how cryoprotectants depressed metabolism nosy neuroprotection · mike perry reminds us of the...

How Cryoprotectants Work Page 3

History of Glycerol Vs.DMSO Page 8

Depressed Metabolismpage 14

Nosy Neuroprotectionpage 18

Alternatives to cryonics page 21

Your Doorwayto the FutureThe Alcor News Blog

Logon today, and everyday, to readabout recent happenings at the Alcor Foundation

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Depressed Metabolism, a technical

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1www.alcor.org Cryonics/Third Quarter 2007

THE SCIENCE OF CRYONICS

2 From the Editor

8 History of DMSO and Glycerol in Cryonics: In thebeginning, the cryoprotectiveagent of choice was DimethylSulfoxide (DMSO). Learn howthe first human cryopreservationprotocol written by DanteBrunol influenced this choiceand what turn of events eventually led to the discoverythat glycerol was a superior cryoprotective agent. MikeDarwin, a leader in cryonics atthe time when these historicalevents were in the making,takes you through the evolutionof early cryonics protocols.

12 Advances in Cryopreservation: Has vitrification closed the chapteron the idea of freezing organs?In his final installment, GregFahy reports on recent researchfindings with some surprisingimplications.

14 Depressed Metabolism: Themost important objective in cryonics after pronouncementof legal death is to drop metabolism as fast as possibleto protect the brain and othertissues from rapid deterioration.Aschwin de Wolf discusses thelimitations of the commonstrategies used in cryonicstoday – general anesthetics andhypothermia – and reviews thepotential of studying hibernatinganimals or administering carbonmonoxide and hydrogen sulfideto induce a “suspended animation”-like state.

IN THIS ISSUE

How Cryoprotectants Work: Water is essential for life processes, butpreventing the damage caused byfreezing of the water in the humanbody is a significant challenge forcryopreservation cases. Brian Wowk,cryobiologist at 21st Century Medicine,explains how chemicals called “cryopro-tectants” greatly reduce freezing injuriesto cells.

CCOOVVEERR SSTTOORRYY:: PPAAGGEESS 33--77

3RD QUARTER 2007 • VOLUME 28:3SPECIAL EDITION

Cover by Randal Fry

18 Nosy Neuroprotection: In 2005 Alcor introducedsternal infusion as an alternative to intravenous medication administration. Taking the discussion a stepfurther, Chana de Wolf reviews the implications ofusing the nose to deliver neuroprotectants to the brain.Will this non-invasive and direct delivery route to thebrain prove superior? Find out.

21 The Road Less Traveled: Alternatives to Cryonics: A demandexists for alternative preservation approaches, beyond present-daycryopreservation. These approaches have the potential to be mucheasier and cheaper than cryonics, and they may be the only option forsome. Are the alternatives good enough to offer a reasonableprospect of eventual restoration to health? R. Michael Perry shareshis own personal story and takes a preliminary look at the options.

2 Cryonics/Third Quarter 2007 www.alcor.org

FROM THE ‘GUEST’ EDITOR

It has been four decades since the firsthuman cryopreservation was performed.

Since then, there have been several advancesin cryopreservation techniques and an accu-mulation of knowledge about the physiolog-ical and cellular events after cardiac arrest.As new and relevant discoveries are made,researchers must integrate information froma variety of scientific fields in order toimprove cryonics protocol and deliver con-sistent quality of care. Though the goal ofcryonics today remains the same as fortyyears ago, we have learned (and forgotten!)a lot since then.

Some people think that Alcor’s emphasis should be on pushing the science ofcryonics forward in terms that scientists will understand. Others believe that Alcor’semphasis should be on outreach and educating its membership. The history of Cryon-ics Magazine reflects both perspectives. The magazine has featured either “dense”technical articles that documented the current state of the art in terms familiar toresearchers and biomedical professionals, or articles that appeal to the average reader.

As the guest editor of this issue, I have incorporated both approaches in this“special edition.” Most of the usual contents have been stripped to make room fora series of articles that discuss the scientific or technical aspects of cryonics.

There are many misunderstandings about vitrification and the cryoprotectiveagents Alcor uses. In his article “How Cryoprotectants Work,” cryobiologist andAlcor director Brian Wowk introduces the reader to the basics of cryopreservationwithout ice formation and clears up some of these misconceptions.

Although long term care at cryogenic temperatures is Alcor’s current mission,Mike Perry reminds us of the possibility of alternative techniques to preserve criti-cally ill patients in his article “The Road Less Traveled: Alternatives to Cryonics.”

We are extremely proud to feature a historical article on the use of DMSO andglycerol by former Alcor president and critical care expert Mike Darwin. On a lesspositive note, this issue will feature Greg Fahy’s final “Advances in Cryopreserva-tion” column. We thank Greg for writing this excellent column and hope to seemore of him in the future.

Don’t forget that Cryonics Magazine has a “Letters to the Editor” section (seeleft-hand column for contact info)! Let us know what you like and what you don’tlike about Cryonics Magazine.

EditorJennifer Chapman

Guest EditorChana de Wolf

Art DirectorJill Grasse

Contributing WritersMike Darwin,

Aschwin de Wolf,Chana de Wolf,

Gregory M. Fahy, Ph.D.,R. Michael Perry, Ph.D.,

Brian Wowk, Ph.D.

Contributing PhotographersTed Kraver, Ph.D.

________________________________

Copyright 2007 by Alcor Life Extension Foundation

All rights reserved.Reproduction, in whole or part, without

permission is prohibited.

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HOW Cryoprotectants WorkBy Brian Wowk, Ph.D.

Life is a complex chemical process that hap-pens in water. Without liquid water, there

is no life, or at least no life process. Cryopro-tectants are chemicals that protect living thingsfrom being injured by water freezing duringexposure to cold. How cryoprotectants workis a mystery to most people. In fact, how theywork was even a mystery to science until just afew decades ago. This article will explain inbasic terms how cryoprotectants protect cellsfrom damage caused by ice crystals, and someof the advances that have been made in thedesign of cryoprotectant solutions.

How Freezing Injures CellsWater expands when it freezes, but con-

trary to popular belief it is not expansion ofwater that causes injury. It is the purification ofwater during freezing that causes injury. Waterfreezes as a pure substance that excludes all else.It is this exclusion process that causes injury.Instead of remaining a solvent that allows themolecules of life to freely mix within it, waterthat freezes gathers itself up into crystals push-ing everything else out. This is illustrated inFigure 1.

Freezing causes damage by two distinctmechanisms. The first is mechanical damageas the shape of cells is distorted by ice crys-tals. The second is damage caused by chemi-cal and osmotic effects of concentrated

solutes in the residual unfrozen waterbetween ice crystals. This is so-called “solu-tion effects” injury.

How Cryoprotectants ProtectCells

Cryoprotectants are chemicals that dis-solve in water and lower the melting point ofwater. For applications outside cryobiology,such chemicals are sometimes called“antifreeze.” Common examples are glyc-

erol, ethylene glycol, propylene glycol, anddimethylsulfoxide (DMSO).

A cryoprotectant concentration of about5% to 15% is usually all that is required topermit survival of a substantial fraction ofisolated cells after freezing and thawing fromliquid nitrogen temperature. Figure 2 showsthe essential concept of cryoprotection dur-ing cell freezing. Growing ice compacts cellsinto smaller and smaller pockets of unfrozenliquid as the temperature is lowered. Thepresence of cryoprotectants makes thesepockets larger at any given temperature thanthey would be if no cryoprotectant were pres-ent. Larger unfrozen pockets for cells reducesdamage from both forms of freezing injury,mechanical damage from ice and excessiveconcentration of salt.

Properties of CryoprotectantsNot all chemicals that dissolve in water are

cryoprotectants. In addition to being watersoluble, good cryoprotectants are effective atdepressing the melting point of water, do notprecipitate or form eutectics or hydrates, andare relatively non-toxic to cells at high concen-tration. All cryoprotectants form hydrogenbonds with water. Since the discovery of glyc-erol as the first cryoprotectant more than 50years ago (1), approximately 100 compoundshave been explicitly identified and studied as

Figure 1A. Cells before freezing.

Figure 1B. Cells after freezing. Cells aresquashed between ice crystals and exposed tolethal concentrations of salt. Contrary to pop-ular belief, slow cooling causes water to freezeoutside cells, not inside cells. Cells are dehy-drated by the growing concentration of salt inthe unfrozen liquid around them.


cryoprotectants, although only a handful areused routinely in cryobiology (2).

The best and most commonly used cry-oprotectants are a class of cryoprotectantscalled penetrating cryoprotectants. Penetrat-ing cryoprotectants are small molecules thateasily penetrate cell membranes. The molec-ular mass of penetrating cryoprotectants istypically less than 100 daltons. By enteringand remaining inside cells, penetrating cry-oprotectants prevent excessive dehydration ofcells during the freezing process.

Vitrification as an Alternative toFreezing

Organized tissue is more damaged byfreezing than isolated cells. Unlike suspen-sions of disconnected cells, tissue doesn’thave room for ice to grow, and cannot easilysequester itself into unfrozen pocketsbetween ice crystals. Organs are especiallyvulnerable to freezing injury. For an organ toresume function after freezing, all the diversecell types of the organ, from parenchymalcells to cells of the smallest blood vessels,have to survive in large numbers. The 25%survival rates often seen in cell freezing arenot good enough. For cryopreservation oforgans, a different approach is required.

In 1984 cryobiologist Gregory Fahy pro-posed vitrification as an approach to cryop-reservation (3). Vitrification, which means“turn into a glass,” was previously known incryobiology as a process that occurred whenwater was cooled too fast to form ice crystals.It was also believed to be the process bywhich cells survived in unfrozen pockets ofconcentrated cryoprotectant between ice crys-tals at very low temperatures. Fahy proposeda way to turn the entire volume of a tissue ororgan into the equivalent of an unfrozenglassy pocket of concentrated cryoprotectant.

To achieve vitrification, it was proposedthat the tissue or organ be loaded with somuch cryoprotectant before cooling that itcould avoid ice formation during the entirecooling process. If cooling is fast, this couldbe done with actually less cryoprotectant con-centration than cells are exposed to during thefinal stages of conventional freezing. Theconcept is illustrated in Figure 3.

By avoiding mechanical distortion causedby ice, and by allowing salts and other mole-cules to remain undisturbed in their natural

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 theentire volume of tissue to become a glassy solid, or “vitrify”, without any freezing at all.

Figure 2. Water when frozen without and with added cryoprotectant. Without cry-oprotectant, almost the entire water volume freezes during cooling. Only salts and otherdissolved molecules prevent water from freezing completely. With cryoprotectant, thepercentage of cryoprotectant present in solution increases as ice grows. At any given tem-perature, ice growth stops when the cryoprotectant becomes concentrated enough tomake the melting point equal to the surrounding temperature. Eventually the cryoprotec-tant reaches a concentration that cannot be frozen. No more ice can grow as the tem-perature is lowered, and there is more room for cells to survive between ice crystals.Below approximately -100ºC, the remaining unfrozen liquid pocket solidifies into a glass,permitting storage for practically unlimited periods of time. Cells survive freezing by exist-ing inside the glassy solid between ice crystals. The larger the starting cryoprotectant con-centration, the larger the unfrozen volume will be at the end of freezing.


locations, vitrification avoids the major damagemechanisms of freezing. The price paid isdamage from cryoprotectant toxicity.

Cryoprotectant ToxicityIn cryopreservation by freezing or vitrifi-

cation, more than half of the water inside cellsis ultimately replaced by cryoprotectant mole-cules. Cryoprotection can be regarded as aprocess of replacing water molecules withother molecules that cannot freeze. When oneconsiders the crucial role that water plays inmaintaining the proper shape and form of pro-teins and other molecules of life, it is astonish-ing that this can be survived.

The toxicity of cryoprotectants adminis-tered at near-freezing temperatures is a differ-ent kind of toxicity than the toxicity experi-enced by living things at warm temperature.For example, to a person under ordinary con-ditions, propylene glycol is non-toxic, whileethylene glycol is metabolized into a poison.However at high concentrations near 0ºC,ethylene glycol is less toxic to cells thanpropylene glycol. Usual rules don’t apply.New rules relating to how life responds whenlarge amounts of water are substituted at lowtemperature remain to be discovered.

Mechanisms of cryoprotectant toxicityare still poorly understood (4,5), but a fewempirical generalizations can be made.Lipophilicity (affinity for fats and oils) strong-ly correlates with toxicity. Molecules with anaffinity for fat can partition into cell mem-branes, destabilizing them. It has also beenrecently discovered that strong hydrogenbonding correlates with toxicity, possibily bydisrupting the hydration shell around macro-molecules. This led to the unexpected resultthat cryoprotectants with polar groups thatinteract weakly with water are best for vitrifi-cation, even if a higher concentration isrequired to achieve vitrification (6). The elec-trical properties of cryoprotectant solutionshave also been related to membrane toxicity(7). Certain cryoprotectants, such as glyceroland possibly DMSO, are also known to haveadverse reactions with specific biochemicaltargets. Finally, mutual toxicity reduction,especially as seen in the DMSO/formamidecombination, has been very useful in vitrifica-tion solution development, although themechanism of this toxicity reduction is stillunknown (5).

Components of CryopreservationSolutions

More than just cry-oprotectants must beadded to cells and tis-sues to protect againstfreezing injury. A cry-opreservation solution,which may be either afreezing solution or vit-rification solution, con-sists of:

Carrier SolutionCarrier solution

consists of solutioningredients that are notexplicit cryoprotectants.The role of the carriersolution is to providebasic support for cells attemperatures near freez-ing. It contains salts,osmotic agents, pHbuffers, and sometimes nutritive ingredients orapoptosis inhibitors. The ingredients are usual-ly present at near isotonic concentration (300milliosmoles) so that cells neither shrink norswell when held in carrier solution. Carrier solu-tion is sometimes called “base perfusate.” Thecarrier solution typically used with M22 cry-oprotectant solution is called LM5.

Different concentrations of cryoprotec-tant may be required at various stages of cry-oprotectant introduction and removal, but theconcentration of carrier solution ingredientsalways remains constant. This constant-com-position requirement can be regarded as thedefinition of a carrier solution. As a practicalmatter, this means that cryopreservation solu-tions must be made by means other thanadding cryoprotectants to a pre-made carriersolution because naïve addition would dilutethe carrier ingredients.

Penetrating CryoprotectantsPenetrating cryoprotectants are small

molecules able to cross cell membranes. Therole of penetrating cryoprotectants is toreduce ice growth and reduce cell dehydrationduring freezing. In vitrification, the role ofpenetrating cryoprotectants is to completelyprevent ice formation. As is shown in Figure

4, penetrating cryoprotectants are the majori-ty ingredients of vitrification solutions.

Non-penetrating Cryoprotectants (optional ingredient)

Non-penetrating cryoprotectants arelarge molecules, usually polymers, added tocryoprotectant solutions. They inhibit icegrowth by the same mechanisms as penetrat-ing cryoprotectants, but do not enter cells.Po lye thy l ene g l yco l (PEG) andpolyvinylpyrrolidone (PVP) are examples.Non-penetrating cryoprotectants are usuallyless toxic than penetrating cryoprotectants atthe same concentration. They reduce theamount of penetrating cryoprotectants need-ed by mimicking outside the cell the cryopro-tective effects of proteins inside the cell. Ithas also been recently discovered that usingnon-penetrating cryoprotectants to increasethe tonicity (osmotically active concentration)of vitrification solutions can prevent a type ofinjury called chilling injury.

Ice Blockers (optional ingredient)Ice blockers are compounds that direct-

ly block ice growth by selective binding withice or binding to contaminants that triggerice formation (ice nucleators). Conventionalcryoprotectants act by interacting with water.

Figure 4. Composition of M22 vitrification solution. All ingredi-ents are penetrating cryoprotectants, except for LM5 carrier solutes,Z-1000 and X-1000 ice blockers, and PVP K12 polymer. M22 is a“sixth generation” vitrification solution, incorporating two decades ofprogress in the development of vitrification solutions for mainstreammedical tissue and organ banking.


Ice blockers compliment conventional cry-oprotectants by interacting with ice or sur-faces that resemble ice. Ice blockers are likedrugs in that only a small amount is requiredto find and bind their target. Low molecularweight polyvinyl alcohol and polyglycerol,called X-1000 and Z-1000, and biologicalantifreeze proteins are examples of iceblockers (8,9). Ice blockers are only used invitrification solutions, not freezing solutions(See Figure 5).

How Cryoprotectants are UsedFreezing solutions containing relatively

low cryoprotectant concentrations near 10%are typically added in a single step. Thiscauses the classic shrink-swell response ofcryobiology in which cells first shrink byosmosis in response to the high solute con-centration outside the cell, and then swell aspenetrating cryoprotectants enter the cell.Within several minutes, or tens of minutesfor thin tissue pieces, the cryoprotectantconcentration inside and outside cells equal-izes, and cells return to a volume defined bythe tonicity of the carrier solution. The cellsor tissue are now ready for freezing. For cry-opreservation by freezing, cooling is doneslowly, typically less than 1ºC per minute.This allows time for water to leave cells asfreezing progresses so that the cryoprotec-tant concentration inside cells rises togetherwith the concentration outside cells. Thisprevents cell interiors from freezing. Freez-

ing can also sometimes succeed even thoughcryoprotectant concentration remains low iffreezing and thawing are done extremely rap-idly so that there is not enough time for iceto grow inside cells.

Vitrification solutions containing cry-oprotectant concentrations near or exceed-ing 50% cannot be added in a single stepbecause the initial osmotic shrink responsewould be too extreme. Instead, material tobe vitrified is successively exposed to severalsolutions containing exponentially increasingconcentrations of cryoprotectant, such as1/8 x, 1/4 x, 1/2 x, 1 x full concentrationvitrification solution, typically for 20 minuteseach step. The addition is done at a temper-ature near 0ºC to minimize toxicity. Thematerial is then ready for vitrification. Forcryopreservation by vitrification, cooling andrewarming are done as quickly as possible.

Unlike cell suspensions or small tissuepieces, organs are too large to absorb cry-oprotectant by just soaking in an externalsolution. For organ cryopreservation, cry-oprotectants are added by perfusion, aprocess in which the cryoprotectant solutionis circulated through blood vessels just asblood would flow through the organ. Thisensures that no cell is more than a few cellsaway from contact with the circulating solu-tion. Rather than adding cryoprotectant indiscrete steps, it is more convenient duringperfusion to increase the cryoprotectant con-centration continuously.

Cryprotectants areremoved by reversing thesteps described above,except that all removalsolutions except for thevery last contain severalhundred millimoles of anosmotic buffer, such asmannitol. The role of theosmotic buffer is to reducethe extent of the initialswell response of cells asthey are exposed todecreased external cry-oprotectant concentration.

Special Considera-tions for Organs

The time required tointroduce and remove cry-

oprotectants from organs is longer than forcells. For vitrification solutions, perfusion timesof hours are typical. This is because cryopro-tectants must move through small spacesbetween cells that line the inside of blood ves-sels, the capillary endothelium. This makes cellsof the capillary endothelium among those mostvulnerable to cryoprotectant toxicity becausethey are exposed to the highest concentrationsof cryoprotectant for the longest time whilewaiting for other cells in the organ to catch up.

The brain has an additional difficulty inthat the spaces between capillary endothelialcells are especially small. This is the so-calledblood brain barrier, or BBB. The BBB caus-es penetrating cryoprotectants to leave bloodvessels even more slowly than other organs,and doesn’t permit water-soluble moleculesbigger than 500 daltons to leave at all. There-fore non-penetrating cryoprotectants do notpass through an intact BBB.

However this doesn’t mean that non-penetrating agents have no effect on braintissue. The osmotic movement of wateracross the BBB is determined by the entirecryoprotectant solution composition. Watermoves to equalize the solution melting point,or “water activity,” on either side of the BBB.This means that any ingredient that lowersthe melting point of the cryoprotectant solu-tion also increases the resistance of tissueoutside the BBB to ice formation by drawingout water and increasing the concentration ofsolutes naturally present in the brain. Thebrain is an organ in which penetratingcryprotectants and dehydration seem to act intandem to provide cryoprotection.

Six Generations of VitrificationSolutions

Vitrification solutions have progressedgreatly since the initial proposal of modern vitri-fication by Fahy in the early 1980s. This progressmay be viewed as occurring in six generationalleaps (10). Generations three through six weredeveloped at 21st Century Medicine, Inc.

Generation 1The simplest vitrification solutions are

single cryoprotectants in carrier solution.

Generation 2It was discovered that higher total cryopro-

tectant concentrations with acceptable toxicity

Figure 5. Effect of ice blockers on ice formation. The flask onthe left contains 55% w/w ethylene glycol solution that wascooled to -130ºC. The flask on the right contains the same solu-tion, except with 1% of the ethylene glycol replaced by 0.9%X-1000 and 0.1% Z-1000 ice blockers. It is almost completely vit-rified, with the majority of the solution being a transparent glassrather than white crystalline ice.


1. C. Polge, A.U. Smith, A.S. Parks, Revival of sperma-tozoa after vitrification and dehydration at low tem-perature, Nature 164 (1949) 666.

2. A.M. Karow, Cryoprotectants—a new class ofdrugs, J. Pharm. Pharmac 21 (1969) 209-223.

3. M. Fahy, D.R. MacFarlane, C.A. Angell, H.T. Mery-man, Vitrification as an approach to cryopreserva-tion, Cryobiology 21 (1984) 407-426.

4. G.M. Fahy, The Relevance of Cryoprotectant “Tox-icity” to Cryobiology, Cryobiology 23 (1986) 1-13.

5. G.M. Fahy, Cryoprotectant Toxicity and Cryopro-tectant Toxicity Reduction: In Search of MolecularMechanisms, Cryobiology 27 (1990) 247-268.

6. G.M. Fahy, B. Wowk, J. Wu, S. Paynter, Improvedvitrification solutions based on the predictability ofvitrification solution toxicity, Cryobiology 48 (2004)22-35.

7. I.B. Bakaltcheva, C.O. Odeyale, B.J. Spargo, Effectsof alkanols, alkanediols and glycerol on red bloodcell shape and hemolysis, Biochem Biophys Acta1280 (1996) 73-80.

8. B. Wowk, E. Leitl, C.M. Rasch, N. Mesbah-Karimi,S.B. Harris, G.M. Fahy, Vitrification enhancement

by synthetic ice blocking agents, Cryobiology 40(2000) 228-236.

9. B. Wowk, G.M. Fahy, Inhibition of bacterial icenucleation by polyglycerol polymers, Cryobiology44 (2002) 14-23.

10. G.M. Fahy, unpublished.

11. G.M. Fahy, B. Wowk, J. Wu, J. Phan, C. Rasch, A.Chang, E. Zendejas, Cryopreservation of organs byvitrification: perspectives and recent advances, Cry-obiology 48 (2004) 157-178.

12. K.G. Brockbank, Y.C. Song, Morphological analy-ses of ice-free and frozen cryopreserved heart valveexplants, J. Heart Valve Dis. 13 (2004) 297-301.

13. Y.S. Song, B.S. Khirabadi, F.G. Lightfoot, K.G.M.Brockbank, M.J. Taylor, Vitreous cryopreservationmaintains the function of vascular grafts, NatureBiotechnology 18 (2000) 296-299.

14. Y.C. Song, Y.H. An, Q.K. Kang, C. Li, J.M. Boggs,Z. Chen, M.J. Taylor, Vitreous preservation ofarticular cartilage grafts, J. Invest. Surg. 17 (2004)65-70.

15. W.J. Armitage, S.C. Hall, C. Routledge, Recovery ofendothelial function after vitrification of cornea at

-110?C, Invest. Opthalmol. Vis. Sci. 43 (2002) 2160-2164.

16. F. Migishima, R. Suzuki-Migishima, S.Y. Song, T.Kuramochi, S. Azuma, M. Nishijima, M. Yokoyama,Successful cryopreservation of mouse ovaries byvitrification, Biol. Reprod. 68 (2003) 881-887.

17. M. Salehnia, Autograft of vitrified mouse ovariesusing ethylene glycol as cryoprotectant, Exp. Anim.51 (2002) 509-512.

18. G.M. Fahy, Vitrification as an approach to cryop-reservation: General perspectives, Cryobiology 51(2005) 348. [Abstract]

19. Y. Pichugin, G.M. Fahy, R. Morin, Cryopreservationof rat hippocampal slices by vitrification, Cryobiol-ogy 52 (2006) 228-240.

20. J. Lemler, S.B. Harris, C. Platt, T.M. Huffman, TheArrest of Biological Time as a Bridge to Engi-neered Negligible Senescence, Ann. N.Y. Acad. Sci.1019 (2004) 559-563.

21. J.M. Baust, M.J. Vogel, B.R. Van Buskirk, J.G. Baust,A molecular basis of cryopreservation failure andits modulation to improve cell survival, 10 (2001)561-571.

could be achieved by combining DMSO withamides such as acetamide or formamide, andthen adding propylene glycol. The combina-tion of DMSO, formamide, and propylene gly-col was the basis of the VS41A (also calledVS55) vitrification solution, the most advancedvitrification solution of the mid 1990s.

Generation 3A breakthrough occurred with Fahy’s dis-

covery that cryoprotectant toxicity correlatedwith the number of water molecules per cry-oprotectant polar group at the critical concen-tration needed for vitrification, so-called qv*(6). This led to the replacement of the propy-lene glycol in VS41A with ethylene glycol,generating the Veg vitrification solution.

Generation 4The use of polymers in vitrification solu-

tions permitted further reductions in toxicity byreducing the concentration of penetrating cry-oprotectants necessary to achieve vitrification.Generation 5

The use of ice blocking polymers permit-ted still further reductions in toxicity by

reducing the concentration of all cryoprotec-tants necessary to achieve vitrification. VM3is a fifth generation vitrification solution (6).

Generation 6It was discovered that chilling injury, a

poorly-understood injury caused by just pass-ing through certain sub-zero temperatureranges, could be overcome by increasing thetonicity of non-penetrating components ofvitrification solutions (11). M22, the cryopro-tectant currently used by Alcor, is a sixth gen-eration solution.

Successful vitrification has now beendemonstrated for heart valves (12), vasculartissue (13), cartilage (14), cornea (15), andmouse ovaries (16, 17). Progress continuesfor the rabbit kidney, with recovery of theorgan demonstrated after cooling to below– 40ºC while cryoprotected with a vitrificationsolution (11), and one reported instance oflong-term survival after vitrification (18). Vit-rification has also shown utility for viablepreservation of diverse tissue slices, includingbrain slices (19), and histological preservationof larger systems (20).

Future generations of cryoprotectantsolutions will have to address many problemsthat are still outstanding, including molecularmechanisms of cryopreservation failure (21),and especially cryoprotectant toxicity. Cry-oprotectant toxicity is emerging as a finalfrontier of cryobiology. The greatest futurebreakthroughs in cryobiology may come frombetter understanding and mitigation of cry-oprotectant toxicity. �

Dr. Wowk is the Senior Physicist at21st Century Medicine, Inc., a compa-ny specializing in the development oftechnology for cold preservation oftransplantable tissue and organs.

Contact the author:[email protected]

References

In The Beginning: Vitrification or Freezing?

In The Prospect of Immortality, the bookwhich launched cryonics, Robert C.W.Ettinger suggests that glycerol might be usedas the cryoprotectant for human cryopreser-vation (1) based largely on the fact that it wasthe dominant cryoprotective agent (CPA) atthat time (1962-64) and most of the positiveresults with sperm and tissues had beenachieved with glycerol. Due largely to the flareand flamboyance of “the Father of DimethylSulfoxide (DMSO*),” Dr. Stanley W. Jacob,who was Assistant Professor of Surgery at theUniversity of Oregon Health Sciences CenterMedical School, DMSO entered the publicconsciousness in a big way in the mid-to-late1960s (2-4). DMSO’s anti-inflammatory, andseemingly incredible skin-penetrating proper-ties, were much talked about.

Sometime between 1966 and 1967,Ettinger asked Dr. Dante Brunol, an Italiannational living in the US, to produce a formal,written protocol for cryopreserving cryonicspatients. Brunol was a biophysicist (Ph.D.),M.D. and surgeon with experience in car-diopulmonary bypass. Brunol, writing underthe nom de plume of Mario Satini, M.D., pro-duced a complicated protocol which, while ithas a number of deficiencies, is really quiteremarkable, and even visionary in severalrespects (5). Brunol opens his protocol withthe following remarks:

“The writer has always favored supercoolingrather than the freezing of humans. Supercool-ing does not lead to the formation of ice crystals.It should be possible to find methods to storehumans at temperatures warmer than -30degrees C, for five years, the time necessary toprotect humans from freezing.

When Professor Ettinger, author of TheProspect of Immortality, asked me to devise amethod to freeze humans, at first I declined theoffer. In my opinion, only a chemical inducingvitrification could save the cells from (ice) crystaldamage.”

The First Human Cryopreservation Protocol

Brunol then goes on to explain how vit-rification is achieved through ultra-rapid cool-ing and, without naming it, introduces theidea of the glass transition point of water(Tg), a temperature below which water hasbecome a glass and cannot organize into crys-tals. He further explains why such ultra-rapidcooling cannot be applied, even to tissues, letalone whole humans. Brunol details his proto-col which consists of the following core ele-ments:

1) Immediate commencement of CPR atthe time medico-legal death is pro-nounced, preferably augmented with anartificial airway and high FiO2 (fractionof inspired oxygen in a gas) oxygenadministration (15 liters per minute). Herecommends at least 30 compressionsper minute, with one ventilation everyfour minutes.

2) Placement of a thermistor in the rectum tomonitor body core temperature. The ther-mistor is affixed to a 10" wooden dowelwith straps to hold it deeply in the rectum.

3) Immersion of the patient in a special tubfilled with ice and 10% DMSO in waterwhile mechanical CPR continues. Thetub supports the patient so that his headremains above the water level allowingmanual ventilation to continue.

4) Use of the Westinghouse Iron Heart (amechanical chest compressor) as soon aspossible to continue CPR during cooling.CPR with the Iron Heart is to continueuntil the patient reaches a core tempera-ture of 15 degrees C, or until extracorpo-real cooling using closed-circuit CPB canbe commenced via femoral-femoralbypass using a heat exchanger.

5) Inject 2 liters of ice-cold 5% Dextran inan isotonic solution via both internal

carotid arteries to hemodilute and coolthe brain.

6) Femoral-femoral cannulation followed byopen circuit perfusion (blood washout)of ~20 gallons (80 liters) of heparinized20% DMSO, 20% glycerol in saline orother isotonic solution at a pressure of120 mm Hg, and a temperature ofbetween 1 degree and 4 degrees C.

7) Using a fairly complex circuit Brunoldemonstrates a good knowledge of phys-iology, and proposes perfusing the pul-monary circulation by turning on theIron Heart and pressurizing the venouscirculation (via retrograde flow throughthe femoral venous cannulae) to 20mmHg at very low flow, while openingthe arterial cannulae to allow effluent toexhaust retrograde into a discard-reser-voir. Perfusion of the pulmonary circuitis to commence when the patient's tem-perature reaches 10 degrees C and is tocontinue for 15 minutes.

8) Preferably, perfusion with the CPA mix-ture should terminate when the patient'score temperature is -4 degrees C.

9) Brunol was very concerned about inter-stitial and intracellular ice crystal damageand he proposed vitrifying the cells byinitiating ice crystal formation in the vas-culature. He proposed doing this by fol-


History of DMSO and Glycerolin CryonicsBy Mike Darwin

* The correct abbreviation for DMSO is Me2SO, as per the International Union of Biochemistry andMolecular Biology (IUBMB) – International Union of Pure and Applied Chemistry (IUPAC) JointCommission on Biochemical Nomenclature.

Robert Ettinger (foreground)demonstrates use of the Iron Heart,

a mechanical chest compressor.

Phot

o by

Ted

Kra

ver

lowing CPA perfusion with the perfusionof a quantity of 10% Dextran at near 1degree C in saline into both the arterialand the pulmonary circulation. The ideawas that this solution would start tofreeze immediately, before the CPA couldequilibrate from the intracellular andinterstitial spaces. Ice would thus formfirst in the vessels and dehydrate the cellsto ~30% of their normal volume.

10) The GI tract, plueural space, and peri-toneum were to be filled with a solution(apparently) chilled to below freezing con-sisting of 20% DMSO, 20% glycerol, and10% ethanol to facilitate core heatexchange. Each pleural space and the peri-toneal cavity are to be filled with 1 liter ofthis fluid. The balance (of up to ~4 gallonsas necessary) was to be used to fill the GItract (upper and lower).

11) Transfer the patient to a container with aperforated bottom to allow the escape ofwater from melting ice, and pack thebody in ice and granular salt: one layer ofsalt, one layer of ice, etc., to achieve atemperature of -20 degrees C for 24hours (to allow for maximal extracellularice growth and intracellular CPA concen-tration).

12) Transfer the patient to an insulated con-tainer and pack in dry ice followed by cool-ing to -196 degrees C as soon as possible.

Minus the post CPA perfusion of 10%dextran-saline, this protocol would have beenvastly better than anything that would be usedin cryonics until at least 1979. Brunol wasincorrect in assuming that ice formationwould start and subsequently outpace diffu-sion of CPA into the 10% dextran-saline solu-tion. However, his idea of initiating andlargely confining ice formation to the largevessels of the vasculature and the body cavi-ties is an intriguing one. Interestingly, the abil-ity to control the location where ice nucle-ation begins may today be possible by addingthe potent ice-nucleation protein produced bythe common soil bacteria, Pseudomonas syringae(6), to the perfusate in the circulatory system.If this was done in addition to ice-blockingpolymers, it might allow for considerable iceformation, but only in the form of very small,non-damaging ice crystals (7). Being able totolerate significant ice formation woulddecrease the concentration of cryoprotective

agents needed for successful preservation,and thus decrease the injury due to cryopro-tective agent toxicity.

When James H. Bedford, the first mancryopreserved, died on 12 January, 1967,Robert F. Nelson of the Cryonics Society ofCalifornia (CSC) had made virtually none ofthe preparations Brunol recommended. SomeDMSO had been acquired, but no carriersolution was available, such as LactatedRinger’s (LR). Similarly, Robert Ettinger hadsent Nelson an Iron Heart, but Nelson hadnot bothered to get oxygen to power it. Thus,Bedford was pin-cushioned with injections ofpure DMSO via syringe, with attempts madeto inject the DMSO directly into the rightinternal carotid artery (8).

Glycerol and the Cryonics Society of New York

Largely because of DMSO’s almostmythical property of being able to penetratecells, it seems to have become the CPA ofchoice amongst cryonicists on the West Coastfrom 1967 until 1979. By contrast, the peopleat the Cryonics Society of New York (CSNY),including Paul Segall, Harold Waitz and Cur-tis Henderson, read Brunol’s protocol anddecided to try to implement those parts of itthat they thought reasonable and practical.Brunol recommended that an Amtec 209industrial roller pump, with a Zero-Max speedcontroller, be used to deliver perfusate. CurtisHenderson, CSNY’s President, purchased oneof these circa 1968 (I still have it to this day).

Zero-Max (mechanical) controllers werethe primary way motor speeds were regulatedbefore solid-state electronic controls cameinto wide use in the 1970s. The Zero-Max isan oil-immersed adjustable speed drive withfour or more one-way clutches that moveback and forth, each rotating the output shafta partial turn for each stroke transmission.Zero-Max controls were used extensively con-trol speed before the widespread applicationof solid state motor controllers in the 1970s.

The bubble trap was designed by a physi-cian associated with CSNY at that time, Dr.Jane Enzman, daughter of the maverickphysicist and engineer Dr. Robert DuncanEnzman.

Unfortunately, this set-up was never usedon a patient. CSNY used a Porti-Boy embalm-ing machine. Paul Segall modified the Brunolprotocol in a number of unfortunate ways.Segall eliminated any attempt at maintainingpost-arrest circulation writing, “No attempt ismade to maintain circulation of the blood for the fol-

lowing reason. It has been observed that if the bloodflow falls under 70 mm Hg for more than 5 minutesthere is irreversible damage (by today's standards) tothe cerebral brain centers. Evidence has shown thatperhaps this [is] due to the blockage of the microcir-culation of the brain (the capillaries become cloggedbecause of the formation of blood clots). In all likeli-hood, artificial circulation after death could not bestarted fast enough to reach the cerebral centers (9).”

Segall, in contrast to Brunol, apparentlynever understood the importance of achiev-ing an adequate intracellular concentration ofcryoprotectant. Brunol actually does the mathand concludes, based on dilution calculations,that the terminal intracellular concentrationof CPA will be 22%. Segall's protocol calledfor an initial flush with 6 liters of ice-chilledheparinized Ringer’s Lactate solution for each30 pounds of body weight (thus, a 150 poundman would be flushed with 30 liters ofRinger’s). This was to be followed by a flushof 8 liters of ice-chilled 20% glycerol inRinger's for a 150 pound man. An additionalliter of 20% glycerol-Ringer’s was to be usedto fill the GI tract. Following this, the patientwas to be transferred to a body bag andpacked in ice and salt for 12 hours and thentransferred to an insulated box and packed indry ice.

This protocol, which was used on CSNYpatients Steven Mandell and Ann Deblasio,would have resulted in negligible concentra-tions of glycerol in the patient’s tissues – lev-els not even cryoprotective for cells in culture.Failure to use CPR and anticoagulation assoon after cardiac arrest as possible resultednot only in massive systemic clotting, butgreatly delayed cooling as well. Segall's ration-ale for using glycerol as the sole CPA wasbased on Suda’s work with cat brains (10).CSNY had acquired DMSO, but did not use it.


Dr. Dante Brunol, 1967

Learning the Hard WayBy contrast, CSC continued to use

DMSO, mostly as a 20% solution in Ringer’s.There is no documentation of the tempera-ture, pressure or volume of solution used. Inthe early to mid-1970s there was an extensiveround of correspondence and a secondattempt to formulate an optimum perfusionprotocol. This time it was Dr. Peter Gouraswho was chosen for this task. Gouras pro-posed using DMSO’s “extraordinary” perme-ation qualities to infiltrate the patient with65% DMSO after Elford and Walter (13) bysoaking him in progressively higher concentra-tions of DMSO as the temperature was con-currently reduced (11). This lead Art Quaife,who was both a gifted mathematician andPresident of Trans Time, to produce a highlysophisticated mathematical analysis of the dif-fusion kinetics showing that equilibration bysoaking the patient in DMSO would take manymonths even at 0 degrees C (12).

During this extensive collaborative corre-spondence a consensus was reached to use20% DMSO in a modified Collin’s solutionbase perfusate, following blood washout withheparinzed Ringer’s Lactate. This was thebeginning of the end of using DMSO in cry-onics. In January of 1973, two patients wereperfused on the same day on opposite coastsof the US using DMSO-Collins by TransTime (San Francisco, CA) and the author(operating as Cryo-Span Midwest) usingDMSO-Ringer’s (Cumberland, MD) (13).Both patients experienced long periods ofwarm and cold ischemia before perfusion waspossible. Almost immediate and massiveedema occurred in both patients with rapiddeterioration of venous return, and ultimate-ly, failure of perfusion. Five-percent ofDMSO, followed by 20% DMSO in modified

Collins solution, was used to perfuse Freder-ick Chamberlain, Jr., (the first neuropatient) in1976. Fred, Jr. had been given immediate andcontinuous cardiopulmonary support, as wellas good external cooling. While edema wasslower to develop, it nevertheless occurred, andagain resulted in failure of venous return (14).

When Jerry Leaf (Cryovita Laboratories)did his first human case for Trans Time,Samuel Berkowitz, in June of 1978 (15),DMSO was again used, but the quantity ofperfusate was small, as was the case whenK.V.M. (initials used for privacy) was perfusedin December of 1978 (16). In both of thesecases, despite the low volume of perfusate,edema was a serious problem. The last casedone with DMSO was L.R., a Trans Timeneuropatient who was perfused (again with avery small volume of 20% DMSO: ~6 liters)in March of 1979 (17). This patient did notexperience noticeable edema, probably owingto the small volume of solution used, promptpost-arrest CPR, and minimal warm or coldischemic injury since she was transporteddirectly from home by ambulance to theTrans Time facility in Emeryville, CA, whereperfusion was carried out.

In the summer of 1979, Jerry Leaf and Ibegan intense discussions, both in writing andby phone, about improving the protocol forhuman cryopreservation. It was during thesediscussions that the issue of both the CPA touse and the proper volume of perfusaterequired to reach an adequate tissue concen-tration arose. I pointed out that the volumesof perfusate being used by Cryovita-TransTime were only achieving “homeopathic” lev-els of CPA in the patients’ tissues. Jerry was incomplete agreement and explained that theCPA protocol he was using had been deter-mined by Paul Segall, of Trans Time.

As of 1977, I had decided that DMSOwas unacceptable, because of the consistentproblems with edema observed, and its docu-mented destructive effects on the vascularendothelium of kidneys being perfused forattempted organ cryopreservation (18). On 10December, 1972 Clara Dostal, a CSNY mem-ber, was perfused with multiple passes ofincreasing concentrations of glycerol in Lac-tated Ringer’s (LR) solution in an attempt toachieve multi-molar equilibration of glycerolin the brain (19). Perfusion was via the rightinternal carotid artery using standard mortu-ary technique. This meant that only one can-nula was available so perfusion had to bealternated between the head and trunk withthe arterial cannula being removed and repo-sitioned after each pass of perfusate. Thepatient’s head was flushed with 6.5 liters ofLR before commencing cryoprotective perfu-sion. Three passes of glycerol in LR wereused: 2.26 M, 4.34 M, and 5.78 M with con-centration on perfusing the brain due to thelimited volume of perfusate available. Twen-ty-seven point two (27.2) liters of perfusatewas used with a perfusion time (combined


Graph A. SP1 Glycerol Concentration vs. Perfusion Time Graph B. SP2 Glycerol Concentration vs. Perfusion Time.

Clara Dostal, 1973 (Note the lack of edema.)


1. Robert C.W Ettinger: The Prospect of Immortality,Doubleday, New York, 1964.

2. Laudahn G. & Jacob S.: Symposium on DMSO Berlin,Germany, 1965.

3. Laudahn G. & Jacob S.: Symposium on DMSO Berlin,Germany, 1966.

4. Jacob SW, Rosenbaum E.E., Wood, D.C.: DimethylSulfoxide (Basic Concepts), Marcel Dekker, Inc., NewYork, 1971.

5. Robert F. Nelson & Sandra Stanley: We Froze TheFirst Man, Dell Publishing Company, New York,1968, pp. 136-56.

6. Graether SP, Jia Z: Modeling Pseudomonassyringae ice-nucleation protein as a beta-helical pro-tein. Biophys J. Mar;80(3):1169-73;2001.

7. Fahy GM. The role of nucleation in cryopreserva-tion. In: Biological Ice Nucleation and Its Applica-tions (R.E. Lee, Jr., G.J. Warren, and L.V. Gusta(Eds.), APS Press, 1995, 315-336.

8. Robert F. Nelson & Sandra Stanley: We Froze TheFirst Man, Dell Publishing Company, New York,1968, pp. 156-9.

9. Darwin M: Dear Dr. Bedford (And those who willcare for you after I do), A thank you note to a pio-neer. Cryonics Volume 12(7) Issue 132 JULY, 1991.

10. Segall P: The body is now ready for storage. Cryon-ics Reports. 4 (3);1969.

11. Suda I, Kito K, Adachi C: Viability of long-termfrozen cat brain in vitro. Nature. Oct15;212(5059):268-70;1966.

12. Elford BC & Walter, CA: Preservation of structureand function of smooth muscle cooled to –79degrees C in unfrozen aqueous media. Nat NewBiol. 15;236(63):58-60;1972.

13. Communication from the Bay Area Cryonics Soci-ety, 1739 Oxford St., #6, Berkeley, CA 94709.“Arthur Quaife, Chairman Jerome B. White, andothers have been working with Dr. Peter Gourasand other scientists to formalize an updated freez-ing protocol.” in Man Into Superman by Robert C.W.Ettinger, Doubleday, New York, 1971.

14. Quaife A: Mathematical Models of PerfusionProcesses, Manrise Technical Review. 2:28-75; 1972.

15. Unpublished Case Data, Alcor Life ExtensionFoundation, Patient A-1056, 09 February, 1973.

16. Unpublished Case Data, Alcor Life ExtensionFoundation, Patient A-1001, 16 July, 1976.

17. Leaf JD: Cryonic Suspension of Sam Berkowitz:Technical Report, Long Life Magazine. 3: (2),March/April, 1979.

18. Leaf JD & Quaife AD: Case Study in Neurosus-pension. Cryonics. 16 November, 1981 pp. 21-28.

19. Jeske AH, Fonteles, MC, Karow AM. Functionalpreservation of the mammalian kidney. III. Ultra-structural effects of perfusion with dimethylsulfox-ide (DMSO). Cryobiology. Apr;11(2):170-81;1974.

20. Bridge SW: Fifteen Years in Cryonics, Part III.Alcor Indiana Newsletter. #5 July/August 1992.

21. Case Report: Two consecutive suspensions, a com-parative study in experimental suspended anima-tion. Cryonics 6:11;1985.

22. Wowk B & Darwin M: Alcor Reaching for Tomor-row, Appendix A: The cryobiological case for cry-onics. Alcor Life Extension Foundation, Riverside,CA; 1988. pp. A-1 to A-9.

23. Ettinger, RCW: Personal Communication, May,1991.

24. Lemler, JB, et al. Alcor cryotransport case report:patient A-1505, 22 March, 2001

head and trunk) of 157 minutes. The finalcranial (right internal jugular) effluent glycerolconcentration was ~4.0 M glycerol.

Despite the comparatively large volumesof perfusate used this patient did not developedema. (Note: until 1981 this volume of cry-oprotective perfusate would have been consid-ered large.) In the winter of 1977, under theauspices of the Institute for Advanced Biologi-cal Studies in Indianapolis, IN, I began researchon brain ultrastructure following perfusion andfreezing of rabbit heads using 2 M glycerol. InNovember of 1978, I perfused my terminally illdog, (a ~16 kg mongrel), with 10 liters of 7%v/v glycerol and 30 liters of 20% v/v glycerol.None of these animals experienced edema.Indeed, the problem was systemic osmoticdehydration. On the basis of these experiencesit was decided that glycerol would be used infuture human cases, with a target terminal tissueglycerol concentration of 3M.

In January of 1980 two consecutive cry-opreservation cases (see Graphs A & B) werecarried out at Cryovita Laboratories for TransTime by Jerry Leaf and myself (20). A total of80 liters of perfusate was used, with the fol-lowing quantities and compositions:

5% glycerol perfusate, 25 liters 10% glycerol perfusate, 10 liters 15% glycerol perfusate, 10 liters 20% glycerol perfusate, 10 liters 25% glycerol perfusate, 10 liters 50% glycerol perfusate, 15 liters

A combination of open and closed circuitperfusion was used. Incredibly, perfusion waspossible in one case for 500 minutes beforecerebral edema became the limiting factor. Inthis patient a terminal venous concentration of2.32 M glycerol was achieved. In the secondpatient, a terminal venous concentration of2.87 M glycerol was achieved after only 133minutes of perfusion, without either systemicor cerebral edema terminating perfusion.

From that time forward it was clear thatglycerol was vastly superior in terms of per-fusability. For the first time it was possible toachieve desired levels of cryoprotection, usingextended perfusion if necessary, even inpatients who had suffered serious warm andcold ischemic injury. In the mid-1980s the tar-get tissue glycerol concentration wasincreased from 3.0M to 4.5M on the basis of

the “Smith Criterion (21),” and, on the basisof dog brain ultrastructural research conduct-ed by BioPreservation in 1995 (22), terminaltissue glycerol concentration was increased to7.5 M (the maximum concentration possibleto perfuse due to viscosity constraints).

An attempt was made by the CryonicsInstitute to switch to a perfusate containingpropylene glycol in November of 1987 (23).This resulted in severe edema which terminat-ed perfusion well before target CPA concen-tration was reached. Until the introduction of21CM vitrification solutions in the summer of2001 (24), all patients were perfused with glyc-erol as a mono-agent. �

Michael G. Darwin was the Presidentof the Alcor Life Extension Foundationfrom 1983 to 1988 and Research Direc-tor until 1992. He was also President ofBioPreservation, Inc., and Director ofResearch of Twenty-First CenturyMedicine from 1993 to 1999. He is cur-rently an independent consultant inthe field of critical care medicine.

References


Advances in CryopreservationBy Gregory M. Fahy, Ph.D., Chief Scientific Officer, 21st Century Medicine, Inc.

Ihave good news and bad news to report atthe top of this installment. The good news

is that, as many readers will know, 21st CenturyMedicine has received a large grant to apply vit-rification methods to whole animals. The badnews is that, because of the increased timecommitments this new initiative will demand, Iwill have to make this my last column install-ment, at least for the foreseeable future. Forthis reason, I would like to depart from my pre-vious plan and shift the direction of the presentinstallment so as to take this one last opportu-nity to give due acknowledgement to recent evi-dence for the contrarian point of view thatcomplex systems can be adequately preservednot by vitrification but by freezing.

It is very interesting that, after a very longperiod of time in which my labs (at the RedCross, the Navy, and 21st Century Medicine)have been the only labs in the world pursuingthe cryopreservation of whole mammalianorgans in any serious way, several new groupshave become interested in the problem andhave published interesting although so far onlypartially successful results with freezing. Theirwork is important and deserves to be noted anddiscussed.

There is a large body of literature on organfreezing from earlier studies, but the only exam-ple of the survival of post-transplant vascularfunction in a whole organ after freezing in liq-uid nitrogen in the past was provided by frozen-

thawed intestines [1]. Nowrecent events have allowed onemore organ to share the intes-tine’s formerly unique status.

The main new initiatives inorgan freezing have beenefforts to successfully freezewhole ovaries to preserve fertil-ity against the risks posed bychemotherapy in youth or byaging. The ovary freezingeffort was kicked off mostprominently by a paper [2] thatwas published in the prestigiousscientific journal Nature in 2002by a consortium headed byRoger Gosden, then of McGillUniversity in Montreal, whohad a long and prominent pre-vious history of studies on thefreezing of ovarian tissue.Seven rat ovaries, still attachedto their fallopian tubes and theupper segment of the uterus,were perfused with 0.1M fruc-tose and dimethyl sulfoxide(DMSO) to attain a finalDMSO concentration of 1.5M(just a bit over 10% by volume)over 30 minutes, frozen slowly,and stored in liquid nitrogenovernight before thawing,

gradual cryoprotectant washout, and vasculartransplantation.

Three of seven thawed grafts had no folli-cles, and the 4 grafts that did have follicles hadreduced numbers of follicles and recipients hadreduced estrogen levels, elevated FSH levels,and reduced uterine weights, but the 4 survivingovaries were able to ovulate and one animal wasable to get pregnant and have two pups(reduced litter size). This was good enough forNature, and, interestingly, the microscopicstructure of the fallopian tubes and uterine por-tion of the transplants was normal. Clearly,freezing was detrimental, and the authors sug-gested that vitrification might work better, butjust as clearly, at least portions of these ovariesand all of the associated structures survivedfreezing and thawing.

The next year, investigators at the Cleve-land Clinic Foundation and Case WesternReserve University reported freezing and trans-planting whole sheep ovaries [3]. Of 11 trans-plants, only three achieved sustained blood flowalthough all reflowed with blood immediatelyafter transplantation. Those three transplantsthat sustained blood flow for 8-10 days permit-ted normal hormone levels postoperatively,whereas there was “immense tissue loss” andhormone levels were elevated when blood flowdid not recover. Follicle survival in the reper-fusing grafts was more than 10 times higherthan in the occluded grafts, but “scattered areasof necrosis” (dead areas) were still seen even inthe patent group, and areas of vascular regener-ation were also observed. These results areremarkably similar to the results with rats in thatin both cases a minority of ovaries “survived”with damage, but, on the other hand, the factthat any survival at all was achieved is remark-able and positive.

In 2004, Amir Arav’s group in Bet Dagan,Israel reported on results of freezing 8 sheepovaries with a novel directional freezing device(US Patent 5,873,254) using 1.4M DMSO (10%by volume) in Viaspan. This device works byslowly advancing the organ, in a test tube,through a tunnel that is colder at the far endthan at the entrance, thereby freezing the organ

advances in organ freezing

Freezing whole ovaries may one day preserve fertility indefi-nitely. Many people would be affected by success in thisendeavor, and the ovary has the large advantage of beingsomething the recipient can live without if it fails.


from one side to the other. After freezing at0.3°C/min to -35°C, the ovaries were plungedinto liquid nitrogen and later thawed by place-ment in a 68°C water bath for 20 seconds andthen in a 37°C water bath for 2 minutes. Trans-plantation permitted immediate blood reflow in5 of 8 grafts, though surgical problems mayexplain two of the failures. Of the 5 trans-plants that had immediate return of blood flow,three were able to support normal progesteronelevels 34-71 weeks after transplantation, andfollicular growth was present at 2 and 3 monthspostoperatively (overall “survival” rate, 3 of 7ovaries). All-in-all, these results resemble thoseof the US group as accomplished withoutdirectional freezing.

The same year, a group in Brussels, Bel-gium, froze three intact human ovaries [4],which are about 10 times larger than sheepovaries. The only measure of success wasmicroscopic appearance without transplanta-tion, but the ovaries did reasonably well by thatstandard. Unfortunately, histological (meaning,as seen in the light microscopic) integrity with-out transplantation generally gives a much bet-ter impression of “survival” than evaluation bytransplantation.

In 2005, Arav’s group reported moredetails on what seems to be the same set ofovaries described in his 2004 paper [5]. Onceagain, 8 ovaries were frozen, 5 were transplant-ed with immediate blood flow restoration, threesupported reasonable progesterone levels post-operatively, and other details also matched

details in the 2004 report. Two ovaries permit-ted retrieval of one viable egg cell each onemonth after transplantation, although 98% ofthe follicles in the frozen-thawed survivorsseemed to be viable based on light microscopeexamination. Four more viable oocytes wereobtained at 4 months post-transplant from oneovary. The histology of the survivors (presum-ably; no comments were given about non-sur-vivors) was also claimed to be normal. Appar-ently one of the three ovaries that was able topermit normal progesterone levels in the 2004study stopped making appreciable progesteroneby week 96 after transplantation. A furtherupdate on Arav’s group’s results was presentedat the 2007 Society for Cryobiology meeting,but the report was very sketchy and did notseem to contain any particularly critical newinformation.

All-in-all, these results are significant inthat they show whole organs can survive freez-ing, thawing, and transplantation, and that isgood to know. However, they all seem to fallvery short of clinically acceptable quality stan-dards, and experiments on the vitrification ofovaries have been far more successful [6]. Nev-ertheless, human clinical trials of frozen-thawed human ovarian transplants are plannedat Yale University, and we will have to wait andsee how these turn out.

At least two groups have, within the lastfew years, reported their interest in freezingwhole testes informally, but have not publishedany results. One remarkable report, however,

noted that freezing whole testes or whole ani-mals with no cryoprotectant allowed viablesperm to be recovered after thawing [7]! (Butthen again, even freeze-dried sperm can suc-cessfully be used to make babies when injectedinto good egg cells!)

The last recent example of new interest inorgan cryopreservation by freezing was report-ed by Ralf Dittrich in 2006 [8]. He was able toslowly freeze whole pig uteri to dry ice temper-ature, thaw them, obtain good contractileresponses to drug stimulation, and use theseresults to challenge the necessity of vitrification[9]! Whether such organs would reperfuse withblood for any length of time in vivo after trans-plantation is still unknown [10], but it is impres-sive nonetheless that coordinated muscularaction in a large, organized structure like thiscan be preserved by freezing. Non-freezingapproaches can also accomplish the samething [11], however. So even though freezingcan work in some cases based on some meas-urements and to some extent, preservationwithout freezing – that is, by vitrification –still seems to be far and away the betteroption, particularly if what one is trying topreserve really matters.

At 21st Century Medicine, we will con-tinue to focus on developing the best possiblemethods for preserving what matters themost, and I look forward to communicatingwith you again about new advances in cryop-reservation from our lab as these advancesunfold and as the opportunity arises. �

1. Hamilton, R., H.I. Holst and H.B. Lehr, Successfulpreservation of canine small intestine by freezing. Journalof surgical research, 1973. 14: p. 313-318.

2. Wang, X., H. Chen, H. Yin, S. Kim, S. Lin Tan andR. Gosden, Fertility after intact ovary transplantation.Nature, 2002. 415: p. 385.

3. Bedaiwy, M.A., E. Jeremias, R. Gurunluoglu, M.R.Hussein, M. Siemianow, C. Biscotti and T. Falcone,Restoration of ovarian function after autotransplantation ofintact frozen-thawed sheep ovaries with microvascular anas-tomosis. Fertility and sterility, 2003. 79: p. 594-602.

4. Martinez-Madrid, B., M.-M. Dolmans, A. van Lan-gendonckt, S. Defrere and J. Donnez, Freeze-thawingintact human ovary with its vascular pedicle with a passivecooling device. Fertility and sterility, 2004. 82: p. 1390-1394.

5. Arav, A., A. Revel, Y. Nathan, A. Bor, H. Gacitua, S.Yavin, Z. Gavish, M. Uri and A. Elami, Oocyte recov-ery, embryo development and ovarian function after cryop-reservation and transplantation of whole sheep ovary.Human Reproduction, 2005. 20: p. 3554-3559.

6. Migishima, F., R. Suzuki-Migishima, S.-Y. Song, T.Kuramochi, S. Azuma, M. Nishijima and M.Yokoyama, Successful cryopreservation of mouse ovaries byvitrification. Biology of Reproduction, 2003. 68: p.881-887.

7. Ogonuki, N., K. Mochida, H. Miki, K. Inoue, M.Fray, T. Iwaki, K. Moriwaki, Y. Obata, K. Morozu-mi, R. Yanagimachi and A. Ogura, Spermatozoa andspermatids retrieved from frozen reproductive organs orfrozen whole bodies of male mice can produce normal off-spring. Proceedings of the National Academy ofSciences, 2006. 103: p. 13098-13103.

8. Dittrich, R., T. Maltaris, A. Mueller, A. Dimmler, I.Hoffmann, F. Kiesewetter and M.W. Beckman, Suc-cessful uterus cryopreservation in an animal model. Hor-mone and Metabolic Research, 2006. 38(141-145).

9. Dittrich, R., A. Mueller, I. Hoffmann, M.W. Beck-man and T. Maltaris, Cryopreservation of complex sys-tems: slow freezing has not had its day yet. Rejuvenationresearch, 2007. 10: p. (in press).

10. Fahy, G.M. and B. Wowk, Response to: “Cryopreser-vation of complex systems: slow freezing has not had its dayyet.” Rejuvenation research, 2007. 10: p. 103-105.

11. Farrant, J., Mechanism of cell damage during freezing andthawing and its prevention. Nature, 1965. 205: p. 1284-1287.

References


depressed metabolismBy Aschwin de Wolf

IntroductionHuman cryopreservation protocol

includes two treatment modalities to modu-late cerebral metabolism; administration of ageneral anesthetic and induction of hypother-mia. Many general anesthetics are believed toreduce cerebral metabolic demand by reduc-ing excitatory brain activity via GABA-Areceptor potentiation. It is not surprising,then, that general anesthetics have been inves-tigated as treatments for medical conditions inwhich cerebral metabolic demand exceedsenergy supply, such as focal and globalischemia. Not unlike other neuroprotectivestrategies explored in ischemia research,results of human clinical trials have been dis-appointing. This is more remarkable in thecase of general anesthetics because reductionof metabolic demand seems to be broader innature than many other neuroprotectivestrategies, which target only specific parts of

the ischemic cascade of harmful biochemicalevents that follows cardiac arrest.

Perhaps one reason why general anes-thetics ultimately do not improve outcome isthat the agent can only be administered duringreperfusion, when blood supply returns to thetissue after a period of ischemia. However, atthis point the energy imbalance has alreadybeen upset during the ischemic period. Or tostate the matter more generally, a neuropro-tective agent can only confer significant bene-fits if the agent intervenes at events that areinitiated at or continued downstream from thetime of reperfusion. Ischemia-induced cellmembrane depolarization is one of the moreupstream events that produces a number ofdifferent pathological changes (e.g., intracellu-lar calcium overload, mitochondrial failure)that can no longer be reversed by just reduc-ing cerebral metabolic demand with a monoa-gent upon reperfusion.

Given the extremely narrow time win-dow for mitigating ischemia-induced energyimbalance, perhaps a general anesthetic canonly provide a benefit in situations wherepost-ischemic resuscitation is initiated almostimmediately but is inadequate to provide suf-ficient cerebral perfusion. This situation mayapply to a number of in-hospital cardiacarrest situations and, of course, many humancryonics stabilization cases. Although admin-istration of a general anesthetic in cryonics iscomplemented by a number of other (down-stream) neuroprotective medications and flu-ids, the search for a more potent inhibitor ofcerebral metabolic demand remains elusive.

It is tempting to look for an agent thatwould drop metabolic demand to zero, butconsidering the fact that most of the energyof the brain is expended on regulating intra-and extracellular ion gradients, reductions inmetabolic demand that would interfere with

Summary: The most important objectivein cryonics after pronouncement of legaldeath is to drop metabolism as fast aspossible to protect the brain and othertissues from rapid deterioration prior tovitrification. To date, most strategies toreduce metabolic demand are of limitedvalue or not practical as an emergencytreatment. General anesthetics may onlywork if they are administered within avery tight time window. Cooling is apotent strategy to protect the brain butpresents a number of practical and clin-ical challenges, including detrimentaleffects at lower temperatures.

Studies of hibernating animals pro-vide an interesting perspective on thebiological mechanisms involved indepressing metabolism and protectionfrom cold injury. So far, practical appli-cations of these studies have been limit-ed to the identification of moleculesinvolved in hibernation and a number ofmodest successes in organ preservationand reduction of heart and brain injury.Although hibernation on demand forhumans remains elusive, recent researchinto carbon monoxide and hydrogensulfide-induced “suspended animation”revives hopes for better protection ofcryonics patients during stabilizationand transport.

Snow White: the first "poison-induced” human hibernator


basic cellular homeostasis without a corre-sponding drop in metabolic rate will produceischemic injury themselves. An intriguingquestion is whether there are broad neuropro-tective strategies that are more potent thangeneral anesthetics in reducing metabolicdemand but do not upset basic cellular home-ostasis or risk damage to the ultrastructuralbasis of identity and memory. Such strategieswould not only include depressing excitatoryactivity but also inhibiting downstream andintermediate steps in protein synthesis, pro-tein transcription, “futile” repair cycles (suchas PARP activation), and immune function.Some of these strategies, or the more radicalvariants thereof, may still remain largely unex-plored in mainstream biomedical researchbecause they would be detrimental in a short-term ischemia-reperfusion recovery model.Examples of these include antioxidant andfree radical interventions that are so thoroughthat they will negatively influence the free rad-ical mediated parts of the immune systemafter reperfusion.

One obstacle for identifying such strate-gies is the lack of proper definitions of termslike metabolic demand, metabolic rate andmetabolic inhibition. Reducing metabolic“demand” can mean the inhibition of specif-ic physiological events with the result that anumber of downstream biochemical reactionsare activated at a lower rate, or not activated atall. This is conceptually different from strate-gies that modulate the rate of biochemicalreactions in general, such as induction of clin-ical hypothermia.

HypothermiaInduction of hypothermia is an interest-

ing case because it not only reduces the rate ofbiochemical reactions, but some (patho) phys-iological are inhibited altogether at specifictemperatures. For example, humans typicallywill experience ventricular fibrillation andasystole (no cardiac electrical activity)between 15 and 25 degrees Celsius, whereassome natural hibernators will continue tolower heart rate at temperatures down to thefreezing point of water. One reason whyhypothermia may even confer neuroprotec-tive benefits when the temperature is onlyslightly lowered is because some parts of theischemic cascade, like excitatory amino acidrelease (excitotoxicity), are inhibited to agreater degree than predicted by the Q10value (2.0) that is often associated with induc-tion of hypothermia1. Variability in the effectsof temperature on protective and pathological

reactions may also explain why hypothermiamay even be beneficial after the ischemicinsult.

Induction of hypothermia presents anumber of challenges as a clinical treatment.The most obvious challenge is that the humanbody will attempt to compensate for unnatu-ral drops in temperature by energy-consum-ing means, such as shivering. In cryonics thismay be prevented by administration of a gen-eral anesthetic. External cooling also causesperipheral vasoconstriction that further limitsheat exchange to the core of the body. Potentalternatives for external cooling in cryonicsinclude extracorporeal cooling and cyclic lunglavage (liquid ventilation).

At very low temperatures (profound andultra-profound hypothermia), induction ofhypothermia itself may produce adverse rhe-ological, metabolic, gastrointestinal, and neu-rological effects because the balance of pro-tective and pathological metabolic modulationchanges in favour of the latter. A good exam-ple of this is that cold impairs ATP-driven ionpumps, but passive transport continues asions move down their electrochemical gradi-ents causing membrane depolarization, intra-cellular calcium overload, and ultimately, celldeath. This is one of the reasons for washingout the blood and replacing it with an “intra-cellular” organ preservation solution inremote cryonics cases with long transporttimes.

Although small decreases in brain tem-perature can confer potent neuroprotectivebenefits, the logistical challenges of externalcooling with ice packs are a concern for cry-onics organizations. Not only does the patientneed to be enclosed in some kind of portableice bath to fully benefit from immersion incirculating ice water, effective cooling is alsodependent on vigorous cardiopulmonary sup-port and administration of vasoactive medica-tions. Even if cyclic lung lavage can be estab-lished promptly as a bridge to extracorporealcooling, cryonics stabilization could benefitfrom practical normothermic methods(applied at normal body temperature) ofmetabolic depression to complement, or as atemporary substitute for, hypothermia.Recent investigations into anoxia tolerance,estivation, and hibernation may guide cryon-ics-specific research to develop these tech-nologies.

HibernationProducing a hibernating state in humans

after cardiac arrest seems to be a formidable

challenge considering the complex and multi-factorial biochemical changes of hibernatinganimals during the hibernation cycle. Hiber-nators prepare for dormancy, or torpor, byincreasing food intake and storage and bydecreasing physical activity. Entrance into tor-por is marked by lowering of the hypothalam-ic temperature setpoint, depression of meta-bolic activity, sequestering of leukocytes, anda decrease in body temperature. During tor-por, heart rate and respirations are substan-tially reduced, or in the case of freeze tolerantanimals, like the wood frog, heart rate and res-piration are stopped completely2. Typicalchanges in metabolic rate can range from 80%to nearly 100% in cryptobiotic animals, whosemetabolic activities come to a reversiblestandstill. Arousal can be rapid and the needfor intermittent euthermic arousal from tor-por may involve the need to eliminate sleepdebt, restore antioxidant defenses, replenishcarbohydrates, and remove metabolic endproducts.

A number of general criteria apply to allanimals that survive long-term hypometabolicsuppression: (1) controlled global metabolicrate suppression, (2) storage and alternateenergy metabolism and limited production oftoxic end products, (3) triggering and signal-ing transduction mechanisms to coordinatemetabolic pathways between cells and organs,(4) reorganization of metabolic priorities andenergy expenditure, (5) coordinated up-and-down regulation of genes, and (6) enhanceddefense mechanisms such as increased pro-duction of antioxidants and stabilization ofmacromolecules3. Hibernating animals pre-vent hypothermia-induced injury by maintain-

The wood frog is primarily found in the north-eastern United States, Canada, and Alaska. In

winter, the wood frog freezes 35-45% of itsbody, and its heartbeat and breathing cease. Inthe spring, the frog’s body thaws with the land

around it, and it returns to normal function.

Source:http://en.wikipedia.org/wiki/Wood_Frog


ing membrane potentials, decreasing bloodclotting, and limiting energy expenditures tobasic physiological necessities at the expenseof protein synthesis, gene transcription, andcell division. Selective up-and-down regula-tion of regulatory enzymes and rapid arousalfrom torpor is achieved by reversible phos-phorylation.

Because aspects of hypometabolismhave been induced in some non-hibernatinganimals by injecting them with the plasma ofhibernating animals, some researchers havespeculated that a “hibernation induction trig-ger” (HIT) may exist that controls entry intohibernation. If such a molecule (or number ofmolecules) exists, it is tempting to think thatadministration in humans can produce hiber-nation on demand. Practical applicationswould range from stabilization of cardiacarrest and stroke victims to long-term spaceflight. Current investigations into HIT-likesubstances indicate involvement of opioidreceptors.

The most promising HIT mimetic so faris the synthetic delta-opioid peptide DADLE(D-Ala2,D Leu5enkephalin). Administrationof DADLE to a normothermic multiorganblock preparation was able to extend survivalof organs to 46 hours, including the heart andliver4. Using the same multiorgan block autop-erfusion method, successful single canine lungtransplantation after 24 to 33 hours wasachieved when the lungs were preserved withwoodchuck HIT-containing plasma5.Hypothermic preservation time of the ratlung has been enhanced by adding DADLE toEuro-Collins solution6. Improved function ofhearts pretreated with HIT or DADLE afterhypothermic storage have been reported for anumber of non-hibernating species includingrats, rabbits and swine7.

Although beneficial effects of DADLEhave been reported in cortical neurons, inves-tigations of DADLE as a neuroprotectant inglobal and forebrain ischemia have been lim-ited to date. A 2006 study didn’t find anyimprovement for pre-ischemic administrationof DADLE in a forebrain ischemia ratmodel8. In 2007 the Safar Center for Resus-citation Research reported that DADLEfailed to improve neurological outcome in adeep hypothermic circulatory arrest rat modeland even produced worse extracerebral organinjury for the highest dose administered (10mg/kg). One explanation for these results ispoor blood brain barrier (BBB) permeabilityof DADLE because of its unfavorablehydrophylicity and charge. A series of cyclicprodrugs of DADLE only improved BBB

permeability in the presence of a P-glycopro-tein inhibitor to prevent P-gP mediated effluxtransporter activation. Bioconversion of theparent drug, however, was low9. Alternatively,pre-ischemic cerebroventricular (ICV) adminis-tration of DADLE did confer neuroprotec-tive benefits in a rat model of forebrainischemia10. As these results indicate, neuro-protective agents with high treatment poten-tial do not necessarily have privileged accessto the brain.

Opioid receptor modulation in cerebralischemia has proven to be a viable researchdirection but the results obtained with HIT-like substances do not seem to produce themulti-factorial and coordinated physiologicaleffects of hypometabolism-mediated cyto-protection that can be observed in hibernat-ing animals. Although induction of artificialhypometabolism in humans may be possibleby pharmacologic modulation of conservedmetabolic pathways with natural hibernators,it is doubtful that hibernation on demand willbe possible anytime soon. This challenge isnot dissimilar to cryobiological research thataspires to protect humans from the extensiveinjury that results from exposure to low (sub-zero) temperatures. Ultimately, advances inmodulation of hypometabolism are necessaryto protect cryonics patients from brain injuryduring stabilization and the descent from nor-mothermia to cryogenic temperatures forlong-term care. The subtle adverse effects ofexposure of the human brain to low temper-atures as such may turn out to be one of thefinal obstacles to be overcome to achieve realsuspended animation.

Carbon Monoxide and HydrogenSulfide

A number of alternative approaches toinduce hypometabolism and hypoxia toler-ance that have been explored in recent yearsinclude administration of carbon monoxideand hydrogen sulfide. The choice of thesetwo gases is remarkable because both areknown to be dangerous poisons at supraphys-iological levels. For example, high levels ofcarbon monoxide can displace oxygen athemoglobin at a rate in excess of 200 timesthe rate of oxygen, causing an acute drop inoxygen levels to the tissues. At physiologicallevels, however, both substances are producedendogenously in the human body where theyperform a number of regulatory and signalingfunctions11,12. Therapeutic administration oflow concentrations of carbon monoxide andhydrogen sulfide have been investigated invarious models of ischemic injury. Low dose

carbon monoxide can enhance protectionagainst hypothermic renal injury and improvefunction of renal grafts13. Hydrogen sulfideincreases glutathione levels in glutamate-mediated oxidative stress14.

Of most interest is administration ofcarbon monoxide or hydrogen sulfide to pro-duce a state of hypometabolism or hypoxiatolerance. C. elegans can survive mild hypoxiaby hypoxia-inducible factor 1 (HIF-1) modu-lated anaerobic energy production and up-regulation of antioxidants. C. elegans can alsosurvive extreme hypoxia by entering a state of“suspended animation.” An intermediate levelof hypoxia, however, is deadly to the organ-ism. Carbon monoxide-induced hypometabo-lism can protect C. elegans embryos against thisintermediate level of hypoxia, even in theabsence of HIF-1 function15.

Following this line of research, in a wide-ly published series of experiments, hydrogensulfide has been found to produce hypome-tabolism and hypoxia tolerance in mice.Although the research to date has not pro-duced much insight into its molecular mecha-nisms, results presented so far indicate thathydrogen sulfide exposure produces a changein energy utilization and physiologicalresponse that is typical of hibernators16.

Concerns have been raised about the lackof proper temperature controls in theseexperiments17. Although it is typical for realhibernators that reductions in metabolic rateprecede hypothermia, biochemical versus tem-perature-induced modulation of metabolismare left implicit in the published results so far.Recent research also indicates that different

"All things are poison and nothing iswithout poison, only the dose per-mits something not to be poison-

ous." Paracelsus, 1493-1541


1. Nakashima Ken and Todd Michael M. Effects ofHypothermia on the Rate of Excitatory AminoAcid Release After Ischemic Depolarization.Stroke, May 1996; 27: 913 - 918.

2. Layne JR, Lee RE, Heil TL. Freezing-inducedchanges in the heart rate of wood frogs (Rana syl-vatica). American Journal of Physiology. 1989 Nov;257(5 Pt 2): 1046-9.

3. Storey KB, Storey JM. Tribute to P. L. Lutz: puttinglife on ‘pause’–molecular regulation of hypometab-olism. Journal of Experimental Biology. 2007May;210(Pt 10):1700-14.

4. Chien S, Oeltgen PR, Diana JN, Salley RK, Su TP.Extension of tissue survival time in multiorganblock preparation with a delta opioid DADLE ([D-Ala2, D-Leu5]-enkephalin). Journal of Thoracicand Cardiovascular Surgery. 1994 Mar;107(3):964-7

5. Oeltgen PR, Horton ND, Bolling SF, Su TP.Extended lung preservation with the use of hiber-nation trigger factors. Annals of Thoracic Surgery.1996 May;61(5):1488-93

6. Wu G, Zhang F, Salley RK, Diana JN, Su TP, ChienS. delta Opioid extends hypothermic preservationtime of the lung. Journal of Thoracic and Cardio-vascular Surgery. 1996 Jan;111(1):259-67.

7. Sigg DC, Coles JA, Gallagher WJ, Oeltgen PR, Iaiz-zo PA. Opioid preconditioning: myocardial func-tion and energy metabolism. Annals of ThoracicSurgery. 2001 Nov;72(5):1576-82.

8. Iwata M, Inoue S, Kawaguchi M, Kurita N, Hori-uchi T, Nakamura M, Konishi N, Furuya H. Deltaopioid receptors stimulation with [D-Ala2, D-Leu5]enkephalin does not provide neuroprotection in thehippocampus in rats subjected to forebrainischemia. Neuroscience Letters. 2007 Mar13;414(3):242-6. Epub 2006 Dec 23.

9. Ohe T, Sato M, Tanaka S, Fujino N, Hata M, Shiba-ta Y, Kanatani A, Fukami T, Yamazaki M, Chiba M,Ishii Y. Effect of P-glycoprotein-mediated effluxon cerebrospinal fluid/plasma concentration ratio.Drug Metababolism and Disposition. 2003Oct;31(10):1251-4.

10. Su Dian-san, Wang Zhen-hong, Zheng Yong-jun,Zhao Yan-hua and Wang Xiang-rui. Dose-depend-ent Neuroprotection of Delta Opioid Peptide [D-Ala2, D-Leu5] Enkephalin in Neuronal Death andRetarded Behavior Induced by Forebrain Ischemiain Rats. Neuroscience Letters. 2007, in press

11. Ryter SW, Alam J, Choi AM. Heme oxygenase-1/carbon monoxide: from basic science to thera-peutic applications. Physiological Reviews. 2006Apr;86(2):583-650.

12. £owicka E, Be³towski J. Hydrogen sulfide (H2S) -the third gas of interest for pharmacologists. Phar-macological Reports. 2007 Jan-Feb;59(1):4-24

13. Neto JS, Nakao A, Kimizuka K, Romanosky AJ,Stolz DB, Uchiyama T, Nalesnik MA, OtterbeinLE, Murase N. Protection of transplant-inducedrenal ischemia-reperfusion injury with carbon

monoxide. American Journal of Physiology: RenalPhysiology. 2004 Nov;287(5):F979-89.

14. Kimura Y, Kimura H. Hydrogen sulfide protectsneurons from oxidative stress. FASEB Journal.2004 Jul;18(10):1165-7.

15. Nystul TG, Roth MB. Carbon monoxide-inducedsuspended animation protects against hypoxic dam-age in Caenorhabditis elegans. Proceedings of theNational Academy of Sciences of the UnitedStates of America. 2004 Jun 15;101(24):9133-6.

16. Blackstone E, Roth MB. Suspended animation-likestate protects mice from lethal hypoxia. Shock.2007 Apr;27(4):370-2.

17. Wowk, B. Is Hydrogen Sulfide the Secret to Sus-pended Animation? Cryonics, July/ August 2005

18. Zwemer CF, Song MY, Carello KA, D'Alecy LG.Strain differences in response to acute hypoxia:CD-1 versus C57BL/6J mice. Journal of AppliedPhysiology. 2007 Jan;102(1):286-93.

References

strains of mice use different metabolic strate-gies to protect themselves from acute hypox-ia18. C57BL/6J (C57) inbred mice, the strainused in the hydrogen sulfide experiments,were found to be more hypoxia tolerant thanCD-1 outbred mice.

Even if hydrogen sulfide would not be ableto induce profound normothermic hypometab-olism, identification of a molecule that couldinduce a hibernation-like state in humans, oreven just confer broad cytoprotection duringinduction of artificial hypothermia, would be anon-trivial therapeutical breakthrough. The bio-medical potential of the gases nitric oxide, car-bon monoxide and hydrogen sulfide are current-ly being investigated by a new biotechnologycompany called Ikaria, which includes the promi-nent NO/PARP researcher Csaba Szabo amongits scientific staff.

Advocates of human cryopreservationmay find the increasing use of the term sus-pended animation for therapeutic interven-tions like whole body (ultra) profound asan-guineous hypothermia and normothermichypometabolism indicative of a lack of pre-cision. But the increased support for andresearch in these areas in mainstream bio-medical science and the media may producea more favorable reception of researchaimed at reversible human cryopreservationand real suspended animation as a result.Another advantage of increased researchefforts in these areas is that cryonicsproviders can benefit from these findings toenhance their own capabilities and initiateinformed research into improved organpreservation solutions and “hibernationmimetics.” �

Aschwin de Wolf holds a master’sdegree in political science from theUniversity of Amsterdam. He movedto the US in 2000 and became a mem-ber of the Alcor Life Extension Foun-dation in 2002. He worked for Sus-pended Animation, Inc. from 2004until 2007 where he was Chief Finan-cial Officer and developed, imple-mented, and documented varioushuman cryopreservation technologies.Aschwin serves as a consultant for anumber of cryonics organizations.

Contact the author:[email protected]


Nosy Neuroprotection:Intranasal Delivery of Neuroprotective Agents to the BrainBy Chana de Wolf

IntroductionIn everyday or emergency medical practice,

intranasal (IN) administration of therapeuticagents (i.e., drug delivery via the nose) offers sev-eral advantages over oral, intravenous, and otherroutes of administration. Drugs can be rapidlyabsorbed through the large surface area of thenasal mucosa, resulting in a rapid onset of actionand avoiding degradation in the gastrointestinaltract and first-pass metabolism in the liver. INdelivery is also non-invasive and essentially pain-less, which helps to increase patient comfort andcompliance. Because of these distinct advan-tages, explorations into the practical applicationsof IN administration are becoming increasinglycommon and IN formulations of a wide varietyof therapeutic agents now exist. Nasal adminis-tration is a logical choice for topical nasal treat-ments such as antihistamines and corticos-teroids, and the nasal mucosa has also receivedattention as a viable means of systemic adminis-tration of analgesics, sedatives, hormones, car-diovascular drugs, and vaccines1.

Importantly, the nose also serves as a directroute to the brain. In 1937, W.F. Faber placedPrussian blue dye in the nasal cavity of rabbitsand later observed the dye in both the olfactorynerve and the brain, demonstrating for the firsttime the existence of the olfactory pathway2.Several decades later, experimenters discoveredthe link between the environment and the brainby performing Faber’s experiment in reverse:dyes injected into the brain ventricles of rabbitsand monkeys showed that the cerebrospinal fluid(CSF) is drained into the lymphatic vessels andthe nasal mucosa via the olfactory neurons3,4.Thus the olfactory neurons, which function pri-marily as sensory cells, also provide a means forthe direct transport of agents to the central nerv-ous system (CNS).

The implications of direct nose-to-braintransport went largely unnoticed until the 1990s,when enhanced public attention to brainresearch compelled scientists to discover andimplement effective treatment strategies in aneffort to combat the upsurge of age-related neu-rodegenerative diseases and related neurologicaldisorders in an increasingly elderly patient popu-lation. A consistent frustration in the treatment

of brain disease and stroke is that many drugsknown to mitigate the damaging effects of suchpathologies are unable to enter the brain fromthe systemic circulation due to the existence of atight membranic structure called the blood-brainbarrier (BBB). However, because the olfactoryreceptor cells are in direct contact with both theenvironment and the central nervous system, theolfactory pathway offers a potential means ofcircumventing the BBB to deliver neuroprotec-tive agents directly to the brain. Accordingly, INdelivery of drugs targeting the CNS is now anarea of great interest5,6.

In cryonics, certain neuroprotective agentsare administered to patients in an attempt to pre-vent (or at least slow down) ischemic damage tothe brain after cardiac arrest and during the low-flow reperfusion provided by cardiopulmonarysupport. Cooling is, of course, our primary lineof defense against such damage because of itsstriking effectiveness in reducing metabolicdemand. However, rapid field cooling still pres-ents considerable logistical and clinical challengesand preferential brain cooling is (at least until thepatient arrives in the operating room) yet to beaccomplished. Therefore, quick and direct pro-tection of the brain is especially important in themoments following pronouncement and duringthe initial stages of cooling, while the patient isstill relatively warm.

Currently, all medications delivered to cry-onics patients are introduced to the systemic cir-culation either via the intravenous (IV) orintraosseous (IO) routes, requiring skilled per-sonnel who have been trained in IV and/or IOtechnique. Due to the blood-brain barrier andfirst-pass metabolism, relatively large volumes ofneuroprotective agents must be given in orderfor an effective dose to enter the brain via the sys-temic circulation. In fact, some of the mostpromising neuroprotective drugs are currentlynot available at all for treatment of cryonicspatients because of poor BBB permeability.

Additionally, IN administration of cardio-vascular drugs has been a growing topic ofinvestigation7. IN propanalol provides immedi-ate β-blockade when taken before exercise bypatients with angina. IN administration ofpropanalol exhibits a pharmacokinetic profile

similar to IV administration and 10 times greaterbioavailibility than oral propanalol (because oralpropanalol undergoes considerable first-passmetabolism in the liver). In cryonics, patientsmay also benefit from IN administration ofvasopressors (i.e., medications for the mainte-nance of blood pressure) during stabilization. INepinephrine has been shown to reach peak plas-ma concentrations in only 15 seconds8, and toimprove coronary perfusion pressure in caninemodels of cardiac arrest and CPR9. Indeed, INadministration of epinephrine is a rapidly obtain-able and feasible route of administration duringany cardiac emergency.

The NoseThe nasal cavity is split into two symmetri-

cal halves by the nasal septum (comprised ofcartilidge and bone) with each side opening atthe face via the nostrils and connecting with themouth at the nasopharynx. The three mainregions of the nasal cavity are the nasalvestibule, the respiratory region, and the olfac-tory region, reflecting the primary nasal func-tions of vocalization, respiration, and olfac-tion10. The main nasal airway passages are nar-row (only 1-3 mm wide), causing inhaled air tocome into contact with the nasal mucosa, whereparticles such as dust and bacteria are filtered bymucociliary clearance. Simultaneously, the air iswarmed by the highly-vascularized nasal epithe-lium and humidified by fluid secreted by themucosa10,11. The epithelial tissue within the nasal

Figure 1. The olfactory bulb, olfactory mucosa,and olfactory nerve cells in humans. Modifiedpicture from the Nobel Prize offical homepage

(Nobelprize.org).


cavity has an extensive blood supply whichdrains blood from the nasal mucosa directly tothe systemic circulation12, thus providing apotential conduit for drug delivery which cir-cumvents first-pass metabolism.

The Respiratory and OlfactoryEpithelium

The respiratory epithelium, making up themiddle and posterior thirds of the nasal cavity,can be described as a pseudo-stratified ciliatedcolumnar epithelium consisting of four main celltypes: ciliated and non-ciliated columnar cells,goblet cells, and basal cells. The epithelial celllayer is covered with mucus, which is producedby the goblet cells and cleared by the beating ofthe cilia. This clearance mechanism protects therespiratory tract and lungs from bacteria andother exogenous compounds. In humans, therespiratory mucosa covers most of the totalnasal surface area and is the major site for drugabsorption into the systemic circulation.

The olfactory epithelium is located at thetop of the nose between the superior turbinateand the roof of the nasal cavity, just beneath thecribiform plate of the ethmoid bone (Fig. 1). Inhumans, it covers only 10-20 cm2, or about 8%of the total nasal surface area, and is composedof three main cell types: olfactory receptor neu-rons, supporting cells, and basal cells13,5. Theolfactory epithelium is more than twice thedepth of the respiratory epithelium, with theolfactory nerve cell bodies typically located in themiddle and deeper regions of the epitheliumwhile nuclei of the supporting cells are organizedin a single layer closer to the mucosal surface.Tight junctions exist between the supportingcells and between the supporting cells and olfac-tory nerve cells14.

The olfactory receptor neurons are bipolarneurons that connect the olfactory bulb of thebrain with the nasal cavity. The single dendritesof the olfactory cells terminate in olfactoryknobs that project above the epithelial surfaceand exhibit 10-25 immobile cilia which containreceptors for binding odorant molecules. Theaxons of the olfactory receptor neurons extendfrom the cell bodies and pass through the lami-na propria and cribiform plate as grouped bun-dles (Cranial nerve I) surrounded by Schwann’scells and perineural cells3. After entering theolfactory bulb, the axons synapse with juxta-glomerular neurons and with tufted and mitralcells in the glomeruli15. Surrounding brain struc-tures include those of the olfactory area (lateralolfactory tract, olfactory tubercle, and olfactorynucleus) and limbic system (amygdala, pre-pyri-form cortex, enthorhinal cortex, hippocampus,thalamus, and hypothalamus)8.

Nasal Transport RoutesAfter nasal delivery drugs first reach the res-

piratory epithelium, where compounds can beabsorbed into the systemic circulation utilizingthe same pathways as any other epithelia in thebody: transcellular and paracellular passiveabsorption, carrier-mediated transport, andabsorption through trancytosis. Althoughabsorption across the respiratory epithelium isthe major transport pathway for nasally-adminis-tered drugs and may represent a potentially time-saving route for the administration of certainsystemic drugs delivered in cryonics medicationprotocols (e.g., epinephrine or vasopressin), theauthor considers the problem of BBB-mediatedexclusion of brain-therapeutic agents to be ofgreater immediate concern. Accordingly, theremainder of this article will deal primarily withthe transport of drugs to the CNS by way of theolfactory epithelium.

When a nasal drug formulation is delivereddeep and high enough into the nasal cavity, theolfactory mucosa may be reached and drugtransport into the brain and/or CSF via theolfactory receptor neurons may occur. The olfac-tory pathways may be broadly classified into twopossible routes: the olfactory nerve pathway (axonaltransport) and the olfactory epithelial pathway13.

Axonal transport is considered a slow routewhereby an agent enters the olfactory neuron viaendocytotic or pinocytotic mechanisms and trav-els to the olfactory bulb by utilizing the sameanterograde axonal transport mechanisms thecell uses to transport endogenous substances tothe brain. Depending on the substance adminis-tered, axonal transport rates range from 20-400mm/day to a slower 0.1-4 mm/day16. Theepithelial pathway is a significantly faster routefor direct nose-to-brain transfer, whereby com-pounds pass paracellularly across the olfactoryepithelium into the perineural space, which iscontinuous with the subarachnoid space and indirect contact with the CSF. Then the moleculescan diffuse into the brain tissue or will be clearedby the CSF flow into the lymphatic vessels andsusequently into the systemic circulation.

Factors Affecting Nasal DrugDelivery to the Brain

The size of the molecule is the major deter-minant in whether a substance will be absorbedacross the nasal respiratory epithelium and/ortransported along the olfactory pathway. Fisher etal. demonstrated an almost linear relationshipbetween the log (molecular weight) and the log(% drug absorbed) of water-soluble compounds(190-70,000 Da)17,18. In general, molecules weigh-ing more than 1000 Da are absorbed far less effi-ciently than smaller molecules19. However, the

bioavailability of larger molecules may beincreased with the use of permeation enhancers.

Other factors affecting delivery to the braininclude the degree of dissocation (determined bythe pKa of a substance and the pH in the sur-rounding area)20, and lipophilicity (higherlipophilicity results in better transport)21. Once adrug is in the brain, it can be further influencedby BBB efflux transporter systems like P-glyco-protein (P-gp)22. Graff and Pollack (2003), how-ever, found that uptake into the brain wasenhanced when drugs were administered incombination with the P-gp efflux inhibitor,rifampin.

Nose-to-Brain ResearchResearching nose-to-brain transfer of drugs

in humans must, for obvious reasons, eitheremploy indirect visualization of drug transfer(e.g., effects on event-related-potentials), meas-urement of drug concentrations in the CSF dur-ing surgery, or simple monitoring of CNSeffects. Such studies have clearly indicated thatdrugs can be delivered to the brain in this man-ner, but they give no clear-cut evidence regardingthe role of transfer. Because of this limitation,studies of the olfactory pathway as a conduit fortransmission of drugs to the CNS have mostlymade use of animals having substantially differ-ent ratios of olfactory-to-respiratory epitheliumthan humans. However, the mechanisms oftransfer remain the same and are worthy of thor-ough investigation. To date, more than 50 drugsand drug-related compounds have been report-ed to reach the CNS after nasal administration indifferent species.

Figure 2. MAD® Nasal drug delivery device by Wolfe Tory Medical, Inc.

(http://www.wolfetory.com/nasal.html)

A growing number of recent reports havedemonstrated the effectiveness of intranasaladministration of neuroprotective agents indecreasing ischemic brain injury. For example,Ying et al. (2007) recently reported that intranasaladministration of NAD+ profoundly decreasedbrain injury in a rat model of transient focalischemia23. Similarly, Wei et al. (2007) showed thatintranasal administration of the PARG inhibitorgallotannin decreased ishemic brain injury inrats24. Such agents are believed to provide neuro-protection by diminishing or abolishing activa-tion of poly(ADP-ribose) polymerase-1 (PARP-1), which plays a significant role in ishemic braindamage. NAD+ was observed to reduce infarctformation by up to 86% even when adminis-tered at 2 hours after ischemic onset. BecausePARP activation appears to be a downstreamischemic event, it may be worthwhile to alsoinvestigate the ability of IN administration ofagents such as antiporters or NMDA receptorblockers to provide neuroprotection against themore upstream events of global ischemia such asmembrane depolarization and excitotoxicity.

Applicability to CryonicsIN administration of neuroprotective

agents would appear to be a possible way to cir-cumvent many of the problems encounteredwhen attempting to administer neuroprotectiveagents during a typical cryonics case. No specialtraining is required to administer drugs to thenasal cavity – IN administration is easy enougheven for relatively unskilled stabilization teammembers and does not require sterile technique.Indeed, using a simple instrument such as theMAD® Nasal drug delivery device (Fig. 2) pro-vides the possibility of self-administration ofneuroprotective and cardiovascular agents inemergency situations (e.g., at the first sign of car-diac arrest). Consequently, time-sensitiveischemia medications can be administered anddistributed throughout the brain more quicklyand, importantly, can provide protection to thebrain before cooling is sufficient to preventmajor ischemic damage.

There is also evidence indicating that manyagents active in the CNS are more effective whengiven nasally than when given by other routes.This suggests that smaller doses may be usedwhen bypassing the BBB in this manner, allow-ing for the possibility of rapid administration ofneuroprotective agents that work in low dosagesimmediately after cardiac arrest.

Limitations on the use of intranasal deliveryas a means to bypass the BBB still exist, includ-ing limitation of the concentrations achievable indifferent regions of the brain, which will varywith each agent. Indeed, data regarding IN trans-

port rates and bioavailability of specific drugs arestill scarce. However, the advantages of INadministration appear considerable and worthfurther investigation to determine the extent towhich they may benefit cryonics. �

Chana de Wolf works as a researchassociate to help Alcor build a sustain-able, multi-faceted cryonics researchprogram. Chana graduated from theUniversity of North Texas with a B.S.in Experimental Psychology in 2001and received an M.S. in Cognitionand Neuroscience from the Universityof Texas at Dallas in 2003. She hasseveral years of experience working asa research assistant in a variety of lab-oratory environments and has taughtcollege-level courses in neurosciencelab methods and biology.

Contact the author: [email protected]


1. Costantino H.R., Lisbeth I., Brandt G., JohnsonP.H., Quay S.C. (2007) Intranasal delivery: Physico-chemical and therapeutic aspects. International Jour-nal of Pharmaceutics 337: 1-24.

2. Faber W.F.(1937) The nasal mucosa and the sub-arachnoid space. American Journal of Anatomy 62:121-148.

3. Jackson R.T., Tigges J., Arnold W. (1979) Subarach-noid space of the CNS, nasal mucosa, and lymphat-ic system. Archives of Otolaryngology 105: 180-184.

4. Yoffey J.M. (1958) Passage of fluid and other sub-stances through the nasal mucosa. Journal of Laryn-gology and Otology 72: 377-383.

5. Illum L. (2004) Is nose-to-brain transport of drugsin man a reality? Journal of Pharmacy and Pharmacolo-gy 56: 3-17.

6. Vyas T.K., Salphati I., Benet L.Z. (2005) Intranasaldrug delivery for brain targeting. Current Drug Deliv-ery 2: 165-175.

7. Landau A.J., Eberhardt R.T., Frishman W.H. (1994)Intranasal delivery of cardiovascular agents: Aninnovative approach to cardiovascular pharma-cotherapy. American Heart Journal 127: 1594-1599.

8. Yamada T. (2004) The potential of the nasalmucosa route for emergency drug administrationvia a high-pressure needleless injection system.Anesthesia Progress 51(2): 6-61.

9. Bleske B.E., Warren E.W., Rice T.L., Shea M.J.,Amidon G., Knight P. (1992) Comparison of intra-venous and intranasal administration of epineph-rine during CPR in a canine model. Annals of Emer-gency Medicine 21(9): 1125-1130.

10. Chien Y.W., Su K.S.E., Chang S.F. (1989) Nasal sys-temic drug delivery. Drugs and the pharmaceuticalsciences. New York, Marcel Dekker, Inc.

11. Proctor F. (1973) Clearance of inhaled particlesfrom the human nose. Archives of Internal Medicine131: 132-139.

12. Mygind N., Pedersen M., Nielsen M.H. (1982) Mor-phology of the upper airway epithelium. In: Ander-sen, D.F.P.a.I. (Ed.) The nose: Upper airway physi-ology and the atmospheric environment. Elsevier,Amsterdam: 71-91.

13. Mathison S., Nagilla R., Kompella U.B. (1998) Nasalroute for direct delivery of solutes to the centralnervous system: fact or fiction? Journal of Drug Tar-geting 5: 415-441.

14. Morrison E.E., Costanzo R.M. (1992) Morphologyof the human olfactory epithelium. Journal of Com-parative Neurology 297(1): 1-13.

15. Shipley M.T., Ennis M. (1996) Functional organiza-tion of olfactory system. Journal of Neurobiology 30:123-176.

16. Vallee R.B., Bloom G.S. (1991) Mechanisms of fastand slow axonal transport. In: Cowan, W.M., Shoot-er E.M., Stevens C.F., and Thompson R.F. (Eds.),Annual Review of Neuroscience, Vol. 14. AnnualReviews, Inc., Palo Alto, CA, USA; 59-92.

17. Fisher A.N., Brown K., Davis S.S., Parr G., Smith D.(1987) The effect of molecular size on the nasalabsorption of water-soluble compounds in the albinorat. Journal of Pharmacy and Pharmacology 39: 357-362.

18. Fisher A.N., Illum L., Davis S.S., Schacht E.H.(1992) Di-iodo-L-tyrosine-labelled dextrans asmolecular size markers of nasal absorption in thealbino rat. Journal of Pharmacy and Pharmacology 44:550-554.

19. McMartin C., Hurchinson L.E.F., Hyde R., PetersG.E. (1987) Analysis of structure requirements forthe absorption of drugs and macromolecules fromthe nasal cavity. Journal of Pharmaceutical Sciences 76:535-540.

20. Sakane T., Akizuki M., Yamashita S., Sekazi H.,Nadai T. (1994) Direct transport from the rat nasalcavity to the cerebrospinal fluid: The relation to thedissociation of the drug. Journal of Pharmacy andPharmacology 46; 378-379.

21. Sakane T., Akizuki M., Yamashita S., Nadai T., Hashi-da M., Sekazi H. (1991) The transport of a drug tothe cerebrospinal fluid directly from the nasal cavity:The relation to the lipophilicity of the drug. Chemicaland Pharmaceutical Bulletin 39: 2456-2458.

22. Graff C.L., Pollack G.M. (2003) P-glycoproteinattenuates brain uptake of substrates after nasalinstillation. Pharmaceutical Research 20: 1225-1230.

23. Ying W., et al. (2007) Intranasal administration withNAD+ profoundly decreases brain injury in a ratmodel of transient focal ischemia. Frontiers in Bio-science 12: 2728-2734.

24. Wei G., et al. (2007) Intranasal administration of aPARG inhibitor profoundly decreases ischemicbrain injury. Frontiers in Bioscience 12: 4986-4996.

References


IntroductionA man lies critically injured on the high-

way, one leg pinned under his battered Harley-Davidson. He had just passed a car whileapproaching an intersection, but he couldn’tquite make it around the pickup stopped in theroad just ahead. The man is alive but uncon-scious; blood dribbles from his mouth as hisfaltering, shallow breathing is assisted by a para-medic who has just arrived on the scene. Forty-five minutes later the man expires at a nearbyhospital, the victim of a brain hemorrhage. Thebrain, which appears during the mandatoryautopsy to be largely undamaged despite thefatal trauma, is placed in a fixative solution aftersome delay at mostly refrigerated tempera-ture…

The above, true-life scenario played itselfout last September for a relative of mine whohad expressed interest in cryonics but not com-pleted any arrangements. He had, however,signed a “Declaration of Intent” expressing awish to be cryopreserved. Unfortunately, with

only that single signed document in place, theusual cryonics procedures could not be appliedand the best that could be attempted was a “sal-vage job.” The brain, as I write this, still rests ina refrigerated (liquid) fixative bath where it isundergoing a slow diffusion of cryoprotectant,after which it will be cooled and placed in liquidnitrogen for long-term care.

Tragic events involving persons who areinterested in cryonics but who have not signedup furnish the type of case where an alternativeto the usual procedures is needed, at least forthe all-important preliminary stages. This pre-sumes that funding can be arranged as neededfor the long-term, cryogenic care. However,there are also many cases where there simply isnot enough funding, and something entirelyapart from the usual expense of cryonics isdesired.

The reasons for considering an alternativeto cryonics includes the potential for much eas-ier, cheaper long-term care and thus greatersecurity against future contingencies such associal unrest, energy and materials shortages, oreconomic hardships affecting the cryonicsprovider. Doubts have been expressed thatalternative procedures would be adequate forthe goal of eventual revival of the cryonicspatient, given the exacting requirements thatmust be met versus the state of the art in cur-rent fixation technology. But this is not enoughreason to give up. A thorough study of existingfixation methods, such as that used in my rela-tive’s case, is called for, along with attempts todevelop better methods.

What follows is a very preliminary report.I have looked into various possible fixativetechniques and their effects. Much further workis called for, both in surveying existing researchand, eventually, in pursuing new research. Ofwhat I have been able to study so far, someapproaches show interesting success. As anoverall impression: tissue ultrastructure, whichis thought to be critical to the problem ofrevival (particularly for brain tissue), appears tobe well-preserved by a number of differentmethods that start with aldehyde fixatives suchas formaldehyde and glutaraldehyde. Some dif-ficulties with fine-scale preservation have beennoted. But these methods, I think, stronglymerit further investigation and, in some cases at

least, provisional use by cryonics providerswhen standard cryonics procedures are unavail-able and the only alternative is to decline the case.

Philosophical IssuesA basic question in cryonics is whether

preservation is good enough to offer a reason-able prospect of eventually restoring a pre-served patient to full health and vigor. At mini-mum there should be enough structural preser-vation so that the healthy state of the patientcan be inferred from the remains. If the healthystate can be known then it should be possible,using future technology, to make appropriaterepairs to bring about the desired recovery sothe patient will return to consciousness and fullfunctionality. The repairs could be extensive, soif, for example, only the brain was preserved theentire rest of the body would need to bereplaced. Repairs to the brain, too, could besubstantial. The issue will arise, for some moreacutely than for others, of whether the resultingpatient, even if similar to the original in everyrespect, really would “be” that original personor, perhaps, just a faithful copy.

While I think there are reassuring answersfor issues such as these, the subject is beyondthe scope of this study. Instead, I will be main-ly concerned with the ability of the variouspreservation techniques to achieve qualitystructural preservation. My “gold standard”will be based on an information-theoretic crite-rion: The healthy brain should be inferablefrom the state or condition of what is pre-served—this I sometimes refer to as a favorablepreservation.1 In practice it is presentlyunknown whether any preservation should beconsidered favorable in this sense, includingstandard cryopreservations. But we can at leastlook at the results and see, in the case of braintissue, whether basic structures such as neuroncell bodies, axons, dendrites, and synapses seemwell-preserved (See Figures 1 & 2 on page 23).

I think, too, that with our present igno-rance of the limits both of preservative meth-ods and of future resuscitation technology,together with philosophical uncertainties, weshould not be too hasty in dismissing any pre-servative technology. Instead we should bewilling to grant some benefit of doubt. Imper-fect or unfavorable preservation could result

The Road less Traveled:Alternatives to CryonicsA very preliminary surveyBy R. Michael Perry, Ph.D.


both from the methods used and from the con-dition of the patient at the time a procedure isstarted. But even cases where significant infor-mation loss occurs the results could be worththe effort. We must keep in mind that futurerepair technology, though it too will have limi-tations, will be powerful in ways hard to appre-ciate today. Debilities other than amnesiashould be fully curable, for example, while evenamnesia could be mitigated through use of his-torical records or in other ways.

On this ground, then, I favor a “no patientleft behind” policy in which preservation isattempted even when certain, expensive meth-ods are out of reach and some damage hasoccurred such as through autopsy or warmischemia.2 (My relative here is an obvious casein point.) The brain, as the seat of the person-ality, is the primary focus of this article,although some interesting studies of other tis-sues also show details at the cellular level, par-ticularly some work with ancient fossils.

Unintentional and ContingencyPreservation

The world of nature may seem an unlikelyplace to look for high-quality, long-term preser-vation of biological tissues, yet something ofthis sort does occur, relatively speaking, in suchremains as natural frozen mummies and speci-mens embedded in amber. Here I include a verybrief summary of some of these unintentionalpreservations, then consider a more recent,intentional but “contingency” preservation inwhich fixation was applied only after long delay.These cases are important, if for no other rea-son than because they show that surprisingamounts of fine structure can be preservedunder adverse circumstances, suggesting thatthe goal of favorable preservation may bereachable through a variety of approaches, notnecessarily cryogenic.

Tyrolean Ice Man. The 5,200-year-oldTyrolean Ice Man discovered in a glacier in1991 is the oldest known frozen humanmummy. Although frozen at high subzero-Cel-sius (non-cryogenic) temperature for most ofthe five millennia since his death, it is clear thatsome thawing and refreezing occurred, includ-ing an incident immediately following discov-ery. Despite the adverse circumstances, a 1998study reports interesting findings for manyparts of the anatomy, in which many details atthe cellular level can be seen, albeit with sub-stantial loss. In the brain there was preservationof the myelin sheaths of axons, even thoughthe material of the axons and the neuron cellbodies were mostly obliterated.3 Details of themyelin sheaths were discernible down to 10

nanometers, or about 65 carbon atom-diame-ters.

Peat Bog Burials. In addition to glaciers,peat bogs have furnished surprising examplesof preservation. One such location, a swampypond near Windover, Florida, was excavated inthe 1980s. Remains of several individuals withrecognizable if greatly shrunken brains werefound. While there was much damage, definitecellular remnants could be seen on light andelectron microscopic examination: axons; cere-bellar purkinje cells in approximately their orig-inal spatial configurations and what appeared tobe remnants of neurons. DNA was recovered,as well. The estimated age of this material was7,000-8,000 years. The preservation, limitedthough it was, is all the more striking since itinvolved above-freezing storage under water,which normally results in rapid decomposition.Apparently the lack of oxygen and neutral pHin the water-soaked peat greatly slowed the nor-mal processes of breakdown, leaving us thistantalizing, if fleeting, glimpse of ancienthumans.4

Amber Fossils. Turning to somethingmuch more ancient still, study was made in2005 of a cypress twig preserved in Balticamber for 45 million years. As reported in theProceedings of the Royal Society, the “Transmissionelectron micrographs revealed highly preservedfine structures of cell walls, membranes andorganelles” with cells more or less normal-appearing. Earlier studies of amber-preservedmaterial also showed fine details of cell struc-ture in both animal and plant tissues. The newstudy used a novel, resin-embedding techniqueto prevent breakup of the extremely fragileancient tissue under microtome sectioning,allowing full cross sections to be prepared formicroscopic examination. DNA and RNA were

not detected but hopes were raised that thesespecies-specific macromolecules were presentat least in inferable form and might be deci-phered with suitable techniques.5

A Recent Case. I conclude this sectionwith a case of recent, intentional preservationthat occurred under adverse circumstances. Thebody of a woman was kept in a mortuary for 2months at 3°C with no fixation, then the brainwas removed and fixed in a phosphate-bufferedformaldehyde solution (4.5%) for 9 weeks. Sub-sequent examination of the brain showedmacroscopically visible surface deterioration;histologically, however, normal brain structureswere preserved including all important celltypes (neurons, astrocytes, oligodendrocytes,microglia), neuropil, axons, and myelin sheaths.From a cryonics standpoint the preservationwas not good but, I would say, not negligibleeither.6

Basic Fixation: Theory, Materials,and Methods7

When a patient undergoes cardiac arrest,blood no longer circulates and cell metabolismis compromised. Without special intervention,deterioration of the tissues soon sets in. Cryon-ics protocols are designed to halt this deteriora-tion by placing the patient’s tissues in a condi-tion of biostasis, in which biological and chemi-cal activity is halted. More specifically, biostasis(1) halts any biological activity within the tis-sues, (2) protects the tissues from attack bymicroorganisms such as bacteria, and (3) stabi-lizes the tissues so that long-term care in anunchanging state is practical. Biostasis in thiscase is achieved through use of low (cryogenic)temperature, –100°C or lower.

An acceptable substitute for a cryonicsprotocol, a non-cryogenic fixation procedure,must likewise accomplish the goals of biostasisand permit long-term care of the patient or thepatient’s brain in an unchanging state. There areseveral possibilities.

Cross-linking fixatives use covalent chemicalbonds to tie down the normally reactive molec-ular components such as proteins and peptidesthat make up tissues. The most widely used fix-ative, formaldehyde, is of this type, as is anoth-er popular fixative, glutaraldehyde. The two aresometimes used in combination to achievewhat is thought to be a better result than eitheralone would accomplish. Formaldehyde hasone aldehyde group per molecule to accomplishthe crosslinking, whereas glutaraldehyde hastwo and also a longer molecule and thus is ableto bond more securely by stretching its bondingover greater distances. However, the extrapower of glutaraldehyde comes at a cost: its

An excavation of a Florida peat bog, similar tothat shown, in the 1980s recovered several indi-viduals, believed to have died 7,000-8,000 yearsago. Apparently the lack of oxygen and neutralpH in the water-soaked peat greatly slowed thenormal processes of breakdown, leaving behind

a fleeting glimpse of ancient humans.4


molecules are larger and thus do not penetrateinto tissue as fast. Using it in combination withformaldehyde means getting what is actually agood fixative, formaldehyde, in fast while theeven-better fixative, glutaraldehyde, can movein at a more leisurely pace and make its extracontribution. As one case in point, my relative’sbrain was preserved in a solution of 2%formaldehyde, 4% glutaraldehyde.

Formaldehyde is the most widely used fix-ative for the various requirements outside ofcryonics; it and glutaraldehyde have also beenmost used in cryonics when called for by specialcircumstances. Other fixatives have more spe-cialized uses, which still may have importance.Precipitating fixatives such as acetone, ethanol,

methanol, and acetic acid achieve their effectsby reducing the solubility of the proteins thatlargely comprise the tissues. Oxidizing fixativesuse oxidation to crosslink proteins; osmiumtetroxide is one fixative in this class that hasuses connected with high-magnificationmicroscopy.

Fixation of a tissue sample requires a reli-able method of delivery. With a whole organsuch as the brain it may be possible to use per-fusion or pumping the fixative solution into theorgan through the vascular system. The alterna-tive is to immerse the sample in a fixative bath,during which the fixative penetrates the tissuemore slowly through simple diffusion. For myrelative, diffusion was used since the brain’s vas-cular system had been traumatized; probablyseveral days of diffusion was adequate for near-ly complete penetration under refrigerated,above-freezing conditions.

When the sample—a brain, say—is fixed,we must ask (1) whether the preservation ofdetail is adequate and (2) whether the samplewill remain stably fixed long enough to accom-plish the desired purpose. These are demandingrequirements for alternative procedures. Sincedecades or centuries may be needed to developtechnology to attempt resuscitation, long-termstability becomes an especially important issue.As with cryonics itself, the basic answers areunknown. Some encouragement is provided bythe high level of detail seen in preserved brainsamples using, for example, formaldehyde fixa-tion. Ultrastructural details under the high mag-nification of electron microscopy (10,000xplus) are quite clear, though this alone is not ademonstration that all the details one would likeare present. However, the same problem existswith tissue preserved cryogenically—theanswer to whether the preservation capturesfine enough details is unknown though thereare at least some encouraging signs along withreasons for concern.

To return to the problem of long-term sta-bility, one consideration is whether the fixativeis uniformly distributed throughout the sample.Preservation could be compromised if thereare “islands” of tissue that were not properlyfixed. (A somewhat analogous, though possiblyless serious, problem occurs with cryopreserva-tion. If cryoprotectant is not uniformly distrib-uted in the tissue prior to lowering the temper-ature to the cryogenic range there could befreezing damage in the “islands” of tissue notprotected, even though the cold penetrates uni-formly everywhere and will prevent deteriora-tion.) So far we have considered “wet” prepara-tions only, in which the sample is to be keptimmersed in a liquid bath. Water molecules inparticular are small and move rapidly, and theirconstant, reaction-prone hammering in, say, a

specimen preserved in an aqueous aldehydesolution could have unwanted long-termeffects. For greater stability and durability itwould thus be desirable to have some form ofdry preparation for long-term care; this willnow be considered.

Dry Preparations for Biostatic CareA number of techniques, some dating

back more than a century, have been developedfor dry storage of preserved biological speci-mens. One motivation for developing thesetechniques has been to provide specimens, suchas whole, human organs, as a teaching aid. Theaim here is to render the organ in a lifelike butinert and touchable form for macroscopic orgross anatomical study or exhibits. One is notconcerned with microscopic details but onlythose features visible to the unaided eye.

Another, very different application, forwhich different embedding methods have beendeveloped, is to render specimens in a formthat can be cut into thin sections for micro-scopic study. Here the focus is on capturing finestructure but not with preserving the wholemass intact. In cryonics, of course, we are inter-ested in both the fine structure and in preserv-ing the whole specimen intact, though not foreither gross anatomical study or microscopicstudy requiring fine slicing. These differencesneed to be borne in mind in the short surveythat follows. The techniques discussed nextlook promising in some cases but could beimproved for our purpose.

Desiccation.8 Nature has pioneered vari-ous forms of preservation through desiccation.In addition to amber fossils (normally quitedehydrated) natural mummies found in desertenvironments capture such detail as DNA andlarge anatomical features, though brain tissue ispoorly preserved. For scientific purposes, desic-cation in the form of freeze-drying has beenused to preserve anatomical specimens in a life-like, inert form for study or exhibits. The spec-imen is first frozen, then allowed to dry out atbelow-freezing temperature, generally underhigh vacuum to accelerate the process. As theice evaporates, the structural shapes remainlargely unchanged, even though the final resultis delicate and will need protection from oxida-tion and moisture. Human brain tissue in largesections (order of 200g or about 15% of awhole brain) has been freeze-dried afterformaldehyde fixation and shows some struc-tural details such as capillaries under the scan-ning electron microscope, though much dam-age has occurred. Some of the damage resultedfrom freezing itself and might be mitigatedthrough use of cryoprotectants; as usual, thepotential of this process is unknown.

Figure 1 (top) & Figure 2 (bottom):Figure 1 (courtesy of Narayanan Kasthuri andKenneth Hayworth) shows a 38-nanometer-thick tissue slice of aldehyde- and osmium-fixedmouse brain cortex embedded in epon resinand sectioned and collected on Harvard's Auto-matic Tape Collecting Lathe Ultramicrotome(ATLUM). This scanning electron microscope(SEM) contrast-reversed image shows synapse(large arrow), synaptic vesicles (V), synaptic cleft(C) separating presynaptic and postsynapticmembranes, and postsynaptic density (D).Brain ultrastructure, including synapses whichare believed important for encoding memory,appears to be well-preserved. Figure 2 (cour-tesy of 21st Century Medicine), showing a SEMimage of a rabbit brain synapse perfused withM22 for 60 minutes, shows comparable ultra-structure preservation.


Early Embedding Techniques.9 Morethan a century ago paraffin embedding wasused to preserve biological specimens in a formthat would be amenable to fine sectioning formicroscopic study, and it is still used. In oneprocedure from a 1902 reference, small tissuesamples fixed in formaldehyde and dehydratedwith ethanol are embedded in melted paraffin,a white or colorless, tasteless, odorless, water-insoluble, solid substance not easily acted uponby reagents, which reaches a temperature of55°C (131°F). The melt is rapidly cooled toreduce crystallization and improve transparencyin the resulting solid embedding. A related tech-nique in the same reference uses a plastic-likematerial, celloidin, in place of paraffin. Thispermits embedding at room temperaturethrough use of an ether-ethanol solvent; it ismore suited to larger specimens, though alsorequiring full dehydration.

Plastination. An embedding techniquedeveloped by Gunther von Hagens in Germanyin the 1970s,10 plastination starts with a previ-ously fixed biological specimen in an aqueousmedium. Water and fats (lipids) are thenreplaced with a liquid plastic resin monomer,which is hardened or cured by polymerization.The resulting, resin-impregnated specimen isdry, odorless, and durable. Silicone resin yields aflexible or rubbery specimen suitable formacroscopic study or exhibits; epoxy resin canbe used to produce thin, rigid sections suitablefor microscopic study. Silicone impregnationhas also been adapted to microscopic study,with good results.11 Other desired effects arepossible using different polymers,12 and sub-stantial further adaptations might be feasible.

A possible drawback of this approach,from the standpoint of preserving the fine struc-tures that are especially important from a cryon-ics standpoint, is the relatively harsh regimenneeded to produce the finished product. Typical-ly, the process starts with an aldehyde-fixed spec-imen in aqueous solution. The specimen isplaced in acetone, and successive changes of thebath remove water and fats. Finally the resinmonomer is introduced, the remaining acetone isremoved by vacuum, and induced catalysis yieldsthe desired polymerization. Concerns have beenraised about whether defatting would obliterateimportant brain information, though there doesnot appear to be strong evidence of this. (Here itis appropriate to mention that lipids neverthelesscould contain important information; preserva-tion of lipids is a difficult process that has notbeen covered in this preliminary survey butdeserves consideration.)

Polyethylene Glycol.13 “Polyethylene gly-col” (PEG) is actually a family of water-soluble

polymers of ethylene glycol in which the mole-cules form long, linear chains. A typical batchwill contain a mixture or distribution of poly-mer chains of varying lengths. Longer chainsresult in a PEG with a higher melting point,which is a waxy solid at room temperature orbelow. Its water solubility and other characteris-tics make it possible to introduce PEG into tis-sue without using dehydration, defatting, heat-ing, or additional polymerization, suggestingthat PEG could overcome possible problemsencountered with many other forms of embed-ding. PEG, however, does not seem much usedfor biological embedding and may not be as sat-isfactory as other methods even for the specialrequirements that relate to cryonics. As usual,there are important unknowns here and furtherinvestigation is called for.

Discussion and ConclusionsThe agonizing question will continually

arise of what to do in an emergency where cry-onics is desired but arrangements are not inplace. An alternative to just giving up is to con-sider some non-cryonics procedures, such aschemical preservation of the brain, as a tempo-rary solution until cryopreservation can bearranged or, if this eventuality is not feasible, asa long-term option itself.

In such a case, the question arises ofwhether the preservation is adequate to meetthe unknown, presumably demanding require-ments if the patient is to be repaired withoutdeficits by future technology. Revival in such a“perfect” form will happen only if enoughidentity-critical structure is present in someinferable form. The high degree of structuralpreservation seen in some fixation methodsraises hopes that in fact such a degree ofpreservation is achievable through non-cryo-genic means.

In addition to the (by appearance) encour-aging results obtainable with water-based fixa-tives in a liquid bath, there is the prospect ofembedding the specimen (normally thepatient’s brain) in a solid matrix for long-termcare. Certainly there are unknowns with such anapproach but one finds this in the standard cry-onics procedures too.

The alternatives discussed herein offer theadditional advantage of more security per unitcost for conditions of social unrest or econom-ic hardship which may occur over the longinterval before revival will be attempted. Inaddition, there is reason to consider preserva-tion methods that knowingly fall short of thebest methods currently possible, when, forexample, the cost of better treatment is prohib-itive. The alternatives must be taken seriously,

then, for a number of reasons, and more workis needed to both assess and enhance theirpotential. Along with this, so long as invalidat-ing difficulties are not discovered, it is impera-tive that such alternatives become more readilyavailable—through some appropriate organiza-tion—and widely advertised. �

Contact the author: [email protected]

1. This essentially is the point of view expressed inMerkle R, “The Technical Feasibility of Cryonics,”Medical Hypotheses 39 6–16 (1992).

2. For a discussion of this issue from a cryonics per-spective (omitting but applicable to other forms ofpreservation) see Wowk B, “Ethics of Non-IdealCryonics Cases,” Cryonics 27(4) 10-12 (Fall 2006),available online at http://www.alcor.org/cryon-ics/cryonics0604.pdf.

3. Hess M W, Klima G, Pfaller K, Künzel K H, GaberO, “Histological Investigations on the Tyrolean IceMan,” Am. J. Phys. Anthr. 106(4) 521-32 (1998).

4. Doran G H, Dickel D N, Ballinger W E, Agee O F,Laipis P J, Hauswirth W W, “Anatomical, Cellularand Molecular Analysis of 8,000-Yr-Old HumanBrain Tissue from the Windover ArchaeologicalSite,” Nature 323, 803-06 (30 October 1986).

5. Koller B, Schmitt J M, Tischendorf G, “CellularFine Structures and Histochemical Reactions in theTissue of a Cypress Twig Preserved in BalticAmber,” Proc. R. Soc. B (2005) 272, 121–26,http://www.journals.royalsoc.ac.uk/content/k6y5c74pbp4g21mf/fulltext.pdf

6. Gelpi E, Preusser M, Bauer G, Budka H, “Autopsyat 2 Months after Death: Brain Is SatisfactorilyPreserved for Neuropathology,” Forensic Sci Int.24;168(2-3):177-82 (May 2007).

7. This section is based in part on the Wikipedia arti-cle Fixation (histology), http://en.wikipedia.org/wiki/Fixation_(histology)

8. This paragraph is based on Hower R O, Freeze-Dry-ing Biological Specimens: a Laboratory Manual, Washing-ton, D. C.: Smithsonian Institution Press (1979).

9. This paragraph is based on Hardesty I, Neurological Tech-nique, Chicago: University of Chicago Press (1902).

10. von Hagens, G, “Impregnation of Soft BiologicalSpecimens with Thermosetting Resins and Elas-tomers,” Anat. Rec. 194: 247-56 (1979).

11. Grondin G, Grondin G G, Talbot B G, “A Study ofCriteria Permitting the Use of Plastinated Speci-mens for Light and Electron Microscopy,” Biotech.Histochem. 69(4) 219-34 (Jul. 1994).

12. For an older but interesting, informative survey seevon Hagens G, Tiedemann K, Kriz W, “The Cur-rent Potential of Plastination,” Anat.and Embr.175(4) 411-21 (Mar. 1987).

13. This paragraph is based on Gao K (ed), PolyethyleneGlycol as an Embedment for Microscopy and Histochem-istry. Boca Raton, Fla.: CRC Press (1993).

I thank Greg Jordan, Aschwin de Wolf, Chana deWolf, Hugh Hixon, Jennifer Chapman, and Brian Wowkfor their generous assistance in tracking down references,proofreading, and/or obtaining material for illustrations.

References

CRYONICS

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Cryonics is an attempt to preserve and protect the gift of human life, not reverse death. It is the spec-ulative practice of using extreme cold to preserve the life of a person who can no longer be support-

ed by today’s medicine. Will future medicine, including mature nanotechnology, have the ability to heal atthe cellular and molecular levels? Can cryonics successfully carry the cryopreserved person forwardthrough time, for however many decades or centuries might be necessary, until the cryopreservationprocess can be reversed and the person restored to full health? While cryonics may sound like sciencefiction, there is a basis for it in real science. The complete scientific story of cryonics is seldom told inmedia reports, leaving cryonics widely misunderstood. We invite you to reach your own conclusions.

The Alcor Life Extension Foundation is the world leader in cryonics research and technology. Alcoris a non-profit organization located in Scottsdale, Arizona, founded in 1972. Our website is one of

the best sources of detailed introductory information about Alcor and cryopreservation (www.alcor.org).We also invite you to request our FREE information package on the “Free Information” section of ourwebsite. It includes:

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Step 1: Fill out an application and submit it with your $150 application fee.Step 2: You will then be sent a set of contracts to review and sign.Step 3: Fund your cryopreservation. While most people use life insurance to

fund their cryopreservation, other forms of prepayment are alsoaccepted. Alcor’s Membership Coordinator can provide you with alist of insurance agents familiar with satisfying Alcor’s current fund-ing requirements.

Finally: After enrolling, you will wear emergency alert tags or carry a specialcard in your wallet. This is your confirmation that Alcor will respondimmediately to an emergency call on your behalf.

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