Cryopreservati on Presented by Husnain Shahid Rida Ali

Cryopreservati on Presented by : Husnain Shahid Rida Ali Adil Riaz Ayaad Mushtaq

What is cryopreservation? • “Cryopreservation is a technique in which the tissues are stored using special media in Liquid nitrogen at -196°C or in vapor phase of nitrogen at -135°C. ’’ • Explants for cryopreservation can be shoot tips, callus cultures , cell cultures , somatic embryos etc. • Once the material is cryopreserved, it can be stored for indefinite period.

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 or those which are already extinct. • Preserving sperms , oocytes & embryos

History of Cryopreservation • More than two centuries ago, in 1776, Spallanzani reported that human spermatozoa 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 living cells and tissues at very low temperatures. He accidentally discovered the cryoprotective properties of glycerol on fowl sperm. Polge reported high pregnancy rates in excess in cattle using sperm that had been frozen for periods of a year.

• In 1953 , the first embryonic development and first offspring was produced from a cryopreserved human spermatozoa. • In 1982 , the first successful cryopreservation of mammalian embryos was achieved and in 1983 an Australian biologist successfully achieved live birth from a cryopreserved human embryo.

• In 1986 , Christopher Chen successfully freezed and thawed human oocytes and first live birth from a cryopreserved human oocyte was recorded. In 1988 , the first attempt was made to cryopreserve immature oocyte. • In 1988 , blastocyst cryopreservation was also developed which led to many viable pregnancies.

Techniques used in Cryopreservation Many techniques or protocols have been developed and utilized with the passage 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 a mixture of highly concentrated penetrating and nonpenetrating Cryoprotective Agents (CPA’s) applied at nonfreezing temperatures, followed by rapid cooling in Liquid nitrogen. • Commonly used cryoprotective agents are glycerol , ethylene glycol , 1 -2 propanediol and dimethyl sulphoxide.

• Due to dehydration and penetration of some CPA’s , there is an increased intracellular solute concentration, combined with rapid cooling, which prevents the nucleation of water and formation of ice crystals both inside and outside the cell thus promoting vitrification of water. • The exposure time to cryoprotective agents or solutions is very vital. Over exposure can be damaging to the cells because higher concentrations of CPA’s can be toxic and can cause excessive dehydration leading to cell shrinkage. • Vitrification method is most commonly used due to its ease of use and its high reproducibility.

Nature’s way of preserving life Natural cryopreservation

Cryopreservation in water- bears: • 1. Water-bears (Tardigrada) are microscopic multicellular organisms, can survive freezing by replacing most of their internal water with the sugar trehalose (natural alpha-linked disaccharide formed by an α, α-1, 1 glucoside bond between two αglucose units), preventing it from crystallization that otherwise damages cell membranes.

• Mixtures of solutes can achieve similar effects. • Some solutes, including salts, have the disadvantage that they may be toxic at intense concentrations. • In addition to the water-bear can tolerate the freezing of their blood and other tissues. • Urea is accumulated in tissues in preparation for overwintering, and liver glycogen is converted in large quantities to glucose in response to internal ice formation. • Both urea and glucose act as "cryo protectants" to limit the amount of ice that forms and to reduce osmotic shrinkage of cells.

Cryopreservation in frogs • Frogs can survive many freeze/thaw events during winter if not more than about 65% of the total body water freezes.

• 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

Salamander one of salamanders called Hynobius keyserlingi

Snake 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 of very low temperatures to preserve structurally intact living cells and tissue.

• Liquid water is considered essential to the structure and function of living cells, it is not surprising that the solidification of water by freezing is usually lethal yet paradoxically freezing can also preserve cells for long periods 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 the remaining liquid phase. • Rival theories of freezing injury have envisaged either that ice crystals pierce or tease apart the cells, destroying them by direct mechanical action, or that damage is from secondary effects via changes in the composition of the liquid phase.

• Cryoprotectants, simply by increasing the total concentration of all solutes in the system, reduce the amount of ice formed at any given temperature; but to be biologically acceptable they must be able to penetrate into the cells and have low toxicity. • Many compounds have such properties, including glycerol, dimethyl sulfoxide, ethanediol, and propanediol. In fact, both damaging mechanisms are important, their relative contributions depending on cell type, 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 to predict the effect of cooling rate on cell survival and the optimum rate will be a tradeoff between the risk of intracellular freezing and effects of the concentrated solutes.

• Ice can be avoided by vitrification (the production of a glassy state with viscosity reaching a sufficiently high value to behave like a solid) but without any crystallization. • Toxicity is the major problem in the use of vitrification methods. Whether freezing is permitted (conventional cryopreservation) or prevented (vitrification), the cryoprotectant has to gain access to all parts of the system.

• However, there are numerous barriers to the free diffusion of solutes (membranes), and these can result in changes (equilibrium) which can be damaging. Hence, the processes of diffusion and osmosis have important effects during the introduction of cryoprotectants, the removal of cryoprotectants, the freezing process, and during thawing. • These phenomena are amenable to experiment and analysis, and this has made it possible to develop effective methods for the preservation of a very wide range of cells and some tissues; these methods have found widespread 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 be a seed or another plant even just a few cells that can be turned into the whole plant. • It contains the information for a species genetic make up a valuable natural resources of plant diversity • For plants, the germplasm may be stored as a seed collection(even a large seed 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 zero metabolism or non-dividing state by reducing the temperature in the presences of cryopreservation. • Broadly it means the storage of germplasm at very low temperature.

Few methods of cryopreservation of germplasm: • • Over solid carbon dioxide(at 79°C) Low temperature deep freezer(at -80°C) In liquid nitrogen( at -196°C) Among these, the most commonly used cryopreservation is by employing liquid nitrogen.

• At the temperature of liquid nitrogen(196°C), the cell stay in a completely inactive state and thus can be conserved for longer period. Infact cryopreservation has been successfully applied for germplasm conservation. Plant species examples • • • Rice Wheat Peanut Sugarcane Coconut.

Mechanism of cryopreservation • The technique of cryopreservation is based on the transfer of water present in the cells from a liquid to solid state. Due to the presence of salts and organic molecules in the cells , the cell water requires much more lower temperature to freeze (68°C) compared to the freezing point of pure water(0°C). When stored at low temperature, the metabolic processes and biological deteriorations in the cells/tissues almost come to standstill.

STAGES OF CRYOPRESERVATION The cryopreservation of plant cell culture followed the regeneration of plants broadly involves the following stages: • • 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 the morphological and physiological characters largely influences the ability of the explants to survive in cryopreservation. Any tissue from a plant can be used for cryopreservation e. g. Meristems Embryos Endosperm plants Ovules Seeds Culture

ADDITION OF CRYOPROTECTANT • Cryoprotectant are the compound that can prevent the damage caused to cells by freezing or thawing. There are several cryoprotectant which include: DMSO GLYCEROL SUCROSE MANNOSE ETHYLENE GLUCOSE PROPYLENE

FREEZING The sensitivity of the cells to low temperature is visible and largely depends on the 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 as important as freezing. • In general, the frozen cells/tissues are kept for storage at temperature in the range of -72 to-196°C. Storage is ideally done in liquid nitrogen refrigerator at 150°C in the vapor phase, or at 196°C in the liquid phase.

• The ultimate objective of storage is to stop all the cellular metabolic activities and maintain their viability for long term storage temperature at -196°C in liquid nitrogen is ideal.

THAWING • Thawing is usually carried out by plunging the frozen sample in ampoules into the warm water (temp 35 -45°C)bath with vigorous swirling. • By this approach, rapid thawing(at the rate of 500 -750°Cmin-1) occurs, and this protects the cell from the damaging effects ice crystal formation. • As the thawing occurs (ice completely melts) the ampoules are quickly transferred to a water bath at temperature 20 -25°C. This transfer is necessary since the cells get damaged if left for long in warm(35 -45°C) water bath.

RECULTURING • In general thawed germplasm is washed several times to remove cryoprotectant. • The material is then cultured in a fresh media.

PLANT REGENERATION • The ultimate purpose of cryopreservation of germplasm is to regenerate the desired plant for appropriate plant growth. • The cryopreserved cell/tissue have to be carefully nursed and grown. • Addition of certain growth promoting substances , besides maintenance of appropriate environmental conditions often necessary for successful plant regeneration.

Applications • Plant materials(cell/tissue) of several species can be cryopreserved and maintained for several years , and used as and when needed. • Cryopreservation is an ideal method for long term conservation of cell culture which produces secondary 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 could develop into frost resistant plant.

Limitations • Expensive equipment is needed to provide controlled and variable rates of cooling/warming temperatures can however be a limitation in the application of in vitro technology for large scale germplasm conservation. • Formation of ice crystal inside the cell should be prevented as they cause injury 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 maximum survival rates and elimination of unwanted factors such as ROS and ice crystal 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 cryopreservation in 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 events during winter if not more than about _____ of the total body water freezes. a) 10 -25% b) 70 -85% c) 45 -65%

3. Germplasm is a living tissues from which new plants can be grown. It can be grown from a) A seed b) Just a few cells c) Both d) None

4. The most commonly used cryopreservation is by: a)Over solid carbon dioxide(at 79°C) b)Low temperature deep freezer(at 80°C) c)In liquid nitrogen( at -196°C)

5. Which is the most effective method to eliminate ice crystal formation: A. Pre cooling treatment B. Vitrification C. Cyclopiazonic acid D. Encapsulation dehydration