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Home , 21st Century Medicine, Brian Wowk, Cryopreservation, Cryoprotectant

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solutes in the residual unfrozen water between . This is so-called “solu- tion effects” injury. How Cryoprotectants Protect Cells Cryoprotectants are chemicals that dis- solve in water and lower the melting point of water. For applications outside , such chemicals are sometimes called “.” Common examples are glyc- ife is a complex chemical process that hap- erol, , , and Lpens in water. Without liquid water, there dimethylsulfoxide (DMSO). is no life, or at least no life process. Cryopro- A concentration of about tectants are chemicals that protect living things 5% to 15% is usually all that is required to from being injured by water during permit survival of a substantial fraction of exposure to cold. How cryoprotectants work isolated cells after freezing and thawing from is a mystery to most people. In fact, how they liquid . Figure 2 shows work was even a mystery to science until just a the essential concept of cryoprotection dur- few decades ago. This article will explain in ing freezing. Growing ice compacts cells basic terms how cryoprotectants protect cells into smaller and smaller pockets of unfrozen from damage caused by ice crystals, and some liquid as the temperature is lowered. The of the advances that have been made in the presence of cryoprotectants makes these design of cryoprotectant . Figure 1A. Cells before freezing. pockets larger at any given temperature than they would be if no cryoprotectant were pres- How Freezing Injures Cells ent. Larger unfrozen pockets for cells reduces Water expands when it freezes, but con- damage from both forms of freezing injury, trary to popular belief it is not expansion of mechanical damage from ice and excessive water that causes injury. It is the purification of concentration of salt. water during freezing that causes injury. Water freezes as a pure substance that excludes all else. Properties of Cryoprotectants It is this exclusion process that causes injury. Not all chemicals that dissolve in water are Instead of remaining a solvent that allows the cryoprotectants. In addition to being water molecules of life to freely mix within it, water soluble, good cryoprotectants are effective at that freezes gathers itself up into crystals push- depressing the melting point of water, do not ing everything else out. This is illustrated in precipitate or form eutectics or hydrates, and Figure 1. Figure 1B. Cells after freezing. Cells are are relatively non-toxic to cells at high concen- Freezing causes damage by two distinct squashed between ice crystals and exposed to tration. All cryoprotectants form hydrogen lethal concentrations of salt. Contrary to pop- mechanisms. The first is mechanical damage bonds with water. Since the discovery of glyc- ular belief, slow cooling causes water to freeze as the shape of cells is distorted by ice crys- outside cells, not inside cells. Cells are dehy- erol as the first cryoprotectant more than 50 tals. The second is damage caused by chemi- drated by the growing concentration of salt in years ago (1), approximately 100 compounds cal and osmotic effects of concentrated the unfrozen liquid around them. have been explicitly identified and studied as

www.alcor.org /Third Quarter 2007 3 cryoprotectants, although only a handful are used routinely in cryobiology (2). The best and most commonly used cry- oprotectants are a class of cryoprotectants called penetrating cryoprotectants. Penetrat- ing cryoprotectants are small molecules that easily penetrate cell membranes. The molec- ular mass of penetrating cryoprotectants is typically less than 100 daltons. By entering and remaining inside cells, penetrating cry- oprotectants prevent excessive dehydration of cells during the freezing process.

Vitrification as an Alternative to Freezing Organized is more damaged by freezing than isolated cells. Unlike suspen- sions of disconnected cells, tissue doesn’t have room for ice to grow, and cannot easily sequester itself into unfrozen pockets between ice crystals. Organs are especially vulnerable to freezing injury. For an to Figure 2. Water when frozen without and with added cryoprotectant. Without cry- resume function after freezing, all the diverse oprotectant, almost the entire water volume freezes during cooling. Only salts and other cell types of the organ, from parenchymal dissolved molecules prevent water from freezing completely. With cryoprotectant, the percentage of cryoprotectant present in increases as ice grows. At any given tem- cells to cells of the smallest vessels, perature, ice growth stops when the cryoprotectant becomes concentrated enough to have to survive in large numbers. The 25% make the melting point equal to the surrounding temperature. Eventually the cryoprotec- survival rates often seen in cell freezing are tant reaches a concentration that cannot be frozen. No more ice can grow as the tem- not good enough. For of perature is lowered, and there is more room for cells to survive between ice crystals. organs, a different approach is required. Below approximately -100ºC, the remaining unfrozen liquid pocket solidifies into a glass, In 1984 cryobiologist Gregory Fahy pro- permitting storage for practically unlimited periods of time. Cells survive freezing by exist- posed as an approach to cryop- ing inside the glassy solid between ice crystals. The larger the starting cryoprotectant con- reservation (3). Vitrification, which means centration, the larger the unfrozen volume will be at the end of freezing. “turn into a glass,” was previously known in cryobiology as a process that occurred when water was cooled too fast to form ice crystals. It was also believed to be the process by which cells survived in unfrozen pockets of concentrated cryoprotectant between ice crys- tals at very low . Fahy proposed a way to turn the entire volume of a tissue or organ into the equivalent of an unfrozen glassy pocket of concentrated cryoprotectant. To achieve vitrification, it was proposed that the tissue or organ be loaded with so much cryoprotectant before cooling that it could avoid ice formation during the entire cooling process. If cooling is fast, this could be done with actually less cryoprotectant con- centration than cells are exposed to during the final stages of conventional freezing. The concept is illustrated in Figure 3. By avoiding mechanical distortion caused Figure 3. Freezing vs. vitrification. Vitrification loads cells and tissue with a high con- centration of cryoprotectant at the very beginning. Cooling quickly then allows the by ice, and by allowing salts and other mole- entire volume of tissue to become a glassy solid, or “vitrify”, without any freezing at all. cules to remain undisturbed in their natural

4 Cryonics/Third Quarter 2007 www.alcor.org locations, vitrification avoids the major damage Components of mechanisms of freezing. The price paid is Cryopreservation damage from cryoprotectant . Solutions More than just cry- Cryoprotectant Toxicity oprotectants must be In cryopreservation by freezing or vitrifi- added to cells and tis- cation, more than half of the water inside cells sues to protect against is ultimately replaced by cryoprotectant mole- freezing injury. A cry- cules. Cryoprotection can be regarded as a opreservation solution, process of replacing water molecules with which may be either a other molecules that cannot freeze. When one freezing solution or vit- considers the crucial role that water plays in rification solution, con- maintaining the proper shape and form of pro- sists of: teins and other molecules of life, it is astonish- ing that this can be survived. Carrier Solution The toxicity of cryoprotectants adminis- Carrier solution tered at near-freezing temperatures is a differ- consists of solution ent kind of toxicity than the toxicity experi- ingredients that are not Figure 4. Composition of M22 vitrification solution. All ingredi- enced by living things at warm temperature. explicit cryoprotectants. ents are penetrating cryoprotectants, except for LM5 carrier solutes, For example, to a person under ordinary con- The role of the carrier Z-1000 and X-1000 ice blockers, and PVP K12 polymer. M22 is a ditions, propylene glycol is non-toxic, while solution is to provide “sixth generation” vitrification solution, incorporating two decades of ethylene glycol is metabolized into a poison. basic support for cells at progress in the development of vitrification solutions for mainstream However at high concentrations near 0ºC, temperatures near freez- medical tissue and organ banking. ethylene glycol is less toxic to cells than ing. It contains salts, propylene glycol. Usual rules don’t apply. osmotic agents, pH 4, penetrating cryoprotectants are the majori- New rules relating to how life responds when buffers, and sometimes nutritive ingredients or ty ingredients of vitrification solutions. large amounts of water are substituted at low apoptosis inhibitors. The ingredients are usual- temperature remain to be discovered. ly present at near isotonic concentration (300 Non-penetrating Cryoprotectants Mechanisms of cryoprotectant toxicity milliosmoles) so that cells neither shrink nor (optional ingredient) are still poorly understood (4,5), but a few swell when held in carrier solution. Carrier solu- Non-penetrating cryoprotectants are empirical generalizations can be made. tion is sometimes called “base perfusate.” The large molecules, usually polymers, added to Lipophilicity (affinity for fats and oils) strong- carrier solution typically used with M22 cry- cryoprotectant solutions. They inhibit ice ly correlates with toxicity. Molecules with an oprotectant solution is called LM5. growth by the same mechanisms as penetrat- affinity for fat can partition into cell mem- Different concentrations of cryoprotec- ing cryoprotectants, but do not enter cells. branes, destabilizing them. It has also been tant may be required at various stages of cry- Polyethylene glycol (PEG) and recently discovered that strong hydrogen oprotectant introduction and removal, but the polyvinylpyrrolidone (PVP) are examples. bonding correlates with toxicity, possibily by concentration of carrier solution ingredients Non-penetrating cryoprotectants are usually disrupting the hydration shell around macro- always remains constant. This constant-com- less toxic than penetrating cryoprotectants at molecules. This led to the unexpected result position requirement can be regarded as the the same concentration. They reduce the that cryoprotectants with polar groups that definition of a carrier solution. As a practical amount of penetrating cryoprotectants need- interact weakly with water are best for vitrifi- matter, this means that cryopreservation solu- ed by mimicking outside the cell the cryopro- cation, even if a higher concentration is tions must be made by means other than tective effects of proteins inside the cell. It required to achieve vitrification (6). The elec- adding cryoprotectants to a pre-made carrier has also been recently discovered that using trical properties of cryoprotectant solutions solution because naïve addition would dilute non-penetrating cryoprotectants to increase have also been related to membrane toxicity the carrier ingredients. the tonicity (osmotically active concentration) (7). Certain cryoprotectants, such as of vitrification solutions can prevent a type of and possibly DMSO, are also known to have Penetrating Cryoprotectants injury called chilling injury. adverse reactions with specific biochemical Penetrating cryoprotectants are small targets. Finally, mutual toxicity reduction, molecules able to cross cell membranes. The Ice Blockers (optional ingredient) especially as seen in the DMSO/formamide role of penetrating cryoprotectants is to Ice blockers are compounds that direct- combination, has been very useful in vitrifica- reduce ice growth and reduce cell dehydration ly block ice growth by selective binding with tion solution development, although the during freezing. In vitrification, the role of ice or binding to contaminants that trigger mechanism of this toxicity reduction is still penetrating cryoprotectants is to completely ice formation (ice nucleators). Conventional unknown (5). prevent ice formation. As is shown in Figure cryoprotectants act by interacting with water.

www.alcor.org Cryonics/Third Quarter 2007 5 Ice blockers compliment conventional cry- ing can also sometimes succeed even though oprotectants from organs is longer than for oprotectants by interacting with ice or sur- cryoprotectant concentration remains low if cells. For vitrification solutions, perfusion times faces that resemble ice. Ice blockers are like freezing and thawing are done extremely rap- of hours are typical. This is because cryopro- drugs in that only a small amount is required idly so that there is not enough time for ice tectants must move through small spaces to find and bind their target. Low molecular to grow inside cells. between cells that line the inside of blood ves- weight polyvinyl and polyglycerol, Vitrification solutions containing cry- sels, the capillary endothelium. This makes cells called X-1000 and Z-1000, and biological oprotectant concentrations near or exceed- of the capillary endothelium among those most antifreeze proteins are examples of ice ing 50% cannot be added in a single step vulnerable to cryoprotectant toxicity because blockers (8,9). Ice blockers are only used in because the initial osmotic shrink response they are exposed to the highest concentrations vitrification solutions, not freezing solutions would be too extreme. Instead, material to of cryoprotectant for the longest time while (See Figure 5). be vitrified is successively exposed to several waiting for other cells in the organ to catch up. solutions containing exponentially increasing The brain has an additional difficulty in How Cryoprotectants are Used concentrations of cryoprotectant, such as that the spaces between capillary endothelial Freezing solutions containing relatively 1/8 x, 1/4 x, 1/2 x, 1 x full concentration cells are especially small. This is the so-called low cryoprotectant concentrations near 10% vitrification solution, typically for 20 minutes blood brain barrier, or BBB. The BBB caus- are typically added in a single step. This each step. The addition is done at a temper- es penetrating cryoprotectants to leave blood causes the classic shrink-swell response of ature near 0ºC to minimize toxicity. The vessels even more slowly than other organs, cryobiology in which cells first shrink by material is then ready for vitrification. For and doesn’t permit water-soluble molecules in response to the high solute con- cryopreservation by vitrification, cooling and bigger than 500 daltons to leave at all. There- centration outside the cell, and then swell as rewarming are done as quickly as possible. fore non-penetrating cryoprotectants do not penetrating cryoprotectants enter the cell. Unlike cell suspensions or small tissue pass through an intact BBB. Within several minutes, or tens of minutes pieces, organs are too large to absorb cry- However this doesn’t mean that non- for thin tissue pieces, the cryoprotectant oprotectant by just soaking in an external penetrating agents have no effect on brain concentration inside and outside cells equal- solution. For organ cryopreservation, cry- tissue. The osmotic movement of water izes, and cells return to a volume defined by oprotectants are added by perfusion, a across the BBB is determined by the entire the tonicity of the carrier solution. The cells process in which the cryoprotectant solution cryoprotectant solution composition. Water or tissue are now ready for freezing. For cry- is circulated through blood vessels just as moves to equalize the solution melting point, opreservation by freezing, cooling is done blood would flow through the organ. This or “water activity,” on either side of the BBB. slowly, typically less than 1ºC per minute. ensures that no cell is more than a few cells This means that any ingredient that lowers This allows time for water to leave cells as away from contact with the circulating solu- the melting point of the cryoprotectant solu- freezing progresses so that the cryoprotec- tion. Rather than adding cryoprotectant in tion also increases the resistance of tissue tant concentration inside cells rises together discrete steps, it is more convenient during outside the BBB to ice formation by drawing with the concentration outside cells. This perfusion to increase the cryoprotectant con- out water and increasing the concentration of prevents cell interiors from freezing. Freez- centration continuously. solutes naturally present in the brain. The Cryprotectants are brain is an organ in which penetrating removed by reversing the cryprotectants and dehydration seem to act in steps described above, tandem to provide cryoprotection. except that all removal solutions except for the Six Generations of Vitrification very last contain several Solutions hundred millimoles of an Vitrification solutions have progressed osmotic buffer, such as greatly since the initial proposal of modern vitri- mannitol. The role of the fication by Fahy in the early 1980s. This progress osmotic buffer is to reduce may be viewed as occurring in six generational the extent of the initial leaps (10). Generations three through six were swell response of cells as developed at , Inc. they are exposed to decreased external cry- Generation 1 oprotectant concentration. Figure 5. Effect of ice blockers on ice formation. The flask on The simplest vitrification solutions are the left contains 55% w/w ethylene glycol solution that was single cryoprotectants in carrier solution. cooled to -130ºC. The flask on the right contains the same solu- Special Considera- tion, except with 1% of the ethylene glycol replaced by 0.9% tions for Organs Generation 2 X-1000 and 0.1% Z-1000 ice blockers. It is almost completely vit- The time required to It was discovered that higher total cryopro- rified, with the majority of the solution being a transparent glass introduce and remove cry- tectant concentrations with acceptable toxicity rather than white crystalline ice.

6 Cryonics/Third Quarter 2007 www.alcor.org could be achieved by combining DMSO with reducing the concentration of all cryoprotec- Future generations of cryoprotectant so- amides such as acetamide or formamide, and tants necessary to achieve vitrification. VM3 lutions will have to address many problems then adding propylene glycol. The combina- is a fifth generation vitrification solution (6). that are still outstanding, including molecular tion of DMSO, formamide, and propylene gly- mechanisms of cryopreservation failure (21), col was the basis of the VS41A (also called Generation 6 and especially cryoprotectant toxicity. Cry- VS55) vitrification solution, the most advanced It was discovered that chilling injury, a oprotectant toxicity is emerging as a final vitrification solution of the mid 1990s. poorly-understood injury caused by just pass- frontier of cryobiology. The greatest future ing through certain sub-zero temperature breakthroughs in cryobiology may come from Generation 3 ranges, could be overcome by increasing the better understanding and mitigation of cry- A breakthrough occurred with Fahy’s dis- tonicity of non-penetrating components of oprotectant toxicity. covery that cryoprotectant toxicity correlated vitrification solutions (11). M22, the cryopro- with the number of water molecules per cry- tectant currently used by Alcor, is a sixth gen- Dr. Wowk is the Senior Physicist at oprotectant polar group at the critical concen- eration solution. 21st Century Medicine, Inc., a com- tration needed for vitrification, so-called qv* Successful vitrification has now been pany specializing in the development (6). This led to the replacement of the propy- demonstrated for valves (12), vascular of technology for cold preservation of lene glycol in VS41A with ethylene glycol, tissue (13), cartilage (14), cornea (15), and transplantable tissue and organs. generating the Veg vitrification solution. mouse (16, 17). Progress continues for the rabbit , with recovery of the Generation 4 organ demonstrated after cooling to below The use of polymers in vitrification solu- – 40ºC while cryoprotected with a vitrification tions permitted further reductions in toxicity by solution (11), and one reported instance of reducing the concentration of penetrating cry- long-term survival after vitrification (18). Vit- oprotectants necessary to achieve vitrification. rification has also shown utility for viable Generation 5 preservation of diverse tissue slices, including The use of ice blocking polymers permit- brain slices (19), and histological preservation ted still further reductions in toxicity by of larger systems (20).

References

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