Cryobiology is the branch of biology that studies the effects of low temperatures on living things. The word cryobiology is derived from the Greek words "cryo" = cold, "bios" = life, and "logos" = science. In practice, cryobiology is the study of biological material or systems at temperatures below normal. Materials or systems studied may include proteins, cells, tissues, organs, or whole organisms. Temperatures may range from moderately hypothermic conditions to cryogenic temperatures.

Contents 1 Definitions/Distinctions 2 Major areas of study in cryobiology 3 Historical background 4 Freezing of living organisms 4.1 Bacteria 4.2 Plants 4.3 Animals 4.3.1 Invertebrates 4.3.2 Vertebrates 5 Cryobiology in Humans 6 Applied cryobiology 7 Scientific societies 8 Journals 9 Notes 10 See also 11 External links

[edit] Definitions/Distinctions

Cryobiology

is the study of life at low temperatures.

Cryogenics

is the branch of physics and engineering that studies the production and use of very low temperatures. Cryogenics is not cryonics, although people often confuse them.

Cryonics

is the low temperature preservation of humans and mammals with the intention of future revival. Cryonics is not part of mainstream cryobiology. Cryonics still depends heavily on speculative future technology which may or may not be invented.

Cryopreservation

is a technology whereby cells, whole tissues, or embryos are preserved by cooling to temperatures below the freezing point of water.

[edit] Major areas of study in cryobiology

6 major areas of study in cryobiology can be identified:

1. Study of cold-adaptation of microorganisms, plants (= cold hardiness), and animals, both invertebrates and vertebrates (= hibernation).
2. Cryopreservation of cells, tissues, gametes, and embryos of animal and human origin for (medical) purposes of long-term storage. This usually requires the addition of substances which protect the cells during freezing and thawing (cryoprotectants).
3. Preservation of organs under hypothermic conditions for transplantation.
4. Lyophilization (freeze-drying) of pharmaceuticals.
5. Cryosurgery, a (minimally) invasive approach for the destruction of unhealthy tissue using cryogenic gases/fluids.
6. Physics of supercooling, ice nucleation/growth and mechanical engineering aspects of heat transfer during cooling and warming.

[edit] Historical background

Cryobiology history can be traced back to antiquity. As early as in 2500 BC low temperatures were used in Egypt in medicine. The use of cold was recommended by Hippocrates to stop bleeding and swelling. With the emergence of modern science, Robert Boyle studied the effects of low temperatures on animals.

In 1949 bull sperm was cryopreserved for the first time by a team of scientists led by Christopher Polge (1926-2006). This led to a much wider use of cryopreservation today, with many organs, tissues and cells routinely stored at low temperatures. Large organs such as hearts are usually stored and transported, for short times only, at cool but not freezing temperatures for transplantation. Cell suspensions (like blood and semen) and thin tissue sections can sometimes be stored almost indefinitely at liquid nitrogen temperature (cryopreservation). Human sperm, eggs and embryos are routinely stored in fertility research and treatments. In the early 2000s a baby was born from a cryopreserved egg fertilized by a cryopreserved sperm.

Cryosurgery (intended and controlled tissue destruction by ice formation) was carried out by James Arnott in 1845 in an operation on a patient with cancer. Cryosurgery is not common.

[edit] Freezing of living organisms

Many living organisms are able to tolerate prolonged periods of time at temperatures below the freezing point of water. Most living organisms accumulate cryoprotectants such as anti-nucleating proteins, polyols, and glucose to protect themselves against frost damage by sharp ice crystals. Most plants, in particular, can safely reach temperatures of −4 °C to −12 °C.

[edit] Bacteria

Three species of bacteria, Carnobacterium pleistocenium, as well as Chryseobacterium greenlandensis and Herminiimonas glaciei, have reportedly been revived after surviving for thousands of years frozen in ice. Certain bacteria, notably Pseudomonas syringae, produce specialized proteins that serve as potent ice nucleators, which they use to force ice formation on the surface of various fruits and plants at about −2 °C^[1]. The freezing causes injuries in the epithelia and makes the nutrients in the underlying plant tissues available to the bacteria.^[2]

[edit] Plants

Many plants undergo a process called hardening which allows them to survive temperatures below 0 degrees C for weeks to months.

[edit] Animals

[edit] Invertebrates

Nematodes that survive below 0 degrees C include Trichostrongylus colubriformis and Panagrolaimus davidi. Cockroach nymphs (Periplaneta japonica) survive short periods of freezing at -6 to -8 degrees C. The red flat bark beetle (Cucujus clavipes) can survive after being frozen to -150 C.[1] The fungus gnat Exechia nugatoria can survive after being frozen to -50 C, by a unique mechanism whereby ice crystals form in the body but not the head. Another freeze-tolerant beetle is Upis ceramboides. [2] See insect winter ecology and antifreeze protein. Another invertebrate that is tolerant to temperatures down to -273 C is the water bear, an extremophile.

The larvae of Haemonchus contortus, a nematode, can survive 44 weeks frozen at -196 C.

[edit] Vertebrates

For the wood frog (Rana sylvatica), in the winter, as much as 45% of its body may freeze and turn to ice. "Ice crystals form beneath the skin and become interspersed among the body's skeletal muscles. During the freeze the frog's breathing, blood flow, and heart beat cease. Freezing is made possible by specialized proteins and glucose, which prevent intracellular freezing and dehydration." [3][4] The wood frog can survive up to 11 days frozen at -4 C.

Other vertebrates that survive at body temperatures below 0 C include painted turtles (Chrysemys picta), Gray tree frog (Hyla versicolor), Box turtles (Terrapene carolina)- 48 hours at -2 C, Spring peeper (Pseudacris crucifer), Garter snakes (Thamnophis sirtalis)- 24 hours at -1.5 C, the chorus frog (Pseudacris triseriata), Siberian salamander (Salamandrella keyserlingii), 24 hours at -15.3 C [5], Antarctic fish such as Pagothenia borchgrevinki and the European common lizard (Lacerta vivipara). [6] [7]

Hibernating Arctic ground squirrels may have abdominal temperatures as low as −2.9 °C (27 °F), maintaining sub-zero abdominal temperatures for more than three weeks at a time, although the temperatures at the head and neck remain at 0 C or above.^[3]

Human embryos can survive cryopreservation at -196 C for 10 years under well-controlled laboratory conditions. [8]

[edit] Cryobiology in Humans

Cryopreservation in humans with regards to infertility is also called vitrification which is a process in which a woman’s eggs are flash frozen in a matter of seconds. Freezing the eggs at such a rapid rate greatly improves their survival rate and prevents ice crystals from forming in the egg that could damage the DNA. Once the couple is ready to conceive the eggs are thawed and through the process of IVF sperm is placed with the eggs and together they are placed back into the uterus. Vitrification has its glitches and is not as reliable as freezing fertilized eggs or embryos because eggs alone are extremely sensitive to temperature. Many researchers are also freezing ovarian tissue in conjunction with the eggs in hopes that the ovarian tissue can be transplanted back into the uterus, stimulating normal ovulation cycles.

Human gametes and 2, 4 and 8-cell embryos can survive freezing and are viable for up to 10 years with cryopreservation.

Therapeutic hypothermia, e.g. during heart surgery on a "cold" heart (generated by cold perfusion without any ice formation) allows for much longer operations and improves recovery rates for patients.

[edit] Applied cryobiology

Cryobiology as an applied science is primarily concerned with low temperature preservation. Hypothermic storage is typically above 0°C but below normothermic (32°C to 37°C) mammalian temperatures. Storage by cryopreservation, on the other hand, will be in the −80°C to −196°C temperature range. Organs, and tissues are more frequently the objects of hypothermic storage, whereas single cells have been the most common objects cryopreserved.

A rule of thumb in hypothermic storage is that every 10°C reduction in temperature is accompanied by a 50% decrease in oxygen consumption^[4]. Although hibernating animals have adapted mechanisms to avoid metabolic imbalances associated with hypothermia, hypothermic organs and tissues being maintained for transplantation require special preservation solutions to counter acidosis, depressed sodium pump activity and increased intracellular calcium. Special organ preservation solutions such as Viaspan (University of Wisconsin solution), HTK, and Celsior have been designed for this purpose^[5]. These solutions also contain ingredients to minimize damage by free radicals, prevent edema, compensate for ATP loss, etc.

Cryopreservation of cells is guided by the "Two-Factor Hypothesis" of American cryobiologist Peter Mazur, which states that excessively rapid cooling kills cells by intracellular ice formation and excessively slow cooling kills cells by either electrolyte toxicity or mechanical crushing^[6]. During slow cooling ice forms extracellularly, causing water to osmotically leave cells, thereby dehydrating them. Intracellular ice can be much more damaging than extracellular ice.

For red blood cells the optimum cooling rate is very rapid (nearly 100°C per second), whereas for stem cells the optimum cooling rate is very slow (1°C per minute). Cryoprotectants, such as DMSO (dimethyl sulfoxide) and glycerol, are used to protect cells from freezing. A variety of cell types are protected by 10% DMSO^[7]. Cryobiologists attempt to optimize cryoprotectant concentration (minimizing both ice formation and toxicity) as well as cooling rate. Cells may be cooled at an optimum cooling rate to a temperature between −30°C and −40°C before being plunged into liquid nitrogen.

Slow cooling methods rely on the fact that cells contain few nucleating agents, but contain naturally-occurring vitrifying substances that can prevent ice formation in cells that have been moderately dehydrated. Cryobiologists are increasingly using mixtures of cryoprotectants for full vitrification (zero ice formation) in preservation of cells, tissues and organs. Vitrification methods pose a challenge in the requirement to search for cryoprotectant mixtures that can minimize toxicity.

[edit] Scientific societies

The Society for Cryobiology was founded in 1964 to bring together those from the biological, medical and physical sciences who have a common interest in the effect of low temperatures on biological systems. As of 2007, the Society for Cryobiology had approximately 280 members from around the world, and one half of them are US based. The purpose of the Society is to promote scientific research in low temperature biology, to improve scientific understanding in this field, and to disseminate and apply this knowledge to the benefit of mankind. The Society requires of all its members the highest ethical and scientific standards in the performance of their professional activities. According to the Society's bylaws, membership may be refused to applicants whose conduct is deemed detrimental to the Society; in 1982, the bylaws were amended explicitly to exclude "any practice or application of freezing deceased persons in the anticipation of their reanimation," over the objections of some members who were cryonicists such as Jerry Leaf.^[8] The Society organizes an annual scientific meeting dedicated to all aspects of low-temperature biology. This international meeting offers opportunities for presentation and discussion of the most up-to-date research in cryobiology as well as reviewing specific aspects through symposia and workshops. Members are also kept informed of news and forthcoming meetings through the Society newsletter, News Notes. The 2008-2009 President of the Society for Cryobiology is John K. Critser.

The Society for Low Temperature Biology was founded in 1964 and became a Registered Charity in 2003^[9] with the purpose of promoting research into the effects of low temperatures on all types of organisms and their constituent cells, tissues and organs. As of 2006, the Society for Low Temperature Biology had approximately 130 (mostly British and European) members and holds at least one Annual General Meeting. The program usually includes both a symposium on a topical subject and a session of free communications on any aspect of low temperature biology. Recent symposia have included long-term stability, preservation of aquatic organisms, cryopreservation of embryos and gametes, preservation of plants, low temperature microscopy, vitrification (glass formation of aqueous systems during cooling), freeze drying and tissue banking. Members are informed through the Society Newsletter, which is presently published 3 times a year. Since 2005, the chair of the Society for Low Temperature Biology has been Tiantian Zhang.^[10][11]

[edit] Journals

CRYOBIOLOGY, (publisher: Elsevier) is the foremost scientific publication in this area, with approximately 60 refereed contributions published each year. Articles concern any aspect of low temperature biology and medicine (e.g. freezing, freeze-drying, hibernation, cold tolerance and adaptation, cryoprotective compounds, medical applications of reduced temperature, cryosurgery, hypothermia, and perfusion of organs).

CRYO LETTERS is an independent UK based rapid communication journal which publishes papers on the effects produced by low temperatures on a wide variety of biophysical and biological processes, or studies involving low temperature techniques in the investigation of biological and ecological topics.

CELL PRESERVATION TECHNOLOGY is a peer-reviewed quarterly scientific journal published by Mary Ann Liebert, Inc. dedicated to the diverse spectrum of preservation technologies including cryopreservation, dry-state (anhydrobiosis), glassy-state and hypothermic maintenance.

[edit] Notes

1. ^ Maki LR, Galyan EL, Chang-Chien MM, Caldwell DR (1974). "Ice nucleation induced by pseudomonas syringae". Applied Microbiology 28 (3): 456–459. PMID 4371331. 
2. ^ Zachariassen KE, Kristiansen E (2000). "Ice nucleation and antinucleation in nature". Cryobiology 41 (4): 257–279. doi:10.1006/cryo.2000.2289. PMID 11222024. 
3. ^ Barnes, Brian M. (30 June 1989). "Freeze Avoidance in a Mammal: Body Temperatures Below 0 °C in an Arctic Hibernator" (PDF). Science (American Association for the Advancement of Science) 244: 1521–1616. http://users.iab.uaf.edu/~brian_barnes/publications/1989barnes.pdf. Retrieved 2008-11-23. 
4. ^ Raison JK (1973). "The influence of temperature-induced phase changes on the kinetics of respiratory and other membrane-associated enzyme systems.". J Bioenerg 4 (1): 285–309. doi:10.1007/BF01516063. PMID 4577759. 
5. ^ Mühlbacher F, Langer F, Mittermayer C (1999). "Preservation solutions for transplantation". Transplant Proc 31 (5): 2069–70. doi:10.1016/S0041-1345(99)00265-1. PMID 10455972. 
6. ^ Mazur P (1977). "The role of intracellular freezing in the death of cells cooled at supraoptimal rates". Cryobiology 14 (3): 251–72. doi:10.1016/0011-2240(77)90175-4. PMID 330113. 
7. ^ Hunt CJ, Armitage SE, Pegg DE (2003). "Cryopreservation of umbilical cord blood: 1. Osmotically inactive volume, hydraulic conductivity and permeability of CD34(+) cells to dimethyl sulphoxide". Cryobiology 46 (1): 61–75. doi:10.1016/S0011-2240(02)00180-3. PMID 12623029. 
8. ^ Darwin, Mike (1991). "Cold War: The Conflict Between Cryonicists and Cryobiologists". Cryonics (Alcor Life Extension Foundation). http://www.alcor.org/Library/html/coldwar.html. Retrieved 2009-08-24. 
9. ^ (Charity Commission for England & Wales No. 1099747)
10. ^ Tiantian Zhang
11. ^ A list of additional scientific societies (mostly using "applied" cryobiology) can be found here.

[edit] See also

• Antifreeze protein
• Cryogenics
• Cryonics
• Cryopreservation
• Cryoprotectant
• Cryosurgery
• Cryptobiosis
• Freezing
• Freeze drying
• Hibernation
• Hypothermia
• Organ transplant
• Perfusion
• Tissue Engineering
• Vitrification
• Ice
• Frost
• Winter
• Insect winter ecology

[edit] External links

• Society for Cryobiology
• Society for Low Temperature Biology
• CRYOBIOLOGY
• CryoLetters
• Cell Preservation Technology
• 43rd Annual Meeting of the Society for Cryobiology in association with the Society for Low Temperature Biology, July 23 - 27, 2006, Hamburg, Germany
• 44th Annual Meeting of the Society for Cryobiology, July 28 - August 1, 2007, Lake Louise, Canada
• 45th Annual Meeting of the Society for Cryobiology, July 20 - 23, 2008, Charlotte, NC, USA
• Cellular cryobiology and anhydrobiology
• An overview of the science behind cryobiology at the Science Creative Quarterly