Cryopreservation is a biological tool sustaining the viability of cell suspensions (cells, tissues, embryos,) for a long period of time at a very low storage temperature-196°C with the help of a cryoprotectant. The chemical substances which are used for cryopreservation are called cryoprotective additives (CPAs). cellular damages occurs in the absence of cryoprotectives in cryopreservation process. The techniques to prevent cryopreservation damages are the combination of two processes namely controlled slow cooling and dynamic process vitrification by which living cells, embryos and oocytes can be stored . both of techniques of cryopreservation, controlled slow cooling and physical process vitrification, the glassy state is essential which occurs at temperature below -100ËšC.This temperature is called the glass transition temperature at which the water becomes highly cooled .This temperature is below -100ËšC and near about -130ËšC . The controlled slow cooling may lead to the intra cellular lattice ice formation, due to these there are high chances of freezing injury and thaw injury. These injuries finally results into lysis ,of the cell (Rawson, 2009). Cryopreservation has major applications in the fields of infertility treatment, animal reproduction of their gametes, embryos of various amphibians, reptiles, Pisces and other domestic and non domestic birds, endangered animal species to many extents which are in cryo banks (Zhang, 2011)
Cyroprotectants (CPAs)
The chemical substances which are used for cryopreservation are called cryoprotective additives (CPAs). Cryoprotectives are of two types namely penetrating and non-penetrating, penetrating CPAs are small, can easily diffuse into the plasma membrane and their toxicity causes cell damage while non-penetrating CPAs are long polymers cannot enter the cytoplasm. the cryopreservation of cells, tissues, gametes, oocytes without cryoprotectives is not much effective .as it leads to injury of cells. The usefulness of the uses of cryoprotectants is that they have a ability to provide viral free medium. The cryoprotectants provide conditions for the cells, tissues and cell lines to store at low temperature (Stacey, 2004). cryoprotectants used to preserve the cells, tissues and cell lines may lead to the increase in toxicity in the cells. The toxicity may increase due to the high concentration of cryoprotectants, and which results into the adverse effect on the cell structure it destroy the cell membrane, cell organelles the cell will lose all the material and it results into the death of cells. The most essential factor is the selection of the cryoprotectants. The composition and concentration of cryoprotectants that have to used for the Vitrification should not play lethal effect on the embryo and oocytes. The cryoprotectants should only permeate the cytoplasm but not to produce the excessive osmotic pressure inside. The cryoprotectants solution should be properly concentrated to avoid crystallization during freezing. The cryoprotectants provide stable conditions for the cells, tissues to preserve at low temperature (Stacey, 2004). The cryopreservation by controlled slow cooling also has negative effect on the cell activity. The controlled rate slow cooling may lead to the intra cellular ice formation, due to formation of ice there are high chances of freezing injury These injury causes death of the cell (Rawson, 2011).
The cooling and warming conditions should be very carefully handled, so that freezing injury, thaw injury and loss of water should not happen. The best cryoprotectants are glycerol, sucrose, dimethylsulphoxide, ethylene glycol, propylene glycol which are used in different cryopreservations.
Controlled slow cooling
The controlled rate slow cooling is performed by sustaining the glassy state at the temperature lower than -100ËšC.At this temperature, The controlled slow cooling may lead to the intra cellular ice formation, due to intra cellular ice formation there are high chances of freezing injury and thaw injury. These injuries might results into the death of the cell. At this stage the kinetic motion of water molecules is prevented (Fuller, 2009).The sample is kept in cooling chamber where temperature is lowered then -80ËšC and cryopreservation is done by storing the samples at this low temperature using a stepwise procedure. This enhances the viability of cells. A programmed procedure is used in the cooling system. The seeding is performed for the nucleation of the sample. The forceps touch the highly cooled straw at a definite temperature or the cryosurgical device is used in this. The controlled release of LN2 is processed in the cooling chamber (Fuller, 2011)
In controlled rate and slow cooling, the cryoprotectants are added step by step by reducing the temperature. The step by step addition of cryoprotectants is useful for the formation of less intracellular ice, less liquid water and less injury to the cells. The controlled rate and slow cooling has tendency to some extent to safely preserve the biological systems because the controlled rate and slow cooling does not provide completely cryoprotection. With controlled rate and slow cooling probably there is mostly chance of intracellular ice formation, removal of water content and freezing injury, which results in damage of cells, embryo and oocytes. Vitrification is other process of cryopreservation of living cells, embryo and oocytes. In vitrification, the amounts of cryoprotectants are used with rapid cooling of the sample are intensively more. The fast cooling of sample might results into the formation of glassy state appearance without any damage occurrence to the living cells, embryos. The process mostly diminishes the chances of formation of intracellular ice, and thaw injury. Vitrification is requisite for living cells, embryos and oocytes in order to preserve the proper structure and retain genetical information in cooling and there by warming after the cryopreservation (Rawson, 2009).
VITRIFICATION
The main objective of vitrification is to prevent the formation of ice crystals in the cells. Vitrification, the glassy state is achieved without the intercellular ice formation by doing two things. First is making the solution is concentrated enough to prevent the rapid excretion of the water which is due to the osmotic gradient formed by external medium. The solutes concentration is increased by adding the cryoprotectants ( (Rawson D, 2009). The second is rapid cooling, which helps to vitrify the water and its contents. The cryoprotactants which are used in the vitrification must have some specific properties. The CPAs have penetrating ability and these helps in dehydrating before the extracellular ice formation. As more than one cryoprotectant is used in vitrification, the proper combination of these is used .In the complex of CPAs at least one of them has penetration ability. The CPAs also induce toxicity which is harmful for the living cells (Watson,2009)
In the physical process of Vitrification of embryos, oocytes and tissues many factors have been taken into the consideration for effective cryopreservation. The essential factor is the selection of the cryoprotectants. The composition and concentration of cryoprotectants that have to used for the Vitrification should not play any lethal effect on the embryo and oocytes. The cryoprotectants should permeate only into the cytoplasm and not to produce the more osmotic pressure inside. The cryoprotectants solution should be properly concentrated to stop crystallization during cooling. The freezing and warming conditions should be very carefully maintained, so that freezing injury, chilling injury and dehydration may not happen. The procedure to dilute embryos and oocytes from the Vitrification solution should carefully handle.
As the water is present in every living systms, the cryoprotectants used in vitrification does not allow water to attain the crystal structure. To stop the water from making the crystal form the high concentration of cryoprotectants used with the rapid cooling of the sample. The high concentration of cryoprotectants makes the solution highly viscous, which leads to the decrease in freezing point. Lowering in the freezing point does not allows the water molecule to set up a crystal form, the process result in the formation of glassy state. The actual reason behind the vitrification is that the amphibians and insects living in the Arctic and Antarctic Circle also create their cryoprotectants in their body during the winter season. The frogs can live many days in cold water without any ice crystal formation due to decrease in freezing point formation of glassy state. Vitrification can be used for the cryopreservation of tissues, blood cells, embryos, oocytes. The oocytes and embryos of mammalian species, embryos of drosophila melongaster and plant embryos of Asparagus officinalis are successful cryopreserved with the help of Vitrification. The first successful cryopreservation of mouse embryo with the help of Vitrification was done in 1985 by Rall and Fery. Vitrification provides large number of application by successful preservation of embryo and oocytes (Best, Cryonics).
Vitrification of oocytes and embryos:
Cryopreservation by vitrification which maintain the glassy state uses different procedures for different species. The oocytes cryopreservation include the addition of various CPAs in varying concentration .It depends upon the stage of maturation of oocytes. The oocytes of mouse and cattle are successfully vitrified (Fuller B, 2011).The cryoprotectants used are ethylene glycol, ficol and sucrose . The mouse oocytes are croypreserved by CPAs include polyethylene glycol (PEG) and 6M Me2 SO (dimethyl sulfoxide) The small amount of samples and specific containers used in vitrification process of mature oocytes which provide favourable results (Fuller B, 2011)..
The aquatic species oocytes have been cryopreserved but there is barrier in embryo cryopreservation of these species (Zhang T et al., 2011).The fish oocytes are cryopreserverd by vitrification. In this the combination of cryoprotectants is used .The Me2SO at all concentration is useful in it and up to 3M propylene glycol is favourable. The use of plastic straw in vitrification and KCl buffer as bathing medium is helpful in achieving the vitrification of fish oocytes (Guan M, 2009).
The embryos of human are successfully cryoperserved through vitrification. The vitrification of human embryos requires small volumes of samples which are cooled rapidly. These samples are placed on loops which are plunged into liquid nitrogen (LN2) and stored in a special storage containers (Fuller B, 2009).The cryoprotectants used are ethylene glycol, sucrose and dimethylsulfoxide. There is no worldwide protocol of achieving successful vitrification with the formation of glassy state. The human embryos are successfully cryoperserved through vitrification.
The oocytes and embryos are effectively preserved by vitrification. The preservation of embryo should be done at the Blastocyst stage. The cryopreservation of embryo and oocytes needs a mixture of cryoprotectants to be used. For vitrification of embryo and oocytes the ethylene glycol and sucrose cryoprotectants are used. The use of ethylene glycol and sucrose solution has good effect on the survival of embryo and oocytes than other cryoprotectants. Other cryoprotectants have toxic effect on the embryo. The use 5.5 mol/l of ethylene glycol and 1.0 mol/l of sucrose quantity, the embryos and oocytes are first treated with this solution and later exposed to liquid nitrogen at -196 °C. In this way the embryos and oocytes can preserve by vitrification (Ali, 2001).
The Vitrification has major applications in the cryopreservation of embryos and oocytes. It is useful in the preservation of endangered species, reproduction of animal species, the production of biomedicines. So, Vitrification has very useful applications in the cryopreservation which leads to production of new varieties of choice. Vitrification is currently making the proposal for new researches on animal and plant species.
The long term storage at very low temperature raises many issues. These may be hazard to the preserved material or the contamination of the tissues or cell lines. The stored samples are kept at very low temperature. Thus there is a regular change in the temperature during the regular maintenance of the vessels in which the samples are stored. This preserving procedure includes the opening of the vessels in area, where all samples are stored at in a one room .This might be due to the heat transfer procedure(Stacey G ,2011). The samples in cryobiology are stored with liquid nitrogen in the procedure of handling these causes excess damage as loss of all the viability of samples.
There is a saying about contamination of the samples in the storage vessels from the microorganisms i.e. fungi etc. The vessels must be cleaned at the bottom surface regularly to restrict from formation of any microbial organisms. The safety measure of the storage vessels in checking of the vacuum formation considered in order to have successful preservation of the biological materials (Stacey G, 2011).
There is also a concern that radiation causes drawbacks in the stored cellular material if the storage vessels are not properly maintained. This may results in the occurrence of mutation sometimes (Stacey G, 2011).
When a huge amount of embryos and oocytes are stored for long time in the cryobanks without any future use. As people stored these germ cells for taking in the future, but they do not need this in their upcoming and did not not receive back these from the cell banks. The investment and human practice as well as storage procedures go in waste without any use. As the oocytes and embryos were cryopreserved for long time in the cell banks, the reproduction can be achieved at any stage of life span.
The requisite for the long term storage of cells and tissues that all the cell banks have the required lab equipments and storage area facilities according to the storage sample to avoid any type of contamination to the samples. The financial strategy of the banks must be strong enough to store the biological sample at a high quality of hygienic conditions. The lab apparatus used in the cryopreservation of cells and tissue are presented according to the sample conditions as if there is any damage to the sample than there must be replacement criteria in which the vessels with least damage are used. In samples stored with liquid nitrogen, the great concern is required .The storage in this medium concerns the more attention in handling and resisting any contamination. The vessels with long storage are used in which there are low nitrogen lost systems. The samples must be stored at the bottom of the vessels. The vessels which have the large opening lids cause more damages (Stacey G, 2011).
In order to check the proper maintenance of the stored sample, there should be routine lab tests like viability test and sterilisation test. The tests are very essential to check the proper conditions of the sample. The issue arising with test is that the test Trypan blue done to findout the viability of the cells. Trypan blue may over estimate the viable cells and which leads to improper use of tissues or cell lines. The tests should have accurate measure of viability to have good results in the applications where they have done. The accident happen at the cell bank due to liquid nitrogen storage is another issue. The liquid nitrogen is very dangerous for the samples preserved nitrogen get interacted with sample material and makes them toxic,. Rise in the level of nitrogen has negative effect on the health of person working at the lab.
