It is the part of biology which studies the effect of life at low temperatures. Cryobiology is derived from Greek words 'cryo' means cold; bio's means life, and logos means science. Not only has that cryobiology meant study of biological materials at below normal temperatures. Biological materials which include organs, tissues, cells, proteins
They are majorly six areas of study of cryobiology;
1. Study of microorganisms, animals, plants at cold adjustment.
2. Cryopreservation of cells, tissues gametes for durable storage.
3. Organ preservation at extreme conditions for transplantation.
4. Freeze-drying (Lyophilization) of drugs.
5. Therapy of unusual and ailing tissue by using severe cold conditions.
6. Usage of physical processes and mechanical engineering aspects through the course of chilling and warming.
HISTORY OF CYIOBIOLOGY:
In the year 1683 there was first documented Cry biological study by Sir Robert Boyle who documented the effects of freezing temperatures on living animals in his monograph "New experiments and Observations touching cold"
Figure:-1. Robert Boyle.
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In the year 1851 Dr. J Arnott used frozen water to successfully treat of headache, acne, and application on cervical cancer provided immediate relief from pain.
Figure:-2. Cancer cells.
In the year 1972 embryo of mouse was successfully frozen, live young were produced from the frozen embryos.
APPLICATION OF CRYIOBIOLOGY IN VARIOUS ASPECTS:
1. In Biotechnology: It is used in the preservation of samples like cell stocks, clones, cell conservatives and biological materials for longer duration of time with minimal contamination.
Figure:-4 Preservation of cells.
2. In Medicine: Used in the preservation of species, embryo's like Humans and Mouse.
Figure:-5 Human embryo.
3. In Cryonics: They are trying to preserve the whole human body used for future technologies that can cure the patient.
For example a well known patient former baseball player Ted Williams, since dead in 2002 held over in liquid nitrogen at Alcor Life Extension Foundation in Scottsdale, Arizona. William's son, John Henry Williams, hopes that future advances will restore him.
Figure:-6 Patient picture
It is nothing but storage of cells and tissues by freezing for longer duration of time. It deals with not only simply preserving, storage of organic material at excessive down temperatures (typically at-196o C it is temperature of liquid nitrogen). From the ancient time, this technique has made important in the field of human in vitro fertilization (IVF), livestock improvement and conservation. Cryopreservation is used mainly in captive breeding programmes, and mainly in genetic source banks for which species are endangered in both animals and plants.
The major breakthrough was reported in the 1947, which granted a major leap for advancing in the field of cryobiology (David, 1947).
APPLICATION OF CRYOPRESERVATION:
1. The vital purpose is freezing and storage of hematopoietic stem cells, which are majorly found in the bone marrow and peripheral blood. The stem cells are collected from the patient's bone marrow for the medication by means of high dose chemotherapy. After completion the patient's cryopreserved cells are thawed and are re infused into the patient's body (David, 1947).
2. Cryopresevation of sperm cells for the further use of treating of malignancies and non-malignant diseases, as well as premortem and non post-mortem cryopreservation. Not only that the cryopreserved sperm cell of men with obstructive azoospermia can yields pregnancy rates similar as the fresh sperm. Mostly the cryopreserved cells are recommended for complicated reconstructive cases with low pregnancy rates which include vasoepididymostomy. (Jenifer et al,. 2003)
SUCESSFUL CRYOPRESEVATION PROTOCOLS:
Characteristically there are mainly five major steps of cryopreservation protocols of cells:
1. Fraternization (mixing) of cells with a CPA(Cryoprotectants).
2. Chilling them to subzero temperatures conditions.
3. Storing the cryopreserved cells in liquid nitrogen.
4. Ejection of CPA.
The above events are done by sequence and inter associated with chilling and warming up and are associated with CPAs. (Fuller et al., 2004)
Cryoprotectants are constituents which are used to protect biological tissues from glacial destruction ( this means damage done by ice formation). For example insects, fishes, reptile's amphibians which leave in Arctic also Antarctic create their own cryoprotectants such as antifreeze compounds and proteins in their bodies to shorten their damage during cold winter periods. Some insects often use sugars or polyols (it is an alcohol containing multiple hydroxyl groups) as cryoprotectants. (Meryman, 1970)
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1. DMSO (dimethyl sulfoxide): It is an organo sulphur compound which is used as cryoprotectants .
2. Ethylene glycol: It is used as anti cooling agent in cryopreservation of cells.
3. Propylene glycol: It is used as re crystallization which occurs quickly in tissues cryopreservation.
Cryoprotectants are mainly classified into two different types:- Those which pass through plasma membrane and other those that don't pass through into the plasma membrane. Those which pass through the membrane protects the cells during the cryopreservation, by depressing the temperature in intra cellular ice forms, and by stopping the unwanted cellular shrinkage and are behaving like secondary solvents for salts.(Adams, 2004).
ROLE OF CRYOPROTECTANT IN VITRIFICATION:
1. During vitrification mainly the plasma lemma (cell membrane) is the exact site where most of the damage is done. Here cryoprotectants play an important role like preventing ice formation, anti freezing not only that, it protects the cell membrane (Conlon, 1998).
2. Cryoprotectants play an important role in virtification it has the capability of reducing the toxicity of the solution and to achieve the necessary concentrations for successful virtification procedure. It has the capability of reducing the temperature in the virtification solution (Santarius, 1988).
ROLE OF CRYOPROTECTANT IN CONTROLLED SLOW COOLING:
During the process of slow cooling the cytoplasm does not get super cool as the same extent the cell slowly gets dehydrated, by adding the cryoprotectant there will be relatively loss of water at a given cooling rate (Fuller, et al., 2004).
Vitrification is defined as conservation of biological materials at extremely cold condition without freezing (Ken and Janet Storey, 1990). Almost all living organisms are composed of large amounts of water, freezing of these microorganisms' results in ice-formation. Insects most often used sugars for cryoprotectant, Vitrifying liquid have been classified as strong or fragile. The tendency to break under mechanical stress, but rather to a highly rapidly rise in viscosity as temperatures approaches from above.
If cooling causes virtification, in practice, vitrification can be divided in two factors based on the high concentrations of solutes which have the capability of glass formation. And the rapid cooling of the sample used for the vitrification. Successful vitrification requires certain steps.
They are mainly based on two components Concentration and composition of the vitrification solution. Vitrification is a process in which liquid starts to perform as a solid during cooling without any periodic change in molecular adjustment or thermodynamic state variables. (Guangzhou, 2009)
APPLICATION OF VITRIFICATION:
1. In Human Embryo freezing vitrification plays an important role for embryo transfer must be cryopreserved for further transfer in the future (Gabriela, 2000).
2. Vitrification will be an alternative method for embryo freezing in the future. The cryoprotectant was removed by washing the embryos in the media which contains two different concentrations of cryoprotectant (Gabriela, 2000).
The term chilling injury is used to characterize to damage of cells which occurs at temperatures above the freezing point of the cells. Direct chilling injury is also known as cold shock occurs immediately upon cooling, whereas in direct chilling injury occurs as a series of temperatures at a period of time. Chilling injury is irreversible depolymerisation of actin and tubilin filaments of the cytoskeleton which in turn affects processes such as cell division.
They are mainly two methods that are generally used to freeze cells. Cooling of cells too slowly can also be damaging. Slow cooling can expose cells to damage in the extracellular and intra cellular solute concentrations. Solute may become so concentrated which can precipitate from solution which, in turn, can use pH fluctuations.
FIGURE: - 7 above diagram showing the physical events occurring in the cell during cooling at different rates. At temperatures around -5o C, ice forms in the extra cellular solution. The build up of solutes outside the cell causes water to exit. At slow cooling rates, water continues to exit the cell and cell dehydrates before eventually vitrifying internally. At faster rates, water loss cannot occur fast enough and so water freezes internally, effectively killing the cell. At ultra rapid rates, small innocuous ice crystals may form, or the cell may be vitrified (Mazur P, 1984).
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1. How susceptible the cell is to chilling injury or cold shock.
2. The size of cell.
3. The fraction of the cell which is opposed to solids.
4. How permeable the cell is to water and cryoprotectants, how temperature affects these permeability's
5. The temperature at which intracellular ice forms in the cell.
In general cells which are large and have a lower surface of volume ratio will have a lower optimal cooling rate than small cell (Mazur P, 1984).
It is a process in which solidification of water which means ice by freezing is usually destruction of viable cells. When we consider freezing is the natural phenomena, there will be surprising to the extent to which is neglected as a subject for basic investigation: the use of freezing to preserve cells and tissues for a variety of research and clinical purposes (Wolfe and Steponkus, 1983).
Freezing is often deadly, on which scale of organs in the formation of ice can impact majorly in the mechanical damage by expansion or rupture by formation of ice crystals which grow through the tissues. They have very few recent reports of cells surveying IIF - Intracellular ice formation (Wolfe and Steponkus, 1983).
FIGURE: - 8 Schematics of proposed freezing injury mechanisms associated with eutectic crystallization. When cell suspension freezes at slow cooling rates, ice forms in the extracellular space first. As the cooling continues, the cell are dehydrated due to the elevated concentration of the unfrozen fraction for eutectic crystallization and temperature of the extracellular solution are crystallized and form the eutectic phase. This can cause mechanical damage directly to the cellular membrane, and/or it can propagate into the intra cellular space across the membrane through pre existing pores or openings formed by the mechanical damage (Hana B and Bischof JC 2004).
SIMILARITIES BETWEEN CONTROLLED SLOW COOLING AND VITRIFICATION:
Both cooling and warming are the methods which are generally used in vitrification and controlled slow cooling. The formation of ice inside the cells is generally considered lethal. When we take the case of vitrification this involves cooling cells at rapid rates that here will be no sign for water molecules to reorganize themselves as ice and instead a glassy or vitrified state is achieved.
In the case of controlled slow cooling there will be formation of intracellular ice must be avoided to ensure cell survival. The cells remain un frozen and super cooled because the cell membranes is going to act as cell barrier in which effectively prevents the relationship between the extra cellular ice and the super cooled intra cellular solution.
When we take the case of warming in vitrification there will be a rapid enough to stop the de -vitrification this means the glassy state changing to ice during warming and the formation of re-crystallization means small ice crystals aggregate to form damaging crystals. In controlled slow cooling also there will be similar damage as slowly there will damaging by exposing the cells to solute concentration (Adams, 2001).
DIFFERENCES BETWEEN CONTROLLED SLOW COOLOING AND VITRIFICATION:
The main difference can be seen in the formation of ice crystals this can be avoided in by reducing the fluid content. This can be seen in both controlled slow cooling and vitrification (Vanderzwalmen et al., 1992).
ADVANTAGES AND DISADVANTAGES OF CRYOPRESERVATION OF BIOLOGICAL MATERIALS:
Advantages using vitrification:
1. Cryopreservation of cells like sperm cells and red blood cells of bulls are successfully cryopreserved by means of vitrification by using glycerol as cryoprotectant (Bratton, 1949). From that time large industries have emerged in producing the cryopreserved of bull sperm and red blood cells.
2. By using DMSO (dimethyl sulfoxide), glycerol and propylene glycol human embryos have been cryopreserved by using vitrification.
3. One most important advantage in vitrification is it can form ice crystals in both cooling and warming. Not only that there will be highest number survival rate and viability of human embryos after thawing and can be cryopreserved (Orief et al., 2005).
Disadvantages of Vitrification:
1. One most important limitation in this method is toxic effects which are caused by high concentrations which are produced by cryoprotectants in liquid- mediated contamination (Orief et al., 2005).
2. Basic disadvantage is cost effective this means it uses the most costly equipment for the cryopreservation and dangerous of handling the equipment because it is stored in liquid nitrogen at very low temperature approximately -196o C (Macfarlane et al., 1990).
Advantages by using Controlled slow cooling:
The main advantage of controlled slow cooling there will be temperature rising approach will be similar in practical system. When we compare the potential advantages the curve deviates significantly (Wild et al., 1982).
Disadvantages by using Controlled slow cooling:
This is not true in for some specialized or large cells such as mammalian Oocytes which require even slower rates of controlled slow cooling. Not only that it is hard to maintain the low rate of cooling through the equipment because the temperature is maintained accurately and controlled by means slow cooling (Ramsay, 1955).
As per my knowledge I conclude cryobiology plays an important role in future preservation of various species and biological materials. By using various techniques like cryopreservation we can develop and preserve the cells by using vitrification. Cryoprotectant plays an important role vitrification but since in some cases it acts in toxic nature in vitrification. Controlled slow cooling is also used for cryopreservation.