Presented by : Husnain Shahid Rida Ali Adil Riaz Ayaad Mushtaq
Cryopreservation Presented by : Husnain Shahid Rida Ali Adil Riaz Ayaad Mushtaq What is cryopreservation?
Cryopreservation is a technique in which the tissues are storedusing special media in Liquid nitrogen at -196C or in vapor phase ofnitrogen at -135C . Explants for cryopreservation can be shoot tips, callus cultures ,cellcultures ,somatic embryos etc. Once the material is cryopreserved,it can be stored for indefiniteperiod . Why we do Cryopreservation?
Preserving those species which can not be stored by any other means . For preserving cultivars of propagated plants. For preserving those species that are going to be extinct in near future orthose which are already extinct . Preserving sperms , oocytes & embryos History of Cryopreservation
More than two centuries ago, in 1776, Spallanzani reported that humanspermatozoa can be maintained after exposing it to low temperatures . In 1886 , Montagazza suggested sperm banks for storage of human sperms . In 1949, Ernest John Christopher Polge discovered how to preserve livingcells and tissues at very low temperatures. He accidentally discovered thecryoprotective properties of glycerol on fowl sperm. Polge reported highpregnancy rates in excess in cattle using sperm that had been frozen forperiods of a year. In 1953 , the first embryonic development and first offspring wasproduced from a cryopreserved human spermatozoa . In 1982 , the first successful cryopreservation of mammalian embryoswas achieved and in 1983 an Australian biologist successfully achievedlive birth from a cryopreserved human embryo . In 1986 , Christopher Chen successfully freezed and thawed humanoocytes and first live birth from a cryopreserved human oocyte wasrecorded . In 1988 , the first attempt was made to cryopreserveimmature oocyte . In 1988 , blastocyst cryopreservation was also developed which led tomany viable pregnancies . Techniques used in Cryopreservation
Many techniques or protocols have been developed and utilized with thepassage of time for doing cryopreservation .Some of them are as follows : Slow cooling or controlled rate cooling. Encapsulation-dehydration Vitrification Vitrification Vitrification is a process which involves the treatment of tissues in amixture of highly concentrated penetrating and non-penetratingCryoprotective Agents (CPAs) applied at non-freezing temperatures,followed by rapid cooling in Liquid nitrogen . Commonly used cryoprotective agents are glycerol ,ethylene glycol ,1-2propanediol and dimethyl sulphoxide . Due to dehydration and penetration of some CPAs , there is anincreased intracellular solute concentration, combined with rapidcooling, which prevents the nucleation of water and formation of icecrystals both inside and outside the cell thus promoting vitrification ofwater . The exposure time to cryoprotective agents or solutions is very vital .Over exposure can be damaging to the cells because higherconcentrations of CPAs can be toxic and can cause excessivedehydration leading to cell shrinkage. Vitrification method is most commonly used due to its ease of use andits high reproducibility . Natures way of preserving life
Natural cryopreservation Cryopreservation in water- bears:
1. Water-bears (Tardigrada) aremicroscopic multicellular organisms, cansurvive freezing by replacing most of theirinternal water with the sugar trehalose(natural alpha-linked disaccharide formedby an , -1, 1-glucoside bond betweentwo -glucose units), preventing it fromcrystallization that otherwise damages cellmembranes. Mixtures of solutes can achieve similar effects.
Some solutes, including salts, have the disadvantage that they may betoxic at intense concentrations. In addition to the water-bear can tolerate the freezing of their bloodand other tissues. Urea is accumulated in tissues in preparation for overwintering, andliver glycogen is converted in large quantities to glucose in response tointernal ice formation. Both urea and glucose act as "cryo protectants" to limit the amount ofice that forms and to reduce osmotic shrinkage of cells. Cryopreservation in frogs
Frogs can survive manyfreeze/thaw events duringwinter if not more than about65% of the total body waterfreezes. The wood frog (Rana sylvatica), has perfected the cryogenic freezing process during its winter hibernation when 35-45% of the frog's body may freeze and turn to ice. Ice crystals form beneath the frog's skin and become interspersed among the skeletal muscles. During the freeze the frog's breathing, blood flow, and heart beat cease. This freezing is made possible by specialized proteins and glucose, which prevent intracellular freezing and dehydration. Some examples of organisms that undergo natural cryopreservation
Five species of frogs which include: Rana sylvatica Pseudacris triseriata Hyla crucifer Hyla versicolor Hyla chrysoscelis one of salamanders called
Hynobius keyserlingi one of snakes called Thamnophis sirtalis Turtles Three of turtles Chrysemys picta Terrapene Carolina
Terrapene ornata Lizard Wall lizards Podarcis muralis Conditions important for cryopreservation
Cryopreservation is the use ofvery low temperatures topreserve structurally intactliving cells and tissue. Liquid water is considered essential to the structure and function of livingcells, it is not surprising that the solidification of water by freezing isusually lethal yet paradoxically freezing can also preserve cells for longperiods of time in a viable state The biological effects of cooling are dominated by the freezing of water,which results in the concentration of the solutes that are dissolved in theremaining liquid phase. Rival theories of freezing injury have envisaged either that ice crystalspierce or tease apart the cells, destroying them by direct mechanical action,or that damage is from secondary effects via changes in the compositionof the liquid phase. Cryoprotectants, simply by increasing the total concentration of all solutesin the system, reduce the amount of ice formed at any given temperature;but to be biologically acceptable they must be able to penetrate into thecells and have low toxicity. Many compounds have such properties, including glycerol, dimethylsulfoxide, ethanediol, and propanediol. In fact, both damagingmechanisms are important, their relative contributions depending on celltype, cooling rate, and warming rate. A consensus has developed that intracellular freezing is dangerous,whereas extracellular ice is harmless. If the water permeability of the cell membrane is known it is possible topredict the effect of cooling rate on cell survival and the optimum ratewill be a tradeoff between the risk of intracellular freezing and effects ofthe concentrated solutes. Ice can be avoided by vitrification (the production of a glassy state withviscosity reaching a sufficiently high value to behave like a solid) butwithout any crystallization. Toxicity is the major problem in the use of vitrification methods.Whether freezing is permitted (conventional cryopreservation) orprevented (vitrification), the cryoprotectant has to gain access to all partsof the system. However, there are numerous barriers to the free diffusion of solutes(membranes), and these can result in changes (equilibrium) which can bedamaging. Hence, the processes of diffusion and osmosis have importanteffects during the introduction of cryoprotectants, the removal ofcryoprotectants, the freezing process, and during thawing. These phenomena are amenable to experiment and analysis, and this hasmade it possible to develop effective methods for the preservation of avery wide range of cells and some tissues; these methods have foundwidespread applications in biology and medicine. Germplasm and its conservation
By cryopreservation What is a germplasm? Germplasm is a living tissues from which new plants can be grown. It can bea seed or another plant even just a few cells that can be turned into the wholeplant. It contains the information for a species genetic make up a valuable naturalresources of plant diversity For plants, the germplasm may be stored as a seed collection(even a largeseed bank) or for trees in a nursery. Animal as well as plant genetics may be stored in a gene bank or cryobank. Cryopreservation of germplasm:
Cryopreservation means in the frozen state. Cryopreservation to bring the plant cells and tissue cultures to a zerometabolism or non-dividing state by reducing the temperature in thepresences of cryopreservation. Broadly it means the storage of germplasm at very low temperature. Few methods of cryopreservation of germplasm:
Over solid carbon dioxide(at 79C) Low temperature deep freezer(at -80C) In liquid nitrogen( at -196C) Among these, the most commonly used cryopreservation is by employingliquid nitrogen. At the temperature of liquid nitrogen(196C), the cell stay in a completelyinactive state and thus can be conserved for longer period. Infactcryopreservation has been successfully applied for germplasmconservation. Plant species examples Rice Wheat Peanut Sugarcane Coconut. Mechanism of cryopreservation
The technique of cryopreservation isbased on the transfer of water present inthe cells from a liquid to solid state. Dueto the presence of salts and organicmolecules in the cells ,the cell waterrequires much more lower temperatureto freeze (-68C) compared to thefreezing point of pure water(0C).Whenstored at low temperature, the metabolicprocesses and biological deteriorations inthe cells/tissues almost come tostandstill. STAGES OF CRYOPRESERVATION
The cryopreservation of plant cell culture followed the regeneration of plants broadly involves the followingstages: Development of sterile tissue culture. Addition of cryoprotectant and pretreatment. Freezing Storage Thawing Reculture Measurement of survival/viability Plant regeneration Development of sterile tissue culture
The selection of plant species and the tissue with particular references to themorphological and physiological characters largely influences the ability ofthe explants to survive in cryopreservation. Any tissue from a plant can beused for cryopreservation e.g. MeristemsEmbryosEndospermOvules Seeds Culture plants ADDITION OF CRYOPROTECTANT
Cryoprotectant are the compound that can prevent the damage caused tocells by freezing or thawing. There are several cryoprotectant which include: DMSOGLYCEROL ETHYLENEPROPYLENE SUCROSEMANNOSE GLUCOSE FREEZING The sensitivity of the cells to low temperature is visible and largely depends onthe plant species. Four different types of freezing are used: Slow freezing method Rapid freezing method Stepwise freezing method Dry freezing method STORAGE Maintenance of the frozen cultures at the specific temperature is asimportant as freezing. In general, the frozen cells/tissues are kept for storage at temperature in therange of -72 to-196C.Storage is ideally done in liquid nitrogen refrigeratorat 150C in the vapor phase, or at -196C in the liquid phase. The ultimate objective of storage is to stop all the cellularmetabolic activities and maintain their viability for long termstorage temperature at -196C in liquid nitrogen is ideal. THAWING Thawing is usually carried out by plunging the frozen sample in ampoulesinto the warm water (temp 35-45C)bath with vigorous swirling. By this approach, rapid thawing(at the rate of Cmin-1) occurs, andthis protects the cell from the damaging effects ice crystal formation. As the thawing occurs (ice completely melts) the ampoules are quicklytransferred to a water bath at temperature 20-25C.This transfer is necessarysince the cells get damaged if left for long in warm(35-45C) water bath. RECULTURING In general thawed germplasm is washed several times to removecryoprotectant. The material is then cultured in a fresh media. PLANT REGENERATION The ultimate purpose of cryopreservation of germplasm is to regenerate thedesired plant for appropriate plant growth. The cryopreserved cell/tissue have to be carefully nursed and grown. Addition of certain growthpromoting substances ,besides maintenance ofappropriate environmental conditions often necessary for successful plantregeneration. Applications Plant materials(cell/tissue) of several species can be cryopreserved and maintained for severalyears ,and used as and when needed. Cryopreservation is an ideal method for long term conservation of cell culture which producessecondary metabolites e.g. medicines. Disease (pathogen) free plant material can be frozen and propagated whenever required. Recalcitrant seeds can be maintained for long. Conservation of somaclonal and gametoclonal variation in culture. Plant material from endangered species can be conserved. Cryopreservation is a good method for the selection of cold resistant mutant cell lines which coulddevelop into frost resistant plant. Limitations Expensive equipment is needed to provide controlled and variable rates ofcooling/warming temperatures can however be a limitation in the applicationof in vitro technology for large scale germplasm conservation. Formation of ice crystal inside the cell should be prevented as they causeinjury to the cell. Sometimes certain solutes from the cell leak out during freezing. Cryoprotectant also effect the viability of cells. Problems related to Cryopreservation Issues in cryopreservation
Crystal formation during liquid nitrogen immersion. Epigenetic and genetic mutations during developmental phase. Non adaptation of plant of the decreasing temperature. Membrane rupturing or alteration of permeability. During rehydration cells damaged undergo lysis.
Cell damage due to dehydration intolerance and osmotic stress. Cyclopiazonic acid (CPA) concentration ROS (reactive oxygen species) formation Solutions and precautions
Vitrification,droplet vitrification. Temperature pre-conditioning. Use of anti-oxidants e.g: Glutathione (GSH) sacrificial oxidation layer. Use of organized tissue results in genetically stable culture. Select non-cryosensitive plants for culture. Use soluble sugars for membrane stability Limitation due to natural factors
All the safety protocols are observed in tissue culture for maximumsurvival rates and elimination of unwanted factors such as ROS and icecrystal formation in culture. Some plants are recalcitrant towards culturing and cryopreservation,these protocols have to be modified. Fundamental studies should be carried out for initiating cryopreservationin these plants. Thank you! 1.Cryopreservation is a technique in which the tissues are stored using special media in: (a)liquid phase (b)vapor phase(c) condensed phase 2.Frogs can survive many freeze eventsduring winter if not more than about_________ of the total body water freezes. 10-25% 70-85% 45-65% 3. Germplasm is a living tissues from which new plants can be grown
3.Germplasm is a living tissues from whichnew plants can be grown. It can be grownfrom A seed Just a few cells Both None 4.The most commonly usedcryopreservation is by:
Over solid carbon dioxide(at 79C) Low temperature deep freezer(at -80C) In liquid nitrogen( at -196C) 5. Which is the most effective method to eliminate ice crystal formation:
Pre cooling treatment Vitrification Cyclopiazonic acid Encapsulation dehydration 6. Which one of the following will yield most genetically stable culture:
Undifferentiated callus Organized shoot tips None of these 7.In _________ Ernest John ChristopherPolge discovered how to preserve living cellsand tissues at very low temperature 1940 1960 1949 1800 P.S. Any means of bribery will BE accepted for answers