Cryopreservation as a means of Suspended Animation

by Ben Best




Cryptobiosis is a state of arrested metabolism by an organism that is protective against harsh environments, such as draught, famine, lack of oxygen or extreme temperature [COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY; PART B; Clegg,JS; 128(4):613-624 (2001)]. Similar physiological mechanisms associated with cryptobiotic protection against dehyration and cold are found in plants as well as animals [JOURNAL OF EXPERIMENTAL BIOLOGY; Storey,KB; 210(Pt 10):1700-1714 (2004)]. Late Embryogenesis Abundant (LEA) proteins protect both plants and animal proteins from drying, cold and osmotic stress [BIOCHEMICAL JOURNAL; Goyal,K; 388(Pt 1):151-157 (2005)]. Cold shock proteins, antifreeze proteins, ice-nucleating agents, and cryoprotectants along with LEA proteins are the chief biochemical mechanisms allowing plants and animals to withstand freezing temperatures [NATURWISSENSCHAFTEN; Margesin,R; 94(2):77-00 (2007)].

The bacteria Pseudomonas and Escherichia coli can survive freezing to liquid nitrogen temperature without mutation [CRYOBIOLOGY; Ashwood-Smith,MJ; 2(1):39-43 (1965)], but multicellular organisms are not so hardy. Polar arthropods survive sub-zero temperatures by dehydration and production of low molecular weight sugars and sugar alcohols that act as cryoprotectants [JOURNAL OF INSECT PHYSIOLOGY; Worland,MR; 49(3):193-203 (2003)]. Some species can tolerate extracellular freezing. The only animal known to survive intracellular freezing is the Antarctic nematode Panagrolaimus davidi. Panagrolaimus davidi experimentally subjected to fast freezing had 54% intracellular ice formation, yet 53% survived [CRYOBIOLOGY; Wharton,DA; 50(1):21-28 (2005)].

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Hibernating marmots maintain a body temperature at 5ºC in ambient temperatures as low as −1ºC [RESPIRATORY PHYSIOLOGY & NEUROBIOLOGY; Heldmaier,G; 141(3):317-329 (2004)]. Arctic ground squirrels have been shown to hibernate for at least two months at temperatures down to −2.9ºC without freezing [SCIENCE; Barnes,BM; 244:1593-1595 (1989)].

Warm-blooded, nonhibernating animals suffer adverse effects when subjected to temperatures below 28ºC, including extreme shivering, high blood viscosity, and cardiac arrest [JOURNAL OF THE AMERICAN VETERINARY ASSOCIATION; Moon,PF; 202(3):437-444 (1993)]. Pharmacological efforts have been made to reduce the shivering threshold and blunt natural thermoregulation in humans to augment the benefits of therapeutic hypothermia [STROKE; Sessler,DI; 40(11):e614-e621 (2009)]. Although bears are capable of depressed metabolism, their body temperature remains above 30ºC — with some shivering helping to maintain muscle strength and temperature [AMERICAN JOURNAL OF PHYSIOLOGY; McGee-Lawrence,ME; 295(6):R1999-R2014 (2008)].

A number of studies have been made of proteins and gene expression in hibernating mammals [MOLECULAR & CELLULAR PROTEOMICS; Shao,C; 9(2):313-326 (2010) and TRENDS IN ENDOCRINOLOGY AND METABOLISM; Melvin,RG; 20(10):490-498 (2009)]. It is likely that genes of hibernating mammals are conserved in the human genome such that induction of these genes could be a means of placing humans in a state of "suspended animation" [BIOESSAYS; Andrews,MT; 29(5):431-440 (2007)].

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In anoxic conditions zebrafish (Danio rerio) embryos enter a state of reversible suspended animation characterized by arrested heartbeat and arrested cell cycle progression [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA); Padillo,PA; 98(13):7331-7335 (2001)]. Caenorhabditis elegans nematodes (which respond to anoxia by entering suspended animation and survive hypoxia by depressing metabolism with hypoxia-inducible factor 1), can be induced into a state of suspended animation with carbon monoxide [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA); Nystul,TG; 101(24):9133-9136 (2004)]. Hypoxia-inducible factor not only depresses metabolism, but depresses apoptotic mechanisms [ANTIOXIDANTS & REDOX SIGNALLING; Snyder,CM; 11(11):2673-2683 (2009)]. C. elegans with lifetime exposure to hydrogen sulfide have mean lifespan increased by 70%, but without any apparent reduction in metabolic rate [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA); Miller,DL; 104(51):20618-20622 (2007)].

The golden-mantled ground squirrel Spermophilus lateralis is a hibernating mammal that can be induced by hypoxia into a state of depressed metabolism and reduced body temperature [JOURNAL OF APPLIED PHYSIOLOGY; Barros,RCH; 91(2):603-612 (2001)]. Hypoxia has also been shown to reduce metabolism in small non-hibernating mammals [AMERICAN JOURNAL OF PHYSIOLOGY; Frappell,P; 262(6 Pt 2):R1040-R1046 (1992)], but hypoxia does not appear to have this effect in large mammals [JOURNAL OF APPLIED PHYSIOLOGY; Korducki,MJ; 76(6):2380-2385 (1994)].

The mouse, a non-hibernating mammal, can be induced with hydrogen sulfide to enter a state of "suspended animation" that drops metabolic rate by 90% and leaves core body temperature only 2ºC above a 13ºC ambient temperature [SCIENCE; Blackstone,E; 308:518 (2005)]. This effect is believed to be due to hydrogen sulfide inhibition of cytochrome c oxidase in mitochondria, thus inhibiting oxidative phosphorylation. A similar effect has been produced in rats using higher hydrogen sulfide concentrations [THE JOURNAL OF TRAUMA; Morrison,ML; 65(1):183-188 (2008)], although another study failed to depress metabolism in rats using hydrogen sulfide [RESPIRATORY PHYSIOLOGY & NEUROBIOLOGY; Haouzi,P; 167(3):316-322 (2009)]. Some researchers have failed to depress metabolism with hydrogen sulfide in sheep [RESPIRATORY PHYSIOLOGY & NEUROBIOLOGY; Haouzi,P; 160(1):109-115 (2008)] or piglets [PEDIATRIC CRITICAL CARE MEDICINE; Zhang,LJ; 9(1):110-112 (2008)], while other researchers have succeeded in reducing metabolism and temperature in pigs [SHOCK; Simon,F; 30(4):359-364 (2008)].

Induction of depressed metabolism (hibernation) in mammals (including humans) with AMP activated protein kinase has recently been suggested [ANNUAL REVIEW OF MEDICINE; Lee,CC; 59:177-186 (2008)].

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When animals lower body temperature their metabolic rate is reduced by nearly half for every 10ºC drop between 40ºC (approximate body temperature) and 0ºC [JOURNAL OF BIOENERGETICS; Raison,JK; 4(1):285-309 (1973) and NATURE; Davidson,EA; 440:165-173 (2006)]. People, especially children, who have fallen in cold water have been protected by hypothermia from possible brain damage after their heart has been stopped for as much as an hour [JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION; Bolte,RG; 260(3):377-379 (1988)]. A pulseless 31-month old girl who had spent nearly six hours in outdoor temperatures below −20ºC and had a rectal temperature of 14ºC survived with full neurological recovery [THE JOURNAL OF TRAUMA; Dobson,JA; 40(3):483-485 (1996)].

Dogs cooled to 34ºC after ten minutes of induced ventricular fibrillation were able to survive 60 minutes of ventricular fibrillation with good neurological outcome [CIRCULATION; Nozari,A; 113(23):2690-2696 (2006)]. Hypothermia has been shown to modulate cerebral concentrations of pro- and anti-apoptotic proteins in favor of prevention of neuronal cell death [ACTA ANAESTHESIOLOGICA SCANDINAVIA; Eberspacher,E; 49(4):477-487 (2005)]. A review of animal models of ischemic stroke found that infarct volume was reduced by about one-third by cooling to 35ºC within 90 to 180 minutes of initiation of permanent  [BRAIN; van der Worp,HB; 130(Pt 2):3063-3074 (2007)]. In a study of swine, most of the pigs cooled 10ºC survived up to 60 minutes of shock without neurological effects [SURGERY; Alam,HB; 132(2):278-288 (2002)]. Six out of six experimental hypothermic dogs having tympanic temperature of 10ºC were shown to endure 90 minutes of cardiac arrest without subsequent neurological damage, and two out of seven endured 120 minutes without evident neurological damage [CRITICAL CARE MEDICINE; Behringer,W; 31(5):1523-1531 (2003)]. Dogs receiving continuous asanguinous perfusion were able to recover from at least four hours below 9ºC and two hours below 5ºC with satisfactory neurological recovery [ALCOR WEBSITE; Asanguinous Ultraprofound Hypothermia; Leaf,JD; Darwin,MG; (1987)].

Human ventricular fibrillation victims subjected to only 1ºC of hypothermia showed a substantial increase in both survival and favorable neurological outcome [CRITICAL CARE MEDICINE; Don,CW; 37(12):3062-3069 (2009)]. Humans have been subjected to deep hypothermic cardiac arrest for aortic surgery for over an hour without gross neurological deficits. The subjects reached complete electrocerebral silence (zero electroencephalographic bispectral index) between temperatures of 16ºC and 24ºC [JOURNAL OF CARDIOTHORACIC AND VASCULAR ANESTHESIA; Hayashida,M; 21(1):61-67 (2007)] and were re-warmed without neurological deficit, confirming that dynamic brain activity can be lost and regained without loss of personal identity.

Many reptiles and invertebrates can withstand lower temperatures than mammals, and thus achieve a more profound state of depressed metabolism and suspended animation. The northern wood frog (Rana sylvatica) can survive in a semi-frozen state down to −6ºC for months with full recovery upon rewarming [THE FASEB JOURNAL; Costanzo,JP; 9(5):351-358 (1995)].

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Larvae of the Alaskan beetle Cucujus clavipes puniceus have been demonstrated to survive temperatures down to −100ºC [THE JOURNAL OF EXPERIMENTAL BIOLOGY; Sformo,T; 213(Pt 3):502-509 (2010)]. The ability to achieve an anhydrobiotic (desiccated) state enables some organisms to naturally tolerate cryogenic temperatures. Larvae of the African chironomid Polypedilum vanderplanki, the largest multicellular animal capable of anhydrobiosis (the only insect capable of anhydrobiosis) reportedly survive temperatures as low as −270ºC [JOURNAL OF EXPERIMENTAL BIOLOGY; Watanabe,M; 206(Pt 13):2281-2286 (2003)]. Vitrification by trehalose and protein has proven to be essential for the chironomid's anhydrobiosis [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA); Sakarai,M; 105(13):5093-5098 (2008)]. Tardigrades, which are microscopic arthropod-like animals capable of anhydrobiosis, can survive cooling to liquid nitrogen temperature (−196ºC) even in a hydrated state [CRYOBIOLOGY; Ramlov,H; 29(1):125-130 (1992)].

Multicellular animals treated with cryoprotectants have survived cooling to cryogenic temperatures. A variety of parasitic helminths have survived liquid nitrogen storage after treatment with DMSO, glycerol or ethylene glycol [CRYOBIOLOGY; James,ER; 49(3):201-210 (2004)].

Many varieties of nematode larvae have survived cryogenic temperatures. The C. elegans nematode is routinely stored in liquid nitrogen. Plant nematodes treated with 25% ethylene glycol, cooled rapidly and held in liquid nitrogen for a few minutes before storage in a −140ºC freezer for a month, showed 85% survival [CRYOBIOLOGY; Irdani,T; 52(3):319-322 (2006)]. Survival rates up to 70% were achieved for parasitic nematode larvae treated with 10% DMSO before 16 month storage in liquid nitrogen [INTERNATIONAL JOURNAL FOR PARASITOLOGY; Gill,JH; 25(12):1421-1426 (1995)]. A majority of a variety of infective nematode species larvae treated with sodium hypochlorite and saline solution survived more than 15 years in liquid nitrogen, although percent infectivity was less than half of percent survival [VETERINARY PARASITOLOGY; van Wyk,JA; 88(3-4):239-247 (2000)]. The stress-sensitive tropical parasitic nematode Radopholus similis treated with a mixture of methanol, glycerol and glucose showed no reduction of infectivity or reproduction after storage in liquid nitrogen [CRYOBIOLOGY; Elsen,A; 55(2):148-157 (2007)].

Complete (100%) survival of entomopathogenic nematode infective juveniles was achieved after treatment with glycerol and Ringer's solution prior to preservation in liquid nitrogen [JOURNAL OF NEMATOLOGY; Bai,C; 36(3):281-284 (2004)]. A 27% survival was achieved for human hookworm (Ancyclostoma ceylanicum) larvae treated with a 10% DMSO, 10% dextran solution prior to 100 days in liquid nitrogen [AMERICAN JOURNAL OF TROPICAL MEDICAL HYGIENE; Duarte,J; 68(1):44-45 (2003)]. More than 50% of ragworm (Nereis virens) larvae treated with 10% DMSO survived liquid nitrogen storage [CRYOBIOLOGY; Olive,JW; 34(3):284-294 (1997)]. Sixty to seventy percent of 50,000-cell fruit fly embryos (Drospophila melanogaster) vitrified with ethylene glycol and cooled to −205ºC hatched upon rewarming, and 40% of the larvae developed into fertile adults [SCIENCE; Mazur,P; 258:1932-1935 (1992)].

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Ice occupies a 9% greater volume than water. Although it is a misconception that water bursts cells by freezing inside, it is nonetheless true that ice expansion in the extracellular space crushes cells [AMERICAN JOURNAL OF PHYSIOLOGY; Mazur,P; 247(3 Pt 1):C125-C142 (1984)]. Vitrification achieved by adding sufficient cryoprotectant can eliminate ice formation during cooling of biological tissues to cryogenic temperatures.

A number of mammalian tissues and organs have been successfully vitrified. Tissue-engineered blood vessels that were vitrified, stored at −135ºC and rewarmed showed comparable metabolic activity to fresh controls [TISSUE ENGINEERING; Dahl,SLM; 12(2):291-300 (2006)]. Rat hippocampal slices vitrified, cooled to −130ºC, and rewarmed showed 100% viability within experimental error [CRYOBIOLOGY; Pichugin,Y; 52(2):228-240 (2006)]. Rabbit trachea that were vitrified, held in liquid nitrogen, and rewarmed were comparable to fresh specimens [CRYOBIOLOGY; Xu,H; 58(2):225-231 (2009)]. Complete mouse ovaries have been vitrified, stored in liquid nitrogen, and rewarmed to produce live pup birth rates comparable to that seen with fresh ovaries [FERTILITY AND STERILITY; Hasegawa,A; 86(Suppl 3):1182-1192 (2006)].

The greatest accomplishment of vitrification to date has been the vitrification of a rabbit kidney, cooling to −135ºC, rewarming, retransplatation into the rabbit, and the kidney able to sustain the rabbit indefinitely as the sole functioning kidney [ORGANOGENESIS; Fahy,GM; 5(3):167-175 (2009)]. But this falls far short of vitrifying a whole rabbit or a whole mouse.

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Although cryogenic storage cannot prevent ionization by radiation or the breaking of chemical bonds, cryogenic storage protects against radiation damage by reducing the mobility and reactivity of reactive species generated by radiation [ULTRAMICROSCOPY; Knopek,E; 10(1-2):71-86 (1982)]. Cooling large molecular hydrated organic compounds to cryogenic temperature has been shown to increase tolerable exposure to ionizing radiation up to ten times in electron microscopy [JOURNAL OF MICROSCOPY; Glaeser,RM; 112(1):127-138 (1978)]. Cells stored in liquid nitrogen subjected to ionizing radiation a hundred times normal background levels show no increase in cell death, nor is there evidence of chromosomal or genetic change after storage in liquid nitrogen [AMERICAN JOURNAL OF PHYSIOLOGY; Mazur,P; 247(3 Pt 1):C125-C142 (1984)]. Frozen 8-cell mouse embryos subjected to the equivalent of 2,000 years of background gamma rays during 5 to 8 months in liquid nitrogen showed no detrimental effect on survival or development [JOURNAL OF REPRODUCTION AND FERTILITY; Glenister,PH; 70(1):229-234 (1984)]. Chinese hamster ovary fibroblasts in 10% DMSO show 3.5 times less damage from 600 rad of x-ray irradiation at liquid nitrogen temperature compared to 22ºC [CRYOBIOLOGY; Ashwood-Smith,MJ; 16(2):132-140 (1979)].

Acetylcholinesterase enzyme subjected to X-ray irradiation shows conformational changes at −118ºC, but no conformational changes when irradiated at −173ºC [PROTEIN SCIENCE; Weik,M; 10(10):1953-1961 (2001)]. X-ray irradiation of insulin and elastase crystals resulted in four times as much damage to disulfide bridges at −173ºC compared to −223ºC [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA); Meets,A; 107(3):1094-1099 (2010)]. Another study showed a 25% crystal diffraction lifetime extension for D-xylose isomerase crystals X-ray irradiated at less than −253ºC compared to those irradiated at −173ºC [ACTA CRYSTALLOGRAPHICA; Chinte,U; 63(Pt 4):486-492 (2007)]. Nonetheless, another study showed negligible differences between frozen hydrated biological samples subjected to X-ray radiation at temperatures between −269ºC and −173ºC [JOURNAL OF STRUCTURAL BIOLOGY; Bammes,BE; 169(3):331-341 (2010)].

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Calling the depressed metabolism by hydrogen sulfide "suspended animation" is hyperbole insofar as metabolism at one tenth normal is still far above absence of metabolism. Even if artificial hibernation of humans could allow for maintenance temperatures close to 0ºC, the metabolic rate would still be one-twentieth normal. A 200 day spaceflight would require 10 days worth of nutrient, oxygen and waste-handling for a mammal hibernating at 0ºC. By contrast, if human cryopreservation were possible at cryogenic temperatures, minutes of metabolism at normal body temperatures would take tens or hundreds of thousands of years [REJUVENATION RESEARCH; Best,B; 11(2):493-503 (2008)]. Moreover, cryopreservation at cryogenic temperature would afford much greater protection against radiation damage. Maintaining cryogenic temperature in space could require modest amounts of energy.

Cryoprotectant toxicity using conventional cryoprotectants remains the single greatest obstacle to vitrification of organs larger than a rabbit kidney [CRYOBIOLOGY; Fahy,GM; 6 June 2009]. If cryoprotectant toxicity could be eliminated, reversible cryopreservation at cryogenic temperature of an entire human could become possible. Alternatively, if all human cells could be induced to produce a non-toxic cryoprotectant such as trehalose (which does not cross cell membranes), whole body vitrification might become possible. Human fibroblasts have already been induced to synthesize cryoprotectant trehalose by genes introduced using recombinant adenovirus vectors [NATURE BIOTECHNOLOGY; Guo,N; 18(2):168-171 (2000)].

Cryonics is the cryopreservation of legally dead humans at cryogenic temperatures in the hope that future technology will not only repair damage resulting from cryopreservation, but cure all diseases and rejuvenate people to the prime of life. Cryonics as it is practiced today uses vitrification, but although ice can be eliminated in many organs, damage is nonetheless done by cryoprotectant toxicity. There may also be freezing damage due to incomplete perfusion of certain organs or tissues. Nanotechnology (nanomedicine) is expected to be able to repair damage on a molecular level. Future technology is not science, but the idea that future technology will be vastly superior to current technology should not be dismissed.

The emphasis of current technology in cryonics is to cryopreserve with the least amount of damage so that future technology has the best chance of repairing the damage. Although some future molecular repair technology could fix damage, but it is more prudent to minimize damage in the first place. Therefore, cryonics organizations first perfuse cryonics patients with anti-freeze cryoprotectants to eliminate ice altogether (vitrification). Electron micrographs of vitrified brain tissue at least indicate that brain structure can be preserved on a fine level, which makes future molecular repair more promising.

Humans in spaceflight would not require rejuvenation or the curing of fatal diseases — nor should there be any repair process require for cryoprotectant toxicity or freezing damage. If something to increase tissue thermal conductivity were developed, less toxic cryoprotectants could be used because faster external cooling would be feasible. Alternatively, something like a reverse microwave technology for cooling rather than warming could have some benefit.

Pure water can vitrify without cryoprotectants if cooled at 3 million degrees Celcius per second [JOURNAL OF MICROSCOPY; Bald,WB;143(Pt 1):89-102 (1986)]. Such a cooling rate would be impractical for mammalian tissues, at least partially because cooling would be applied externally. If something could be invented that acted like an inverse microwave that cools tissue uniformly the way microwave heating warms uniformly, less cryoprotectant could be used. Adiabatic demagnetization is something like an inverse microwave effect. A cryonics patient could be perfused with a solution composed not only of cryoprotectant but of a paramagnetic salt such as ferric ammonium alum in a magnetic field. By gradually decreasing the strength of the magnetic field the salt ions become disordered, uniformly drawing heat throughout the body of the cryonics patient, not just from the surface. This or other novel mechanisms of cooling could potentially be of great benefit in solving the problem of rapidly cooling large biological objects.

Microwaves rotate water molecules to generate heat, and this technology has been used to improve temperature uniformity during rewarming of vitrified tissues [CRYOBIOLOGY; Wusterman,M; 48(2):179-189 (2004) and PHYSICS IN MEDICINE AND BIOLOGY; Robinson,MP; 47(13):2311-2325 (2002)]. An oscillating electric field has been used to reduce the amount of ice formed when plunging 1.5 microliter samples of ethylene glycol (EG) into liquid nitrogen. Ice formation was reduced about 56% for a 3.5 Molar EG solution, and reduced about 66% for a 4.5 Molar EG solution [CRYOBIOLOGY; Jackson,JH; 34(4):363-372 (1997)]. A Japanese application called the Cells Alive System (CAS) [SCIENCE LINKS JAPAN; Episode 21; ABI Inc] is using a rotating electric field in food processing [PATENT 6,250,087; Norio Owada and Satoru Kurita and PATENT 7,237,400; Norio Owada]. Why this effect works is a matter of dispute, but it is known that static electric fields enhance ice nucleation [PURE AND APPLIED GEOPHYSICS; Pruppacher,HR; 104(1):623-634 (1973)], so perhaps oscillating electric fields have the opposite effect. Another Japanese group reported subjecting rat ovaries to a magnetic field during cooling, and observing better cryopreservation than controls, despite the absence of cryoprotectant [ACADEMIC COLLABORATIONS FOR SICK CHILDREN; Mihara,M; 1:34-37 (2009)]. If these, or similar technologies, could be perfected to allow for vitrification with little or no cryoprotectant, cryogenic organ preservation and human suspended animation through cryopreservation at cryogenic temperatures could become possible.

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