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Study of life at lower temperatures is known as cryobiology, majorly cryobiology studies preservation of embryos, cells and their tissues and gametes. It also studies the conditions to be provided for organ transplantation at sub zero temperatures, and this preserving at ultra low temperatures is known as cryopreservation. Cold adaptation, lyophilazation, cryosurgery and physics of ice nucleation are few major areas in the field of cryobiology.
Preserving the organs or tissues or cells at extreme low temperature without letting them becoming ice is called preservation. Usual freezing forms ice/crystals which certainly damage cells and their tissues like blood cells etc. on the other hand Vitrification forms glassy or amorphous solid which never damages living systems.
Two factors are required for successful process of Vitrification
Solutes in high concentration (mixed with cryoprotectants) provided in bathing medium and which can form glassy solid.
Very high rapid cooling
First successful cryopreservation of the mouse embryos by the process of vitrification was done in 1985 (By Rall and Fally)
The temperature -130 0 C is known as glass transition temperature and the temperature maintained in the process of vitrification is -196 0C. Many steps have to be optimized for successful vitrification. Concentration and composition plays an important role in the survival of embryos.
Formation of Ice:
If the ice forms inside the cells, then the cells get damaged, this happens due to embryos get cooled in solution physiologically. In order to prevent ice formation within the cell, the concentration of embryos should be maintained high. As the embryos are bigger in size there are many chances of ice formation inside their cytoplasm. In some cases the cells get damaged even though they are dehydrated due to extracellular ice formation and this is because concentrated embryos occupies unfrozen channel by which embryos become smaller (Schneider et al.1987)
Cryoprotectants like propylene DMSO etc are added to prevent the ice formation. The osmolality of the solution is raised by the addition of cryoproctectants as the unfrozen space is occupied by embryos as soon as ice is formed in the cell. This whole process prevents the ice formation in the cell when cryoprotectant is added.
Effect of Temperature:
Normally cells get damaged when they are cooled below 0 oc temperature (Martino et al.1996).High concentrated fracture plane damages the cell which is present in the solution and also they are damaged during cooling and warming of embryos and also while glass transition state is taking place. This damage during the glass transition phase can be avoided by placing straw in the same phase.
The toxicity of the cryoprotectants should be low, as their concentration is high in this process. And for this purpose ethylene glycol is used as it has low toxicity(kasai et al.1998). The cells are also gets damaged by the osmotic injuries. Before the external ice formation the cryoprotectants helps in dehydration. They also act as protective layers on membranes etc by reducing the effect of salt. They also have ability to penetrate into the cells.
Optimized treatment of embryos is said to have high survival when they undergone vitrification.
Equipments used in vitrification are cheaper when compared to other processes
Process is quite simple; we can do this process by using simple freezers instead of programmable freezers.
It is even simple to get embryos transferred to liquid nitrogen as they can be transferred directly.
Controlled slow cooling:
The process of controlled slow cooling is preserving the cells at lower temperatures without getting them damaged.
Cells are very prone to damage during the controlled slow cooling process. During the thawing process ice formation, toxicity of cryoprotectants and other injuries can damage the cells. While cooling, human oocytes may get effected and mouse oocytes were seen to be effected by the presence of sodium ions aswell.
The results of controlled slow cooling were better than vitrification in case of survival of embryos. If the concentration of cryoprotectants becomes very low then the cell damage will be less than ice formed. Where as, if the concentration of the solution becomes higher, then more damage is seen with ice formation in the cell.
High concentration of sodium cause cell damage
During the process of vitrification, ice formation inside the cells take place but where controlled slow cooling gets effected by higher solution concentration.
Controlled slow cooling successfully cryopreserves the embryos
Controlled slow cooling induces the formation of ice outside by letting the water comes out of the cell and hence there wont be any ice formation inside the cell (Mazur p 1977) this process is done by increasing the concentration of solute.
Cells in this process are cooled very slowly and which enables the water present inside to come out before it crystallizes.
Glycerol, DHSO permeates in the cell by replacing the water present in it, which is then transformed into ice hence by enabling the concentration of the cryoproctectants and decreasing the freezing point . The formation of crystal and its growth is prevented (lovelock j 1954) and the cell surface are responsible for damage of cells ( Mazur p et al 1993)
Very expensive equipments are needed in case of controlled slow cooling
It is slow process.
In order to cryopreserve embryos successfully by using either of the process( controlled slow cooling or vitrification) we should make sure that we maintain proper concentrations of cryoprotectants and are done at their optimum temperatures. The other important aspect is the usage of proper and more appropriate /suitable cryoprotectants should be made.
One of the best cryoprotectants available is ethylene glycerol because of its low toxicity and has high penetration ability when compared with others. It is always a better idea to maintain the temperature at -100 degrees in both of the processes to cryopreserve the embryos.
2. Issues encountered in establishing viable long term storage of cell banks of tissue, cells and cell lines:
One of the major importance of cell lines and tissues is in producing the vaccines. The vaccines are also produced from the diploid fibroblast cells lines. Storing the cell lines has its own advantages and disadvantages. Genetic variations in the cells and contamination are included in its disadvantages. Many cells are now a days stored at lower temperatures, mammal cell lines are one of those which were successfully stored by cryopreservation at lower temperatures. The cells should be maintained making sure that they are free from any kind of contamination. The growth media used should be free from contamination as there are many chances of producing bacteria or fungus on growth media which would damage the cell lines which ultimately leads which to cell death. One of the typical for of contamination is mycoplasma which is related to membrane if cells. It is very tiny in size and it prevents turbidity in colonies. Mycoplasma is very smaller that it cannot be seen under electron microscope hence proper care is need to be taken. The cells in mycoplasma are infected by virus which is very dangerous for the workers in lab, not only that but the virus also affects the cell cultures badly. The virus contamination cause is due to the composition of growth media ( doblhoff et al 1991)
The other issues related to cell bank are:
Survival of cell lines for longer times
Consistent preserving of all samples
Other major issue is homogenizes
Backup should be kept away from the site
Cross contamination etc.
Cell cultures can be stored successfully with the help of cryoprotectants and freezing which results in greater viability of the cells with no contamination of virus in it. Using the above method virus present in the cultures can be identified and taken off the cultures. Serial passaging is good only for limited number of times as longer passaged cells have risk of research and also risk in the manufacture of the product. There are many chances for cells to undergo variation genetically if they are stored for longer time. Other reason for this can be cross contamination due to accidents in labs which ultimately gets infected by micro organisms.
Wrong labeling is also a major issue for scientists, it is usually due to lacking in proper attention( Mac lead et.al). The solution for above problem is careful considerations are to be taken while cryopreserving the cell banks. While producing the stock excess of 3-4 ampoules should be taken in order to prevent the loss of material in laboratory accidents.
It is required to be careful while noting down the growth conditions, full details of the medium. To determine viability and sterility and even mycoplasma presence quality control test is required (cord et al 1992). In order to avoid the issues of quality, the samples are required to undergo control tests which are of 4 kinds:
TEST1, TEST2, TEST3 and TEST4
Test1 checks the viability using tryphan blue dye, Test2 checks viral antibody response in animals, Test3 is bio-activity non viable test. And Test4 shows the growth characteristics.
Cells should be thawed in cell culture when they are recovering and cryopreserved. During the preservation at very low temperatures there are many chances of getting contamination and they should undergo proper temperature cycles. Labeling of vials should be carefully done at room temperatures. Warming may occur even while they are stored in storage containers as they are also good conductors of heat. Vessels are needed to be checked and filled properly in order to keep the cells alive. Environment also causes the micro organism contamination ( Fountain et al. 1997)
Natural radiations plays an important role in mutations occurring in preserved cells(Glenister et al 1984). A very high amount of nitrogen is also lost by the large lid vessels. The cycling temperature has an important role in production of sentinel banks as they are used to track the cell growth in the storage vessels( Stacey 1998)
All the cells and cell lines are preserved for future production of vaccines by following few guidelines:
Sterilization and TSE contamination/cell-media virus
It should be always made sure that cells do not loose genetic stabilization.
One of the major problems in preserving the cell lines is associated with liquid nitrogen as it acts as carrier for many types of contaminations like bacteria, virus, fungi etc (Schafer et al 1976). The submerged lids of plastic tubes are also one of the cause for infection. The contamination was found to be similar in both the cases( freezing and during culture media). Hence LN2 can be said to be the source for contamination when cryopreserved ( Prince et al 1995)
cryopreservation has many applications in storage of various organs like cells and tissues, and organ preservation. cryopreservation has many applications in the fields of biotechnology, biomedicine, animal reproduction and various organs conservation.
Cryopreservation is again the best option for storage of hepatocytes, using the help of this technique sufficient and permanent cell supply can be achieved.
in biomedical sciences cryopreservation has a great use in storage and transport of large organs like kidneys and hearts but these can be stored only for short times but where are blood and other cell suspensions can be stored for very longer times at -196 degrees in liquid nitrogen. frequently infertility treatments are done using the cryopreserved eggs and embryos of human beings.
cryobiology has much importance in animal reproduction aswell