Using Cryopreservation To Store Embryos Biology Essay

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Fuller et al (2004) explained that Early ovarian tissues Cryopreservation studies were carried out in animal starting in 1950s in London under supervision of Sir Alan Parakes after discovery of cryoprotective properties of glycerol for sperm by Polge in 1949,and investigative work in human ovarian tissues has been started in last five years. 15% glycerol for rabbit granulosa cella with controlled slow cooling with storage temperature -79 showed some success. Series of investigations showed that controlled slow cooling and rapid warming was more successful than fast coolling and gave important principles for ovarian tissue cryopreservation…… while Vitrification has been applied in different species apart from human beings. Above 200 babies are born after oocyte cryopreservation with commonly used method of controlled slow cooling in presence of 1,2 propane diol and sucrose and many thousands from cryopreserved embryos becaue Cryopreserved embryo has longer history with higher success rate than oocytes with wider applications in biotechnology and in biomedicine.

Primary oocytes developed from primordial germ cells during development of foetus , are present in female at birth at prophase stage of meiosis 1 surrounded by single layered squamous epithelium. At puberty one oocyte increase in diameter up to 100 microns and cumulus cells are loosely packed, enter in to second meiotic division controlled by systemic and hormonal systems ,with complex structure of follicles making granulosa and thecal cells. Embryo development with two stages pre and post implantation occurs after fertilization. Technique has been applied to preserve all stage

embryos. 8 cell stage human embryo was preserved by controlled slow cooling in 1983 and blastocyst stage was preserved with glycerol in 1985. 1,2 propanediol/ sucrose for 1-cell fertilised zygote or later stages has also been used for cryopreservation during same time period. Slow coolling methods provided great success in cryobiology reducing the rate of intracellular ice formation(IIF) and ultimately chilling injury dilution shock during warming to embryos.

Eight cell embryo was placed in 1.5M 1,2 -propane diol in PBS containing 20% supplement at 20C for 15 minutes. Transferred Into 20 micro liter straw containing 1.5M 1,2 -propane diol and 1.5 0.1 sucrose solution for 10 minutes. Cooled down to -7C at the rate 2C/min after seeded. Oocytes were plunged in to liqid nitrogen after cooling up to -35C at the rate of -.3C/min. During thawing process samples warmed in air for 40 seconds and then transferred to water bath at +30 C until all ice has melted. Oocytes were exposed in solutions of 1.0 M p- diol, 0.2M sucrose and 0.5M p- diol + 0.2 sucrose, 0.2 sucrose. At the end washed with fresh embryo culture medium( 10% serum supplement and HTF)

1M-1.5M Me2SO or glycerol has been used in Whittingham/ Wilmut methods for preservation of mouse embryos at different stages of development during 1970s. Samples transferred to liquid nitrogen or liquid helium after cooled down up to -7C with seeding and following process with slow cooling up to -80C. Process require slow warming from -80C to+1, +12and +60C/min. (Fuller lectures 2009) Fuller et al (2004)

Cryopreservation technique can be used to preserve, oocytes, embryos and ovarian tissues. Oocytes are very sensitive to cryopreservation due to unique structures and large surface/volume ratio , with essential components and features e.g cell size, Zonapallucida, cortical granules and chromosomes on the spindles with out nuclear membrane. Suffer more osmotic damage during addition and removal of cryoprotectants due to less water and cryoprotectants permeability. Oocytes are very sensitive to higher concentrations of CAPs. Different combinations of CAP 40% Ethylene Glycol , Ficoll and sucrose(EFS) can be used for different cellular combinations to achieve higher success. .(Fuller lectures 2009) Fuller et al (2004)

Chilling injury may maximum up to 0C in oocytes with higher lipid contents.

Before cryopreservation of mature oocytes at meiotic division 11, eggs must hyaluronidase and kept in a culture for 5 hours. And then place them into warm modifies HTF. Replace them at room temperature for 10 minutes cooling up to 22C. Transfer mature oocytes for 20 minutes to HFT with 15% HSA, 1.5M 1,2- propanediol. Further placing oocytes for 10minutes in to 1.5 M PROH and 0.2M sucrose. 0.3 ml of PROH and sucrose medium with oocytes must be filled in cryovial and cooled down to -5 C at the rate of 2C/min and hold there for 15 minutes for establishment of seeds. Further cooling up to -38 at the rate of 3C/min and plunge in to liquid nitrogen for storage. Sucrose function as osmotic buffer controls volume fluctuations during CPA exposure and increase CPA concentrations before freezing and reduce osmotic damage on warming.(Fuller lectures 2009) Fuller et al (2004)

Small quantity of sample can be vitrified in special chambers for better results. Chamber with open pulled straws can also be used for Vitrification but there would be complications on devitrification. In straw method sample to be vitrified can be directly exposed to liquid nitrogen in open containers. There is risk of contamination in this technique. Mukaidaet al(1998) has reported some success in vitrified embryo in EFS containing 40% ethylene glycol, 30% ficoll and 0.5M sucrose solution at cleavage stage. Michelle Lane et al 1999 developed cryoloops to vitrify by direct plunging in to liquid nitrogen and storage in small vials and thawing small volumes in ul of . Fluid volume of blastocoele can be reduced by using micro pippet to avoid ice formation. In clinical practice oocyte maturation up to M11 stage and xenografting is very difficult process. Recently method being used is cut 70 cubes of 2x2mm and one strip of 12x4mm biopsy samples of cortex of ovarian tissue and exposed in cryoprotective medium then transfed in to pre cooled 2ml cryogenic vial with Leibovitz medium containing 4mg/ml of human serum Albumin and 1.5MMol/L of DSMO. Programeable freezer can be used for cooling from 0C to -8C at the rate of -2C/min. Precooled forceps can be used for seeding by touching the sample manually and then cooled -40 at the rate of -0.3C/min, follow cooling to-150C at the rate of -30C/min and for storage for 6 years replace in -196C.

For thawing cryogenic vials can be warmed for 2 minutes at 2C-23C at room temperature then transfer in to water bath at 37C for 2 minutes following washing with fresh medium three times.

Seeding is crucial step in cryopreservation can be done by automated seeding, which induce rapid influx of cryogen in to the chamber, or by using natural nucleators more information is required to determine chemical toxicity.

Cryoprotectants are essential for survival but with toxic effects like cortical granule release, cellular shrinkage and cytoskeleton damage. Depolymerisation of tubulin in spindle and chromosome dispersal can occur during meiosis due to exposure to cryoprotectants (CPAs)and slow cooling creating abnormalities in the embryo. But there is evidence for reformation of tubulin spindle after thawing. Spindle depolymerisation may be prevented by CPAs. Low concentrations must be introduced to the samples at the room temperatures. Maternal mitochondria plays vital role in young embryo, there activity and dispersal during cryopreservation must be noticed. .(Fuller lectures 2009) Fuller et al (2004)

During the process of controlled slow cooling water molecules can move out of the cell slowly to reduce osmotic stress.

Cortical granules release and distribution of mitochondria can also occurs by actin cytoskeleton.

Recent research has been carried out synthetic proteins which can bind on the surface of ice crystal reducing its ability to grow during warming. .(Fuller lectures 2009) Fuller et al (2004) Vitrification of human oocyte is still in experimental stages. Techneue may be applied in future. More research is required for improved techniques for greater success.

(2)problems and how these are encountered and ehthical issues. Process of cryoinjury applications in biomedicine and biotechnology, long term storage and tissue banking.

There are several ethical issues for cryopreservation of human embryo. E.g ownership of the baby in case of death or divorce of couple, religion and society and time limit for storage of embryos.

MSc in Biotechnology Cryobiology, Citations - Chapter 18, Life in the Frozen State

(Tiantian Zhang lecture 1)Cryobiology is the study of living system at low temperatures to develop techniques for preservation of cells, tissues, embryos and transplantation organs hypothermically. Viability of cells tissues and multiple organ systems can be maintained for longer periods at ultralow temperatures at original state of cells including, physical, chemical and genetic state with out any change. ChrisPolge(1928-2006)invented technique in 1949 by preserving sperms, developed more recently with routine applications in biomedicine for heart , kidney stored at cooltemperatures not freezing, animal reproduction and conservation of rare species. Small tissues and blood can be stored for extended period in liquid nitrogen. Technique is routinely being used in infertility treatments to preserve human sperms, eggs aqnd embryos.

Low temperatures induce two type of injury (1) Freezing injury(2) Chilling injury. Life is water dependent. Water crystallisation occurs with supper cooling which is very lethal. Most cells freeze at -0.5C thermodynamically due to protective solvents, which depress freezing point. Extracellular ice formation occurs between -5 to -15C at higher cooling and change the composition of remaining extracellular liquid. decrease in temperature induce chemical potential imbalance. Intracellular supper cooled water has higher chemical potential than extracellular with partly frozen solution. Supper cooled water flows out of the cell and crystalise. Cooling rate has major impact on cellular integrity. At lower cooling rate water molecules move out of the cells by the process of exosmosis and increase the intracellular solute concentration and eliminate supper cooling effect to maintain intracellular chemical equilibrium of water in accordance with extracellular water.Dehyration occurs with out freezing. At higher cooling rate chemical potential of extracellular water is faster than the rate of diffusion of water out of the cell, which lead in to intracellular ice formation(IIF) with lethal effects on the cell may be causing cellular death. There are three possible ways of IIF.(1) Homogenenous(1um pure water nucleation point is -39) and with each 10 folds increase in diameter increase 2C. Increased solute concentration depress homogenous nucleation temperature, which is 3.3C for each unit increase in osmolality. Cellular homogenous nucleation temperature range is approximately -38C to -44C(2) Hetrogenous or seeding by extracellular ice dependent on intracellular nucleating agents with range of -31C to -38C. If ice nucleation occurs below -30C it could have different circumstances. (3) IIF at median temperatures(-10C to -20C) in cell is mostly due to seeding mechanism. There are different theories of intracellular ice nucleation above -30C.1= Due to growth of ice in extracellular area and move through cellular membrane external ice with direct impact. 2= May be lipid bilayer function as catalyst in nucleation with change in plasma membrane with indirect impact of external ice. 3= Osmotic imbalance by physical and chemical changes during cooling process damage plasma membrane and induce intracellular ice formation(IIF).

Freezing injury at lower cooling rate is due to increased concentration of electrolytes in remaining liquid in intracellular and extracellular spaces. Probably due to decrease in cell volume. There are different dehydration theories at slower cooling rate. Cells are unable to shrink osmotically at super cooled level. Cellular injury occurs to achieve osmotic equilibrium. Cellular injury can occur during warming. Small ice in rapidly cooled cells may recrystalise in bigger size with slow warming, causing lethal damage to living system. Rapid thawing in some cells with IIF show higher survival rate with out any swelling. It was mentioned by Ashwood Smith(1980) and Farrant(1980) that cellular damage associated with rapid thawing may be due to small fraction of intracellular ice sealing the cells effecting osmotic damage in the beginning of thawing process. There is another suggestion by Vorotiln et al(1991), rapid heating of frozen living system may be associated with mechanical burden creating more damage due to thermal conductivity. Survival rate can be increased with combination of slow warming and rapid warming.

(Lecture 2 Tiantian) Decrease in temperature close or below 0C with out freezing of living systemis associated with cellular damage is known as chilling injury or temperature chilling injury originated during early 18th century during the effect of cooling on plants. Temperature shock is another term used to describe cooling effect on mammalian sperm with irreversible damage in 1934. There are two categories of chilling injury. 1= Direct chilling injury dependent on cooling rate2= indirect chilling injury. Term "cold shock" can be used to described both. Exposure to low temperatures for extended period is indirect chilling injury. Some time it is difficult to classify the situation.Cold shock can occur in all cell types during the process of rapid cooling at sub zero levels. Viability of living cells can be affected by rate of cooling and direct chilling injury or cold shock does not depend upon warming rate.More injury is observed during incubation at lower temperatures for longer. Permeability of cells can be damaged after fast cooling some time reversible process on warming. Process of injury can be reduced with modified culture conditions. Cold shock occurs due to lipid phase transition. E.g studied in sperms, oocytes and platelets. Zebrafish sperms, eggs and embryos can be studied easiliy due to short generation time, genetic resemblance with human genome, easy sequencing to study human diseases, cryopreservation success for fish eproductive material may help in research field. Indirect chilling injury is independent of rate of cooling but can effect proteins and lipids with mammalian oocytes or embryos and insect embryos, structural and functional changes in proteins and enzymes affecting metabolic pathways.

(Controlled slow cooling by David Rawson)

Cryopreservation is process of storage of living cells, tissues and organs in viable state for extended period, with out contamination and genetic changes. Cells can die rapidly at suppercooling stage and during thawing process. Successful cryopreservation stop biological time keeping biological material at original state. Chemical processes and movement of molecules release energy and require energy. Internal energy of the system can be measure in form of temperature. Optimal temperature provide sufficient energy for faster molecular movements. As temperature is lowered functional energy will be reduced reducing molecular movement and nearly stop at -130C stage known as glass transition temperatures when there is no diffusion or liquid water existence. Cryobiology has exploited this phenomenon. Optimal temperatures are essential for living systems. Homeostasis can not be maintained at lower temperatures and cells can face severe damage like membrane imbalance due to lipid phase transitions, damage to enzymes and other proteins , depolarization of microtubules, cytoskeletons and lots of other destructive events at extremely low temperatures. Intracellular ice crystalisation(IIC), osmotic damage due to water loss and higher salt concentrations in extracellular spaces. It was very challenging to freeze cells in viable state for long time with out any severe damage. Normally cells die during cooling up to 0C due to cold shock. Cells contain 70-90% water. Liquid nitrogen(-196C boiling and -210Cfreezing point) can be used for freezing. Different cells have different chilling requirements in terms of IIC & osmosis. Danger zone starts from 0C. Cryobiology has developed techniques to maintain cells viability with out any physical, chemical or genetic damage. There are still adverse effects on some cells and small organisms,e.g bacteria and viruses can be stored at -60C for longer. other biological systems can be stored below -130C in liquid nitrogen(-196C) or above liquid nitrogen in gaseous state(-178Cto-150C).

During cooling process below 0C metabolic rate of the cells slows down and ion channels are disrupted but the cell will survive if there is osmotic balance. Further cooling from 0C to -20C ice crystalisation will occur with increased salt concentration in the remaining liquid in extracellular space. To balance the solute concentration with respect to intracellular concentration water moves out of the cells causing dehydration and shrinkage and some time cellular damage and death can occur with dehydration due to un limited water loss and damaging level of solute concentration. In rapid cooling water molecules are unable to move out quickly from the cells and IIF occurs. Balanced cooling rate is required to avoid IIC and dehydration. With out cryoprotectants(CPAs) it is impossible in most of the cells super cooling of bathing medium must be avoided to maintain the cooling rate. Size and structure of the cells must have effect on the cooling rate of the cells. Complex membrane system also effects the cooling.

There is not much work done on cryopreservation of oocytes and embryos. About 20 fish species have been studied with very low success rate. Embryos and oocytes may have some tolerance up to -35C.

Cryoprotectants are chemicals with three major functions. (1) protect cellular structure from salt(2) Reduce water activity level in case of incomplete dehydration(3) Helpful in dehydration. Cryoprotectants can be toxic at different concentrations, temperatures 20,10 and 4C, dehydration properties and ability to penetrate the cells.There are some factors which must be considered during controlled slow cooling selection and condition of the cells, no contamination, no physical damage, optimal cryomedium, optimal cell population, optimal PH with out any change. Programmed freezing units can be used for controlled slow cooling with controlled liquid nitrogen(LN2) supply with cooling rate -1C to -3C/min and the slowest rate is-0.5C/min or faster -5C to -10C/min. Samples are exposed to low temperatures with known cooling rate holding above LN2. To recover the samples back to normal temperature inn viable state warming rate selection, removal of CPAs , optimal bathing solution medium and to avoid osmotic shock are crucial points. Different cells and tissues require different protocol for cryopreservation and thawing. Viability test can determine motility, cell division, metabolism and vital staining. Cells quality must be tested and then must be exposed with cryoprotectants and then cells can be freezed by controlled slow cooling below -80C after wards can be stored at -196C. Controlled process can be use for warming and thawing and cryoprotectants can be removed at the end to bring the cells into original normal living state.

(BARRY FULLER & SHARON PYNTER the rational basis of controlledrate slow cooling)

Preservation and storage of unfertilised mammalian oocytes is essential for modern reproductive technologies for infertility treatments, cancers or other abnormalities in humans. In vitro Fertilization(IVF), donations, screening and delayed births in humans. Conservation of domestic animals, transportation of important strains all over the world, disease control, preservation and storage of genetically modified strains and to protect in danger species. But there are several ethical and legal issues in clinical practice e.g parental and ownership of the child or consent of using of unfertilized gametes. Cryopreservation of human embryo is prohibited or limited in some countries. But rules are slightly flexible in case of animal embryo preservation. The history of mature oocyte cryopreservation is 30 years back with 1st live birth in 1977 in mice and other species after wards e.g. rabbits, cows, and 20 years back in humans with very low success rate in most of the species.

Morphological and physiological diversities exist in between mammalian oocytes creating difficulties for successful cryopreservation. IIF increases due to bigger cell size and less water permeability creating intracellular water retention during cooling process. One cryopreservation protocol can not be applied on all mammalian oocytes due to different membrane permeability and cryoprotectants and solute concentrations in different species and strains.Successful cryopreservation of oocytes require to preserve all components including zona pellucida a glycoprotein, cortical granules, mitochondria and microtubules spindles. During natural process of fertilization single sperm bind with receptors on zona pellucida which surrounds whole oocyte and also release cortical granules which bind on other similar receptors on zona pellucida blocking further attachment of other sperms. If these receptors on zona pellucida are damaged during cryopreservation, there will not be sperm oocyte interaction even if other components of oocytes are undamaged. This problem can be solved by intra cytoplasmic sperm injection(ICSI) which is quite expensive technique mostly used in humans. Condensed chromosomes aligned on microtubular spindles of fertilized egg for segregation during cell division and embryo formation. Any damage occurd in cryopreserved oocyte may lead to aneuploidy, because there is an evidence that these microtubule spindles disassemble during cooling but reform on wrming but there is no clue for after effects in the embryo. Selection of immature oocyte before spindle formation stage before meiosis 1 called" germinal vesicle breakdown" stage may solve the problem. But the problem is how to obtain oocytes in immature stage, while in vitro maturation technique is still in early stages. Cryopreservation and storage of immature oocyte is still being under research. Species based morphological factors are hurdle in successful cryopreservation. E.e. porcine oocytes are prone to cooling due to high lipid contents.

Different factors should be controlled for successful cryopreservation for example formation of ice crystals to avoid biological injury during thermal transition. All biochemical and physiological conditions need to be preserved under extreme dehydration with balanced conditions, because hydration with water molecules of biomolecules and other structures is essential to avoid any denaturation of essential components of the cells. All biological systems contain different nature of macromolecules carbohydrates, lipids and nucleic acids suspended in aquous solution with solute particals creating complexity for cryopreservation. Dehydration due to cooling process solution will become viscous amorphous state locking up water molecules movement to avoid crystal formation. Water molecules movement is stopped at -100C may be with presence of small crystals, which can grow in to bigger size when slow temperature rise occurs. Different research based studies provided the evidence.

Different techniques rapid Vitrification and controlled slow cooling can be applied to achieve stable cold dehydrated state. Different factors play significant role during the process of controlled slow cooling. Heat transfer can be controlled by temperature gradient, thermal nature of the sample and housing chamber and then relative volumes are compared to the coolant with -100C end temperature with limited choice of coolant. Passive cooling which is non linear process with greater temperature gradient at the beginning with initial faster cooling rate. Sample can be placed in the cabinet of a -80C in cold chamber freezer or In vapour form liquid nitrogen. The linear slow cooling has more advantage like sample can be monitored and reduce the injury and high risk of IIF in oocytes that can occur during rapid cooling at sub zero temperatures. Passive slow cooling chambers are being used commercially in laboratories for cryobanking but not suitable for oocyte preservation due to sensitivity of temperatures of the samples. Cooling chambers can be monitored and sample,s quality can be assessed during cooling.

Oocyte preservation require more sophisticated laboratory equipment, developed earlier by using stirred alcohol bath in evacuated dewars suspended in liquid nitrogen. Flow of cold nitrogen vapors in closed environment can be balanced below -100C with an electrical heater for good performance. Similar principle based nitrogen vapour cooling technology is being used recently with constant flow of nitrogen vapour. Cleaning of machinery is essential to avoid any contamination. Nitrogen vapour is another issue which is expelled from cooling madchine in to environment.

Controlled linear cooling 0.5C/min can be performed with machine suitable for oocyte preservation avoiding liquid nitrogen. Cooling process between +30C to -100C can be controlled……………………….add.from notes

Arrangements can be used where liquid nitrogen is not available e.g. field cryopreservation in veterinary and agriculture and where clean room environment can be altered with nitrogen vapours.

Osmotic stress in unfertilized oocytes may reduce embryonic development in several species with more sensitivity in bovine oocytes during hypertonic conditions in mature oocytes. More disrupted spindle structures have been observed in porcrine oocytes during anisotonic conditions. Human mature oocytes can be exposed to range osmolalities for five minutes at 37C during in vitro development following ICSI further development is not studied whether tolerance applies & longer exposure times at lower temperatures. Permeability of cell to water and cryoprotectants must be determined by measuring the cell volume change. To reduce the osmotic stress during cryoprotectant exposure……………………………………….

Lcture vitrification

Preservation of cells and tissues at sub zero temperatures with out freezing in to glassy amorphous solid state with minor damage. Crystalisation occurs during freezing, which damage the blood vessels and other delicate structures. High solutes including cryoprotectants concentrations in bathing medium and very rapid cooling are two key factors for Vitrification. Technique was used in 1985 for cryopreservation of mouse embryo and several other applications after wards to preserve tissues, blood cells , plant somatic embryos Asparagus officinalis, Drosophila melanogaster oocytes and embryos of several mammalians. There are some reports for successful cryopreservation of mammalian systems with controlled freezing. Technique require several controlled steps. Composition, cooling and warming conditions and concentration of Vitrification solution plays vital role in viability of the embryo. Embryo need to be equilibrated in that solution,Vitrification of dilute solution with embryo. Cooling of pure water below or near 0C induce crystalisation known as freezing. In Vitrification it does not happen. Temperature drop reduce molecular movement and chemical reaction until there is less thermal energy available for cellular function. At super cooling temperatures system with liquid composition transform in to glassy amorphous state with out any liquid, known as Vitrification. Water and soluble chemicals like cryoprotectants can vitrify faster than pure water. Cryoprotectants possess tendency to protect cells during freezing and thawing injury. CPAs are helpful in dehydration before external ice formation(EIF) occurs and rduce water movement in side the cell after dehydration and plays role to maintain tne integrity of the membrane. CPAS must be able to penetrate well through cell membrane showing less toxic effects. Vitrification require solution with one or more CPAs with low toxic level.Physiochemical natureof Vitrification solution plays vital role in Vitrification and sufficient concentration stop crystal formation during cooling and vitrification

There are chemicals known as cryoprotectants which protects the cells from injury during cooling.


FULLER, B (2009) MSc in biotechnology Lectures at university ofBedfordshire,Cryobiology BHS…,Crobiology of Female reproductive Potential Oocytes.embryo.

FULLER, B LANE, N BENSON, E.E(2004) Life in The Frozen State, Ch 18 FULLER, B PAYNTER, S. J, WATSON, P Cryopreservation of Human Gametes and Embryos, PP 505-530, CRC Press, Uite State ofAmerica