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ASYMPTOTE GUIDE TO CRYOPRESERVATION 2nd Edition ‐ October 2007

©2007 Asymptote Ltd

John Morris Asymptote Ltd St Johns Innovation Centre Cowley Road Cambridge CB4 0WS, England Tel:00 44 (0)1223 421 161 Fax:00 44 (0)1223 421 166 www.asymptote.co.uk info@asymptote.co.uk.

Table of Contents

EF600 Controlled Rate Freezer ...................................................................................................................................... 3 Introduction to the Asymptote Guide to Cryopreservation .......................................................................................... 4 Physics of Freezing ......................................................................................................................................................... 5 The Effects of Freezing on Cells in Suspension .............................................................................................................. 9 Supercooling and Cell Survival ..................................................................................................................................... 16 Procedures for Ice Nucleation ..................................................................................................................................... 20 Long‐Term Storage ...................................................................................................................................................... 24 Contamination ............................................................................................................................................................. 26 Thawing & Post Thaw Handling ................................................................................................................................... 29 Cryopreservation Protocols ......................................................................................................................................... 30 Cryopreservation of Peripheral Blood Mononuclear Cells (PBMCs) ....................................................................... 30 Cryopreservation of Embryos ................................................................................................................................. 30 Cryopreservation of Spermatozoa. ......................................................................................................................... 32 Reproducibility of Protocols ........................................................................................................................................ 35 Appendix A: Definitions of terms as applied to Cryobiology ....................................................................................... 38 Appendix B: Reference;ps ............................................................................................................................................ 40

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EF600 CONTROLLED RATE FREEZER

The EF600 controlled rate freezers are designed to apply the basic principles of cryopreservation explained in this guide, particularly on the subject of the temperature control (accurate and reproducible) and of the ice nucleation.

Avoidance of the liquid nitrogen as cryogen minimizes any risk of contamination and allows the EF600 to be used in clean rooms, laminar flow hoods and isolation cabinets. Additional features of this equipment include:

• • • • • • •

• • • •

Pre‐programmed freezing protocols Data logging Easy access to samples for manual nucleation

Visual control of ice nucleation Quiet ‐ no noisy solenoid valves Easy to clean sample holder (may be sterilised by water based disinfectant preventing cross contamination) Removable sample plates which can either be sterilized or be used as disposables. Allows safe cryopreservation of contaminated samples (e.g. HIV or hepatitis infected samples) or samples of unknown microbial quality.

Ergonomic ‘Bench Top’ Design Portable for conservation and veterinary applications Optional temperature logging of sample temperature for quality control Optional full programming to allow user specified protocols in addition to pre‐programmed protocols.

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INTRODUCTION TO THE ASYMPTOTE GUIDE TO CRYOPRESERVATION

Cryopreservation protocols are generally simple and readily undertaken in commercially available equipment.

However, an understanding of the basic principles of cryobiology is desirable to ensure that the methodology is correctly and successfully applied to minimise cell damage during the processes of freezing and thawing.

This Guide aims to provide this understanding by explaining the physical principles underlying cryopreservation and setting them in the context of cryobiology. Examples are presented relating to embryo and spermatozoa cryopreservation for use in In Vitro Fertilization (IVF) and Assisted Reproduction Technologies (ART) and Peripheral Blood Mononucelar Cell (PBMCs) cryopreservation important for many clinical studies. The basic principles described also apply to other cell types.

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PHYSICS OF FREEZING

During freezing cells are exposed to a variety of stresses. In this section, we discuss some relevant aspects of the physical changes that are associated with the formation of ice, firstly the temperature changes that occur, and secondly the consequent changes in concentration of the unfrozen fraction.

The temperature changes observed during the freezing of an aqueous solution are shown below, with reference to the well‐documented system of glycerol and water. (Figure1).

“latent heat plateau”

0

Temperature (°C)

-1 -2 -3 -4 -5

Nucleation

-6 -7 -8 0

5

10

15

Time (minutes)

Figure 1. Measured temperature changes following nucleation and ice growth in an aqueous solution of glycerol. The sample was maintained isothermally at ‐7°C until ice nucleation was initiated (arrowed).

Water and aqueous solutions have a strong tendency to cool below their melting point before nucleation of ice occurs; this undercooling is often referred to as supercooling. In the aqueous solution of glycerol shown in Figure 1, the solution, which has a melting point of about ‐1.3°C, has been maintained at ‐7°C with no nucleation occurring until deliberately initiated.

The tendency of a system to undercool is related to a number of factors including temperature, holding time, rate of cooling, volume and purity from particulates. In the cryopreservation of cell suspensions there is a strong likelihood of undercooling occurring in the suspending medium, and this can damage the cells in suspension (This is further explained in the ’Supercooling and Cell Survival’ section on page 17). To avoid the damaging effects of supercooling in cells and in particular embryos, oocytes etc., it is usual to initiate ice formation in the suspending

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medium in a controlled manner relatively close to the melting point. This deliberate nucleation is commonly referred to as “seeding” although, strictly, seeding means the introduction of a crystal to an undercooled solution.

One effect of ice crystallisation in an aqueous solution is to remove water from solution. The remaining aqueous phase becomes more concentrated and a two‐phase system of ice and concentrated solution then co‐exists. As the temperature is reduced, more ice forms and the residual unfrozen phase becomes increasingly concentrated. This is illustrated in Figure 2, again for glycerol and an isotonic solution of NaCl (300 mOsm).

100

Percentage value

80

60

40

20

0 0

-10

-20

-30

-40

-50

-60

-70

Temperature (°C)

Figure 2. The equilibrium freezing process in an aqueous solution of glycerol (12% w/v) in isotonic NaCl (300 mOsm) nucleated at its melting point (‐2.9°C). Following ice nucleation the amount of ice formed (‐‐‐‐ ‐) changes in a non‐linear manner with temperature. The glycerol concentration of the residual unfrozen fraction (‐‐‐‐‐) increases as the temperature is reduced to the eutectic temperature ‐64°C)

At temperatures between the melting point of this solution (‐2.9°C and ‐64°C), shown in figure 2, the two phases of crystalline ice and an aqueous solution containing glycerol and sodium chloride co‐exist. The amount of ice changes non‐linearly with temperature, and in the case of the initial glycerol concentration illustrated, approximately 50% of the ice forms between ‐2.9°C and ‐6°C. Ice formation is an endothermic process and the large fraction of ice formed following nucleation explains the existence of the “latent heat plateau” (Figure 1), where during the change of phase, the temperature decreases only slightly. At ‐64°C, the eutectic temperature, the unfrozen phase solidifies.

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The data in Figure 2 is that of the equilibrium phase diagram: equilibrium conditions will only occur when there is sufficient time available, for example following very slow rates of cooling such as those used for the cryopreservation of embryos. At faster cooling rates, different non‐equilibrium values will exist. It may be noted that following the formation of ice in an aqueous solution, other physical parameters of the residual unfrozen solution may change, e.g. the soluble gas content increases resulting in the formation of gas bubbles, the viscosity may increase dramatically and the pH changes in a complex manner.

The equilibrium relationship between glycerol concentration and temperature shown in the phase diagram is independent of the initial concentration of the glycerol in the solution. However, the fraction of water which remains unfrozen at a given temperature is dependent on the initial glycerol concentration as shown in Figure 3. It may also be noted that the melting point (100% unfrozen fraction) is reduced with increased solute concentration.

100 90

Unfrozen percentage

80 70 60 50 40 30 20 10 0 0

-10

-20

-30

-40

-50

-60

Temperature (°C)

Figure 3. The effect of initial concentration on the fraction of water unfrozen following cooling below the melting point in different aqueous solutions of glycerol. 5% glycerol (‐‐‐‐‐), 10% glycerol and 15% glycerol (‐ ‐‐‐‐) all solutions contain 300 mOsm NaCl (‐‐‐‐‐).

A number of compounds, so called cryoprotective additives, are used to reduce cellular damage following freezing and thawing. They achieve this by increasing the unfrozen fraction at a given temperature and thereby reducing the ionic composition. It is clear from Figure 3 that glycerol would have this effect. The effect on the ionic (sodium chloride) composition during freezing of adding glycerol to the growth media is illustrated in Figure 4. Without the

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glycerol, the increase in ionic composition following ice formation is dramatic and by ‐10°C the ionic concentration reaches approximately 3 mol/l which is, not surprisingly, lethal to cells. The other commonly employed additives (propanediol, dimethlysulphoxide etc.) act in a similar manner to glycerol. Cells are exposed to a high concentration of the cryoprotective additive during freezing rather than a high ionic concentration, which is less damaging. Cells are permeable to all of the commonly employed cryoprotective additives, and it is standard practice to “incubate” cells in the cryoprotective additive before freezing commences to allow them to attain an equilibrium intracellular concentration.

Sodium Chloride concentration (g/100g)

25

20

15

10

5

0 0

-10

-20

-30

-40

-50

-60

Temperature

Figure 4. The increase in ionic concentration following freezing in 300 mOsm NaCl (‐‐‐‐‐), or a 300 mOsm Nacl solution containing 5% glycerol (‐‐‐‐‐), 10% glycerol (‐‐‐‐‐) or 15% glycerol (‐‐‐‐‐).

When compared at the same (molar) concentration, all cryoprotective additives have a very similar effect to that described above. However the protective efficiency of these compounds may vary from cell‐type to cell‐type: for example it has been reported that human embryos are best frozen with propanediol whilst human blastocysts are optimally frozen with glycerol. This may be related to the relative cellular toxicity or the differing permeability of these additives to differing cell‐types, but, because experiments to compare them have been mostly carried out at a single freezing protocol, the explanation may be more complex than these experiments suggest.

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THE EFFECTS OF FREEZING ON CELLS IN SUSPENSION

This section examines the effects of freezing on cells suspended in cryoprotectants, with the special effects of supercooling examined in detail.

It should be noted that, following ice nucleation in the suspending medium, cells in suspension are not punctured by ice crystals nor are they mechanically damaged by ice. This is clearly shown in Video Sequence 1, which shows the growth of extracellular ice induced at a low level of undercooling around a human oocyte.

Video sequence 1

In undercooled solutions, ice crystals grow by the migration of water molecules to the ice crystal lattice. They are not sharp icicles pushing through the solution. The ice crystals do not penetrate the membrane of the oocyte, rather their growth simply deflects around the cell.

Following ice formation, cells partition into the unfrozen fraction where they are exposed to increasingly concentrated solutions: cells are not normally ‘captured’ within the ice crystal lattice. The distribution of ice crystals, freeze concentrated material and cells following cryopreservation of human sperm in a 0.25ml straw is shown in the Figures 5, 6, and 7 each with increasing magnification. These images are obtained by freeze fracture followed by a deep etching, which reveals the structure of ice crystals, with cells entrapped within the freeze concentrated material and few cell structures are evident.

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Figure 5. Cross fracture of a whole straw (0.25ml capacity) of sperm cryopreserved in glycerol; low magnification. Etching to remove ice crystals reveals the structure of the freeze concentrated glycerol.

Figure 6. Cross fracture of a whole straw (0.25 ml capacity) of sperm cryopreserved in glycerol. Sperm tails are shown extending from the freeze concentrated glycerol into the spaces previously occupied by an ice crystal.

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Figure 7. Cross fracture of a whole straw (0.25 ml capacity) of sperm cryopreserved in glycerol. Frozen sperm cell with head entrapped in freeze concentrated glycerol with tail extending into an ice void.

The technique of freeze substitution followed by sectioning shows cells within the freeze concentrated matrix.

Figure 8. Light microscopy of a thin sectioned freeze substituted straw of sperm cryopreserved in glycerol.

Sections through sperm cells can be seen as dark stained bodies within the freeze concentrated matrix.

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More detail of an area of freeze concentrated glycerol containing sperm cells is shown in Figure 9 where sections through different parts of seven or eight sperm cells are visible.

Figure 9. Transmission electron microscopy of a thin sectioned freeze substituted straw of sperm cryopreserved in glycerol. The freeze concentrated matrix is electron dense, due to the protein components of eggs yolk included in the cryoprotectant. Frozen sperm are entrapped within the freeze concentrated matrix.

The above figures illustrate that cells rarely come into direct contact with ice crystals; rather they become concentrated into the unfrozen fraction, where they are exposed simultaneously to a number of physical stresses.

These are listed below in Table 1.

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Stress Encountered

Potential Cellular Response

Reduction in temperature

Membrane lipid phase changes

Increase in solute concentration

Depolymerisation of the cytoskelton Osmotic shrinkage

Increase in ionic concentration

Direct effects on membranes, including solubilisation of membrane proteins

Dehydration

Changes in pH

Destabilisation of the lipid bilayers Demonstrated to be damaging, but the mechanism is not well defined Mechanical damage Diffusion processes, including osmosis may become limited Denaturation of proteins etc

Cells may become closely packed

Membrane damage

Precipitation of salts and eutectic formation Gas bubble formation Solution becomes extremely viscous

TABLE 1 Some stresses encountered by cells during “slow” freezing.

However, it is generally considered that the osmotic response of cells is the primary determinant of viability. The hypertonic conditions the cells encounter lead to an osmotic loss of water, the extent of which is dependent on the rate of cooling. At ‘slow’ rates of cooling, cells may remain essentially in equilibrium with the external solution, reaching low temperatures, osmotically shrunken with the intracellular compartment sufficiently viscous so that the cells eventually vitrify. As the rate of cooling is increased, there is less time for water to diffuse from the cell, which becomes increasingly supercooled until eventually intracellular ice formation occurs – this is inevitably lethal. Video Sequence 2 shows intracellular ice formation within a human oocyte.

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Video Sequence 2

With many cell‐types, an optimum rate of cooling has been found for reasons that have been explained qualitatively above.

• • •

At rates of cooling slower than the optimum, cell death is associated with long periods of exposure to hypertonic conditions – essentially the cells become “pickled” As the rate of cooling is increased the exposure time to hypertonic conditions is reduced and damage due to this stress component is minimised. But there is less time available for osmotic shrinkage At rates of cooling faster than the optimum, cell death is associated with intracellular ice formation

The optimum rate of cooling may be considered to be the fastest rate of cooling at which intracellular ice formation does not occur. The response of cells to the hypertonic conditions encountered during freezing is determined by a number of biophysical factors:

• • • • •

Cell volume and surface area Permeability of the cell to water – the ability of the cell to respond, by loss of water, to an increase in extracellular concentration Arrhenius activation energy – the temperature dependence of the water permeability Type and concentration of cryoprotective additives Cooling rate

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Thus, to avoid the probability of intracellular ice damage to embryos, which have low surface area to volume ratio and low water permeability, slow rates of cooling are required. Other cells, with a large surface area to volume ratio and a higher value for water permeability may be cooled faster before the intracellular compartment becomes significantly supercooled.

Most approaches to cryopreservation have used linear rates of temperature reduction. An alternative approach has been reported (Morris et al., 1999) which is based on the fact that most physical parameters which the cells are exposed to during freezing vary in a non‐linear manner with temperature. Modifications of the freezing process, to take more account of the parameters the cell encounters and responds to, have been demonstrated to give better recovery on thawing than linear rates of cooling.

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SUPERCOOLING AND CELL SURVIVAL

It has been long recognized that a key factor in determining the viability of embryos following freezing and thawing is the initiation of ice formation in the suspending medium. In a controlled series of experiments (Whittingham, 1977), embryo suspensions deliberately nucleated below ‐9°C were found to have a low viability following a conventional cryopreservation protocol, whilst deliberate nucleation at higher sub‐zero temperatures (‐5°C to ‐ 7.5°C) gave much higher viability on thawing (Figure 10).

100

Survival (%)

75

50

25

0

-4

-5

-6

-7

-8

-9

-10

-11

-12

Nucleation Temperature (°C)

Figure 10. The dependence of the survival of mouse 8 cell embryos after seeding as a function of sub‐zero nucleation temperature (Redrawn from Whittingham 1977). Following ice nucleation the embryos were cooled to ‐196°C by a conventional protocol.

In IVF, embryos etc. are normally frozen in straws and an analysis of the spontaneous nucleation behaviour (Figure 11) clearly demonstrates that, if nucleation is not deliberately initiated, the recovery of embryos would be expected to be very low because most of the spontaneous nucleations occur below ‐8°C.

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100%

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80%

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60%

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40%

20

20%

0

Cumulative frequency

Frequency (%)

100

0% -7

-8

-9

-10

-11

-12

-13

-14

-15

Nucleation Temperature (°C) Figure 11. The observed spontaneous nucleation temperatures within 0.25 ml straws.

If embryos had been processed in the population of straws illustrated in Figure 11, the recovery rate upon thawing from liquid nitrogen would be predicted to be less than 20% compared with 80% if nucleation had been induced at ‐6°C.

The physical basis of this injury is clear from examination of the thermal histories of supercooled straws, as shown in Figure 12.

Current, standard practice is to cool straws initially to a temperature of about ‐7°C, allow thermal equilibration at ‐ 7°C, then deliberately nucleate ice in the straw by touching the outside of the straw with cold forceps, cryopen or ultrasonic probe etc. The temperature rises on nucleation to near its melting point (approximately ‐2°C) and then immediately following ice formation the temperature returns, at a rate observed to be approximately 2.5°Cmin‐1, to ‐7°C (Figure 12 (a)). The cell is then cooled slowly, during which cellular dehydration occurs. By contrast, in a straw supercooled in an environment held at ‐15°C (Figure 12 (b)), deliberate or spontaneous ice formation again results in a temperature rise followed this time by a more rapid rate of cooling (10°C min‐1) to ‐15°C. This rapid rate of cooling over a large difference in temperature between the melting point and the environment does not permit cellular dehydration to occur and lethal intracellular ice formation is then inevitable.

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Temperature (°C)

0

-5

-10

-15

-20 0

5

10

15

20

25

30

Time (minutes)

Figure 12a. Measured temperatures within straws during an embryo freezing protocol. During conventional embryo cryopreservation the straws are held at a temperature of ‐7°C and deliberately nucleated, the resultant rise in temperature following ice nucleation is small.

Temperature (°C)

0

-5

-10

-15

-20 45

60

75

Time (minutes)

Figure 12b. Measured temperatures within straws during an embryo freezing protocol. In the absence of deliberate nucleation, straws may reach much lower temperatures before spontaneous nucleation occurs.

A large rise in temperature to the melting point of the suspending medium then occurs followed by a rapid reduction in temperature. This rapid reaction will inevitably result in intracellular ice formation within embryos or oocytes.

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Intracellular ice formation following extreme supercooling is illustrated in Video Sequence 3 in which a mouse two‐ cell embryo has been supercooled to ‐15°C, spontaneous ice nucleation around the cell is followed by intracellular nucleation (cells become black due to the formation of many small ice crystals).

Video Sequence 3

Also note that the pattern of extracellular ice crystal growth at a high level of undercooling is very different to that observed at a smaller level of undercooling (as shown in the first Video Sequence 1).

Although it is standard practice to initiate ice formation in the suspending medium for embryos and oocytes, with other cell‐types this practice is not always considered essential. However, the recovery of spermatozoa (Songsasen & Leibo, 1977; Zavos & Graham 1983) erythrocytes (Diller 1975), granulocytes (Schwartz and Diller 1984) and bacteria (Fonseca et al., 2006) has been demonstrated to be improved by the controlled nucleation of ice during cooling. Deliberate initiation of ice would be expected to maximise the recovery of all cell types and, in all cases, would reduce any sample to sample variation.

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PROCEDURES FOR ICE NUCLEATION

General

Many cell types are processed in a continuous phase of fluid, >0.5ml; for example sperm in straws, cell suspensions in cryovials and bags. (For the special case of embryos and oocytes in straws, see below). To allow ice nucleation to be induced, the sample is cooled to a temperature of 2°C to 3°C below the melting point of the liquid. Following thermal equilibration at the nucleation temperature ice formation is initiated by touching the outside of the cryocontainer with a liquid nitrogen cooled spatula, forceps, cotton bud or a nitrous oxide cryopen. This causes a local cold spot at the wall, which leads to ice nucleation. Once an ice crystal has been formed it then propagates through the remaining undercooled fluid. It is also possible to induce nucleation using a small ultrasonic probe – including an ultrasonic toothbrush! A number of chemicals, for example purified membrane protein from the ice nucleating bacteria Pseudomonas syringae, crystals of silver nitrate and certain crystalline forms of cholesterol are known to induce ice formation in undercooled fluids. In principle these may be added to a cell suspension before freezing and ice formation will occur when the relevant temperature is reached during cooling – in this case there is no need to use any of the physical methods listed above.

Figure 13. Ice nucleation in an undercooled cryovial using a cryopen

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The spec cial case of embryos e and d oocytes in straws s

As describ bed above, to obtain high viability v of em mbryos, oocytees etc. in a cryopreservatio on programmee, it is essential th hat ice nucleattion is deliberaately initiated. The differencees between tho ose laboratoriees that achievee good results and d those that are less successfful can often bee attributed to o this simple prractical step.

Most IVF laaboratories fre eeze embryos in straws, in w which the embrryo is containeed in a small vo olume of liquid d, with adjacent co olumns of liquid (Figure 14).

Airr

Air

Diluent or growth medium

StrawP lug

Embryyos Cryoprotectant

inDiluent orr growth medium

Fiigure 14 Schem matic of straw lloading for ma ammalian emb bryo cryopreserrvation.

Most laborratories freeze e straws horizo ontally, whilst aa few freeze sttraws verticallyy: this has no effect on viabiility or ease of icee nucleation. EEmbryos sink in n the cryoproteective additivee and will be fo ound at the waall of the straw w when frozen horrizontally or at the bottom o of the column o of liquid when n frozen verticaally. Oocytes h have a lower d density and will tend to be more e buoyant. Following thermal equilibration at the nucleattion temperatu ure (commonlyy ‐7°C) ice formation is initiated by touching th he outside of tthe straw with a liquid nitroggen cooled spatula, forceps, ccotton bud or a nitrous oxide crryopen. This caauses a local ccold spot at thee wall, which leads to ice nucleation. Depeending on the labo oratory, ice nu ucleation may be initiated in the centre of the column off liquid contain ning the embryyos, at the menisccus of this column or in the diluent or gro owth medium.. If ice nucleation is initiateed in the diluent, ice growth pro opagation into o the embryo containing fluiid occurs via the continuouss liquid pathway along the w wall of the straw.

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Immediately following ice nucleation the temperature will rise where ice has formed (see Physics of Freezing section above) and an ice front can be observed to propagate along the straw, resulting in a “wave” of so called ‘nucleation peaks’ (Figure 15).

0

Temperature (°C)

-1 -2 -3 -4 -5 -6 -7 0

2

4

6

8

10

12

Time (minutes) Figure 15. Temperature measured by three fine thermocouples placed 1 cm apart within a straw (0.5ml capacity) containing 17.5% glycerol. Following ice nucleation successive nucleation peaks were observed as the ice front moves along the straw. Under these experimental conditions the rate of ice front propagation was relatively slow (approximately 2 cm min‐1)

Practical difficulties with ice nucleation of embryos in straws may arise from a number of points:

•

Because straws have a large surface area, a small diameter and a thin wall, very rapid warming occurs when they are removed from a cold environment (Figure 16). If straws are removed from the controlled rate freezer to initiate nucleation it is likely that the temperature will rise above the melting point, thus preventing ice formation. It may also then happen that ice can form locally at the cold nucleating tool, whilst the temperature of rest of the fluid remains above the melting point, so preventing ice crystal growth to propagate through the sample. In both cases actual nucleation will occur spontaneously during the later cooling, leading to reduced viability.

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Temperature (°C)

0 -2 -4 -6 -8 0

5

10

15

20

25

Time (seconds) Figure 16. Measured temperature within a single straw following removal (arrowed) from a controlled rate freezer at ‐7°C into ambient air at 20°C. Prior to nucleation the temperature rise within 5 seconds is sufficient to prevent ice nucleation.

•

•

•

In some laboratories it is common practice to check visually that ice propagation has occurred throughout the sample. If straws are removed from the controlled rate cooling equipment, this in itself may cause melting of the nucleated ice.

The thermal control of the freezing equipment may not be sufficiently accurate or stable at the nucleation temperature (‐7°C). Also any thermal fluctuations within the freezing apparatus may lead to re‐melting of the ice.

It has been observed in straws that ice nucleation at temperatures very close to the melting point results in a very slow propagation of ice through the sample. In some cases the ice propagation can actually become blocked and embryos are then effectively supercooled and would not be expected to survive further cooling.

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LONG‐TERM STORAGE

Cryopreserved material is ideally stored at the temperature of liquid nitrogen (‐196°C). The viability of biological material stored at ‐196°C is essentially independent of the period of storage. The oldest sample available, bovine spermatozoa that has been stored for over 50 years, shows no reduction in viability. The stresses associated with cryopreservation are not mutagenic: millions of cattle have been produced from frozen sperm and the incidence of abnormalities is identical to that observed with fresh sperm.

Samples are immersed in liquid nitrogen or stored in the vapour immediately above nitrogen. Immersion in liquid nitrogen guarantees a stable storage temperature but there is a potential risk of contamination. Vapour phase storage reduces the risk of contamination. However, very large temperature gradients may exist within the vapour phase above liquid nitrogen and these gradients are made worse by opening Dewars for transfer of samples etc.

The relatively high temperatures encountered by samples stored in the vapour phase together with temperature cycling may result in a reduction in viability. Vapour phase storage would be expected to cause problems with vitrified samples.

A wide variety of cryocontainers are available for cryopreservation. These vary in the manner in which they are sealed and whether they are suitable for storage in liquid nitrogen or the vapour phase (Table 2).

Cryocontainer

Liquid or Vapour phase Potential for Contamination storage

Conventional straws – sealed with a plug, PVA powder or ball bearing Heat sealed straws – for example CBS high security straw Cryovials Heat sealed glass amoules Specialist Bags – for example Baxter cryocyte, Pall etc

Vapour phase only

High

Liquid or vapour

Low

Vapour phase only Liquid or vapour Liquid or vapour

High Low Low

TABLE 2 Cryocontainers used for cryopreservation – suitability for liquid or vapour phase storage and the potential for contamination during long term storage.

Cryocontainers which are not sealed (e.g. cryovials, conventional straws) will leak during storage. This results in a high probability of contamination during long term storage. In addition any accumulation of liquid cryogen within the cryocontainer will lead to problems on thawing. When the temperature is raised above ‐190°C, 1 volume of liquid nitrogen turns into approximately 1500 volumes of nitrogen gas, which can lead to an explosive shattering of the cryocontainer.

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Because straws have a large surface area, a small diameter and a thin wall, very rapid warming occurs when they are removed from liquid nitrogen. Figure 17 shows the rapid warming that can occur following the removal of a single straw (0.25 ml capacity) from liquid nitrogen. Although this is an extreme example, it demonstrates that great care must be taken in the handling of cryopreserved material, for example during audit.

Temperature (°C)

0

-50

-100

-150

-200 0

10

20

30

40

50

60

Time (seconds) Figure 17. Measured temperature in a single straw (0.25 ml capacity) following removal from liquid nitrogen (arrowed) into ambient air at 20°C. After approximately 40 seconds the straws are transferred back into liquid nitrogen

Successful frozen storage at temperatures above ‐150°C is also possible. Storing biological material in a mechanical freezer is more convenient than using liquid nitrogen and a number of freezers are available which maintain temperatures down to ‐150°C. As a first approximation, cells should be stored at temperatures below the glass transition temperature (Tg) of the solution they are frozen in. For example, the tertiary system 300 mOsm salts, water and dimethyl sulphoxide has a Tg of ‐123°C. It is the Tg of the solution that the cells are suspended in which determines low temperature stability, not that of the “glass transition temperature” of water (See Appendix A definitions)

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CONTAMINATION

Recently there has been a clear demonstration of microbial contamination of samples within liquid nitrogen storage tanks (Bielanski et al., 2003; Fountain et al., 1997; Piasecka‐Serafin 1972;Teddar et al.,1995). For example, approximately 2% of stem cell cultures in cryovials stored in liquid nitrogen were found to be contaminated with micro organisms (Table 3). The two main potential sources of contamination are, firstly other cryopreserved samples stored in the same vessel, and secondly the liquid nitrogen itself.

Generally when liquid nitrogen is manufactured it has a very low microbial count. However contamination may occur during storage and distribution. In addition serious problems may occur with any portion of the cold chain which periodically warms up. In particular ‘transfer’ Dewars and dry shippers, which are allowed to warm, may accumulate pools of condensate which may become heavily contamined with bacteria and fungi. When refilled with liquid nitrogen this microbial ‘soup’ is effectively cryopreserved and then deposited onto samples.

The addition of contaminated liquid nitrogen to top up a storage vessel or the introduction of contamination from other sources will lead to increased contamination of the vessel with time. The potentially high level of contamination in storage vessels is not only a hazard for cryopreserved samples, it may also be a hazard to operators. CryoSEM of the ice detritus from a liquid nitrogen storage tank in routine use in an IVF clinic is shown in Figure 18, this material when thawed was demonstrated to contain high levels of viable micro organisms (Table 3).

Figure 18. The ultrastructure of ice contamination collected from a Dewar used for the long‐term storage of IVF samples

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Viable micro organisms isolated from: Thawed samples (1)

Liquid Nitrogen (1)

Liquid Nitrogen (2)

Propionibacterium acnes Acinetobacter calcoaceticus Staphylococcus (not S. aureus) Gram‐positive cocci

Aspergillus species Penicillium species Paecilomyces Non‐fermenting Gram‐negative rods Bacillus sp.

Corynebacterium Staphylococcus. Coagulase‐ negative a‐hemolytic Streptococcus Gram‐positive rods

Acinetobacter baumannii Klebsiella oxytoca Micrococcus spp.

Chrysenomonas luteola

Staphylococcus aureus Penicillum species Staphylococcus. coagulase‐negative Candida parapsilopsis Corynebacterium minutissimum Bacillus species Citrobacter freundil Enterococcus faecium Candida glabrata Pseudomonas paucimobillis Pseudomonas aeruginosa Gram‐variable rods

Sphingobacterium spiritivorum Weaksella virosa Non‐hemolytic streptococcus

TABLE 3 Viable micro organisms isolated from cryopreserved samples and from liquid nitrogen storage dewars, (1) fountain et al 1997; (2) from morris 2005.

•

•

Within the freezing equipment: vapour phase controlled rate freezers spray non‐sterile liquid nitrogen directly onto the samples. Sterility may be further compromised by any liquid condensate accumulated within ducting between freezing runs. Ideally, freezing equipment should have the capability of being sterilised between freezing runs. Operation of a vapour phase controlled rate freezer will also deposit viable micro organisms into the laboratory environment – this is important for critical environments, such as clean rooms.

During storage: straws may be contaminated on the outside, seals and plugs may leak allowing particulates to transfer via the liquid nitrogen within the storage vessel.

A number of solutions have been proposed to reduce contamination:

• •

•

Filtration of liquid nitrogen – possible, but requiring high specification equipment that is expensive and is not applicable to the general laboratory.

Storage of cryopreserved samples in the vapour phase – this would be expected to reduce the probability of contamination but not avoid it – contamination in the vapour phase has been clearly demonstrated (Fountain et al 1997).

Sealed cryocontainers – recently straws which may be sealed have been developed (CBS high security straws) – these may be stored in the liquid phase or vapour phase without leakage of external contaminants (Bielanski et al., 2003).

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THAWING & POST THAW HANDLING

During thawing of cryopreserved samples the physical processes which occured during freezing will be reversed.

The solidified system partially melts and cells again become suspended into hypertonic solutions which become more dilute during thawing. Cryoprotective additives and water may be transported across cell membranes and any intracellular ice may grow before it finally melts. In most cases examined, rapid rates of thawing are generally better than slow rates of warming. In standard practice, material cryopreserved in straws is thawed by being held in air for 40 seconds, during which time the temperature will rise rapidly to approximately –50°C (as shown in Figure 17) before the straw is transferred to a water bath held at 30°C for 1 minute. One of the reasons for holding the straw in air is to allow evaporation of any liquid nitrogen trapped within the straw. Direct immersion of straws containing entrapped liquid nitrogen into warm water would lead to rapid boiling of the liquid nitrogen with possible fracture of the straw or violent expulsion of the plug. However, the development of sealed straws now makes this step unnecessary and frozen straws could be directly immersed in a water bath.

After thawing the cryoprotective additives are diluted out, either in a single step or in two steps. This dilution is necessary because cells containing cryoprotective additive will tend to expand upon exposure to normal growth medium. To prevent swelling of cells, shrinkage is induced by using wash out solutions which contain hypertonic sucrose (0.2 M); this sucrose is then diluted away by washing with growth medium.

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CRYOPRESERVATION PROTOCOLS

This section is not intended to be a ‘recipe book’ but is included to highlight specific points of relevance to the cryopreservation of spermatozoa and embryos. Practical details of cryopreservation of cells are contained in several sources (Day and McLellan, 1995; Fuller and Grout, 1991; Fuller et. al., 2004 Hunter‐Cevera, and Belt,1996); for human IVF Cells (Dale and Elder, 2000; Karow and Crister, 1997); for veterinary and conservation IVF (Watson and Holt, 2001)

CRYOPRESERVATION OF PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMCS)

The use of cryopreserved PBMCs is well established as a routine procedure for clinical laboratory testing. Frozen PBMCs are used for various diagnostic purposes; for example HLA typing, detection of HLA antibodies in patients on waiting lists for organ/bone marrow transplantation, and mixed lymphocyte reactions. Frozen PBMCs are also used in “look back procedures” in transfusion medicine or diagnosis of patients. For example, no difference in isolation rates is found between fresh and frozen PBMCs regarding the human immunodeficiency virus.

Several methods for the cryopreservation of PBMCs have been reported (reviewed in Sputtek and Korber, 1991).

In general the cell concentration ranges from 5 x 106 ml‐1 to 50 x 106 ml‐1. The most frequently used medium is RPMI supplemented with human or fetal calf serum or plasma (up to 20% v/v) and the cryoprotectant of choice is 5% to 10% w/v dimethylsulphoxide. Cryopreservation is generally carried out in cryovials which are cooled at 1°C min ‐1 to temperatures of ‐60°C or below. To avoid major risks of contamination cryovials are stored in the vapour phase above liquid nitrogen. Seeding is not considered to be essential and recoveries on thawing range from 60% to 90% depending on the criteria used to assess viability.

CRYOPRESERVATION OF EMBRYOS

The pioneering studies on mammalian embryo cryopreservation used either glycerol or dimethylsulphoxide as cryoprotectants. This has been superseded by the protocol of Lasalle et al., 1985, which uses 1,2 propanediol, and is now employed by almost all IVF laboratories. Human embryos are considered to have a higher permeability to propanediol than either glycerol or dimethylsulphoxide and propanediol is less toxic than dimethylsulphoxide.

Much of the detail described in the earlier sections of this Guide is of direct relevance to embryo cryopreservation.

Embryos are generally cryopreserved in straws, increasingly in high security straws, to reduce any possibility of contamination during controlled rate freezing and storage. Some laboratories use glass ampoules or cryovials for embryo cryopreservation.

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In summary, the standard protocol consists of the following steps:

1.

2.

3.

4.

5.

6.

7.

8.

1,2 Propandiol (1.5 M) is used as cryoprotectant. Embryos are equilibrated in the cryoprotectant at room temperature to allow uptake of the cryoprotectant into the cell.

Embryos are loaded into straws or ampoules.

The samples are then cooled at 2°C min ‐1 to ‐7°C and allowed to thermally equilibrate before ice nucleation.

Deliberate nucleation, ‘seeding’.

Following ice crystal growth the temperature is then reduced at a slow rate of cooling (0.3°C min ‐1) to ‐ 30°C. The samples are then cooled rapidly to liquid nitrogen temperatures (see below).

Thawing is carried out in a two‐stage manner; straws are held in air for 40 seconds and then transferred to a water bath (30°C) for a further minute.

The cryoprotectant is then removed by dilution through solutions containing sucrose (0.2 M) and washed in culture medium.

This method has been reported to yield 70%‐80% survival rates with 12% implantation rate per embryo transferred. Lower survival rates are generally associated with failure to deliberately nucleate ice (step 4)

A variation in the technique between laboratories is at step 6, the manner of rapid cooling from ‐30°C to liquid nitrogen temperatures. In veterinary IVF it is common practice to transfer the straws directly from ‐30°C to liquid nitrogen. Some IVF clinics also transfer in this manner but most IVF clinics continue to cool straws within the controlled rate freezer to temperatures of ‐100°C before transfer to liquid nitrogen. Either method can yield equally good results. Direct transfer to liquid nitrogen at ‐30°C is the simplest solution but needs some care in the transfer. Samples taken from a ‐30°C environment warm very quickly in ambient air (Figure 19) and so the transfer must be complete within 5 seconds, otherwise straws may warm as illustrated, resulting in damaging rapid cooling once placed into liquid nitrogen. Cooling to temperatures of ‐100°C within the controlled rate freezer carries less risk of damaging temperature excursions during transfer to the final storage temperature.

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Temperature (C)

-5

-15

-25

-35 0

10

20

30

40

Time (seconds) Figure 19. Measured temperature in straws removed from a controlled rate freezer at ‐30°C into ambient air.

CRYOPRESERVATION OF SPERMATOZOA.

Spermatozoa, suspended in a cryoprotective additive such as glycerol are relatively insensitive to the linear rate of cooling during freezing. A very broad response curve exists with little difference in survival observed following cooling at 1°C min‐1 to 100°C min ‐1, as shown here for human spermatozoa (Figure 20). This curve is typical of that observed for many species of mammalian spermatozoa (Leibo and Bradley, 1999).

100

Recovery (%)

75

50

25

0 1

10

100

Cooling Rate (°C/min)

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1000

Figure 20. Measured recovery of sperm suspended in glycerol at different rates of cooling (Redrawn from Henry et al., 1977).

The relatively insensitive response illustrated above is unusual for mammalian cell types. Furthermore, the recovery of viability is comparatively low, with typically less than 60% of cells retaining motility on thawing. The cooling rate dependency of cell recovery of many cell‐types may be predicted from computer models of their osmotic behaviour during freezing. However the predicted results with spermatozoa have not been in agreement with experimental observations. Recently, it has been demonstrated that unlike many other cells, the observed reduction in viability of spermatozoa at rapid rates of cooling is not caused by the formation of intracellular ice rather than by an osmotic imbalance during warming (Morris 2006).

It is clear that spermatozoa have unusual cryobiological behaviour and improvements to their survival have not been amenable to conventional approaches of cryobiology. Glycerol has been the cryoprotectant of choice for spermatozoa, and historically egg yolk has also been included. Vapour freezing of straws, by suspending the straws in a tray at a defined height above liquid nitrogen has been the usual method of sperm cryopreservation and controlling ice nucleation has not been considered to be critical.

During vapour freezing a large variation is observed from straw to straw in respect of the rate of cooling and the ice nucleation temperature (Figure 21).

Temperature (°C)

40

20

0

-20

-40 0

5

10

15

20

Time (minutes) Figure 21. Measured temperatures within 3 straws (0.5ml) during vapour freezing

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When controlled rate freezing has been employed, 10°C min‐1 has been assumed to be ‘optimal’ and ice nucleation has not been deliberately initiated.

Temperature (C)

0

-10

-20

-30

-40 2

3

4

5

Time (minutes) .

Figure 22. Measured temperatures within straws (0.5 ml capacity), freezer environment temperature (‐‐‐‐), straws (‐‐‐‐‐) and (‐‐‐‐‐).

In non‐nucleated samples a large straw‐to‐straw variation is observed (Figure 22). The environment temperature within the controlled rate freezer decreases at 10°C min‐1 and before nucleation the samples track this temperature. However, following ice nucleation a large deviation is observed. One straw nucleates at ‐8°C: its temperatures rises close to the melting point of the glycerol solution, there is a latent heat plateau and then the sample cools to the environment temperature of ‐25°C at 28°C min‐1. In the other sample ice nucleation occurs at ‐ 16°C: the sample temperature rises close to the melting point than falls rapidly (36°Cmin‐1) to ‐34°C. This straw‐to‐ straw variation and the consequent loss of viability may not be important in samples where sperm counts are normal. However, in the case of oligozoospermic or asthenozoospermic samples these losses may be highly significant. With the development of intracytoplasmic sperm injection and the availability of techniques for surgical sperm removal, there is an increased need to store low numbers of sperm and therefore to improve freezing methods to maximise survival. Recently, methods which allow the cryopreservation of very low numbers of sperm have been developed (Cohen et al., 1997).

New approaches to sperm cryopreservation have been successfully demonstrated (Morris et al, 1999). These new protocols may be applied in the Asymptote EF600 and provide considerably improved recovery rates after thawing.

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REPRODUCIBILITY OF PROTOCOLS

A source of some confusion is the widespread practice of quoting cryopreservation protocols in terms of the control parameters programmed into nitrogen controlled rate freezers. In all cell freezers the temperature being controlled is that of a sensor within the machine not the sample temperature. The sample temperature will follow the temperature of the machine, with more or less lag depending on its size and location (Figure 23).

5

Temperature (°C)

0

-5

-10

-15

-20 00:00

15:00

30:00

45:00

00:00

Time (minutes) Figure 23 Measured temperatures within straws and ampoules containing 12.5% glycerol cooling at 0.3°C min‐1 by a standard embryo cryopreservation protocol in a conventional nitrogen controlled rate freezer.

Detail of the temperatures following nucleation, 1.0 ml sample in a plastic ampoule (‐‐‐‐‐), in a 0.5 ml straw (‐‐‐‐‐) and measured chamber temperature (‐‐‐‐‐)

It can be seen that under the same cooling conditions different capacity freezing containers may have very different thermal histories. These differences are maintained through the whole cycle but are particularly evident in the initial cooling phase following ice nucleation. The effects are more pronounced at faster rates of cooling (Figure 24).

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0

Temperature (°C)

-20

-40

-60

-80

-100 15:00

20:00

25:00

Time (minutes)

30:00

Figure 24. Reproducibility of sperm freezing protocol: Measured temperatures within straws and ampoules containing 12.5% glycerol cooling at 10°C min‐1 by a standard sperm cryopreservation protocol in a conventional nitrogen controlled rate freezer. Detail of the temperatures following nucleation at –7°C, 0.5 ml sample in a plastic ampoule (‐‐‐‐‐), 0.5 ml straw (‐‐‐‐‐), 0.25 ml straw (‐‐‐‐‐), measured chamber temperature (‐‐‐‐)

Different controlled rate freezers have different heat transfer coefficients which again will result in different thermal histories even when machines are programmed to carry out an identical freezing cycle.

The geometry of the sample is important. The thermal history of a 0.5 ml sample frozen within a straw (high surface area to volume ratio) is different to that achieved within an ampoule. The surface area to volume ratio of several containers used in cryopreservation are presented in Table 4.

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Cryocontainer

Surface area to volume ratio (m‐1)

0.25 ml straw 0.5 ml straw 1.0 ml fluid in a round bottomed 2 ml capacity cryovial 2.0 ml fluid in a round bottomed 2 ml capacity cryovial 20 ml fluid in a Baxter cryocyte bag

2.87 2.17 2.07

1.09

1.04

TABLE 4 The surface area to volume ratio for various containers used in cryopreservation.

All this has led to inaccuracy when protocols have been transferred, for example, from straws to plastic ampoules.

It has been demonstrated that when the same freezing protocol is used, the recovery of mouse embryos is much lower when frozen in plastic ampoules instead of conventional straws. Examination of the above figures clearly illustrates the reason for this. It is of course possible to redefine the protocol to achieve the desired thermal history within the ampoule and under these new conditions it would be anticipated that the recovery would be identical.

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APPENDIX A: DEFINITIONS OF TERMS AS APPLIED TO CRYOBIOLOGY

Eutectic A eutectic is a mixture of such proportions that the melting point is as low as possible, and furthermore all the constituents crystallize simultaneously at this temperature from the molten liquid. For example an aqueous solution of sodium chloride will remain a two phase system of ice and a concentrated solution of sodium chloride until the temperature reaches ‐21.4°C at which temperature the sodium chloride solution will solidify.

Freezing point The freezing point is the temperature at which the first crystal of ice appears during freezing. In special circumstances this can be the same temperature as the melting point. However water and aqueous solutions have a tendency to supercool and ice formation can be delayed to temperatures significantly below the melting point.

For example, in carefully controlled conditions water may be cooled to ‐40°C before ice nucleation becomes inevitable. Because the freezing point depends on the method of freezing, it is more useful to refer to the melting point.

Glass A solid with the molecular structure of a liquid, strictly an extremely viscous liquid with many mechanical properties of a solid.

Glass transition temperature (Tg) The temperature at which a material transforms from a liquid to a glass – this is usually taken as the temperature at which the viscosity of the liquid exceeds 1012 poise.

The solutions that cells are cryopreserved in have well defined Glass Transition temperatures – for example the tertiary system of 300 mOsm NaCl, water and glycerol, Tg is ‐ 64°C whilst that of the equivalent tertiary system with dimethylsulphoxide is ‐123°C. It is the Tg of the solution that the cells are suspended in which determines low temperature stability. The glass transition temperature for water is ‐132°C and erroneously it has been suggested that it is essential to store cells at temperature below ‐132°C and also that during any controlled rate freezing that cells must be cooled at a controlled rate to a temperature below ‐132°C before transfer to liquid nitrogen. It is correct that fusion (sintering) of ice crystals has been demonstrated to occur rapidly (within minutes) at ‐100°C (Petrenko and Whitworth, 1999). But this is not relevant to the cryopreservation of cells in complex solutions.

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Latent heat plateau Following initial ice formation in an aqueous solution, the temperature rises close to the melting point, and decreases slowly below this temperature for a significant time as more ice forms.

Melting point Melting point is the temperature at which the last crystal of ice disappears during warming. For example, the melting point of water is 0°C and for a 10% solution of glycerol, –2.3°C.

Nucleation Nucleation is the initiation of ice crystal formation, by physical or chemical methods, in undercooled water or aqueous solutions.

Seeding Seeding is the special case of initiation of ice crystal formation in undercooled water or aqueous solutions by the introduction of ice crystals.

Supercooling see undercooling

Undercooling The tendency of water and aqueous solutions to cool below their melting point before nucleation occurs. The extent of undercooling is the difference between the temperature of unfrozen system and the melting point. Also referred to as supercooling.

Vitrification The formation of a glass.

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APPENDIX B: REFERENCE;PS

Bielanski A., Berferon H., Lau, P.C.K, and Devenish, J. (2003) Microbial contamination of embryos and semen during long term banking in liquid nitrogen. Cryobiology 46 146‐152.

Cohen, J., Garrisi, G.J., Congedo‐Ferrara, T.A. et al., (1997) cryopreservation of single human spermatozoa. Human Reproduction 12 994‐1001.

Dale, B and Elder, K (2000) In Vitro Fertilisation. Cambridge University Press, second edition.

Day J.G. and McLellan M.R. (1995) Cryopreservation and freeze drying protocols. Methods in Molecula Biology 38, Humana Press, Totowa NJ.

Diller, K.R. (1975) Intracellular freezing: effect of extracellular supercooling. Cryobiology 12 480‐485.

Fonseca F, Marin M. and Morris G.J. (2006) Stabilization of frozen Lactobacillus bulgaricus in glycerol suspensions: freezing kinetics and storage temperature effects. Applied and Environmental Microbiology 72 6472‐6482.

Fountain, D., Ralston, M., Higgins, N., Gorlin, J.B., Uhl, L., Wheeler, C., Antin J.H., Churchill, W.H. and Benjamin R.J. (1997) Liquid nitrogen freezers: a potential source of microbial contamination of hematopoietic stem cell components. Transfusion 37 585‐591.

Fuller, B.J and Grout B.W.W. (1991) Clinical Applications of Cryobiology, RCR Press, Boca Raton, FL.

Fuller, B.J, Lane, N. and Benson, E.F. (2004) Life in the Frozen State, CRC Press, Boca Raton

Gilmore J.A., Liu, J., Woods, E.J., et al., (2000) Cryoprotective agent and temperature effects on human sperm membrane permeabilities: convergence of theoretical and empirical approaches for optimal cryopreservation methods, Human Reproduction 15 335‐343.

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Henry, M.A., Noiles, E.E., Gao, D., et al., (1993) Cryopreservation of human spermatozoa. IV The effects of cooling rate and warming rate on the maintenance of motility, plasma membrane integrity and mitochondrial function. Fertility and Sterility 60 911‐918.

Hobbs, P.V. Ice Physics. (1974) Clarendon Press. Oxford. Pp. 586‐589.

Hunter‐Cevera, J.C. and Belt, A. (1996) Maintaining cultures for biotechnology and industry. Axademic Press, San Diego

Karow, A.M. and Crister (1997) Reproductive Tissue Banking; scientific principles. Academic Press, San Diego.

Lassalle, B., Testart, J. and Renard, J.P. (1985) Human embryo features that influence the success of cryopreservation with the use of 1, 2, propanediol. Fertility andSterility 44 645‐651

Leibo, S.P. and Bradley L. (1999). Comparative cryobiology of mammalian spermatozoa. In Cagnon C. (ed.) The Male gamete: From basic knowledge to clinical applications. Cache River Press, Vienna IL, USA pp 501‐516.

Morris, G.J. (2005).The origin, ultrastructure and microbiology of the sediment Accumulating in liquid nitrogen storage vessels. Cryobiology 50 231‐238.

Morris G.J. (2006). Rapidly cooled human sperm: no evidence of intracellular ice formation. Human Reproduction 21 2075‐2083

Morris, G.J., Acton, E. and Avery, S. (1999) A new approach to sperm cryopreservation. Human Reproduction 14 1013‐1021.

Petrenko, V.F. and Whitworth R.W (1999. Physics of Ice pp. 233‐240 Oxford University Press.

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Piasecka‐Serafin M. The effect of the sediment accumulation in containers under experimental conditions on the infection of semen stored directly in liquid nitrogen (‐196°C). Bulletin Academy Polish Science, Biology 20 263‐267.

Schwartz G.J. and Diller K.R. (1984) Intracellular freezing of human granulocytes. Cryobiology 21 654‐660.

Songsasen N. and Leibo S.P. (1997). Cryopreservation of mouse spermatozoa 1. Effect of seeding on fertilizing ability of cryopreserved spermatozoa. Cryobiology 35 240‐254.

Sputtek A and Korber B (1991) Cryopreservation of red blood cells, platelets, lymphocytes and stem cells In Fuller, B.J and Grout B.W.W. Eds Clinical Applications of Cryobiology, RCR Press, Boca Raton, FL, pp 95‐147

Teddar, R.S., Zuckerman, M.A. Goldstone, A.H., Hawkins, A.F., Fielding, A., Briggs, E.M. Irwin, D., Blair, D., Gorman, A.M., Patterson, K.G. Linch, D.C., Heponstall, J., and Brink N.S, Hepatitis B transmission from contaminated cryopreservation tank, Lancet 346 137‐140.

Watson, P.F. and Morris, G.J. (1987) Cold shock injury in animal cells. In Temperature and Animal cells (K. Bowler, B.J. Fuller eds). Symposia of the Society for Experimental Biology 41 Company of Biologists, Cambridge, UK. pp.

311‐340.

Watson, P.F. and Holt W.V. (2001) Cryobanking the Genetic Resource. Wildlife conservation for the future. Taylor and Francis, London.

Whittingham, D.G. (1977) Some factors affecting embryo storage in laboratory animals. In The Freezing of Mammalian Embryos (K Elliot, J Whelan eds). Ciba Foundation Symposium 52 Elsevier, Amsterdam. pp 97‐108.

Zavos P.M. and Graham E.F. (1983) Effects of various degrees of supercooling and nucleation temperatures on fertility of frozen turkey spermatozoa. Cryobiology 20 553‐559

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