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The cell or the embryo when they are exposed to very low sub level temperatures they will under suffocation which results in the physical and the chemical imbalance inside the cell. And finally the cells or the embryos rapture due to the formation of intra and extra cellular ice which are developed by the extremely low temperatures. No matter if the cells are dropped to the sub zero level by slowly cooling the cell or treating the cell to the sub zero temperatures by sudden drop of the temperature the cells will die because they are not tolerant to the extreme cold temperatures so we cannot store living tissues, embryos and oocytes alive in the cold temperature. This process can be achieved through inducing the cryopreseravatives into the cell, by replacing it with water which is readily present inside the cell. These cryopreseravatives will help the cells to transform into the glassy or amorphous state so that cell resist from the crystalline ice formation which helps the cell from rupturing and to store them alive for ages. This process can be done through two methods they are controlled slow cooling and another is through vitrification. With the help of these processes we can be able to cryopreserve the animal oocytes and the embryos and store them in the banks for the vitro fertilization or to conserve the germ line of the species.
Controlled Slow Cooling
The oocyte will undergo chilling sensitivity and osmotic instability when they are cooled to the sub zero temperatures. The cell in the oocyte or embryo can be effect with the temperature stress starting from the zero degrees centigrade. The cryopreseravative is designed such that the cooling process will takes place inside but cell will be viable and damage is nullified. The cryopreseravatives depend upon the type of embryo and the oocyte. For example the fish embryos are very highly sensitive to the chilling injuries especially zebra fish because they contain yoke in their embryo. The fish embryo is about like 1 - 3mm in diameter and it is more than 1000 times larger than the mammalian embryos.
And the embryo is well protected by the thick cell of blastoderm which is surrounded by Chorion which is a protective membrane. The special thing about the fish embryo and the oocyte is it can tolerate the sub zero level temperature up to - 35°C.
When the cell is cooled down to the 0°C it will affect the different aspects of the cell it affects cell metabolism, it transport system in cell and ionic transfer. During the cooling period from 0°C to -20°C the ice crystals will start producing which will make the solute medium present around the oocyte will turn more concentrated so the water present inside the cell due the concentration difference occurred in the medium. So the cell will begin to shrink due to the dehydration caused by the water loss inside the cell which can lead to the cell death.
The control rate can be maintained by avoiding the bathing medium and the sample is introduced at the temperature at which the extracellular ice is will start forming with the help of the super cooled solution to avoid the sudden change in the temperature.
The controlled slow cooling process will be on the based on the type of embryo or oocyte selected the optimal conditions of the cells must be studied and the they must be free from the contamination and any type of the physical damage to the embryo or oocyte. According the above mentioned conditions taken into the consideration we must design the cryopreseravatives and determine the optimal concentration on the cryo medium. The average amount of the embryos that are alive for the take sample is also must be estimated to prevent the possible cell loss during the process and finally the pH must be optimised and must maintain the pH without changing.
The apparatus used in the controlled rate slow cooling is a unique pre programmable freezing system to which the liquid nitrogen [LNâ‚‚] is circulated through its cooling chamber to achieve the super low cooling temperatures. Its cooling rate will be normally -1 to -3°C per min the max. Cooling rate will be like -5 to -10°C per min. And on the other hand we will have a -80°C freezer which we can use to place the sample in it.
The selection of the cryoprotectant will be based on the embryo penetration ability and its dehydration properties. According to it the toxicity of the cryo protectant is judged and it is serial dilute to avoid the osmotic shock to the embryo. Based on mainly on the cell viability the freezing protocol is developed on the each stage of the process. The viability tests will be different for different materials which includes the metabolic activity, cell division and staining of the cell.
After these steps the embryos are subjected to the low temperatures -100°C by using controlled rate slow cooling technique by feeding the optimum temperature date to the computer and the cryoprotected embryo or oocytes are placed in the equipment and the remaining process is automated by the machine. Thus the embryo or oocyte is sent to - 100°C and turned into glassy state without intra cellular ice formation.
Vitrification consists of two main things they are high concentration of solutes and extreme rapid cooling of the sample. The vitrification process depends upon the cryopresevatives they must be capable of dehydrating the embryo before the ice formation they must try to nullify the water activity in the cell. It must have the ability to penetrate into the cell of the embryo or the oocyte with good dehydration properties, but the concentration of the cryoprotectent is very high.
Barry, J. Fuller, et al. (2005) Life in the frozen state Dawsonera [Online] Available at: http://library.beds.ac.uk (Accessed: 08 February 2010)
Barry, F. and Sharon, P. (2010) Week 9 Controlled slow cooling [Online]. Available at: http://breo.beds.ac.uk (Accessed: 20 January 2010).
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Julia, K. et al. (2006) 'Preliminary study on modification of yolk sec of zebrafish embryos using microinjection' 27(5) pp 319 - 328 PMC [online] Available at: http://www.ncbi.nlm.nih.gov (Accessed: 08 February 2010)
Fre´de´ric, D. et al. (2004) Cell Size and Water Permeability as Determining Factors for Cell Viability after Freezing at Different Cooling Rates 70(1) pp 268 - 272 PMC [online] Available at: http://www.ncbi.nlm.nih.gov (Accessed: 08 February 2010)
(2) Different issues encountered in the establishing viable long term storage
The cell lines of the animal are most important for the in vitro techniques for the production of the vaccines and many other things; they are the important substrates of the critical biological assays. The mammalian cell lines are generally can be cryopreserved and they are stored in the cell banks in the ultra low temperatures so in order to protect them, banks will be regularly searching for the infectious agent and to prevent them so that they can preserve the cell lines safely for the other application like producing vaccine to the disease etc. The cell banking procedures will be carefully looking after the key issues in the safe preservation of the cell lines of tissues.
The will maintain the principles of cell banking which are mainly associated with safety and quality controls associated with some regulatory guidelines. In which it includes the establishment issues, maintenance issues, patent applications, public collection for the research and development.
ESTABLISHMENT OF THE CELL BANK PROCEDURE
In the preservation of the mammalian cell lines it involves very critical protocols before transferring to the ultra low temperature storage. In the preservation of the animal cell culture initially one must take the validate procedure in the culture the first step is the toxicity testing of the cryoprotectent and condition for the cryoprotectant profile must be investigated. The cell line profile is studied very deeply and known its optimal conditions and the temperatures which are used for the cell condition maintenance when it undergoes cryopreservation. Choose the unique technique which is standard and works well with the type of cell line.
ISSUES ENCOUNTERED IN THE LONG TERM STORAGE
They are many lethal potential hazards are involved in the long term storage of cells in the low temperature the main is contamination which is caused due to the variation in the temperature in the storage banks. This by the routine access into the storage room this may make the cell or the cell lines temperature fluctuations. The frequent access into the storage room because of the temperature variations will take place in the vessel containing the cells, so the individual storing system must be completely withdrawn and the storage can be opened only after the temperature is stabilised.
The storage vessels must also be good conductors of heat because the storage vessel that we are using to store the cells will also play the important role in the storage. If the storage vessel itself is not good means there is a possibility of huge amount of loss in the product and the money. The storage vessel also must be properly arranged and filled correctly. These may look like a silly thing to study about but any kind of mistake is done in the process of storing will result in the great amount of loss that one cannot expect, so we must be able to take care about every aspect.
The microbial contamination will occur mainly through the surrounding environment and it is its main source so long term storage vessels will be better if we clean the surrounds regularly and the substance which is accumulated under or surface of the storage vessels and the storage boxes must be properly sealed. And the room surrounding must be disinfected is important for the prevention of the culture contamination. The cell viability can also be lost by the natural radiation that is present which may sometimes also cause mutation in the cells and the tissues. The storage vessel must away from the radiation even thought there are is no affect on the long term cell storages as they are stored in the nitrogen vessels but the safety precaution must be taken to avoid the mutation to take place. It also depends on which type of the cell or the tissues that we are storing as there some of the tissues which are highly sensitive such as embryos.
The banks must be well equipped by the help of the 24 hrs temperature monitoring system which help in the regulation of the temperature all the time. And also with the liquid nitrogen level monitoring system which are useful in the data monitoring and providing us with the critical analysis all the time so that we can identify the emergency cases previously and resolve the problem before it is out of the hands. The data filling and the cautions maintenance procedure must be maintaining very strictly in the regular bases. Even the electricity is one of the important factor here and it is highly important must be checked thoroughly.
During the first establishment of the cell line there different aspect that we need to take care of one of the main thing is to protect them from the possible loss of the culture. And also must achieve reliable supply of the culture. The bank must be as a well working system and must securely take care of the every possible aspect in the bank system so that the storage must be in the good condition without any contamination. Any time of small defect in the system can lead to the harmful threats and also can lead to the huge amount of loss. Must readily able to provide the valuable information to the research and development purpose and the development depend on the preserved cell lines or the tissues.