Cryobiology

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Cryobiology is the study of living organisms, organs, biological tissues or biological cells at biologically low temperatures, typically below the triple point of water. This knowledge is practically applied in cryopreservation and cryosurgery. The word cryobiology (from the Greek words "cryo" = cold, "bios" = life, and "logos" = science) literally signifies the science of life at low temperatures.


The Scope of Cryobiology

Cryobiology comprises the study of any biological material or system (e.g., proteins, cells, tissues, organs, or organisms) subjected to any temperature below normal (ranging from moderately hypothermic conditions to cryogenic temperatures). At least 6 major areas of cryobiology can be identified:

1. Study of cold-adaptation of microorganisms, plants (= cold hardiness), invertebrates, and animals (= 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.

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 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 in 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. Although not very widespread, cryosurgery has its benefits. 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.

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[1]. 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[2]. 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[3]. 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[4]. 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.

Scientific Societies

One of the two leading scientific societies in the field of cryobiology is the Society for Cryobiology. This society 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 November 2006 the Society for Cryobiology had approximately 300 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, this includes explicitly any practice or application of freezing deceased persons in the anticipation of their reanimation. This is the characteristic difference between "cryobiology" (which is a scientific discipline) and "cryonics". 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 2007-2008 President of the Society for Cryobiology is Andreas Sputtek[5].

The Society for Low Temperature Biology was founded in 1964 and became a Registered Charity in 2003 (Charity Commission for England & Wales No. 1099747) 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. The 2005-2006 Chair of the Society for Low Temperature Biology has been Tiantian Zhang [6].

A list of additional scientific societies (mostly using "applied" cryobiology) can be found here.

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).

There is an independent UK based rapid communication journal named CRYO LETTERS 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.

Notes

  1. 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. PMID 4577759.
  2. Mühlbacher F, Langer F, Mittermayer C (1999). "Preservation solutions for transplantation". Transplant Proc 31 (5): 2069-70. PMID 10455972.
  3. Mazur P (1977). "The role of intracellular freezing in the death of cells cooled at supraoptimal rates". Cryobiology 14 (3): 251-72. PMID 330113.
  4. 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. PMID 12623029.
  5. Andreas Sputtek
  6. Tiantian Zhang