. If the blood brain barrier is intact, mannitol will suck water out of the extravascular space. The brain is the only place that mannitol can do this, and that is why a mannitol is eff
ective for inhibiting edema of the brain but only if there is not extensive ischemic damage to the blood brain barrier. (Mannitol has yet another benefit in that it scavenges hydroxyl radical [CHEM.-BIOL. INTERACTIONS 72:229-255 (1989)]).

(For the formula of MHP-2 see TableII of CryoMsg4474 or TableVII of CryoMsg2874 which also contains the formula for Viaspan in TableV.)

The initial perfusate can also contain other ingredients to assist in reducing damage to the cryonics patient. Anticoagulants can reduce clotting problems, and antibiotics can reduce bacterial damage. Damaging effects of ischemia can be reduced with antioxidants, antiacidifiers, an iron chelator and a calcium channel blocker.

Both Alcor and Suspended Animation, Inc. use an Air Transportable Perfusion(ATP) system of equipment which allows them to do blood washout in locations remote from any cryonics facilty by using equipment that can easily be carried on an airplane. There is a video demonstration of an ATP on YouTube.

[For further details on organ preservation solution see ORGAN TRANSPLANTATION SOLUTION]

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Cryoprotectants are used in cryonics to reduce freezing damage by prevention of ice formation (see Vitrification in Cryonics ). Cells are much more permeable to water than they are to cryoprotectant. Platelets & granulocytes, for example, are 4,000 times more permeable to water than they are to glycerol[CRYOBIOLOGY; Armitage,WJ; 23(2):116-125 (1986)]. When a cell is exposed to high-strength cryoprotectant, osmosis causes water to rush out of the cells, causing the cells to shrink. Only very gradually does the cryoprotectant cross cell membranes to enter the cell (the "shrink/swell cycle"). For isolated cells, the halftime (time to halve the difference between a given glycerol concentration in a granulocyte and the maximum possible concentration) is 1.3minutes[EXPERIMENTAL HEMATOLOGY; Dooley,DC; 10(5):423-434 (1982)] but tissues & organs would require more time because their cells are less accessible. Even after equilibration, however, the concentration of glycerol inside neutrophilic granulocytes never rises above 78% of the concentration outside the cells.

As shown in the diagram for mature human oocytes placed in a 1.5molar DMSO solution, the shrink/swell cycle is highly temperature dependent, happening with slower speed of recovery and with greater volume change at lower temperatures[HUMAN REPRODUCTION; Paynter,SJ; 14(9):2338-2342 (1999)]. This creates tough choices in cryonics, because cryoprotectants are more toxic at higher temperatures.

Proliferation of cultured kidney cells declines linearly with increasing osmolality due to urea & NaCl above 300mOsm/kgH2O, but the effect of added glycerol on cell growth is much less[AMERICAN JOURNAL OF PHYSIOLOGY; Michea,L; 278(2):F209-F218 (2000)]. Kidney cells which invivo can tolerate osmolalities of around 300mOsm/kgH2O do not survive over 300mOsm/kgH2O invitro, possibly because of more rapid proliferation[PHYSIOLOGICAL REVIEWS; Burg,MB; 87(4):1441-1474 (2007)].

Cells subjected to high levels of cryoprotectants can be damaged by osmotic stress. Quantifying osmotic damage has been a challenge for experimentalists who must distinguish between electrolyte damage, cryoprotectant toxicity, cell volume effects and osmotic stress. Concerning the last two, osmotic damage due to cell shrinkage may be distinguished from osmotic damage as a result of the speed at which the cryoprotectant crosses the cell membrane, ie, by the membrane permeability to the cryoprotectant. Cryoprotectants with lower permeabilities can cause more osmotic stress than cryoprotectants with high permeability.

Membrane permeabilities of a variety of nonelectrolytes (including cryoprotectants) have been studied on a number of cell types, including human blood cells[THE JOURNAL OF GENERAL PHYSIOLOGY; Naccache,P; 62(6):714-736 (1973)]. Critical factors determining membrane permeability are lipid solubility of the substance (which increases permeability) and hydrogen bonding (which decreases permeability). In general, permeability decreases as the molecular size of the substance increases. In contrast to blood cells, human sperm is more than three times more permeable to glycerol than to DMSO[BIOLOGY OF REPRODUCTION; Gilmore,JA; 53(5):985-995 (1995)]. For both blood cells and sperm cells permeability to ethylene glycol is very high compared to the other common cryoprotectants. Yet for mature human oocytes propylene glycol has the highest permeability and ethylene glycol has the lowest permeability of the most commonly used oocyte cryoprotectants[HUMAN REPRODUCTION; Van den Abbeel,E; 22(7):1959-1972 (2007)]. In contrast to human oocytes, however, for mouse oocytes ethylene glycol(EG) permeability is comparable to that of DMSO, propylene glycol(PG), and acetamide(AA), but not glycerol(Gly)[JOURNAL OF REPRODUCTION AND DEVELOPMENT; Pedro,PB; 51(2):235-246 (2005)].

Water and cryoprotectants both cross cell membranes more slowly at lower temperatures. Cryoprotectants slow the passage of water across cell membranes. Glycerol, DMSO and ethylene glycol all reduce the rate at which water crosses human sperm cell membranes by more than half[BIOLOGY OF REPRODUCTION; Gilmore,JA; 53(5):985-995 (1995)].

Aside from the choice of cryoprotectants, a major concern is the way cryoprotectant is administered. For example, glycerol (the standard cryoprotectant used in cryonics for many years) can either be administered full-strength or it can be introduced in gradually increasing concentrations. Under optimum conditions, glycerol results in 80% vitrification and 20% ice formation. Glycerol has been replaced by better cryoprotectants that can vitrify without any ice formation, but I will typically use glycerol as my example cryoprotectant. A patient should probably not be perfused with a 100% solution of glycerol or other cryoprotectant because of the possibility of osmotic damage. It is prudent to begin perfusion with low concentrations of cryoprotectant because water can diffuse out of cells thousands of times more rapidly than cryoprotectant diffuses into cells. Using gradually increasing concentrations of cryoprotectant (ramping) prevents the osmotic damage this differential could cause.

Human granulocytes (which are more vulnerable to osmotic stress or shrinkage than most other cell types) can experience up to 600mOsm/kgH2O hypertonic solution (which shrinks cells to 68% of normal cell volume) for 5minutes at 0C with no more than 10% of the cells losing membrane integrity. But at about 750mOsm/kgH2O (NaCl) or 950mOsm/kgH2O (sucrose) less than half of granulocytes display intact membranes when returned to isotonic solution. Nonetheless, the cells did not display lysis if retained in hyperosmotic medium. In fact, granulocytes could tolerate up to 1400mOsm/kgH2O if not subsequently diluted to less than 600mOsm/kgH2O[AMERICAN JOURNAL OF PHYSIOLOGY; Armitage WJ; 247(5Pt1):C373-381 (1984)]. A subsequent confirming study showed that rehydration of PC3 cells shrunken by NaCl solution creates more osmotic damage than the initial dehydration[CRYOBIOLOGY; Zawlodzka,S; 50(1):58-70 (2005)]. Cell survival after rehydration was higher at 0C than at 23C.

Although toxic effects of 2M (17%w/w) glycerol on granulocytes are quite evident at 22C, almost no toxic effect is seen at 0C[CRYOBIOLOGY; Frim,J; 20(6):657-676 (1983)]. For no mammalian cells other than granulocytes is 2Molar glycerol toxic. Nonetheless, abrupt addition of only 0.5Molar glycerol at 0C resulted in only 40% of granulocytes surviving when slowly diluted to isotonic solution a
nd warmed to 37C. Only 20% of granulocytes survived this treatment when 1Molar or 2Molar glycerol were added (there was no difference in survival between the two concentrations). But if sucrose or NaCl was added to keep the granulocytes shrunken to 60% of normal cell volume, almost all granulocytes survived when incubated to 37C. Insofar as the transient shrinkage of granulocytes due to glycerol is not less than 85% of normal cell volume, it seems unlikely that cell shrinkage can account for the damage[AMERICAN JOURNAL OF PHYSIOLOGY; Armitage WJ; 247(5Pt1):C382-389 (1984)].

Human spermatazoa tolerate much higher osmolality than granulocytes. Sperm cells can experience up to 1000mOsm/kgH2O hypertonic solution for 5minutes at 0C with no more than 10% of the cells losing membrane integrity. At about 1500mOsm/kgH2O (NaCl, white circles) or 2500mOsm/kgH2O (sucrose, black circles) less than half of sperm cells display intact membranes when returned to isotonic conditions. But 80% of sperm cells showed intact cell membrane after exposure to 2500mOsm/kgH2O at 0C if maintained at hypertonicity rather than restored to isotonic solution (NaCl & sucrose, triangles). Sperm cells gradually returned to isotonic solution following exposure to 1.5Molar glycerol at 22C showed only 3% lysis, whereas 20% of sperm cells lysed if the return to isotonic was sudden. No lysis was seen for sperm not returned to isotonic medium. At nearly 5000mOsm/kgH2O glycerol (about 4.5Molar) 17% of sperm cells showed lysis (had loss of membrane integrity) at 0C and 10% had lysis at 8C if not returned to isotonic media[BIOLOGY OF REPRODUCTION; Gao,DY; 49(1):112-123 (1993)]. For cryonics purposes it would be best to maintain cells in a hypertonic condition to maximize potential viability during cryogenic storage.

Cells from mouse kidney (IMCD, Inner Medullary Collecting Duct) can be killed by NaCl or urea that is 700mOsm/kgH2O, but the death is apoptotic and takes up to 24hours. The IMCD cells can tolerate up to 900mOsm/kgH2O of urea and NaCl in combination because of activation of complementary cellular defenses (including heat-shock protein)[ AMERICAN JOURNAL OF PHYSIOLOGY; Santos,BC; 274(6):F1167-F1173 1998)].

Nearly half of mouse fibroblasts displayed cell membrane lysis after restoration to isotonicity following exposure to the equivalent of 3600mOsm/kgH2O of osmotic stress from rapid addition of 4Molar (30%w/w) DMSO at 0C. Few cells were damaged by slow addition of the DMSO[BIOPHYSICAL JOURNAL; Muldrew,K; 57(3):525-532 (1990)].

Human corneal epithelial cells could tolerate 4.3M (37%w/w) glycerol with only 2% cell loss at 4C if the cells were subjected to gradually increasing (ramped) concentration (doubling osmolality in about 13minutes), but for stepped increases of 0.5M every 5minutes above 2M (17%w/w) to 3.5M (30%w/w) glycerol at 0C there was a 27% cell loss. For the same ramped method with DMSO there was a 6% cell loss at 2M (15%w/w) and a 15% cell loss at 3M (23%w/w). The same stepped method for DMSO resulted in a 1.5% cell loss for cells stepped from 2M to 3.5M (27%w/w) and a 22% cell loss for cells stepped from 2M to 4.3M (33%w/w). In all cases cell viability was assessed after washout and three days of incubation at 37C[CRYOBIOLOGY; Bourne,WM; 31(1):1-9 (1994)]. (Conversion of glycerol molarity to %w/w was approximated by multiplying by 8.6 and for DMSO was approximated by multiplying by 7.6)

In the context of cryonics it should be remembered that cells are not being returned to body temperature and need not be returned to isotonicity before cryoopreservation. There would be little time for apoptosis, and most cells would be far better preserved at low temperature and in hyperosmolar solution. Future technologies may be able to prevent apoptosis and have better methods for restoring irreplaceable cells to normal temperatures and osmolalities. For neurons, even abrupt stepped perfusion with cryoprotectant is likely to effectively result in ramped perfusion when allowances are made for the diffusion times required across blood vessels (blood brain barrier) and interstitial space. A more worrisome effect from the point of view of cryonic cryoprotectant perfusion is the effect of the cryoprotectants on vessel endothelial cells notably the effect on edema and vascular compliance.

Cell shrinkage may directly damage the cell (and cell membrane) due to structural resistance from the cell cytoskeleton and high compression of other cell constituents[HUMAN REPRODUCTION; Gao,DY; 10(5):1109-1122 (1995)]. Aside from membrane damage, other forms of cellular damage occur due to hypertonic environments, including cross-linking of intracellular proteins subsequent to cell dehydration. Bull sperm lose motility (often only temporarily) in a less hypertonic medium than one causing membrane damage[JOURNAL OF DAIRY SCIENCE; Liu,Z; 81(7):1868-1873 (1998)]. Osmotic stress can depress mitochondrial membrane potential in a manner that is mostly reversible after restoration to isotonic conditions[PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA); Desai,BN; 99(7):4319-4324 (2002)]. Human oocytes subjected to 600mOsm/kgH2O sucrose showed 44% of metaphaseII spindles having abnormalities[HUMAN REPRODUCTION; Mullen,SF; 19(5):1148-1154 (2004)]. Hypertonic solutions can trigger apoptosis[AMERICAN JOURNAL OF PHYSIOLOGY; Copp,J; 288(2):C403-C415 (2005)].

Despite these other types of damage due to hyperosmolality, the greatest risks in cryoprotectant perfusion in cryonics are those associated with membrane damage and edema due to cell swelling. The evidence that maintaining hypertonicity is more protective of cells than returning to isotonic conditions, and the desire to minimize edema during perfusion seem to make it advisable in cryonics to perfuse in hypertonic conditions.

(return to contents)

Once the patient is at the cryonics facility the transport solution can be replaced with a cryoprotectant solution. A perfusion temperature of 10C gives the best tradeoff of avoiding the high viscosity of lower temperatures and at the same time limiting the ischemic tissue degradation, chilling injury, and cryoprotectant toxicity that would be seen at higher temperatures. (Cryonicists usually worry more about ischemic damage than cryoprotectant toxicity due to a belief that ischemic damage has a greater likelihood of being irreversible irreparable by future molecular-repair technology.)

Cryoprotectants should be sterilized to prevent the growth of bacteria. Sterilization of cryoprotectants by heating can cause the formation of carbon-carbon double-bonds, which are evident by a yellowing of the cryoprotectant. Only a few such double-bonds can produce the yellow appearance, so the fact of yellowing is not evidence that the cryoprotectant is no longer serviceable. But a preferable method of cryoprotectant sterilization is filtration through a 0.2micron filter.

Rapid addition of cryoprotectant causes endothelial cells to shrink thereby breaking the junctions between the cells[CRYOBIOLOGY; Pollock,GA,; 23(6):500-511 (1986)]. On the other hand, endothelial cell shrinkage by hypertonic perfusate can increase capillary volume, thereby increasing blood flow as long as excessive vascular damage does not occur. Blood and clots are often observed to be dislodged during cryoprotectant perfusion in cryonics cases. For cryonics purposes some vascular damage may actually be an advantage insofar as it increases diffusion and vascular repair may be an easy task for future science. In fact, the breakdown of the blood-brain barrier in the 1.8-2.2 molar glycerol range is essential for perfusion of the brain as long as damag
ing tissue edema (swelling) can be avoided. Aquaporin (water channel) expression in the blood-brain barrier could be a safer means of allowing cryoprotectants into the brain[CRYOBIOLOGY; Yamaji,Y; 53(2):258-267 (2006)].

Closed-circuit perfusion (with perfusion solution following a circuit both inside & outside the patient's body) is contrasted with the open-circuit perfusion used by funeral directors for embalming. In the open-circuit perfusion of embalming, fluid is pumped into a large artery of the corpse and forces-out blood from a large vein and this blood is discarded.

A closed-circuit perfusion, as illustrated in the diagram, can be set up at low cost for gradual introduction of cryoprotectant into cryonics patients. As shown in the diagram, the perfusion circuit bypasses the heart. Perfusate enters the patient through a cannula in the femoral (leg) artery and exits from a cannula in the femoral vein on the same leg. Flowing upwards (opposite from the usual direction) from the femoral artery and up through the descending aorta, the perfusate enters the arch of the aorta (where blood normally exits the heart), but is blocked from entering the heart. Instead, the perfusate flows (in the usual direction) through the distribution arteries of the aorta, notably to the head and brain. Returning in the veins (in the usual direction), the perfusate nontheless again bypasses the heart and flows downward (opposite from the usual direction) to the femoral vein where it exits. A better alternative to the femoral circuit, however, is to surgically open the chest to cannulate the heart aorta (for input) and atrium (for output).

Although it is not shown in the diagram, there will be a pump in the circuit to maintain pressure and fluid movement. A roller pump, rather than an embalmer's pump, should be used. A roller pump achieves pumping action by the use of rollers on the exterior of flexible tubing that forces fluids through the tube without contaminating those fluids. Embalmer's pumps may use pressures much higher than those suitable for cryonics, resulting in blood vessel damage. Embalmer's pumps are also easily contaminated (and hard to clean), unless a filter is used. Contamination doesn't matter much in embalming, but in cryonics contaminants entering the patient through the pump can damage blood vessels, interfering with perfusion. If an embalmer's pump is used for cryonics purposes, ensure that the pressure can be lowered to a suitable level and that it is cleaned and sterilized. The main advantage of roller pumps, however, is the fact that they provide a closed circuit, whereas embalmer's pumps are open-circuit. Roller pumps are generally calibrated in litres per minute. Depending on the viscosity of the solution, a flow rate of 0.5 to 1.5litres per minute will be necessary to achieve the desired perfusion pressure of approximately 80mmHg to 120mmHg (physiological pressures).

Gaseous and particulate microemboli can produce ischemia in capillaries and arterioles. A study of patients having routine cardiopulmonary bypass surgery showed that 16% fewer patients had neuropsychological deficits eight weeks after the surgery when a 40micrometer arterial line filer had been used[STROKE; Pugsley,W; 25(7):1393-1399 (1994)]. Both roller pumps (peristaltic pumps) and centrifugal pumps can generate particles up to 25micrometers in diameter through spallation, although centrifugal pumps generate fewer particles[PERFUSION; Merkle,F; 18(suppl1):81-88 (2003)]. Filtration of perfusate with a 0.2micrometer filter prior to perfusion is a recommended way of removing potential microemboli, including bacteria. At room temperature 20micrometer diameter air bubbles take 1to6seconds to dissolve in water, although high flow rates and turbulence can increase microbubble formation[SEMINARS IN DIALYSIS; Barak,M; 21(3):232-238 (2008)]. De-airing of tubing before perfusion considerably reduces the possibility of microbubbles entering the patient[THE THORACIC AND CARDIOVASCULAR SURGEON; Stock,UA; 54(1):39-41 (2006)].

Mean Arterial Pressure (MAP) for an normal adult is regarded as being in the range of 50 to 150mmHg, and Cerebral Perfusion Pressure (CPP) is in the same range[BRITISH JOURNAL OF ANAETHESIA; Steiner,LA; 91(1):26-38 (2006)]. Vascular pressure normally drops to about 40mmHg in the arterioles, to below 30mmHg entering the capillaries, and is down to 3 to 6mmHg (Central Venous Pressure, CVP) when returning to the right atrium of the heart. Perfusing a cryonics patient at about 120mmHg should open capillaries adequately for good cryoprotectant tissue saturation without damaging fragile blood vessels.

Outside the patient, some of the drainage is discarded, but most is returned to a circulating (stirred) reservoir connected to a concentrated reservoir of cryoprotectant. The circulating reservoir is initially carrier solution which gradually becomes increasingly concentrated with cryoprotectant as the stirring and recirculation proceed. The circulating reservoir can be stirred from the bottom by a magnetic stir bar on a stir table and/or from the top by an eggbeater-type stirring device. The stirring will draw cryoprotectant from the cryoprotectant reservoir, and pumping of the perfusate should also actively draw liquid from the cryoprotectant reservoir. Gradually a higher and higher concentration of cryoprotectant is included in the perfusate and the osmotic shock of full-strength cryoprotectant is avoided.

The carrier solution for the cryoprotectant should perform similar tissue preservation functions as is performed by the transport solution, and should be carefully mixed with the cryoprotectant so as to avoid deviations from isotonicity which could result in dehydration or swelling & bursting of cells. The carrier solution will help keep cells alive during cryoprotectant perfusion.

An excellent carrier solution for cryonics purposes would be RPS-2 (Renal Preservation Solution number2), which was developed by Dr. Gregory Fahy in 1981 as a result of studies on kidney slices. More recently Dr. Fahy used RPS-2 as the carrier solution in cryopreserving hippocampal slices an indication that it is well-suited for brain tissue as well as for kidney. RPS-2 not only helps maintain hippocampal slice viability, it reduces the amount of cryoprotectant needed because it has cryoprotectant (colligative) properties of its own. The formulation of RPS-2 is: K2HPO4, 7.2mM; reduced glutathione, 5mM; adenine HCl, 1mM; dextrose, 180mM; KCl, 28.2mM; NaHCO3, 10mM; plus calcium & magnesium[CRYOBIOLOGY; Fahy,GM; 27(5):492-510 (1990)]. LM5 (Lactose-Mannitol5) is a carrier solution for use in vitrification solutions that include ice blockers. LM5 does not contain dextrose, which is believed to interfere with ice blockers.

The cryoprotectant reservoir will not in general contain pure cryoprotectant (although in principle it could), but rather a "terminal concentration" solution of cryoprotectant that is equal or slightly above the final target concentration. As perfusion proceeds and drainage to discard proceeds, the level of both reservoirs drops in tandem until both reservoirs are nearly empty, at which point the circuit concentration will have reached the cryoprotectant reservoir concentration. Provided that the two reservoirs are the same size and same vertical elevation, the gradient will be linear over time (if the drainage rate to discard was constant).

For cryoprotectant to perfuse into cells there must be constant exposure to cryoprotectant surrounding the cells and there must be pressure to maintain that exposure. In a living animal the heart maintains blood pressure that for
ces blood through the capillaries and forces nutrients into cells. A dead animal with no blood pressure and which is being perfused with cryoprotectant also requires pressure for the capillaries to remain open and for cryoprotectant to be maintained at high concentrations around cells.

Alcor found that closed-circuit perfusion must be maintained for 5-7 hours for full equilibration of glycerol, because the diffusion rate of water out of cells is thousands of times the rate at which glycerol enters cells. Of course, it would be possible to pump glycerol into a patient for 5-7 hours with open-circuit perfusion, but only by using thousands of dollars worth of glycerol. The newer vitrification cryoprotectants used by Alcor are vastly more expensive than glycerol. When using expensive cryoprotectants it makes far more sense to recirculate in a closed circuit. Closed-circuit perfusion also has the benefit of allowing for ongoing monitoring of physiological changes occurring in the patient's body during the perfusion process. Open-circuit with an inexpensive cryoprotectant has the advantage of avoiding recirculation of toxins.

Cryoprotectants, particularly glycerol, are viscous and cryoprotectants in high concentration are particularly viscous. The introduction of air bubbles into cryoprotectant solutions during pouring and mixing should be avoided because air emboli that enter the cryonics patient can block perfusion. Elimination of air bubbles from viscous cryoprotectant solutions is extremely difficult. Prevention is more effective than cure. Cryonicist Mike Darwin wrote about this problem and possible solutions in a 1994 CryoNet message.

Improper mixing of perfusate containing high levels of cryoprotectant can result in a phenomenon that appears to be high viscosity, but in reality is edema. If, for example, isotonic carrier solution is mixed half-and-half with cryoprotectant solution an open circuit perfusion may have to be halted when no further perfusate will go into the patient. The problem is caused not by viscosity, but by the fact that the isotonic solution became hypotonic due to dilution with cryoprotectant causing the cells to swell and forcing perfusion to end. In closed-circuit perfusion, the cryoprotectant concentrate reservoir contains cryoprotectant at about 125% the terminal concentration in a vehicle of isotonic carrier solution so that when reservoir concentrate is mixed with isotonic carrier there is no change in tonicity.

Newer cryoprotectants are less viscous than glycerol, so perfusions can be done in less time. After 15 minutes of perfusion with carrier solution, cryoprotectant concentration linearly increases at a rate of 50millimolar per minute until full concentration is reached in about two hours (a protocol developed on the basis of minimizing osmotic damage when perfusing kidneys). Perfusion is increased for an additional hour or two until the cryoprotectant has fully diffused into cells (as indicated by similarity of afflux and efflux cryoprotectant concentrations).

Only after a few hours of closed-circuit perfusion is the concentration of cryoprotectant exiting the cryonics patient equal to the concentration of cryoprotectant entering the patient. Only an extended period of sustained pressure will keep capillaries open, and otherwise facilitate diffusion of cryoprotectant into cells. And the exiting cryoprotectant concentration will equal the entering cryoprotectant concentration only when the tissues are fully loaded with cryoprotectant. A refractometer is used to verify that terminal cryoprotectant concentration has been reached in the brain.

(A refractometer measures the index of refraction of a liquid, ie, the ratio of the speed of light in the liquid and the speed of light in a vacuum (or air). Light changes speed when it strikes the boundary of two media, thus causing a change in angle if it strikes the new medium at an angle. Because the refractive index is a ratio of two quantities having the same units, it is unitless. Sodium vapor in an electric arc produces an excitation between the 3s and 3p orbitals resulting in yellow-orange light of 589nm what Joseph Fraunhofer called the "Dline". Insofar as the sodium "Dline" was the first convenient source of monochromatic light, it became the standard for refractometry. The refractive index of a liquid is thus a high-precision 5-digit number between 1.3000 and 1.7000 at a specific temperature, measured at the sodium Dline wavelength. For example, the refractive index of glycerol at 25C nD25 is 1.4730.)

Closed-circuit perfusion may be necessary for removal of water as well as loading of cryoprotectant if it is true that open-circuit perfusion cannot remove water effectively.

One could imagine that the additional time spent doing closed-circuit (rather than open-circuit) perfusion means increased damage due to above-zero temperature. But most cells are still alive and metabolizing very slowly at 10C. Viaspan, RPS-2 and other organ preservation solutions are designed to keep tissues alive for extended periods at near-zero temperatures certainly for the time required for closed-circuit perfusion. Ramping (slowly increasing concentration) of cryoprotectant should be done in such a way that the ion and mannitol or lactobionate concentration remains unchanged in the perfusate. Ramping is not an osmotically neutral process, however, because cryoprotectant is expected to dehydrate tissues.

Read more from the original source:
Perfusion & Diffusion in Cryonics Protocol - BEN BEST

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cryonics – The Skeptic's Dictionary – Skepdic.com

Posted: February 2, 2016 at 4:48 pm

Cryonics claims it can store a dead human body at low temperatures in such a way that it will be possible to revitalize that body and restore life at some unspecified future date. One hook the cryonics folks use is to give hope that a cure for a disease one dies of today will be found tomorrow, allowing that cure to be applied to the thawed body before or while bringing the dead person back to life. Cryonics might be called resurrection by technology and believers in it might be classified as suffering from the Moses syndrome. The simple fact is once you are dead, you are dead forever. This fact may seem horrifying, but it is not nearly as horrifying as the thought of living forever.

The technology exists to freeze or preserve people and that technology is improving and will probably get better. The technology to revivify a frozen body exists in the imagination. Nanotechnology, for example, is a technology that supporters of cryonics appeal to. Someday, they say, we'll be able to rebuild anything, including diseased or damaged cells in the body, with nanobots. So, no matter what disease destroyed healthy cells in the living body before preservation and no matter what damage was done to the cells of the frozen body during storage, nanotechnology will allow us to bring the dead back to life. This seems like wishful thinking. Nanotechnology might rebuild a mass of dead tissue into a mass of healthy tissue, but without a complete isomorphic model of the brain it will be impossible to return a mushy brain to the exact state it was in before death occurred. (Of course, since this is an exercise in imagination, one can posit that some day we will be able to preserve the brain without any decomposition or transformation at all.) In any case, some other jolt, probably electricity, will be needed to get the heart beating and the brain working again, assuming, of course, that the mush brain has been reconstructed into a healthy brain.

Some preserved by cryonics have the head severed from the body after death. Then, either the head alone is preserved, or both the head and the body are preserved separately. Maybe some future technology will allow the head to be attached to an artificial body. It can be imagined without contradiction, as the philosophers say, so it is not logically impossible that some day our planet will be inhabited by bodiless heads that are connected to machines that allow either actual or virtual experiences of any kind imaginable without requiring the head to leave the room. Of course, when that times comes medical science will have advanced to the point where the aging process can be reversed or maintained in stasis.

A business based on little more than hope for developments that can be imagined by science is quackery. (Cryonics should not be confused with cryogenics, which is a branch of physics that studies the effects of low temperatures on the structure of objects.) There is little reason to believe that the promises of cryonics will ever be fulfilled. Even if a dead body is somehow preserved for a century or two and then repaired, whatever is animated by whatever process will not be the same person who died. The brain is the key to consciousness and to who a person is. There is no reason to believe that a brain preserved by whatever means and restored to whatever state by nanobots will result in a consciousness that is in any way connected to the consciousness of the person who died two centuries earlier.

For those who want to live forever, cloning might be a more realistic possibility but I wouldn't bank on it. First, there is the aging problem. Even if cloning is successful, you won't be able to clone yourself as younger. Of course, you can hope that future technology will have solved the aging problem. Perhaps your body can be cloned repeatedly until science can assist you to overcome aging. However, there is no reason to believe that your clone would be a continuation of you. Your bodies might have identical looking cells, but the only way your minds could be identical is if you had no experience. (It is logically impossible for your bodies to have identical experiences since they occupy different spatial and temporal coordinates.) In that case, you would be as good as dead.

origin of cryonics

Teacher Robert Ettinger (physics and math) brought cryonics into the intellectual mainstream in 1964 with The Prospect of Immortality. Ettinger founded the Cryonics Institute and the related Immortalist Society. He got the idea for cryonics from a story by Neil R. Jones. "The Jameson Satellite" appeared in the July 1931 issue of Amazing Stories. It told the tale of

one Professor Jameson [who] had his corpse sent into earth orbit where (as the author mistakenly thought) it would remain preserved indefinitely at near absolute zero. And so it did, in the story, until millions of years later, when, with humanity extinct, a race of mechanical men with organic brains chanced upon it. They revived and repaired Jameson's brain, installed it in a mechanical body, and he became one of their company.*

Thus was born the idea that we could freeze our bodies, repair them at a later date, and bring them back to life when technology had advanced sufficiently to do the repairs and the reviving.

ethical & other issues

I will leave to others to discuss most of the ethical, legal, political, and economic issues of cryonics. I'll conclude with some comments about the cryonics case of Ted Williams.

Williams died in 2002 at the age of 83. According to his estranged daughter, Barbara Joyce (Bobby-Jo Ferrell) Williams, he left a will in which he expressed his desire to be cremated and have his ashes spread over his favorite fishing grounds in the Florida Keys. His son (Barbara Joyce's half-brother), John Henry Williams, arranged for Williams's body to be processed by Alcor LIfe Extension Foundation. A story in SportsIllustrated.com (SI) stated:

Hall of Famer Ted Williams' head and body are being stored in separate containers at an Arizona cryonics lab that is still trying to collect a $111,000 bill from Williams' son [he had already paid $25,000], according to a story by Tom Verducci in the latest issue of Sports Illustrated.

Alcor still has Williams's head in a canister and his body in a tank, both filled with liquid nitrogen (to keep the remains at a cool -321 degrees Fahrenheit). According to SI, Alcor representatives met with John Henry Williams, but not Ted Williams, about a year before Ted's death. Furthermore, SI reported that the Consent for Cryonic Suspension form submitted to Alcor after Williams had died had a blank line where his signature should have been.

There was a lawsuit by the estranged daughter that fizzled, allegedly for lack of funds, but no legal action by the authorities was taken against John Henry or Alcor. There is a movement still going to right this ship (see the Free Ted Williams website.) Larry Johnson, who worked briefly at Alcor, is leading the crusade to get Congress and a couple of state legislatures to regulate the cryonics industry and have Ted Williams cremated. A video interview with Johnson on "Good Morning America" discussing the disposition of Ted Williams's body at Alcor can be viewed by clicking here. Johnson's book on the subject, Shiver: A Whistleblower's Chilling Expose of Cryonics and the Truth Behind What Happened to Ted Williams, is scheduled to be published in May 2009.

See also Ralian and my comments on cryonics in Mass Media Funk.

Problems Associated with Cryonics – Cryonics: Alcor Life …

Posted: December 27, 2015 at 9:44 pm

(and some possible solutions)

When you buy a house, the seller is legally obliged to disclose any known defects. When you review a company's annual report, it tells you every problem that could affect the corporate share value. Since arrangements for cryopreservation may have a much greater impact on your life than home ownership or stock investments, we feel an ethical obligation to disclose problems that affect cryonics in general and Alcor specifically. We also believe that an organization which admits its problems is more likely to address them than an organization which pretends it has none. Thus full disclosure should encourage, rather than discourage, consumer confidence.

As of 2011, Alcor is nearly 40 years old. Our Patient Care Trust Fund is endowed with more than 7 million dollars and is responsible for the long-term care of over 100 cryopatients. In almost every year since its inception Alcor has enjoyed positive membership growth. We are the largest cryonics organization in the world yet in many respects we are still a startup company. We have fewer than a dozen employees in Scottsdale, Arizona and approximately 20 part-time independent contractors in various locations around the USA, mostly dedicated to emergency standby and rescue efforts. We serve fewer than 1,000 members and the protocols that aid our pursuit of the goal of reversible suspended animation continue to be developed. At the present time the technology required for the realization of our goal far exceeds current technical capabilities. Cryonics will not be comparable with mainstream medicine until our patients can be revived using contemporary technology, and we expect to wait for decades to see this vision fulfilled. Nevertheless, we have made important progress by introducing brain vitrification to improve patient tissue structure preservation.

Alcor shares some of the characteristics of startup companies. The organization is understaffed in some important areas and lacks as much capitalization as would be desired to support maximum growth. Limited resources prevent the organization from hiring as many highly qualified and experienced personnel as desired, and sometimes we have to postpone enhancements to equipment and procedures.

Because Alcor must react quickly to circumstances, it cannot always handle multiple tasks simultaneously. We feel a significant impact if, for example, several members experience legal death in quick succession. A heavy caseload generally means that administrative and even technical development work is postponed while member emergencies take precedence.

On the other hand, Alcor staff believe very strongly in the mission of the organization and are extremely dedicated. Alcor transport team members feel that they are saving lives, and behave accordingly. Most of all, everyone at Alcor is concerned with insuring the security of the patients who have been cryopreserved for the indefinite future. The organization's powerful sense of purpose is reinforced by the fact that all Alcor directors and most staff members have made arrangements to be cryopreserved themselves in the future.

Unlike most startups, Alcor is unlikely to fail for financial reasons. Due to the legally independent status of the Patient Care Trust from Alcor, patients can be maintained indefinitely through its portfolio of cash, investments, real estate, and capital equipment. Some wealthy Alcor members have contributed gifts and endowments to help the organization to advance, and in the event of a financial crisis, many of the people who hope ultimately to be cryopreserved would probably provide assistance. In this sense Alcor benefits from its small size, since it maintains an intimate relationship with many members which would be more problematic if our membership was ten times as large.

Inability to Verify Results

When a conventional surgical procedure is successful, usually the patient recovers and is cured. If the same surgical procedure is unsuccessful or a surgeon makes a serious error, the patient may die. These clear outcomes provide prompt feedback for the people involved. A physician may feel deeply satisfied if a life is saved, or may be deeply troubled (and may be sued for malpractice) if errors cause a death that should have been avoidable.

Clear feedback of this type does not exist in cryonics, because the outcome of our procedures will not be known definitively until decades or even a century from now. We have good reason to expect future technologies capable of repairing cellular damage in cryonics patients, but we feel equally certain that if a patient experiences very severe brain damage prior to cryopreservation, repairs may be delayed, may be incomplete, or may be impossible. The dividing line between these positive and negative outcomes cannot be established clearly at this time.

Suppose a patient experiences 30 minutes of warm ischemia (lack of blood flow at near-normal body temperature) after legal death occurs. Will this downtime create damage that is irreversible by any imaginable technology? Probably not. But what if the ischemic interval lasts for an hour or two hours, or a day? We simply don't know where to draw the line between one patient who is potentially viable, and another who is not.

Of course we can refer to experimental work that has evaluated the injury which occurs when cells are deprived of essential nutrients. These studies provide some guidance regarding the likely damage that a patient may experience, but they still cannot tell us with certainty if future science will be able to reverse that damage.

Another problem afflicting cryonics cases is that many uncontrolled variables prevent us from developing objective criteria to compare one case with another. Consider these two examples:

In the first case, will the long transport time negate the advantage of a rapid initial response and replacement of blood with a chilled preservation solution? In the second case, will the initial hours of warm ischemia outweigh the advantage of the rapid transport to Alcor? We can make educated guesses, but we cannot answer these questions definitively. We have no certain way of knowing which case will work out better, because we have no evidence no outcome.

We do have some simple ways to determine if a patient's circulatory system allows good perfusion with cryoprotectant. Personnel in the operating room will notice if blood clots emerge when perfusion begins. The surface of the brain, visible through burr holes which are created to enable observation, should be pearly white in color. The brain should shrink slightly as water is replaced with cryoprotectant. When perfusion is complete the patient's features should have acquired a sallow color indicating that cryoprotectant has diffused through the tissues.

These simple observations are helpful, but still the people who work hard to minimize transport time and maximize the rate of cooling can never enjoy the satisfying payoff that a physician receives when one of his patients recovers and returns to a normal, active life. This lack of positive outcome can cause feelings of frustration and futility, sometimes leading to disillusionment and burnout.

Conversely, if a case goes badly, team members will be protected from negative feedback. A team leader can never say to one of the personnel, "Because of your error, the patient has no chance of recovery."

The lack of a clear outcome also prevents us from refuting people who claim that future science will be able to undo almost any degree of damage. The danger o
f this extreme positive thinking is that it can lead to laziness. Why bother to make heroic efforts to minimize injury, if nanotechnology will fix everything?

Alcor's stated policy firmly rejects this attitude. Team members are very highly motivated to minimize injury because we believe that our members should not bet their lives on unknown capabilities of future science. Alcor generally hosts a debriefing after each case, encouraging all participants to share complaints, frustrations, and suggestions for improvement. Ideally, each case should be a learning experience, and participants should welcome criticism as an opportunity to identify weaknesses and overcome them in the future.

Still the lack of a clear outcome remains one of the biggest weaknesses in cryonics, since it encourages complacency and prevents accountability. The antidote to this problem is a better set of objective criteria to evaluate cases, and Alcor is working in consultation with brain ischemia experts to develop such criteria.

Volunteer Help

During the 1960s the first cryonics organizations were run entirely by volunteers. The field was not sufficiently reputable to attract qualified medical staff, and no one could have paid for professional help anyway.

Today cryonics is making a transition to professionalism, but financial limitations are prolonging the process. Some paramedics are associated with Alcor, and we hope for more in the future. We have an MD medical director, access to three contract surgeons, access to a hospice nurse, and assistance from an ischemia research laboratory in California where staff has extensive experience in relevant procedures such as vascular cannulation and perfusion. Alcor also communicates with a cryobiology laboratory that has made the most important advances in organ preservation during the past decade. Still, most transport team members who work remotely from the facility are volunteers who receive a week or two of training and modest payment for their work.

In the future, as Alcor becomes more financially secure and is able to offer higher salaries, the organization will attract more medical professionals. At this time, the transition is incomplete.

Limited Support from Mainstream Science

In the 1960s scientists in mainstream laboratories investigated techniques to cryopreserve whole organs. By the end of the 1970s most of this work had ended, and the field of cryobiology separated itself very emphatically from cryonics. The Society for Cryobiology has discouraged scientists from doing work that could advance cryonics, and has adopted a bylaw that threatens to expel any member who practices or promotes cryonics. Consequently the few scientists who are willing to do cryonics-related research live in fear of being excluded from the scientific specialty that is most relevant to their work.

The rift between cryonics and cryobiology may have been caused initially by fears among mainstream scientists that cryonics had a "tabloid journalism" flavor incompatible with science. In addition many scientists have been dissatisfied with the idea of applying procedures without a complete and full understanding of their outcome. Generally, in medicine, first a technique is studied, validated, and perfected, and then it is applied clinically. Cryonics has, of necessity, done an end-run around this formal approach by rushing to apply a technique based on theoretical arguments rather than validated clinical effectiveness.

During the past decade our knowledge and procedures have advanced far beyond the crude freezing methods imagined by most cryobiologists, and experts in molecular nanotechnology have voiced strong support. As more papers are published describing technical advances, we expect that cryobiologists and other scientists will revise their negative assessment of cryonics. In the future we believe that the arbitrary barrier between cryonics and cryobiology will gradually dissolve, and cryonics research will be recognized as a legitimate specialty of the field. However, for the time being the dim view taken of cryonics by most cryobiologists remains problematic, impairing Alcor's ability to achieve respectable status among other relevant groups such as prospective members, regulatory officials, and legislators.

Limited Legal and Government Support

Cryonics is not explicitly recognized in the laws of any state in the United States (see The Legal Status of Cryonics Patients). This does not mean that cryonics is illegal or unregulated. In fact, Alcor must comply with state laws controlling the transport and disposition of human remains, and we make arrangements with licensed morticians to insure that these requirements are met. Alcor also complies with federal regulations established by agencies such as OSHA and EPA.

Still, the lack of specific enabling legislation for cryonics can cause problems. In the late 1980s the California Department of Health Services (DHS) asserted that because there was no statutory procedure for becoming a cryonics organization, human remains could not be conveyed to a cryonics organization via the Uniform Anatomical Gift Act (UAGA), and therefore cryonics was illegal. Fortunately, the courts were unimpressed by this argument. In 1992 the legality of cryonics, and the legality of using the UAGA for cryonics, were upheld at the appellate level.

In 1990 the Canadian province of British Columbia enacted a law that specifically banned the sale of cryonics services in that province. In 2002 the Solicitor General (Canadian equivalent of a state Attorney General) issued a written clarification stating that the law only prohibited funeral homes from selling cryonics arrangements. Cryonics could still be performed in the province, even with the paid assistance of funeral homes, provided they were not involved in the direct sale of cryonics. This position is affirmed by the Business Practices and Consumer Protection Authority of British Columbia. Despite these assurances, anxiety about the law remains.

In 2004 a bill was passed by the Arizona House of Representatives to place cryonics and cryonics procedures under the regulation of the state funeral board. In its original form this law would have prevented our use of the UAGA. The bill was ultimately withdrawn, but may be revived at a later date. Very hostile comments were made about cryonics during the floor debate of this bill. We cannot guarantee that any future legislation will be friendly to cryonics or will permit cryonics to continue in Arizona.

Despite these uncertainties, the United States enjoys a strong cultural tradition to honor the wishes of terminal patients. We believe that the freedom to choose cryonics is constitutionally protected, and so far courts have agreed. We are hopeful that we will be able to continue performing cryonics without technical compromise, under state supervision where necessary, for the indefinite future.

Limited Mainstream Medical Support

Cryonics is not an accepted or recognized "therapy" in the general medical community. To the average medical professional, cryonics is at best an unusual anatomical donation. At worst it can be viewed by some physicians as fraud upon their patient. Hospitals have sometimes deliberately delayed pronouncement of legal death, delayed release of patients to Alcor, or forbade the use of cryonics life support equipment or medications within their facilities. On one occasion in 1988 Alcor had to obtain a court order to compel a hospital to release a patient to Alcor promptly at legal death and permit our stabilization proce
dures on their premises.

Relations with hospitals and their staff are not always difficult. Usually when nurses and physicians learn that cryonics is a sincere practice that is overseen by other medical professionals, they will be willing to accommodate a patient's wishes, or at least will not interfere with them. Sometimes medical staff will even assist with cryonics procedures such as administering medications and performing chest compressions if Alcor personnel are not present when legal death occurs.

The lack of formal medical recognition or support for cryonics generally means that cryonics patients remote from Alcor must be moved to a mortuary for blood replacement before transport to Alcor. Ideally these preparatory procedures should be performed within hospitals, not mortuaries. Hospitals presently allow organ procurement personnel to harvest organs from deceased patients (a fairly elaborate procedure) within their walls. We are hopeful that similar privileges will be extended to cryonics more often as the process becomes better understood and accepted, but we cannot predict how quickly this change will occur.

High Incidence of Poor Cases

In more than 50 percent of cryonics cases legal death occurs before Alcor standby personnel can be deployed, and is often followed by hours of warm ischemia. This downtime may cause severe cellular damage.

The threat of autopsy, in which the brain is routinely dissected, is an even greater danger. Any person who suffers legal death under unexpected circumstances, especially involving accidents or foul play, is liable to be autopsied. Alcor strongly urges members living in California, Maryland, New Jersey, New York, and Ohio to sign Religious Objection to Autopsy forms.

Sometimes cryonicists perish under circumstances resulting in complete destruction or disappearance of their remains. Cryonicists have been lost at sea, suffered misadventures abroad, or even disappeared without a trace. Two members of cryonics organizations were lost in the 2001 collapse of the World Trade Center towers. One was a policeman performing rescue operations.

Cryonics is not a panacea or a "cure" for death. The cryonics ideal of immediate cooling and cardiopulmonary support following cardiac arrest cannot be achieved in the majority of cases. We have good reasons to believe that molecular records of memory persist in the brain even after hours of clinical death, but only future physicians using medical technology which we do not yet possess will be able to determine, finally, whether such a person is really still "there."

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Cryonics | Evidence-Based Cryonics

Posted: December 20, 2015 at 10:44 pm

CryonicsMagazine, July 2013

[The following is a text adaptation of a PowerPoint presentation given on Sunday, May 12, 2013at the Resuscitation and Reintegration of Cryonics Patients Symposium in Portland, Oregon]

An understanding of probable futurerepair requirements for cryonicspatients could affect current cryostoragetemperature practices. I believe thatmolecular nanotechnology at cryogenictemperatures will probably be required forrepair and revival of all cryonics patientsin cryo-storage now and in the foreseeablefuture. Current nanotechnology is far frombeing adequate for that task. I believe thatwarming cryonics patients to temperatureswhere diffusion-based devices couldoperate would result in dissolutionof structure by hydrolysis and similarmolecular motion before repair could beachieved. I believe that the technologiefor scanning the brain/mind of a cryonicspatient, and reconstructing a patient fromthe scan are much more remote in thefuture than cryogenic nanotechnology.

Cryonicists face a credibility problem.It is important to show that resuscitationtechnology is possible (or not impossible)if cryonicists are to convince ourselvesor convince others that current cryonicspractice is not a waste of money and effort.For some people it is adequate to know thatthe anatomical basis of the mind is beingpreserved well enough even if in a veryfragmented form that some unspecifiedfuture technology could repair and restorememory and personal identity. Otherpeople want more detailed elaboration.

Books have detailed whatnanotechnology robots (nanorobots) willlook-like and be capable-of, including(notably) Nanosystems by K. Eric Drexler(1992) and Nanomedicine by Robert A.Freitas, Jr. (Volume I, 1999; Volume IIA,2003). The online Alcor library containsarticles detailing repair of cryonics patientsby nanorobots at cryogenic temperature,in particular, A CryopreservationRevival Scenario using MolecularNanotechnology by Ralph Merkle andRobert Freitas as well as RealisticScenario for Nanotechnological Repairof the Frozen Human Brain. Despitethe detailed descriptions, calculations, andquantitative analyses that have been given,any technology as remote from presentcapabilities as cryogenic nanotechnology iscertain to be very different from whateveranyone may currently imagine. It is difficultto argue against claims that all suchdescriptions are nothing more than handwaving,blue-sky speculations.

Current medical applications ofnanotechnology are mainly limited to theuse of nanoparticles for drug delivery.1Nanomachines are being built, but they arelittle more than toys including a rotor thatcan propel a molecule2 or microcantileverdeflection of DNA by electrostatic force.3In classical mechanics and kinetictheory of gases, on a molecular level,temperature is defined in terms of theaverage translational kinetic energy ofmolecules, which means that the lowerthe temperature the slower the motion ofthe molecules. According to the ArrheniusEquation, the rate of a chemical reactiondeclines exponentially with temperaturedecline. It would be wrong to concludethat nanomachines would barely be able tomove at cryogenic temperatures, however.Nanomachines operate by mechanicalmovement of constituent atoms, a processthat is temperature-independent. In fact,nanomachines would probably operatemore effectively at cryogenic temperaturebecause there would be far less jostlingof atoms in the molecular structuresupon which nanomachines would operate.Nanomachines would also be less vulnerableto reactions with oxygen at cryogenictemperature, although it would nonethelessbe preferable for cryogenic nanorepair tooccur in an oxygen-free environment.

Although under ideal circumstances iceformation can be prevented in cryonicspatients, circumstances too often result inat least some freezingsuch as inability toperfuse with vitrification solution, or poorperfusion with vitrification solution becauseof ischemia due to delayed treatment.Past cryonics patients were perfusedwith the (anti-freeze) cryoprotectantglycerol, whereas cryonics patients arecurrently perfused with cryoprotectantsolutions that include ethylene glycoland dimethylsulfoxide (DMSO). Unlikewater, which forms crystalline ice whensolidifying upon cooling, cryoprotectantsform an amorphous (non-crystalline,vitreous) solid (a hardened liquid) whensolidifying upon cooling. The hardenedliquid is a glass rather than an ice. Thetemperature at which the solidification(vitrification) occurs is called the glasstransition temperature (Tg).

For M22, the cryoprotectant used byAlcor to vitrify cryonics patients, Tg istypically between 123C and 124C(depending on the cooling rate). Tg isabout the same for the cryoprotectant(VM-1) used for cryonics patients at theCryonics Institute.Although freezing can be reduced oreliminated by perfusing cryonics patientswith vitrification solution before coolingto Tg, eliminating cracking is a moredifficult problem. Cryonics patients arecooled to cryogenic temperatures byexternal cooling. Thermal conductivity isslow in a cryonics patient, which meansthat the outside gets much colder thanthe inside. When the outside of a samplecools more quickly than the inside of thesample, thermal stress results. A vitrifiedpatient subjected to such thermal stresscan crack or fracture. No efforts have beenmade to find additives to M22 that wouldhave a similar effect as boron oxide hason allowing Pyrex glass to reduce thermalstress.

If a vitrified sample is small enough,and if cooling is slow enough, the samplecan be cooled far below Tg down toliquid nitrogen temperature withoutcracking. A rabbit kidney (10 millilitervolume) can be cooled down to liquidnitrogen temperature in two days withoutcracking/fracturing.6 Cryonics patientsare much too large to be cooled to liquidnitrogen temperature over a period ofdays without cracking. The amount oftime required for cooling vitrified cryonicspatients to liquid nitrogen temperaturewithout cracking is unknown, and wouldprobably be much too long.

In 1990 cryobiologist Dr. Gregory Fahypublished results of cracking experimentsthat he performed on samples of thecryoprotectant propylene glycol.4 Tg forpropylene glycol is 108C, but in RPS-2carrier solution the Tg is 107C. In oneexperiment he demonstrated that crackingbegan at lower temperatures for smallersamples, specifically: 143C for 46 mL,116C for 482 mL, and 111C for 1412mL. (The last volume is comparable to thevolume of an adult human brain.) Dr. Fahyalso demonstrated that cracking could bedelayed by cooling at slower cooling rates.But when cracking did occur, the cracksformed at the lower temperatures werefiner and more numerous.

Based on evidence that large cracksformed at higher temperatures by morerapid cooling results in a relief of thermalstress that prevents the fine and morenumerous cracks formed when crackingbegins at lower temperature, the CryonicsInstitute (CI) altered its cooling protocolfor cryonics patients. CI patients arecooled quickly from 118C to 145C,and then cooled slowly to 196C.5In order to minimize or eliminatecracking in cryonics patients, proposalshave been made to store the patients attemperatures lower than Tg (124C), buthigher than liquid nitrogen temperature(196C).6 Such a cryo-storage protocolis described as Intermediate TemperatureStorage (ITS). Alcor currently cares for anumber of ITS patients at 140C, but aconsensus has not yet been reached aboutwhat ITS temperature will be chosen whenthis service is made available to all Alcormembers.

Although Alcors vitrification solutionM22 can prevent ice formation with somesamples and protocols, M22 cannot preventice nuclei from forming at cryogenictemperatures. Ice nuclei are local clustersof water molecules that rotate into anorientation that favors later growth of icecrystals when a solution is warmed. Icenuclei are not damaging, but the fact that icenuclei can form indicates molecular mobilitywhich could be damaging. Specifically,between the temperatures of 100C and135C, ice nuclei can form in M22, withthe maximum ice nucleation rate occurringnear Tg. At 140C the ice nucleation ratefor M22 is undetectable. But nuclei will beprobably formed in cooling to 140C.

Although cryostorage at 140C is anattempt to minimize cracking and minimizenucleation, this ITS neither eliminatescracking nor ice nuclei formation.Cryonics patients slowly cooled from Tgto 140C will surely experience someice nucleation. Alcor places a listeningdevice (crackphone) under the skullof its cryonics patients for the purposeof monitoring cracking events. Myunderstanding is that for most Alcorpatients the crackphone detects crackingat Tg or only slightly below Tg, althoughthere was reportedly one M22-perfusedpatient for which the first fracturing eventoccurred at 134C. The propylene glycolexperiments would support the view ofcracking occurring slightly below Tg, butvitrified biological samples resist crackingbetter than pure cryoprotectant solutions.

With ice formation, cracking could occurat temperatures higher than Tg. AlthoughITS may prevent the formation of crackingthat could occur in cooling below 140C,it does not prevent the cracks that occur incooling from Tg to 140C.I have wondered whether there areforms of damage which would occurin a cryonics patient stored at 140Cthat would not occur during storage at196C. A solid cryogenic state of matterdoes not prevent molecular motion.Molecular motion in a biological sampleheld at cryogenic temperature could resultin damage to that sample.

Ions generated by radiation aremuch more mobile than molecules.An ionic species (probably protons) intrimethylammonium dihydrogen phosphateglass is nine orders of magnitude moremobile than the glass moleculesandsodium ions in sodium disilicate glass aretwelve orders of magnitude more mobilethan the glass molecules.9

Cryobiologist Peter Mazur has statedthat below 130C viscosity is so high(>1013 Poise) that diffusion is insignificantover less than geological time spans. Headds that there is no confirmed case ofcell death ascribed to storage at 196Cfor some 2-15 years and none even whencells are exposed to levels of ionizingradiation some 100 times background forup to 5 yr.10 Frozen 8-cell mouse embryossubjected to the equivalent of 2,000 yearsof background gamma rays during 5 to8 months in liquid nitrogen showed noevident detrimental effect on survival ordevelopment.11

In attempting to evaluate damagingeffects of temperature and radiation, itcould be valuable to analyze chemicalalterations, rather than complete cell deathor viability. Acetylcholinesterase enzymesubjected to X-ray irradiation showsconformational changes at 118C, but noconformational changes when irradiatedat 173C.12 X-ray irradiation of insulinand elastase crystals resulted in four timesas much damage to disulfide bridges at173C compared to 223C.13 Anotherstudy showed a 25% crystal diffractionlifetime extension for D-xylose isomerasecrystals X-ray irradiated at less than 253Ccompared to those irradiated at 173C.14

One study showed that lettuce seedsshow measurable deterioration when storedat liquid nitrogen temperature for periodsof 10 to 20 years. Rotational molecularmobility was quantified. A graphical plotwas generated showing increasing timesfor when 50% of lettuce seeds would failto germinate as a function of decreasingtemperature. Those times were estimated tobe about 500 years for 135C and about3,400 years for 196C.15 Translationalvibrational motion has been given as anexplanation for seed quality deterioration atcryogenic temperatures.16 The mean squarevibrational amplitude of a water moleculeis not even zero at 0 Kelvins (273C), andhas been determined to be 0.0082 squareAngstroms. The mean square vibrationalamplitude is 0.0171 square Angstroms at173C and 0.0339 square Angstroms at73C.17

Realistically, however, 3,400 years ismuch longer than cryonics patients arelikely to be stored. Storage in liquid heliumat 269C or in a shadowed moon craterat 235C18 would certainly be moretrouble than it is worth. Northern woodfrogs spend months in a semi-frozen stateat 3C to 6C, and are able to revivewith full recovery of heartbeat uponre-warming.19 An empirical study of acryoprotectant very similar to M22 (VS55) showed viscosity continuing to increaseexponentially below Tg, just as viscosityincreases exponentially with temperaturedecrease above Tg.20 The exponentialdecrease in viscosity (molecular mobility)that makes ice nucleation cease at 135Cindicates that there is probably littlemolecular mobility at 140C, despite thepossibility of damage from ionic species orvibrational motion. All things considered,however, my personal preference is forstorage in liquid nitrogen, rather than someintermediate temperature above 196C. Iwould also prefer for cryogenic nanorobotrepair to be at liquid nitrogen temperature.

I am by no means a nanotechnologyexpert, but I can give a brief descriptionof my own views of how cryogenicnanotechnology repair of a cryonicspatient would proceed. I must thank RalphMerkle for his assistance in allowing me toconsult with him to formulate and clarifymany of my views.I believe that repair of cryonics patientsat cryogenic temperature would be acombination of nano-mining and nanoarcheology.Nanorobots (nanometer-sizedrobots) would first clear blood vessels ofwater, cryoprotectant, plasma, blood cells,etc. The blood vessels would becomemining shafts that would provide access toall body tissues. Nanometer-sized conveyorbelts or trucks on rails could removeblood vessel contents. Where freezingor ischemia had destroyed blood vessels,artificial shafts would be created. Unlikethe nano-mining that simply removes allblood vessel contents, the creation ofartificial shafts would have the characterof an archeological dig. Care would betaken in removing material to avoiddamaging precious artifacts that mightindicate original structure which could be discovered at any unexpected moment.

Section 13.4 of K. Eric Drexlers bookNanosystems provides diagrams and detailsof a nanorobot manipulator arm. Such adiamondoid component would containabout four million atoms, and could befitted with a variety of tools at the endof the arm. A variety of tips with varyingdegrees of chemical reactivity couldallow for reversible, temporary chemicalbonds that could be used for grabbingand moving molecules. These could rangefrom radicals or carbenes that would formstrong covalent bonds, to boron thatcan form relatively weak and reversiblebonds to nitrogen and oxygen, to simpleO-H groups that can form even weakerhydrogen bonds. Tools for digging neednot be so refined. The manipulator arm isdepicted as being 100 nanometers long and50 nanometers wide, although nanorobotswould need to be larger to includecapability for locomotion, computation,and power. A complete nanorobot couldbe as large as a few thousand nanometersin size. A capillary is between 5,000 to10,000 nanometers in diameter, so thereshould be plenty of room for many suchnanorobots to operate. Ralph Merkleestimates that 3,200 trillion nanorobotsweighing a total of 53 grams could repaira cryonics patient in about 3 years.21,22 Likemany of the calculations associated withnanotechnology, I take these figures with apound of salt. It is certainly true, however,that it could take years to repair a patient,and that there should not be a rush tofinish the job.

Merkle & Freitas have suggested thatnanorobots be powered by electrostaticmotors. Stators and rotors would be electricrather than magnetic. Tiny moving chargedplates are easier to fabricate than tiny coilsand tiny iron cores, but more fundamentally,magnetic properties do not scale well withreduced size (i.e., molecular-scale magneticmotors dont work), whereas electrostaticproperties do scale well with reduced size.Electrostatic actuators are already beingused in microelectromechanical systems(MEMS).23 High density batteries couldprovide power for days, and rechargingstations could be located throughout thepatient. Alternatively, nanotube cablescould bring power to the patient fromthe outside. Such cables could also bea means of transmitting and receivingcomputational data. Nanotube cablescould also be used to reunite fracture faces created by cracking. Scanning and imageprocessing capabilities would need toevaluate what needs to be fixed.

As much as possible I would favorreplacement rather than repair, whichwould greatly simplify the process. Itwould be much easier to replace a kidneythan to repair the diseased kidney ofan elderly patient who died of kidneydisease. Curing disease and rejuvenationwould thus become part of the repair of acryonics patient. Of course, neuro patientswould require an entirely new body. Thebrain would be the major exception toreplacement strategy because the braincould not be replaced without loss ofmemory and personal identity.

Even within the brain, however, it couldbe feasible to replace many componentswithout loss of memory and personalidentity. It could be feasible to replacemany organelles such as mitochondria,lysosomes, etc., and many macromoleculessuch as proteins, carbohydrates, and lipids.DNA could be repaired, and possiblyeven modified to cure genetic disease,but epigenetic expression in neurons maybe critical for reconstruction of synapticstructure. Synaptic connections wouldnot only be restored, but the quantityand quality of neurotransmitter contentsshould be restored. It is not simply a matterthat some neurotransmitters are inhibitoryand others are stimulatory. There are morethan 40 different neurotransmitters used inthe brain, and there must be a good reasonwhy such variety is necessitated.

Part of the repair process could involveremoval of ice nuclei, nearly all of whichwould be extracellular. Re-created bloodvessel contents would include freshcryoprotectant, water, plasma, and bloodcells without the original ice nuclei. Althoughsome repair scenarios favor different typesof repair above cryogenic temperature, Idoubt that this is necessary or desirable.Alternative repair scenarios involvesplitting the brain in half, and halvingthe halves repeatedly at cryogenictemperaturewith digitization at eachstepuntil the brain has been totallydigitized.21,22 Or digitization could bedone by repetitive nano-microtomes atcryogenic temperature. The digital datacould be used for full reconstruction. Somepeople might object that if one individualcould be created from digital data, manysuch individuals could be createdraisingquestions of which are duplicates and which is the original. There is detaileddiscussion of the duplicates problem/paradox in the philosophy section of mywebsiteBENBEST.COM.

Although other repair scenarioscould prove to be feasible, I believethat cryogenic nanotechnology will berequired for all cryonics patients in theforeseeable future until the problem ofcryoprotectant toxicity can be solved.With effective nontoxic cryoprotectants,sufficient cryoprotectant could be usedto prevent ice nuclei formation at alltemperatures, prevent devitrification(freezing) upon rewarming, and eliminateall toxic damage. In such a case, therecould be true reversible cryopreservation(suspended animation).

What is needed to create thenanotechnology required for repair ofcryonics patients? Small machines willneed to build parts for smaller machines,which would in turn build even smallermachines. Many details of machine operation must be perfected at each stage.Current modern technological civilizationbegan with cave people pounding on rocks.Ralph Merkle has said that compared tofuture technology, current technology ispounding on rocks.

References

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2. Kudernac T, Ruangsupapichat N,Parschau M, Maci B, Katsonis N,Harutyunyan SR, Ernst KH, Feringa BL.Electrically driven directional motion of afour-wheeled molecule on a metal surface.Nature. 2011 Nov 9;479(7372):208-11.

3. Zhang J, Lang HP, Yoshikawa G,Gerber C. Optimization of DNAhybridization efficiency by pH-drivennanomechanical bending. Langmuir. 2012Apr 17;28(15):6494-501.

4. Fahy GM, Saur J, Williams RJ. Physicalproblems with the vitrification of largebiological systems. Cryobiology. 1990Oct;27(5):492-510.

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10. Mazur P. Freezing of living cells:mechanisms and implications. Am JPhysiol. 1984 Sep;247(3 Pt 1):C125-42.

11. Glenister PH, Whittingham DG,Lyon MF. Further studies on the effectof radiation during the storage of frozen8-cell mouse embryos at -196 degrees C. JReprod Fertil. 1984 Jan;70(1):229-34.

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13. Meents A, Gutmann S, Wagner A,Schulze-Briese C. Origin and temperaturedependence of radiation damagein biological samples at cryogenictemperatures. Proc Natl Acad Sci U S A.2010 Jan 19;107(3):1094-9.

14. Chinte U, Shah B, Chen YS, PinkertonAA, Schall CA, Hanson BL. Cryogenic(<20 K) helium cooling mitigates radiationdamage to protein crystals. Acta CrystallogrD Biol Crystallogr. 2007 Apr;63(Pt 4):486-92.

15. Walters C, Wheeler L, Stanwood PC.Longevity of cryogenically stored seeds.Cryobiology. 2004 Jun;48(3):229-44.

16. Wowk B. Thermodynamic aspectsof vitrification. Cryobiology. 2010Feb;60(1):11-22.

17. Leadbetter AJ; The Thermodynamicand Vibrational Properties of H$_2$O Iceand D$_2$O Ice. 1965 Sep;A287:403-425.

18. Paige DA, Siegler MA, Zhang JA,Hayne PO, Foote EJ, Bennett KA,Vasavada AR, Greenhagen BT, SchofieldJT, McCleese DJ, Foote MC, DeJong E,Bills BG, Hartford W, Murray BC, AllenCC, Snook K, Soderblom LA, Calcutt S,Taylor FW, Bowles NE, Bandfield JL,Elphic R, Ghent R, Glotch TD, WyattMB, Lucey PG. Diviner Lunar Radiometerobservations of cold traps in the Moonssouth polar region. Science. 2010 Oct22;330(6003):479-82.

19. Costanzo JP, Lee RE Jr, DeVries AL,Wang T, Layne JR Jr. Survival mechanismsof vertebrate ectotherms at subfreezingtemperatures: applications in cryomedicine.FASEB J. 1995 Mar;9(5):351-8.

20. Noday DA, Steif PS, Rabin Y.Viscosity of cryoprotective agents nearglass transition: a new device, technique,and data on DMSO, DP6, and VS55. ExpMech. 2009 Oct;49(5):663-672.

21. Merkle, RC. The Molecular Repair ofthe Brain. Cryonics. 1994 Jan;15(1):16-31.

22. Merkle, RC. The Molecular Repair ofthe Brain. Cryonics. 1994 Apr;15(2):18-30.

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Cryonics | Evidence-Based Cryonics

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Cryonics | Evidence-Based Cryonics

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CryonicsMagazine, July 2013

[The following is a text adaptation of a PowerPoint presentation given on Sunday, May 12, 2013at the Resuscitation and Reintegration of Cryonics Patients Symposium in Portland, Oregon]

Although Alcors vitrification solutionM22 can preve
nt ice formation with somesamples and protocols, M22 cannot preventice nuclei from forming at cryogenictemperatures. Ice nuclei are local clustersof water molecules that rotate into anorientation that favors later growth of icecrystals when a solution is warmed. Icenuclei are not damaging, but the fact that icenuclei can form indicates molecular mobilitywhich could be damaging. Specifically,between the temperatures of 100C and135C, ice nuclei can form in M22, withthe maximum ice nucleation rate occurringnear Tg. At 140C the ice nucleation ratefor M22 is undetectable. But nuclei will beprobably formed in cooling to 140C.

Section 13.4 of K. Eric Drexlers bookNanosystems provides diagrams and detailsof a nanorobot manipulator arm. Such adiamondoid component would containabout four million atoms, and could befitted with a variety of tools at the endof the arm. A variety of tips with varyingdegrees of chemical reactivity couldallow for reversible, temporary chemicalbonds that could be used for grabbingand moving molecules. These could rangefrom radicals or carbenes that would formstrong covalent bonds, to boron thatcan form relatively weak and reversiblebonds to nitrogen and oxygen, to simpleO-H groups that can form even weakerhydrogen bonds. Tools for digging neednot be so refined. The manipulator arm isdepicted as being 100 nanometers long and50 nanometers wide, although nanorobotswould need to be larger to includecapability for locomotion, computation,and power. A complete nanorobot couldbe as large as a few thousand nanometersin size. A capillary is between 5,000 to10,000 nanometers in diameter, so thereshould be plenty of room for many suchnanorobots to operate. Ralph Merkleestimates that 3,200 trillion nanorobotsweighing a total of 53 grams could repaira cryonics patient in about 3 years.21,22 Likemany of the calculations associated withnanotec
hnology, I take these figures with apound of salt. It is certainly true, however,that it could take years to repair a patient,and that there should not be a rush tofinish the job.