To get the reproducible outcomes in biomedical research, genetic stability is essential and it is achieved via cryopreservation technique.
Technique of cryopreservation involves the preservation of viable cells, living tissues, gametes, embryos, organs and also some organisms on cooling at low sub-zero temperatures, characteristically at -196°C for a prolonged time to implement the applications of these biological materials over biomedicine, conservation and animal reproduction (Mazur., 1970). Long time storage is achieved by using this technique (Pereira and Marques., 2008).The cryopreservation technique is carried out in two different ways:
Vitrification and 2. Controlled slow cooling (Frederickson., 2000).
To proceed with these two protocols, several steps need to be taken and also we must look its advantages and limitations. Alteration in temperature induces main two damages – Freezing injury and chilling injury and these injuries are reduced greatly by using the cryoprotectant. Detailed analysis of these and its role in both vitrification and slow cooling techniques is described below (Fuller et al., 2004).
TWO APPROACHES OF CRYOPRESERVATION:
Preservation of biological materials under hypothermic condition with devoid of freezing is called as vitrification (Rall, W. F. and Fahy., 1985). Vitrification induce glassy formation instead of formation of ice crystal, thus it is not causing essential damage to the living system (Fuller et al., 2004).
Preserving cells from room temperature upto the temperature of liquid nitrogen is called as slow cooling. Damage associated with this are reduced using cryoprotectant
(Gao and Critse., 2004 and Guan et al., 2008).
MAJOR DAMAGES ASSOCIATED WITH THIS TECHNIQUE:
During cryopreservation, major injury that induces damage to the cell survival is:
Chilling injury. (Gao and Critser., (2000).
FREEZING INJURY: with the significant preservation at hypothermic temperature, water becomes solidify and it causes the cell damage, even to unviability. (Fuller et al., 2004).
This diagram is reproduced from the material belongs to (Ashwood-Smith and Farrant., 1980).
At high rate of freezing, ice nucleation provokes. Most cells has thermodynamic freezing point above -0.5°C. But the freezing of cell developed only after reaching – 5°C. Unfrozen state of cell and its environment occurs due to the protective solute’s super cooling and freezing point depression. External medium impulsively induce ice ‘seeding’ formation between – 5°C and – 15°C, but composition of cell persist in a super cooled and unfrozen state. Extracellular solution remains in unfrozen fraction and that influences the ice formation in external medium. Concentration of solute in extracellular solution rises in respect to the decrease in temperature. So, ice formation developed and encourages probable imbalance between the cell and external solution. Water present inside the cell is in super cooled state than extracellular region; due to the potential imbalance, water migrates to extra cellular region and freezes. Entire event of cell relays over the cooling. Decrease in cooling induces the dehydration of cell and the intracellular freezing is prohibited. Rapid cooling induces intracellular ice formation as a result of rapid decrease in extra cellular solution than the water diffusing out from the cell. Ice formation inside the cell is certainly lethal (Fuller et al., 2004).
INTRACELLULAR ICE NUCLEATION:
Homogenous nucleation, seeding by extracellular ice and heterogeneous nucleation are the possible ways IIF.
When the rate of cooling decreases, electrolytes concentration on freezing relate to unfrozen section of water. It is classified into intra and extracellular electrolytes.
CELL VOLUME DECREASE:
Volume decrease whilst freezing induces injury to cells by minor tonicity solution. Decrease in cell volume whilst freezing concerns cell damage.
DEHYDRATION THEORIES (Meryman):
Inability of cell to shrink osmotically below perspective level whilst it tries to reaches osmotic equilibrium. This is called as ‘minimum volume’ hypothesis over damage of slow-freezing.
Different cell type reaches damage upon cooling around 0 °C without freezing, i.e without ice formation. Damage occurs irreversibly on chilling temperature. If this happens in sperm cells, it is termed as temperature shock. Direct and indirect chilling injuries are the major two categories of chilling injury. These injuries are expressed upon lower temperature and it is termed as ‘cold shock’. It depends over the rate of cooling. Indirect chilling injury occurs on exposure to reduced temperature for a prolonged time and it is independent of rate of cooling. It is sometimes difficult to distinguish cold shock and indirect chilling injury (Fuller et al., 2004).
‘ The above plot is reproduced from the material belongs to (Muldrew et al., 2004).
Cells become sensitive to cold shock as it rapidly cooled at low temperature for long time. Viability of cell and its severity of injury are relays over the ”rapid” or “slow” cooling. Also this cold shock s not depends on warming rate but it depends on rate and duration of cooling (Tsai et al., 2009). Membrane permeability is injured upon rapid cooling and chance of reversibility is available for some cases. Addition of specific compounds and cell former cooling condition influences the response of cell. Thermotropic activity of lipid membrane is suspected to identify the injury due to cold shock. Lipid phase transitions of cell membrane influence the injury of cold shock in many species.
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INDIRECT CHILLING INJURY:
Long exposure of biological materials at low temperature causes indirect chilling injury and this injury is cooling rate independent. Lipids and proteins are changed by means of its activity and structure. Eg: changes in enzyme activity and protein denaturation. Also the metabolic pathway and enzyme linked reactions face some alterations as the co-ordination is decreased according to the decrease in temperature rate (Fuller et al., 2004).
ROLE OF CRYOPROTECTANT:
Cryoprotectant enhances the dehydration process formerly formation of external ice. The activity of water is greatly reduced during the lack of water loss. By reducing the effect of salts, it acts as a protective influence on structure of the cell.
Freezing protocol progression needs consistent method to detect the cell viability (Fuller et al., 2004).
Cryopretectant may be a chemical additive that is added to the solution before freezing to ensure the high survival rate after post thawing.
Role of cryoprotectant is to support and protect the survival of biological material upon cooling to hypothermic temperature for long duration of time. Property of an effective cryoprotectant is high solubility with decreased toxicity. Cryoprotectant can be classified according to chemical class and mode of action. Each categorized cryoprotectant plays a vital role upon thawing and cooling.
Freezing point depression is promoted by permeating cryoprotectant due to the presence of electrolytes. Non-permeating cryoprotectant promotes decreased formation of ice crystal upon freezing by prior dehydration of biological material.
Reduced deviation of volumes and solutes damage concentration is enhanced by the cryoprotectant. Eg: DMSO (Fuller et al., 2004).
Cell protection is also achieved by fluctuating formation of ice crystal into harmless shape and size during thawing and freezing.
It is necessary to look the toxicity of cryoprotectant over cells and its permeability. High concentration of cryoprotectant itself injured. Direct exposure of cryoprotectant with membranes and proteins induce ionic pumps disruption over trans membrane and also causes enzyme inactivation. But more amount of cryoprotectant in vitrification ensures viscous and amorphous medium. The possible approach to overcome this problem is achieved by using mixture of cryoprotectant at definite concentration (Tsai et al., 2008 and Fuller et al., 2004).
SIMILARITY AND DIFFERENCES OF VITRFICATION AND CONTROLLED SLOW COOLING: -ADVANTAGES AND DISADVANTEGES:
Effective vitrification demands enormous sample cooling and solute with high concentration with combination of cryoprotectant (Bielanski, and Lalonde., 2009).
Successful vitrification was enhanced in 1985 to cryopreserve the mouse embryo and this technique is also effectively applied to preserve the blood cells, tissues, embryo and oocyte of Drosophila melanogaster, Asparagus officinalis plant as well as embryos of numerous mammalians. Cryopreservation of mammalian system report entails the success achieved through the technique of controlled freezing. However in the case of fruit fly, vitrification occupies a success where the controlled freezing failed. Efficient vitrification technique relays on an optimization of some specific steps that includes appropriate composition and concentration of provided vitrification solution with specific cooling/warming environments. Also this technique induces equilibration of living cells present and to dilute the cells present in the vitrification solution (Fuller et al., 2004).
A series course of freezing and warming of bovine in-vitro matured, fertilized and cultured blastocysts using electron microscope (EM) grids (A-F)’ ( Reproduced from park et al., 1999)
The use of slow cooling includes several ranges of rates of cooling when we compared vitrification with rapid and ultra rapid cooling. The ultimate goal of both techniques is to produce a glass like state of cells to prevent the damage caused by formation of ice crystal upon cooling (El-Danasouri, and Selman., 2005). At first, vitrification procedure involves lengthy pre-equilibrium procedure. Currently, combination of penetrating and non-penetrating solutes is used with non-toxic property with several ranges of cooling rates. Both the technique result in successful cryopreservation of embryos and oocytes of humans (Borini, and Coticchio., 2009). Even these procedures resulted good, slow cooling technique applied for cryopreservation of oocytes shows very less successive rates when compared to vitrification. Vitrification acts as a promising technique in many areas in reproductive technology, even though its positive rates need to establish further.
Vitrification is an easy procedure and that consumes less time duration. Also this vitrification technique is safer and cheaper when compared to control slow cooling. ( Kuleshova, L.L. and Lopata, A., 2002).
Cryopreservation of cell faces relative damage due to cooling and thawing. Mostly damage occurs whilst storing the cells at hypothermic conditions. Maintaining healthier cells for further use are very essential and we need to prevent it from genetic drift and contamination. To stop the biological action of the cell and to maintain that in its preserved state is the role of cryostorage. In fluid system, molecular motion is achieved via temperature (Fuller et al., 2004).
The molecular motion get reduce according to the decrease in temperature.
Biological species are designed to be viable and active at maximal temperature but it lost its activity at hypothermic condition. At that instance, lipid phase transition, structural and enzymatic damage and de-polymerization occurs (Kiefer et al., 2005). Major damaging phenomenon upon cooling are: Intracellular ice crystallisation and osmotic damage. Chilling sensitivity or cold shock leads the cell to death at the temperature below 0°C. These effects differ from one cell type to another. Bacteria and some viruses can sustain in – 60 degree but the holding temperature for most of the biological sample is below -130°C (Fuller et al., 2004).
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Conventional cryopreservation method is established to overcome the formation of ice whilst cooling. Formation of ice crystals are avoided by vitrification via its usage of concentrated solution and rapid cooling. This vitrification method contains a potential advantage as it is rapid and this technique does not require rate cooling equipment. Vitrification results in good survival rate of preserved oocytes and embryos. Cryopreservation widely applicable to retain genetic resources and protect the endemic species (Tsai et al., 2010). Vitrification acts as an alternative method to slow cooling. This provides higher survival of pregnancy range and embryo viability. This vitrification acts as a suitable procedure in infertility clinics. In this, cryopreservation of numerous embryos is maintained within short period and thus it acts as a simple method. Still, less number of controlled studies and childbirths are concerned over vitrification technique. Multiple pregnancy risk associated with freezing using controlled slow cooling is restricted using vitrification. Also it works with high efficacy (Kuc et al., 2010 and Trounson and Mohr.,1983). Vitrification acts as an attractive cryopreservation method when compared with controlled slow cooling technique. In contrast to slow cooling method, this vitrification technique is precise and in this each and every step is visualized. Vitrification reduces the time duration of exposure to sub-physiological environments. It requires only less than 10 minutes carrying out while slow cooling takes nearly two hours. Vitrification is simpler and it does not need costly programmable freezing equipment. In some cases, chilling injury also prevented by vitrification (Fuller et al., 2004).
Needle immersed vitrification requires less concentrated and minimum volume of vtrification solution. Maximize cooling rate, reduce toxicity of vitrification solution with low volume of less concentration cryopreservation.
In vitrification, upon freezing, only numerous ice crystals are formed and so less mechanical disruption results by ice crystal (Wang et al., 2008). Vitrification technique is accompanied without the withdrawal of more amount of water. So, less chemical damage only exist. But the chemical damage due to cryoprotectant is a complicated matter. ( long). Common variation held between vitrification and controlled slow freezing is due to the numerous additions of cryoprotectants. Implementation of maximum equilibration condition and dilution are expected from the vitrification media. It is necessary to use low toxic agents in the vitrification solution.
To achieve an efficient vitrification, formulation of 2 things over the vitrification technique are essential.
1. physicochemical properties : Concentrated vitrification solution induce glassy solid formation and it helps to devoid of crystallization whilst cooling.
2. cyoprotectant: using low toxic cryoprotectant with an intrinsic permeability.
Vitrification protects the cell from ice formation while cryopreservation.Both the vitrification and slow cooling are used to preserve human oocytes (Fuller et al., 2004).
In case of human ES cell cryopreservation, improved efficiency is noted in vitrification than in traditional cryopreservation (Zhou et al. 2004 and Peng-Fei et al., 2006). Analysis of colonies after vitrification yields rapid growth and differentiation when compared with slow freezing technique. Vitrification acts as a promising approach to cryopreserve the multi cellular tissue. Even, vitrification achieved certain merits; it is associated with several problems. In the state of vitrified, ‘glass’ is susceptible to cracking. Care is essential on warming to neglect the formation of ice. Heat transfer rate occurring during vitrification process may vary depends on device.Vitrification include the rapid cooling protocol and it is difficult to maintain at certain temperature with the available equipment. Very rapid and even rewarming requires avoid of devitrification.
During slow cooling, increase solute concentration to glass transition needs while prevent by cooling slow enough to allow the cells to dehydrate to protect intracellular supercooling (Youssry et al., 2008).
Vitrification requires higher and potentially cytotoxic concentration of cryoprotective agents for one hour before its immersion into liquid nitrogen at specific temperature. To reduce its toxicity, pre equilibrium performed at 4°C. It allows the direct visualizaton of cell by the operator (El-Danasouri, and Selman., 2005)
Eventhough this vitrification entails with meritful approaches, this technique still been experimental. Also, it requires more additives to reach and it is potentially cytotoxic. This technique highly depends on operator. Timing takes to cover all the steps and it is critical. In contrast to slow freezing, this vitrification needs enough level of training.
If the vitrified solution stars to devitrify, (crystalise into ice), viability will be lost. This happens when thawing or extended time of storage persists (Fuller et al., 2004).
Viability of vitrified samples is not certain for lengthy period of time but in case of slow cooling, preserved cells can be viable for many years, even to thousands of years.
Direct exposure of cryogen can be achieved by fast cooling. As it is so, this process may carry possible contamination of organism from the liquid nitrogen. So, this process cannot be applicable for therapeutic cells.
Vitrification technique is applied only to cooled cell suspensions in minor quantities. This method is not projected to apply in large quantities like cryovials, matrix tubes, bags, microtitre plates etc. Quality control measurement via this vitrification technique is made to be impossible as we need to take experiments for all straws. (Fahy et al., 2004)
Usually the slow cooling procedure is used in infertility centers. But it is associated with documented limitations. Also sometimes, it damages sensitive parts of the cell ( eg- zona pellucida) and it induce biological changes. Because of these changes, we will get a depleted outcomes. To overcome this, Modifying cryopreservation procedure is attained- freezing and thawing by polymers. This also enhanced with changing the time duration of the cooling protocol and it is looked as same as the path to simplify and fast up cryobanking procedures to get beneficial results. As the vitrification technique connected with some problems, it acts as a challenging technique for reproductive medicine. The slow freezing technique serves as an effective method for humans too (Mandelbaum, J., 2000). An alternative method for cryopreservation was developed and it is called as vitrification.
Comparative study has been taken between controlled slow cooling and vitrification techniques with patients undertaking controlled ovarian stimulation in GnRH agonist to determine efficacy. The rate of pregnancy after vitrification reveals more than higher successive rate than result achieved via slow cooling. Efficacy of vitrification yields (50.4%), and slow cooling results in (25.9%) successive rates.Human ovarian tissue also cryopreserved (Noriko et al 2009)
Both cryopreservation as well as cryostorage contains budding advantages, especially in invitro fertilization. Ultimate goal of cryopreservation is to achieve maximum persistence rate and sustainability of biological system after thawing. In slow cooling procedure, clinically satisfactory result has not been attained. Slow cooling procedure needs costly equipment and also it is time consuming.
One of a significant advantage of vitrification process is its tendency to form any ice crystals during both cooling and warming. In contrast, its limitation held in toxic effects due to addition of cryoprotectants and contamination via liquid nitrogen.
In slow cooling technique, toxicity of cryoprotectant is relatively less. But many research outcomes supports the vitrification process rather than slow cooling in fertility treatment(Tsai et al., 2010). Blastocyst cells can be preserved by both the cryopreservation techniques. Among these, vitrification promotes increasing chance for future development. A reliable advancement is needed for vitrification to enhance the preservation of supernumerary blastocysts. Unsatisfactory results have been produced for the blastocyst preservation through slow freezing method. Vitrification acts as an alternative principle which is allied with capability of inducing more pregnancy rate and increased survival of embryo upon cryopreservation (Trounson and Mohr ., 1983 and Fuller et al., 2004).
Approach taken by Kolibianakis et al results in the comparative analysis of both vitrification and controlled slow cooling. And its outcome provides similar results are given by both of these techniques. But comparatively, post thawing survival frequency is better in vitrification than slow cooling. Finally, they suggested that the there is no link between the vitrification process in giving high rate of pregnancy but it displays the successful post thawing survival both in the cleavage stage and in the blastocyst stage (Youssry et al., 2008 and Porcu et al 2000). According to Balaban et al survival rate of human 3 day embryo preservation reported the percentage of survival rate by vitrication as 94.8% whereas slow cooling provides 88.7%. (Kuc et al., 2010). Vitrification study over the embryo in cleavage stage testified 80% of survival rate and 22-35% of pregnancy rate. These results are more significant than the slow cooling procedure. Although the two main approaches of cryopreservation contains signficant results, Vitrification gains more positive outcomes. Even in both the cases, limitations persist. All of its limitations can be always overcome by its positive side.
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