Cooling And Vitrification In Cryopreservation Of Biological Materials Biology Essay

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In the world of science this two procedures Vitrification and Slow cooling are use to keep the biological materials such as cells, bone marrow , DNA etc at the low temperature , when compared to their normal temperatures. These two procedures will come under the Cryobiology.


It is the study of life below the low temperature.


In the centuries 2500 BC this was used by the people of Egypt for the medical purpose. They used to stop the bleeding and injuries during the injuries. In the latter centuries this was brought into popular by Robert Boyle. For the first time it was the Christopher Polge who used the bull sperm in cryopreservation. The 1970's brought great development in cryobiology by Zeo Layland who brought Slow Cooling technique which laid a path to the birth of first human embryo frozen, which latter used all over the world for the animals, cells and human biology. In the year 1986 Dr. Christopher Chen in Australia used the slow frozen oocytes for the pregnancy in the world for the first time.

Advantages of cryobiology:

Helps in the preservation of biological materials.

By this the biological materials can be preserved for long time.

Sperm, gametes, embryos, tissues, bone marrow, organ can be preserved.

Helps to study the adapting nature of plants and animals under the low temperature.


This is the process, which come under the Cryobiology. This is the process in which the cell is kept under the very low temperature which causes the cell to stop its biological chemical reactions and finally the cell leads to death. But sometimes the cell which is kept under the process of cryopreservation may get damage, when it is taken to the low temperature. Some of the biological materials are kept under very low temperature which is the liquid phase of the liquid nitrogen. Because it is the best process for the preservation some complex biological compounds which lead to stop their biological chemical reactions. In order to be free from the risk the most two techniques used are the Slow Cooling and Vitrification.


James Lovelock is the important person who made the Gaia theory fame. Using this theory he said that the damage that occurs to the red blood cells is due to the osmotic stress during the process of the freezing. In the early years of 1950s he said that when the cell faces the increase of salt concentration make it to dehydrate for the loss of water to the external ice which may cause the damage of the cell. In the year of 1950s they are rapid development of the freezing techniques which made helping in bringing the pregnancies. Before this the insemination of frozen sperm was brought into live. Latter in the 1957 the scientist of the United Kingdom started the cryopreserving the fowl sperm.

In the year of 2000s the baby was born by the cryopreservation egg, Laina Beasley born in July 2005. Not only in the human beings, this is brought into the animals which made to the result of A Ocelots kitten born in Cincinnati Zoo in 2001. As freezing damage in the cells are of two aspects. The primary one is that cell gets damage due to the ice crystal, and the second is the damage of cell when more ice is formed due to the concentrate of the solute. Latter in the USA they made a solution for this aspects of the damage in cell by the typical rate of cooling 1C/min but this rate of cooling depends on the size of the cell and the water content in the cell. In this they are a form of anti-freeze known as the cryprotectant which is used to equalize the physical optimal parameter osmotic. Cryoprotectants have ability to protect the cell to face the freezing injury which was discovered accidentally.


When the biological materials are kept under the preservation they are need to be protect for the long time. At same time the protected material should be able to function for a long time when they are rewarmed to the sub zero level. During the process of the preservation some chemicals are used to preserve them in low temperature and in the same way they are rewarmed, and should have the ability to function for a long time. But in some cases of preservation chemicals are not used such as in fungi, yeast. The cryoprotectants are used in this cases, now a day's some chemicals like dimethyl sulfoxide, glycerol. But in some of the specimens the dimethyl sulfoxide affects the preservation due to the toxicity nature. (Smith, 1983) This toxicity can be reduces to some level by use of glucose

Advantages of Cryoprotectants:

Helps the material from rapid cooling

Prevents from formation of ice in the intracellular region.

When the cell undergoes high concentration of solute it helps to prevent from dehydration (Mazur, 1984).

Helps the cell to function even after the rewarming.

Slow cooling:

This is the early technique used in the cryopreservation which is used to prevent to the cell from the damage in the freezing



It is the control rate technique which was developed in the 1970s which has been enabled the first human embryo birth. From then this technique is used all over the world for the biological materials. And some machines which are used in the cryopreservation bring the cell to the freezing point such as the liquid phase of the liquid nitrogen. This technique machines are used to freeze the oocyte, blood products, sperms, skin, embryo, general tissues and stem cells preservation in research labs, hospitals all over the world. But in the slow cooling the cell gets dehydrate


This is the new technique used in the cryopreservation which is used to prevent to the cell from the damage in the freezing. It is the preservation at extremely low temperature without any freezing. In this process can be done without the involvement of the cryoprotectants.


Right from the development of the slow cooling the glycerol is used to cryobiology as the cryoprotectant for the bull sperm and blood cells. But however it is know that glycerol is not helpful to prevent the whole organ from the damage. For more suitable cryoprotectants in those cases many of the biotech companies worked to develop. In the 21st century the kidney of rabbit is preserved at -135oC, which made as the vitrification cocktail, because latter the kidney which is preserved at the -135oC was again planted back into the body of rabbit, the kidney was found to be functioning without any failure. At present preservation of the brain is under the progress, they are looking to prevent the brain from damaging such as damage to the tissues and loss of the memory in the brain which was encoded.

The Institute of Cryonics are working to preserve the whole body without damage in the cells, tissue and all the organs which should again function properly when they are transplanted, this is in the progress. In this the freezing involves in ice crystal formation, which lead to the damage of the sensitive structures such as the blood vessels. For a successful vitrification it needs combinations of the two factors, one is the high concentration of solutes in the bathing medium capable of glass formation, and the other is the extreme rapid cooling of the samples. In the year 1985 for the first time the cryopreservation of mouse embryos by Rall and Fahy. Steps that to be followed for the successful vitrification are

concentration and composition of the vitrification solution

The procedure used to equilibrate cells in this solution

The cooling/warming conditions

The procedure used to dilute cells from the vitrification solution

Freezing injuries:

In a living cell the liquid water is most important to maintain its structure and function, when this cell is kept in the freezing preservation, due to the low temperature then to its survival then the cell faces the freezing injuries which may lead the living cell to destruction. When the cell is under the preservation the injury that effect is shown in the figure the inverted 'U' in this the position of the cell which it can function normally is shown as the survival point , when this cell is put on to the freezing beyond its limit, that is a cell has its own capability for a certain limit of low temperature or high temperature, when this cell exceeds the limit of low temperature the solution around the cell makes it injury, in such cases the intracellular ice formation will be occurred, at this stage the cell leads to the injury and destruction occurs. In some cases like the high cooling rate the cell undergoes both the extracellular and intracellular.

Freezing injuries at high cooling rate:

When we take most of the cells they have the thermodynamic point at -0.5oC. But when we need to preserve the cell the cell must get freeze, to do this the cell will be undertaken below -5oC. At this position the cell undergoes the super cooling at which the medium around the cell and the cell remain unfrozen, due to the protective solute that is bounded around and within the cell.

The cell which is taken to the low temperature between the -5oC and -15oC the ice forms in the external medium. At which the cell content remain super cooling in an unfrozen state. The ice which is formed in the external medium will affect the extracellular solute. The solution concentration in the extracellular solution will increase when the temperature gets decreases and the ice will be grown, this increase of ice is the ice phase. Due to this the chemical imbalance is occurred between the biological material and the unfrozen external solution.

The external part of the cell gets frozen when the water flows off, this occurs when the higher chemical potential then the water of the partly frozen solution outside the cell. And this subsequent physical event in the cell depends on the rate of cooling in the cell. If the cooling is sufficiently slow, the loss of water rapidly by exosmosis. When this occurs the result of the cell will dehydrate and will not freeze intracellular. TZ p3

When the cooling is too rapid the rate at which the chemical potential of water extracellular solution decreases is much faster than to the rate which water can be diffuse out of the cell and they will be the end result in the intracellular ice formation. In the shown figure the cell under the preservation will have the outflow of the intracellular water which may lead to shrink of the cell and the extracellular ice will be formed which leads to the shrunken cell with little or no ice formed internally. It is the indirect assumption that the formation of the ice inside the cell is unpreventable. At present many of the studies have been suggested that intracellular ice formation during the process of the freezing causes the death or damage of the cell. In the process of the intracellular ice formation they are three possible ways which it can be occurred.

Chilling injuries:

Chilling injury is defined as the low temperature stress on the absence of freezing. Actually the word chilling injuries is used in the botany, in the early 18th centuries to describe the plants which are subjected to the low temperature that is chilling temperature above the 0oC were often damaged irreversibly. The temperature shock was first used in 1934 to show the irreversibly damage to mammalian sperms that occurred when these cell undergo rapid cooling below the body temperature at which few degree fall down rapidly in a minute of time. At these both sperm cells and the plant cells the chilling injury are similarly related mechanism. In the process of chilling injuries they are two type's direct chilling injury and the indirect chilling injury.

Direct chilling injuries:

This is also known as the Cold shock. This is mostly used to describe both phenomena, which is expressed quickly upon reduction in temperature and Dependent on cooling rate. Cold shock injury is almost independent of the rate of warming. Injury is increased as the period incubation at the reduced temperature is extended.

Indirect chilling injuries:

Indirect chilling injuries are usually evident following a relatively long exposure period at the time of the reduced temperatures, and its enable to the independent of the rate of cooling.

Metabolic and enzymatic processes can find in the fast developing embryos. Especially in Drosophila and zebrafish the injury get more rapid at the low temperatures. This is due to the co-ordination is increased lost with decreasing temperature. The reduction in temperature will affect the enzyme rate reaction to a different extent.


In the process of preservation both the techniques have the similarity of freezing during the process of preservation.

In slow cooling the cooling is done intracellular and extracellular and in the same way in vitrification, but little change at place where ice crystal formation is occurred in slow cooling and not in vitrification

Somehow both techniques are similar with slight changes during the process of the preservation of biological materials.


Vitrification techinque

Slow cooling technique

This is simple technique

This is complex technique

This safer technique

This is risky technique

This more costly technique

This cheaper compare to vitrification

Ice crystal don't form in the process of freezing

In this ice crystals formation is seen

This is most successful technique

Not much success then vitrification

Cell death will not occur

Have the chances to the cell death


In the cryopreservation the both techniques vitrification and controlled cooling techniques are used to preserve the biological materials for a long time. Vitrification technique has the uniqueness for the preservation of the oocytes, because the oocytes brought under this technique have more capable to the fertilization. This oocytes lead to the normal pregnancy. In process of the vitrification the ice crystal formation is not occurred both in the intracellular and the extracellular. In vitrification the whole cell including the medium solidify (freeze). In the process of vitrification the cell doesn't get any damage and don't lead the cell to death (Kasa, 2004). The preservation of materials at a controlled slow cooling, we can store the materials at -196oC, best example is storage of hematopoietic cells (Hill et al., 1972). The main advantages of cooling and warming rates are that it contains very less amount of cryoprotectants, with this it can reduce toxic effect and also osmotic injury (Orief et al., 2005).


When we come to the vitrification we don't face any unfavarable conditions during the process of preservation, because of cryoprotectants which toxic in nature and more cost(Chi, 2001). Ice crystals are occurred in the intracellular and the extra cellular region of cell in the process of preservation in slow cooling technique. This is the major disadvantage in controlled slow cooling. (Kasa, 2004).

Main Outcome Measure:

As per the reported number of pregnancies done after transfer of embryos which were cryopreserved by vitrification. Both slow cooling and vitrification procedures have successful cryopreservation of human embryos and oocytes. Both procedures have healthy births, but slow cooling of oocytes gives very low success rates. Vitrification is a promising novel technique in reproductive technology


As per the reference and my knowledge controlled slow cooling and as well as vitrification are useful techniques for the preservation of biological materials, when compared vitrification technique is more useful technique for the preservation as slow cooling technique. Vitrification is a simple procedure that requires less time, safer and more cost effective than slow cooling.