hum. reprod. update 2003 shaw 583 605

Terminology associated with vitri®cation and othercryopreservation procedures for oocytes and embryos

J.M.Shaw1 and G.M.Jones

Monash Institute of Reproduction and Development, Monash University, Clayton, Victoria 3168, Australia

1To whom correspondence should be addressed. E-mail: [email protected]

Over the past 30 years many cryopreservation procedures have been applied to oocytes, embryos, sperm, ovarian

and testicular tissue. Over this time many, often specialized, terms have been developed for all aspects of these pro-

cedures. This can make it dif®cult for readers who are not familiar with the terminology or protocols to compare

and evaluate different procedures. This paper describes the main cryopreservation procedures, the terminology

associated with them, and brie¯y explains the underlying physical, chemical and biological processes. The aim is to

help readers understand and interpret other papers on slow cooling, rapid cooling, ultrarapid cooling and vitri®ca-

tion.

Key words: cooling/cryopreservation/embryos/oocytes/vitri®cation

Introduction

Cooling to low subzero temperatures (cryopreservation), is used to

store human oocytes, embryos, sperm and gonadal tissues. There

are several well-established cryopreservation protocols for human

sperm and embryos, most of which were established >15 years ago

and are classi®ed as `slow cooling' or equilibrium cooling

procedures (Elliott and Whelan, 1977; Leibo, 1986; Mazur,

1990). In these slow cooling procedures, cryoprotectant (anti-

freeze) is added to the solution surrounding the cell and the sample

then cooled at a rate dictated by the size and permeability of the

cell. The thawing rate and protocol for cryoprotectant removal also

needs to be appropriate for the cooling procedure and the cell's

characteristics. The outcome is thought to be best when the rate of

cooling allows an equilibrium to be established between (i) the rate

of water loss from the cell (dehydration) and (ii) the rate at which

this water is incorporated into growing, extracellular, ice crystals.

For human oocytes and embryos, the optimum rate is thought to lie

between 0.3 and 1°C/min, for sperm 10°C/min, hamster cells

100°C/min and for red blood cells >1000°C/min, i.e. several orders

of magnitude faster (Elliott and Whelan, 1977; Critser and

Mobraaten, 2000). The period of slow cooling is usually ended

once the temperature has fallen to between ±30 and ±80°C, after

which they are moved to a low temperature storage tank (<±

130°C). This review does not aim to give details on how to

perform slow cooling procedures or their outcomes, as several

other articles and reviews already do this (Elliott and Whelan,

1977; Friedler at al., 1988; Karow and Critser, 1997; Critser and

Russell, 2000; Shaw et al., 2000b.

Oocytes, embryos and cells can also be cryopreserved by non-

equilibrium procedures such as `vitri®cation', `rapid cooling' and

`ultrarapid cooling'. We will collectively refer to the non-

equilibrium protocols as rapid protocols. Rapid protocols all differ

from slow protocols in that (i) most dehydration and cryoprotec-

tant permeation takes place before cooling commences, and (ii)

cooling is usually performed in a single step in which the specimen

is cooled directly from a temperature >0°C to low subzero

temperatures (<±130°C). Rapid protocols are commonly used for

animal embryos as they can give as good or better outcomes than

`slow cooling', yet use less equipment, less liquid nitrogen and less

processing time. Rapid protocols started to be evaluated for human

oocytes and embryos in the 1980s (Trounson, 1986) but were, until

recently, rarely used by human IVF units. Now several large IVF

units have started to publish results for rapidly cooled oocytes and

embryos. The outcomes (survival and pregnancies) of rapid

cooling need further improvement, but the convenience and low

cost of rapid cooling is likely to ensure increasingly widespread

use of these protocols for human embryos.

Because rapid protocols have not been widely used, relatively

few clinicians and embryologists are fully conversant with the

technical terms speci®cally used for rapid cooling protocols. This

can make it dif®cult to read and understand papers describing any

rapid cooling (`vitri®cation', `rapid cooling' and `ultrarapid

cooling') protocol. This has in part arisen because many of the

terms used in cryopreservation have been acquired from low

temperature physics, chemistry and engineering. This paper aims

to allow readers to more easily and effectively read and interpret

papers on cryopreservation literature, and in particular papers

Human Reproduction Update, Vol.9, No.6 pp. 583±605, 2003 DOI: 10.1093/humupd/dmg041

Human Reproduction Update Vol. 9 No. 6 ã European Society of Human Reproduction and Embryology 2003; all rights reserved 583

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describing rapid cooling procedures. To achieve this aim, in the

following sections we have summarized the terminology for the

ice and physical events associated with cryopreservation (Tables I,

II, VI), the chemicals and solutions (Tables III, VII), and the

procedures and processing steps (Table VIII), brie¯y outlined the

factors that in¯uence the outcomes of these cryopreservation

procedures (Tables IV, V, IX) and listed the equipment and

materials employed (Figure 1; Appendix 1, Table X). Words

associated with safety issues when working with liquid nitrogen

are also included (Appendix 2, Table XI). This review does not

aim to give details on how to perform rapid cooling procedures or

their outcomes, as several other articles and reviews already do

this (Shaw et al., 2000b; Vajta, 2000; Shaw and Kasai, 2001;

Kuleshova and Lopata, 2002).

Water, ice and physical changes within cryopreservationsolutions

Cooling to low subzero temperatures (e.g. ±196°C the temperature

of liquid nitrogen) without protection will kill most cells

immediately from damage caused by the growth of intracellular

ice crystals (see Tables VI±XI which list de®nitions of these

terms). Cryopreservation procedures are designed to minimize

damage caused by ice crystal formation and growth. To understand

how cryopreservation procedures achieve this goal, it is helpful to

know how they modify ice crystal formation and growth.

Ice dynamics in pure water

Ice formation is a stochastic process that starts when individual

water molecules join to form microscopic ice nuclei (also called

embryos or germs). In pure water the highest temperature at which

ice can form at normal pressure is 0°C, but ice nuclei form only

very reluctantly at this temperature, with the result that nuclei do

not usually form until the temperature falls below 0°C. Solutions

that remain free of ice, even though they are below the temperature

at which ice can form, are said to be in a supercooled state. The

lowest temperature at which pure water can be held without

freezing is thought to be ~±40°C, but ice normally forms at

temperatures between ±5 and ±15°C through spontaneous (due to

homogeneous or heterogeneous nucleation) or induced (by

seeding) ice nucleation. Once an ice nucleus has formed, further

water molecules can very readily bond onto this frozen surface,

allowing it to grow in size. Ice nuclei and the smallest ice crystals

are too small to see even with a microscope, but these usually grow

into ®rst microscopic and subsequently macroscopic ice crystals.

If ice forms in water at the highest possible temperature it only

spreads slowly and results in relatively few large crystals. When

ice forms in a supercooled solution it grows more rapidly. When

the ice forms there is a release of heat (the latent heat of fusion or

crystallization), as the change from liquid to crystal releases

energy. The amount of energy released may be considerable and

commonly brings the temperature of the whole sample back up to

0°C. With pure water the temperature will remain at 0°C until the

freezable water has formed ice, after which it will re-equilibrate

with the ambient temperature. Following the crystallization of

pure water, very little remains unfrozen even at temperatures as

high as ±5°C (>80% ice) or ±10°C (>90% ice) (Karow, 2001).

When water crystallizes, the space occupied by each individual

water molecule increases with the result that 1 g of ice (density

0.92) occupies more space than 1 g of water (density 1).

Consequently ice ¯oats.

The shape of the ice crystals is largely dictated by the shape and

behaviour of individual water molecules. Normal ice, i.e. that

which develops when water freezes, is also known as hexagonal

ice (or Ice 1h) because multiple groups each comprising six

individual water molecules bond to each other to form sheets.

Although crystals can grow along either the a-axis (parallel to the

hexagonal face or basal plane) or the c-axis (perpendicular to the

hexagonal face or prism plane), the individual molecules most

readily bind to incomplete hexagons, thereby extending existing

sheets. Water molecules do not attach onto the `side' of a sheet. As

a result crystals form characteristic angles. To thaw all this ice the

temperature has to rise above the freezing point. Pure water

therefore melts at 0°C, the same as the highest temperature at

which ice crystals can ®rst form. During the thawing process, the

change from crystal to liquid requires energy input.

Ice dynamics in cells and aqueous solutions containing solutes

Adding salts and other solutes to pure water lowers the tempera-

ture at which ice forms/melts. There is no limit to how low the

freezing/melting point can be depressed by solutes, with the result

that solutions with very high concentrations may never form ice,

Table I. General classi®cation of current cryopreservation procedures

Slow cooling (equilibrium)

procedures

Rapid cooling (non-equilibrium) procedures

Non-vitrifying De-vitrifying Vitrifying

State during cooling

Ice present (usually seeded

at ±5 to ±9°C)

Variable amount of ice

(spontaneous seeding)

Glass-like state with

no ice

Glass-like state with

no ice

Cooling rate usually 0.3 to

1°C/min to between ±30 and

±80°C then >200°C/min

Cooling rate depends on container and cooling strategy usually 200 to 20 000°C/min (tested range 5 to 130 000°C/min)

(e.g. Rall and Fahy, 1985; Rall, 1987; Hochi et al., 2001; Arav et al., 2002)

State during warming

Ice present More ice forms Ice forms as the glass

de-vitri®es

Maintains glass-like,

ice-free state

Warming rate usually 4 to

100°C/min

Warming rate depends on container and warming strategy usually 300 to >12 000°C/min (e.g. Rall and Fahy, 1985;

Rall, 1987; Hochi et al., 2001; Arav et al., 2002)

J.M.Shaw and G.M.Jones

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rather they will solidify into an ice-free (vitreous) state. Even when

salts and solutes are present, they rarely become incorporated into

the ice crystals themselves, because of the very speci®c shape and

bonding requirements of the growing ice crystals. Molecules other

than water molecules are instead excluded from the growing ice

crystals, and tend to accumulate between crystals or are physically

pushed ahead of an advancing ice front. The formation and growth

of ice crystals can, however, be modi®ed by compounds such as

antifreeze proteins (O'Neil et al., 1998), ice blocking agents (e.g.

Wowk et al., 2000) and cryoprotectants.

Rapid cooling solutions for oocytes and embryos use ingredients

and procedures which aim to reduce or eliminate both intra- and

Table II. Temperature table

°C °F

Approximate human body temperature +37 +98

Approximate room temperature +20 +68

Temperature below which oocytes and embryos of chill-sensitive species can suffer lethal

damage, attributed to membrane phase transitions (e.g. Drobnis et al., 1993; Pollard and Leibo,

1994; Martino et al., 1996). Pure DMSO is frozen at this temperature

+16 +61

Temperature of domestic refrigerator +2 to +6 +41

Freezing/melting point of pure water 0 +32

Approximate temperature for intracellular ice formation in human oocytes (Trad et al., 1999).

Seeding of the external solution is most commonly performed at between ±4 and ±9°C

±5 +23

Approximate temperature for intracellular ice formation in mouse embryos (Toner et al., 1991) ±10 +14

Temperature of domestic freezer; temperature at which a saturated sodium chloride solution

will freeze. Vitri®cation solutions stored at this temperature should remain liquid

±20 ±4

Highest routinely used plunging temperature for slow cooled specimens ±30 ±22

Plunging temperature routinely used for, e.g., materials slow cooled in DMSO ±35 ±31

Plunging temperature routinely used for slow cooled ovarian tissue. The lowest temperature at

which pure water can be held as a supercooled liquid without freezing

±40 ±40

Temperature of low temperature scienti®c freezers and approximate temperature of dry ice

(solid CO2), lowest plunging temperature routinely used for slow cooled specimens

±80 ±112

Lowest temperature at which growth of damaging ice crystals during warming has been

documented (see e.g. Shaw et al., 1995a,b)

±90 ±130

Temperature of ultralow-temperature electric freezers. Most solutions have glass transition

temperatures higher (warmer) than this

±130 ±202

Temperature of coldest ultralow-temperature electric freezers currently available ±150 ±238

Boiling point of liquid nitrogen at normal air pressure ±196 ±320

Boiling point of liquid nitrogen exposed to a reduced pressure (see `nitrogen slush' in text) ±197 to ±210 ±349

Absolute zero; 0 on the Kelvin scale of absolute temperatures ±273 ±459

Nitrogen vapour can be any temperature, but needs to be at low subzero temperatures (under ±150°C) for storage or rapid cooling.DMSO = dimethylsulphoxide.

Table III. Glass-forming properties of some chemicals used in cryoprotectant solutions; expressed as theminimum weight percentage, % wt/v (molarity in parentheses), of the chemical that has to be added to givea glass-forming solution

Chemical Fahy (1989) Ali and Shelton (1993) Penetrating?

Most effective glass formers

Butylene glycol (2,3-butanediol)a 46 (5.1) (3.0) Yes

Propylene glycol (1,2-propanediol)b 44 (5.7) (4.0) Yes

1,3-Butanediola 49 (5.4)

Dimethylsulphoxideb 49 (6.3) (5.0) Yes

Glycerol (1,2,3-propanetriol)b 65 (7.1) (5.0) Yes

1,3-Propanediola 57 (7.5)

Ethylene glycol (1,2-ethanediol)b 55 (8.9) (6.5) Yes

Acetamidea 61 (10) No

Least effective glass formers

aNot usually used for oocytes or embryos.bThese are routinely used for oocytes and embryos.The chemicals are listed in order of effectiveness as glass formers from the most effective to the leasteffective.

Terminology for cryopreservation techniques

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extracellular ice formation. These are discussed in greater detail

later in this review. All slow cooling procedures by contrast use

solutions which form ice, as the formation of ice crystals is integral

to the success of the slow cooling procedure.

An unprotected cell will form intracellular ice when cooled to

low zubzero temperatures. The ice undergoes uncontrolled growth

and forms large ice crystals which kill the cell. Slow cooling

procedures reduce the likelihood of this happening by letting

extracellular ice draw water out of the cell until so little free water

remains in the cell that only small (non-lethal) ice crystals can

form. This form of dehydration can only occur if ice is present in

the extracellular solution and does not form inside the cell. To

ensure that the solution forms ice before the cell, these procedures

rely on the difference in ice nucleation temperature between cells

and their surrounding solutions. Cells contain more solutes and

less free water than most physiological solutions in which they are

held. Ice therefore tends to form more readily in a physiological

solution than in cytoplasm. A solution containing solutes which is

cooled at a steady rate may, however, like pure water, supercool

and not nucleate until it reaches a temperature below that at which

the cytoplasm can nucleate. This is now known to be particularly

likely with oocytes, as those of several species start to form

intracellular ice at unusually high subzero temperatures (e.g. ±5°C)

both with and without added cryoprotectants (Toner, 1991; Trad

et al., 1999; Koseoglu et al., 2001). To ensure that extracellular ice

always forms at a temperature above that at which the cell can

spontaneously form intracellular ice, most slow cooling protocols

actively seed (induce ice formation in the solution) at temperatures

between ±6 and ±9°C and thereby avoid supercooling. When these

cryoprotectant solutions are seeded the solution freezes and energy

is released (latent heat of fusion), raising the temperature to the

solution's melting point. But the temperature does not (as in pure

water) rise to and remain at this point, rather the it falls as the

solute concentration of the remaining unfrozen fraction rises, as

this lowers the solution's melting point. Human oocytes may be

particularly vulnerable to freeze±thaw damage during slow

cooling because they start to form intracellular ice at such high

temperatures (e.g. ±5°C) that they contain ice before the solution is

seeded. It is not known why this occurs but they may contain

structures or molecules that act as ice nucleators. Even now we do

not fully understand the interactions between ice and cells. It is

thought that extracellular ice remains outside the cell and does not

usually cross/penetrate the cell membrane (possibly because the

membrane has a layer of bound water). However, there is evidence

that the dynamics of ice inside the cell is different depending not

only on whether extracellular ice is present, but also on whether

the cells are present singly in suspension or as monolayers or when

gap junctions are present (Acker and McGann, 2003; Armitage

and Juss, 2003). In rapidly cooled straws, the solution nearest the

walls both cools and warms fastest. The difference can be

biologically signi®cant as embryos rapidly cooled in straws with 3

mol/l (22%) dimethylsulphoxide (DMSO) showed high survival

and development rates when located close to the edge of the straw

in a region which only formed ice on warming, but very poor

development if they were located in the middle of the straw where

ice formed both on cooling and warming (Shaw et al., 1991a).

Ice nuclei on their own are unlikely to cause damage to a cell,

even if they are located within it because they are so small (they

start as pairs of water molecules). However, water molecules near

an ice nucleus readily bond with it, allowing it to rapidly develop

into a large ice crystal that can cause signi®cant intracellular

damage (Miller and Mazur, 1976; Elliott and Whelan, 1977; Leibo

et al., 1978; Mazur, 1984, 1990; Acker and McGann, 2003). An

advancing ice front (crystal surface) binds water molecules, while

physically excluding other molecules; it therefore causes damage

in part because it acts like an agricultural plough but also because

this leads to the accumulation of salts, solutes and gases that can

have osmotic and toxic effects. This damage may be exaggerated

by the conformational change that makes individual H2O (water)

molecules each occupy a greater volume in the frozen than in the

liquid state. The magnitude of these forces is exempli®ed by old

mining practices in which rocks were split by repeated freeze±

thaw cycles. Thawing frozen material will cause further damage in

particular if large ice crystals are present because the ice melts to

form areas of pure water, which can cause osmotic shock. There is

clear evidence that oocytes and embryos can be damaged both

during cooling and warming. Ice nucleation, ice crystal growth and

recrystallization does not just occur at around the melting point of

a solution, rather it occurs over a wide temperature range. For

example, if straws containing different solutions are moved from

liquid nitrogen to room temperature air or a waterbath at 37°C,

those with vitrifying (ice-free) solutions will warm at a relatively

Table IV. Survival and development of 8-cell mouse embryos rapidly cooled (>500°C/min) in vitrifying or de-vitrifying solutions is more dependent on thewarming rate than on the presence of ice crystals during warminga

Cryoprotectant Physical state

after cooling

Warming rate and physical state during warming

Fast Moderate Slow

(2500°C/min) (300°C/min) (10°C/min)

VS1 (~13 molal) Vitri®ed Vitreous (glass) Vitreous (glass) De-vitri®ed (ice)

22±87.7% blastocysts 80.7% blastocysts 0% blastocysts

3% fetuses

90% of VS1 (~12 molal) Vitri®ed De-vitri®ed (ice) De-vitri®ed (ice) De-vitri®ed (ice)

80±84.6% blastocysts 87.4% blastocysts 0% blastocysts

26% fetuses

aFrom Rall and Fahy (1985), Rall et al. (1987), Rall and Wood (1994).The vitri®cation solution was termed `VS1' and was used either at full or reduced concentration (here 90% of VS1).


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uniform rate. Straws containing solutions with ice will initially

warm at the same rate as those with vitri®ed contents, but from

temperatures of ~±100 or ±90°C will warm to the solutions melting

point at increasingly slow rates relative to the vitri®ed straw. Once

all ice has melted, the solution in the straw will again warm

rapidly. This pattern develops because, in addition to the energy

(heat) from the surroundings needed to raise the temperature, the

solution with ice has to take up extra energy to convert ice into

water. This implies that the temperature of a slow cooled straw

only has to rise to ±100°C (which can be reached within 5 s in air)

for changes in the ice to occur. Although there are many different

warming protocols for slow cooled specimens, it is possible to kill

up to 100% of embryos by using a warming protocol that is

inappropriate for the type of cryoprotectant or the cooling protocol

used (see e.g. Elliott and Whelan, 1977; Shaw et al., 1995a,b). In

samples of pure water the difference in density between ice (0.92

g/ml), the water that forms when the ice melts (1 g/ml) and the

remaining non-frozen fraction (>1 g/ml) can lead to separation in

particular in large samples in which the water and ice can ¯oat to

the top, leaving the (now more concentrated) remainder at the

bottom. Gentle agitation of the vial or straw throughout the

thawing step can help minimize the separation. During cooling,

Table V. Review showing how rapid cooling with vitrifying, de-vitrifying and non-vitrifying solutions/protocols affects the development of 8±16-cell mouseembryos (comments in parentheses)

Reference Cryoprotectant Blastocyst formation (%) Fetus formation (%)

Vitrifying solutions/protocols

Rall and Fahy (1985) VS1 (100% = ~13 molal) 87.7 (¯ >2000°C/min)

~82 (¯ <20°C/min)

Rall and Wood (1994) VS3a (BSA) 91 (¯ >2000°C/min) 65±73

92.9 (¯ <20°C/min)

Dinnyes et al. (1995) VS3a 85±97 (strain dependent) 18±71 (strain dependent)

Rall (1987) VS3 >95

Ishida et al. (1997) EFS 45±60

Mukaida et al. (1998) 30 versus 40% DMSO + FS 0±63 versus 37±53

30 versus 40% acetamide + FS 0±5 versus 0±10

30 versus 40% PROH + FS 8±82 versus 0±63

30 versus 40% EFS 0±96 versus 70±92

30 versus 40% GFS 0±32 versus 44±74

Bautista et al. (1997) 7 mol/l EG 93±100

De-vitrifying solutions/protocols

Rall and Fahy (1985) 90% of VS1 (~12 molal) ~84.6 (¯ >2000°C/min) 26

~80 (¯ <20°C/min)

Rall and Fahy (1985) 85% of VS1 (~11 molal) ~88 (¯ >2000°C/min)

~73 (¯ <20°C/min)

Rall and Fahy (1985) 80% of VS1 (~10.4 molal) ~80 (¯ >2000°C/min)

~60 (¯ <20°C/min)

Trounson et al. (1988) ~26% DMSO (3.5 mol/l) 65 32 (per embryo frozen)

0.25 mol/l sucrose 42 (per embryo transfer)

Shaw et al. (1991b) 33% DMSO (4.5 mol/l) 80±99 75±88 (per embryo frozen)

8.6% sucrose (0.25 mol/l)

Krivokharchenko et al. (1996) 30% glycerol, 0.7 mol/l sucrose

(from abstract only)

59.3±90.2 34

Wilson and Quinn (1989) ~26% DMSO (3.5 mol/l) 87 62

0.25 mol/l sucrose

Kuleshova et al. (2001) 25% EG (4.8 mol/l), 35% Ficoll 96

Kuleshova et al., 2001) 15% EG (3.08 mol/l), 40% Ficoll 97

Kuleshova et al. (2001) 10% EG (2.08 mol/l), 50% Ficoll 92

Protocols/solutions forming ice on cooling

Trounson et al. (1988) DMSO ~15% + sucrose (0.25 mol/l) ~47 8 overall

10/embryo transfer

Rall and Fahy (1985) 75% of VS1 ~48 (¯ >2000°C/min)

0 (¯ <20°C/min)

Rall and Fahy (1985) 70% of VS1 ~48 (¯ >2000°C/min)

0 (¯ <20°C/min)

Rall and Wood (1994) VS3a 6% BSA

48% glycerol (6.5 mol/l) 75 (¯ 5°C/min 20°C/min)

In most studies, straws were warmed rapidly.¯ = cooling rate, = warming rate; DMSO = dimethylsulphoxide; EG = ethylene glycol; PROH = 1, 2-propanediol; G = glycerol; F = Ficoll; S = sucrose;FS = Ficoll and sucrose; EFS = ethylene glycol and Ficoll and sucrose; GFS = glycerol and Ficoll and sucrose; BSA = bovine serum albumin.


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compounds other than the water can crystallize out of the solution.

If phosphate-buffered saline is placed in a normal domestic

freezer, crystallization/precipitation of some non-aqueous

components can occur. Once these components have come out

of solution they may not readily re-dissolve, with the result that the

overall ionic balance is different from that before freezing.

Free versus bound water and dehydration injury

In considering the dynamics of ice crystal growth, it is important to

recognize that in addition to free water, which crystallizes quite

readily, biological systems also contain `bound' water. The

`bound' water molecules are intimately hydrogen-bonded to the

atoms within molecules such as proteins, RNA, DNA or

membrane phospholipid head groups. These water molecules

form a substantial `coat', or hydration shell (one-third of their

weight) around the biological molecule and are essential for

maintaining their structure and function. Cryopreservation has to

try to remove free water to minimize damage from ice crystal

growth, without causing damage by uncontrolled removal of the

bound water providing structural support to proteins, DNA and

membranes.

Both slow and rapid cooling procedures reduce the likelihood of

the free water forming large (damaging) intracellular ice crystals

by enforcing both water loss (dehydration) and cryoprotectant

uptake. The presence of penetrating cryoprotectants and additives

such as sugars can help cells recover from dehydration. The cell

membrane comprises a phospholipid bilayer. Under normal

conditions the hydrophilic headgroups form the inner and outer

surface while the hydrophobic tail group lies between the two.

Drying can disrupt this arrangement (e.g. Bronshteyn and

Steponkus, 1993) by forcing the hydrophilic head groups to

coalesce or the hydrophobic portions to be exposed. Water

molecules form a hydration shell around proteins. The removal of

this shell can cause the irreversible collapse of the protein.

Cryoprotectants and sugars are thought to counteract dehydration

injury by acting as substitutes for water molecules at the surface of

proteins. Sugars can intercalate into the phospholipid head groups,

making them less likely to undergo an irreversible phase transition

(Anchordoguy et al., 1987; Crowe et al., 1988). To minimize

dehydration injury, a cell needs to be equilibrated with

cryoprotectant before drying commences. In both slow and rapid

cooling, cells will die from ice formation if they are frozen with

too high water content. Very severe dehydration (<80% of the

original water content) can also cause damage. Optimal cryopre-

servation procedures therefore have to establish a balance between

(i) too high and too low water contents and (ii) having enough

cryoprotectant present to replace water around the cell molecules

without causing excess toxicity (see e.g. Szell and Shelton, 1987).

The way in which the cryoprotectant is loaded into a cell

appears to in¯uence the cell's ability to tolerate dehydration and

may explain why protocols in which the concentration of

cryoprotectant is increased gradually can be more effective than

addition in a single step. Most cryoprotectant solutions are at a

signi®cantly higher osmolarity than physiological solutions

(mouse sperm freezing solutions are the exception). Each time a

cell is placed in a solution with a higher osmolarity than the one at

which the cell is currently equilibrated, it will shrink. The extent to

which it shrinks and the rate at which it recovers re¯ects the

difference in osmolarity between the old and the new solution, but

also the type of cryoprotectant, the presence of cryoprotectant

additives, temperature and cell type (Elliott and Whelan, 1977;

Critser et al., 1997; Paynter et al., 1997a, 1999a; Songsasen et al.,

2002). Critser et al. (1997) placed mouse oocytes in 1 mol/l DMSO

at 20°C. These oocytes shrank within seconds, by 25% (to ~75% of

their original volume) through water loss across the membrane,

after which an in¯ux of DMSO entered the cell replacing the lost

water, causing the oocyte to return to its original cell volume

within 20±25 min. Mouse oocytes which were placed directly into

higher DMSO concentrations shrink more and those in 6 mol/l

DMSO shrank to 25% of their original size within a few seconds,

and took longer to return towards their normal size. It is often

dif®cult to assess the degree of dehydration because many cells

collapse or ¯atten rather than shrink symmetrically, but is likely

that this extreme shrinkage results from the loss of both free and

some bound intracellular water, and may as a result be associated

with irreversible damage to the cell. Cells which are pre-

equilibrated with a low cryoprotectant concentration (e.g. 0.5±1

mol/l) can be placed at very high cryoprotectant concentrations

without exhibiting this extreme shrinkage, and recover better from

the treatment. The bound water around the proteins and other

intracellular structures would already have been partially replaced

(and as a result be partially protected from further water loss) by

penetrating cryoprotectant.

Chemicals and solutions used for cryopreservation

Small (<100 mol. wt) hydrophilic, membrane-penetrating com-

pounds which hydrogen bond with water molecules and can form

hydrogen bonds, with e.g. a protein (in place of a water molecule),

are particularly effective as antifreeze agents. The strength of the

bonding can be measured as the release of heat (heat of solution)

when the cryoprotectant is added to the solution. The presence of

these compounds in an aqueous solution lowers the solution's

tendency to crystallize, and lowers the highest subzero tempera-

ture at which ice crystallization occurs. High concentrations of

cryoprotectant compounds make the solution form a wholly ice-

Figure 1. Liquid nitrogen and vapour storage tanks. In liquid storage tanks,specimens are immersed in liquid nitrogen. In vapour storage tanks,specimens do not contact liquid nitrogen. Some vapour storage tanks havethe coolant within the walls, not within the storage space. The insulation inthe walls and the size of the opening in¯uence both the storage capacity(samples) and insulation properties.


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free (glass-like/vitreous) state when cooled. The capacity for

aqueous solutions with high concentrations of cryoprotectants to

reduce, or even abolish, ice nucleation forms the basis for their use

in vitri®cation solutions. Although there are many compounds

which can act as antifreeze agents, many are too toxic to use with

living cells, in particular at the concentrations which are needed

for vitri®cation to occur. As a result the only antifreeze chemicals

which are routinely used for vitri®cation or rapid cooling of cells

are polyols such as glycols (glycerol, propanediol, ethylenediol or

ethylene glycol), and DMSO. Other compounds such as alcohol

can also protect embryos from cryoinjury. However, all these

chemicals are toxic at the concentrations used in cryopreservation

solutions (usually >10%), so it is important that the composition of

the solution and the equilibration times and temperature are

balanced against the permeability characteristics and sensitivity of

each cell type (e.g. Shaw et al., 1991b; Paynter et al., 1997a,b).

Studies on liposomes indicate that the cryoprotective properties of

DMSO may be attributable to an electrostatic interaction between

its polar sulphoxide moiety and phospholipid membranes

(Anchordoguy et al., 1991), but also showed that at increased

temperatures there was a hydrophobic association between DMSO

and phospholipid bilayers (Anchordoguy et al., 1992).

Components of cryopreservation solutions

Most cryopreservation solutions used for oocytes and embryos are

made up in a physiological solution to which is added: one or two

cryoprotectants capable of permeating the cell membranes (usu-

ally propanediol, DMSO ethylene glycol or ethylene glycol), one

additive that is non-penetrating to the cell membrane (usually a

sugar), and a protein (which improves handling characteristics,

membrane stability (Anchordoguy et al., 1987; Crowe et al., 1988)

and can reduce toxicity). The permeable cryoprotectants are all

toxic and can cause osmotic shock, particularly at high concen-

trations. Vitri®cation solutions commonly also contain a polymer

(e.g. Ficoll) to make the solution more likely to vitrify at a given

concentration of penetrating cryoprotectant. The permeating

cryoprotectants lower the freezing point, and replace some of the

bound water molecules in and around proteins, DNA and other

intracellular components. The non-penetrating additives stay

outside and aid in dehydration before and during preservation;

they may also help stabilize the membrane by stabilizing the

phospholipid headgroups. Compounds such as amino acids vary in

their effect. Studies on liposomes have shown that most amino

acids increased damage; some such as glycine and alanine were

protective in the presence of salts, while amino acids with charged

amine group side-chains were cryoprotective whether salt was

present or not (Anchordoguy et al., 1988). In general, cryoprotec-

tant solutions used for slow cooling contain ~10% penetrating

cryoprotectant, whereas most of those used for rapid cooling or

vitri®cation contain >30% penetrating cryoprotectant. The con-

centration of non-penetrating sugar usually ranges from 0.1 to 1

mol/l (in the case of sugar this is 0.342 to 3.42 g/10 ml). It is

important to realize that the permeability of cell membranes to the

penetrating cryoprotectants varies greatly and depends not only on

which chemical it is and the temperature, but also which species

the cell is from and at what stage of development it is at (see e.g.

Paynter et al., 1997a,b; Paynter et al., 1999a,b).

Some cryopreservation procedures allow cells to be added

directly to the cryoprotectant and to also remove this solution, in a

single step, with physiological media. Most procedures for oocytes

and embryos, and in particular vitri®cation procedures, require

gradual or stepwise addition and gradual or stepwise dilution. The

composition of the solutions used before cells are placed in the

®nal cryopreservation solution varies, but generally the aim is to

replace some of the bound water in the cell's proteins, DNA and

other components with a penetrating cryoprotectant. The concen-

tration of the penetrating cryoprotectant is kept low to minimize

toxicity. The composition of the solutions used after warming also

varies but the aim is to ensure that all the penetrating

cryoprotectant is removed without causing osmotic shock.

It is not easy to control osmotic shock. The semipermeable

membrane that surrounds each cell lets water, cryoprotectants and

some other solutes cross these membranes, but not at the same

speed. Water is a very small molecule and crosses the membrane

more readily than the cryoprotectants currently used in cryopre-

servation. Cells which are equilibrated with cryoprotectant are

therefore at risk of osmotic injury when they are placed in a less

concentrated solution. In early slow cooling procedures this was

most commonly avoided by stepwise addition and dilution steps.

Rapid cooling and vitri®cation protocols use signi®cantly higher

cryoprotectant concentrations than the slow cooling procedures

(>4.5 mol/l as opposed to 1±2 mol/l). To counteract osmotic shock,

equilibration times for rapid cooling protocols are often short and

dilution is commonly performed in solutions in which an

impermeable non-toxic compound such as sucrose or trehalose

has been added to counteract osmotic stress. Following vitri®ca-

tion, the cells are particularly fragile and vulnerable to osmotic

shock (Pedro et al., 1997; Edashige et al., 1999), and there is

evidence of major changes in membrane permeability with the

result that normally impermeant molecules (e.g. propridium

iodide) can enter the cell (Kaidi et al., 1999; Newton et al.,

1999). It is possible that sodium chloride may enter the cell at this

time because replacing sodium chloride with choline chloride can

improve the post thaw outcome (Stachecki et al., 1998; 2002).

Serum and extracellular physiological solutions contain phos-

phates and glucose as well as high concentrations of sodium

chloride (e.g. 68±126 mmol/l) and calcium chloride (e.g. 0.3±2.04

mmol/l) and a low concentration of potassium chloride (2.5±5.5

mmol/l) (Gardner and Lane, 2000). By contrast the cytoplasm of

embryos (see e.g. Baltz et al., 1997), or oocytes (e.g. Dick, 1978)

contains less sodium (~10 mmol/l) and calcium and more

potassium (70±266 mmol/l).

Optimal cryopreservation procedures need to minimize osmotic

shock to the cell at all phases of the cryopreservation procedure.

The rate of cryoprotectant in¯ux and ef¯ux is in¯uenced by the

species, cell type, stage of development, solution composition and

temperature. This makes it dif®cult to extrapolate pre- and post-

cryopreservation equilibration conditions from one cell type or one

species to another. Our own impression is that an optimized

equilibration procedure shrinks the embryo (often very rapidly)

before cooling, but after warming, the cells exhibit a gradual return

to their original size, and do not swell beyond their original size.

What are vitri®cation solutions?

Procedures in which a solution/specimen solidi®es to form a glass-

like, or vitreous, state without any ice crystal formation during

cooling and remains in this state throughout the warming step are

called vitri®cation procedures. Whether vitri®cation will occur is


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Table VI. Terms used to describe ice, physical changes and water movement during cryopreservation procedures

Activation energy Here, in this context: the minimum amount of energy needed to transport water or other molecules across a cell membrane

Annealing Holding a specimen at a speci®c subzero temperature. Used in studies which aim to achieve a steady state of ice nucleation

(number and distribution) and ice crystal size and structure

Bound water Osmotically inactive water, usually bound to cell components such as proteins

Bubble formation On warming, solutions may contain bubbles. These form at advancing ice fronts, but may also develop when fractures or

cracks occur in frozen or vitri®ed samples. Can be symptomatic of poor cryopreservation conditions (Shaw et al., 1988). It is

not known what the bubbles contain but they form as readily in normal and degassed solutions

Colligative properties Properties of a liquid that can be altered by the presence of a solute (molecules or ions), but are not dependent on the type of

solute (these are: vapour pressure, freezing point depression and boiling points, and osmotic pressure). All of these properties

ultimately relate to the vapour pressure

Cooling A decrease in temperature

Cracking Most frozen and vitri®ed solids and the containers in which they are held become very brittle and prone to fracturing at low

temperatures. As cracking is most evident when a steep temperature gradient (large temperature difference) forms across the

specimen, it is most likely with fast cooling and warming (see e.g. Kasai et al., 1996; Poledna and Berger, 1996; Shi et al.,

1998; Liu et al., 2000; Arav et al., 2002)

Critical cooling rate Slow cooling at this rate dehydrates the cell without any intracellular ice formation, and can allow the intracellular concentration

of solutes and cryoprotectants to reach such high concentrations that the cell will vitrify when plunged into liquid nitrogen

Cryoinjury Cryopreservation, because it in¯icts such a range of chemical, biological and physical insults, can damage almost any part of a

cell. Although cryopreservation can cause severe injury leading to instant death/lysis, several fully optimized protocols maintain

the cells/embryos viability and developmental potential (no statistical difference as compared to non-treated controls). See also

Table IX and text

Crystallization In this context, refers to the transition of water molecules from a liquid state into ice. Crystallization releases heat (see heat of

fusion below). Crystals grow as spikes or ®ngers probably because the tips more effectively disperse the heat of fusion and

therefore grow faster than ¯at surfaces

Cuboidal ice A metastable form of ice obtained, e.g., when water vapour condenses at very low temperatures (e.g. ±130°C) also known as

Ice 1c. Density 0.92. One of several less common molecular con®gurations which water molecules can adopt. All atoms have

four tetrahedrally arranged nearest neighbours and 12 second neighbours. Under some conditions cuboidal ice slowly converts

to hexagonal ice

Dehydration Reducing the water content of a cell to reduce the likelihood of damaging intracellular ice formation when the cell is cooled

(see text on equilibrium and non-equilibrium methods and free versus bound water)

Dehydration curve A graph plotting water content of cells (often assessed by measuring cell volume) against another parameter such as time in

the cryoprotectant solution, or temperature (during a slow cooling ramp)

Dehydration injury Proteins and other cellular components are readily damaged by extreme water loss, as this removes water which is integral to

their conformation (see hydration shell). The hydrogen bonding properties of cryo- and lyo- protectants allow them to

reversibly substitute/replace the position/function of water molecules, thereby reducing injury

De-vitri®cation An event seen in vitri®ed solutions, usually during warming, characterized by the formation of ice nuclei or ice crystals.

The physico-chemical causes and its effect on viability of frozen cells or tissues have not yet been fully established

(see e.g. Karlson, 2001).

Differential scanning

calorimeter

Apparatus used to measure small changes in temperature (as calories). Sensitive enough to measure the release of heat

during ice crystallization, and the uptake of heat during melting

Eutectic point The minimum melting temperature of a mixture of two compounds. As an example: When DMSO is added to water, the

melting point becomes lower than for either of the pure ingredients (0°C for water and +18°C for DMSO). DMSO solutions

at concentrations routinely used for mouse embryo cryopreservation (e.g. 4.5 mol/l DMSO) are liquid even at <±30°C. It is also

the temperature at which a mixture of two compounds starts to melt, but in practice this ®rst melting is almost always

undetectable

Exosmosis Movement of water out of the cell by osmosis (because of a chemical gradient)

Fracture See cracking

Freezing The formation of ice crystals. The term freezing is therefore inappropriate for vitri®cation solutions which do not form ice

Freezing point 0°C is the equilibrium freezing point for water, i.e. the temperature at which water, ice and vapour can coexist at atmospheric

pressure. Ice can nucleate, or be seeded at this temperature, but forms more easily at temperatures <0°C

Glass A solid-like phase which does not contain any ice nuclei or ice crystals. Glass formation can occur in aqueous solutions used

in cryopreservation (vitri®cation) under appropriate cooling conditions. It is solid, not just a supercooled, very viscous liquid

Glass transition The temperature at which vitrifying solutions change to/from the solid, stable, glass-like state

Heterogeneous

nucleation

The formation of ice nuclei triggered by surfaces or impurities

Hexagonal ice The normal form of ice obtained when water freezes, also known as `Ice 1h'. Density 0.92. Protons are disordered but the

molecules form hexameric box-like structures (slight asymmetry 0.25% shorter in the c-direction) arranged as stacked sheets.

Classi®ed in internet sites as `space group P63/mmc, 194; Laue class symmetry 6/mmm with open, all-gauche, low-density

structure, with lower packing ef®ciency than simple cubic (~1/2) or face-centered cubic (~3/4) structures' (adapted from

http://www.lsbu.ac.uk/water/ice1h.html) (terms such as mmm and mmc form part of the nomenclature de®ning the symmetry of

crystals/space groups)

Homogeneous

nucleation

The spontaneous formation of ice nuclei not triggered by surfaces or impurities

Hydration shell It is estimated that proteins and DNA have to contain ~0.3 g water/g to remain functional. This water is not free, but bound

(e.g. to phosphates and bases) and is integral to the conformation of the biological molecule


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Table VI Continued

Hydraulic conductivity Movement of water across a membrane (Lp). Permeability

Hysteresis A solution is said to exhibit hysteresis if it melts at a temperature that is different from it's highest possible ice nucleation

temperature. Compounds such as antifreeze proteins can cause hysteresis. Not in¯uenced by supercooling

Ice Ice can form in any of 12 different crystal structures, plus two amorphous states. The oxygen atom of each molecule is strongly

covalently bonded to two hydrogen atoms, while the molecules are weakly hydrogen-bonded to each other. In all solid ice the

water molecules are hydrogen-bonded to four neighbouring water molecules: there are two hydrogen atoms near each oxygen,

one hydrogen atom on each O±O bond. When ice and water coexist at the freezing point, the amount of ice remains constant as

long as no heat is added or removed (e.g. Schulson, 1999). Heat is released as water changes from the liquid to crystal phase.

Energy needs to be added to convert ice back to water

Ice-axis Ice crystals have structure dictated by the shape of each water molecule and the way it bonds to neighbouring water

molecules. This leads to crystals with hexagonal symmetry with an a- and a c-axis

Ice nucleation See homo- and heterogeneous nucleation and crystallization above (c.f. manual `seeding' as de®ned in Table VIII)

Kedem±Katchalsky

coef®cient

Membrane transport can be modelled using principles of non-equilibrium thermodynamics. Kedem±Katchalsky equations model

the ¯ux of both water and cryoprotectant through a cell membrane when driven by concentration gradients. See e.g. Liu et al. (2000)

Lp Hydraulic conductivity. See e.g. Liu et al. (2000)

Melting Put simply, the process in which ice turns into water. As a solution melts there is a constant interchange of water molecules

between ice and water. The rate of exchange is lowest where the molecules form a ¯at (planar) ice surface and highest where

the crystal structure is incomplete or damaged (e.g. at corners). Corners, or smaller crystals, usually melt at a temperature at

which a large planar crystal surface is stable. Energy (heat) has to be added in order for each molecule to change from ice

(a low energy state) to water (a higher energy state)

Melting point/melting

point range

The temperature at which the solid and liquid phases of a given compound or mixture have the same vapour pressure. Melting

point range: the temperature at which a solid sample begins to melt and the temperature at which melting is complete. For water

the melting point range is from ±100 to 0°C

Membrane phase

transition

Membranes are phospholipid bilayers. A phase transition alters the membrane ¯uidity because individual components or

regions of the membrane change between a liquid and a solid state. The transition temperature is dictated by the chain length

and saturation level of the individual fatty acids

Modelling Many important aspects of cryopreservation can be modelled mathematically (see e.g. Liu et al., 2000). Parameters to take

into account include: cell diameter, cryoprotectant molarity (initial and ®nal), ambient temperature. The cell's osmotically

inactive volume, the re¯ection coef®cient in Kedem±Katchalsky equations, cell membrane water (hydraulic) and

cryoprotectant permeability, molar volume of the cryoprotectant, and time

(e.g. http://www.me.umn.edu/divisions/tht/bhmt/tutorial/CPAload)

Nucleation/nucleation

temperature

The ®rst event in ice crystal formation. At the molecular level, individual water molecules join. The change from a liquid to a

crystalline state is associated with a loss of energy (latent heat of crystalization). Nucleation is a stochastic process

Osmotic shock Cell shrinkage and swelling which can lead to irreversible damage or lysis caused by excess water loss/gain (see text for full

description). See e.g. Pedro et al. (1997)

Permeability How much and how fast chemicals and water move across the cell membrane (can be denoted as P). Water permeability is

expressed as hydraulic conductivity

Phase diagram: phase

transition diagram

A plot (commonly of temperature against pressure or temperature against solute concentration) showing when the solution

undergoes a speci®c physical change e.g. glass transition, melting, de-vitri®cation, nucleation

Re-crystallization A complex process, usually associated with warming, in which individual ice crystals within a solution change size. Small

crystals commonly shrink while large crystals become larger (see also crystallization and melting)

Re¯ection coef®cients Also written as `s'(sigma), gives a measure of how well solutes cross a membrane

Supercooling A solution which remains liquid, even though it is below the temperature at which it can support ice, is said to be in a

supercooled state. Ice crystallization will occur rapidly if a supercooled solution is seeded

Td Temperature at which de-vitri®cation (if any) occurs. The composition of the solution will in¯uence the temperature and

extent of this event

Tg Temperature at which glass transition occurs; characterized by a sharp exotherm because of heat loss when metastable clusters

form. Below Tg, it is not merely a viscous liquid, but a solid in a stable thermodynamic state. The glass transition temperature

varies considerably and is dependent on the composition of the solution

Thawing Melting of ice crystals. The term thawing is therefore inappropriate for vitri®cation solutions which do not form ice

Thawing rate/warming

rate

The rate at which the specimen is warmed. Specimens slow cooled to between ±30 and ±40°C are usually warmed rapidly

(>200°C/min) while those slow cooled to <±60°C are usually warmed slowly (<20°C/min). Specimens containing ice do

not warm as uniformly or rapidly as ice-free samples as energy is taken up by the melting ice crystals

Time±temperature

transformation curve

A plot of crystallization temperature against time. Generally gives a <-shaped plot which shows the temperature at

which crystallization is fastest. In¯uenced by concentration and viscosity (see e.g. Sutton, 1992)

Tm Melting temperature. The melting temperature varies considerably and is dependent on the composition of the solution

Triple point The temperature and pressure at which water can be present as a liquid, crystal or gas. Adding solutes alters the triple point.

Increased pressure can also lower the triple point

Vapour pressure The pressure (e.g. in Pascals) that needs to be applied in order to maintain the amount of water vapour constant in the

presence of pure water or ice

Vapour pressure

depression

Change in vapour pressure (in Pascals) caused by adding e.g. solutes to the water

Vitri®cation See `Glass' (above)

Warming An increase in temperature


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Table VII. Terminology for chemicals and solutions used for cryopreservation

Albumin A protein ~68 000 Da. Used to reduce the `stickiness' of oocytes and embryos during handling, but it can also promote the

vitri®cation of cryoprotectant solutions (Rall and Wood, 1994; Shaw and Trounson, 1998)

Antifreeze Most cryoprotectants act as `antifreezes' in that they depress the ice nucleation temperature

Antifreeze protein Molecules which reduce the growth of ice crystals. A common source is from Arctic or Antarctic ®sh. Some act by having

a tertiary structure that produces one hydrophobic face which preferentially binds to crystalline ice, (i.e. the ice front) and a

hydrophilic face on the opposite side which preferentially binds liquid water. This `coating' reduces the likelihood of water

molecules in the liquid phase becoming crystalline by attaching to an existing ice surface. Antifreeze proteins have been added

to slow and rapid cooling procedures with highly variable success (Shaw et al., 1995a,b; Wang, 2000)

Buffer The solutions in which cells are cryopreserved include commonly available physiological buffers (e.g. phosphate-buffered

saline, HEPES, or MOPS buffered media), media (T6, Medicult, HTF), special media (e.g. choline chloride-based buffer

Stachecki et al., 1998, 2002), serum or even pure water. To this are added permeating and non-permeating cryoprotectants

and other additives. The choice of buffer can affect the outcome (Vasuthevan et al., 1992). Most buffers stabilize the pH.

In the semen cryopreservation literature the solutions (usually buffered and with cryoprotectant) that are added to the semen

before/after cryopreservation are referred to as `extenders'

Choline chloride A salt, mol. wt 139.6. It has been used instead of sodium chloride (NaCl) in slow cooling and vitri®cation procedures

(Stachecki et al., 1998, 2002) as this may help reduce the adverse effects of high NaCl concentrations on the hydration

shell of proteins and reduce the risk of raising cytosol NaCl levels

Colloidal pressure The osmotic pressure exerted by colloids (e.g. proteins). Colloid osmotic pressure (Donnan pressure) is the pressure

difference which has to be established between a colloidal system and its equilibrium liquid to prevent material transfer

between the two phases when they are separated by a membrane, permeable to all components of the system, except the

colloidal ones

Composition The composition of a cryoprotectant solution can be expressed on the basis of weight (wt%, wt/wt, g etc.) or by volume

(ml, v/v) or a combination of the two (e.g. wt/v). A mole is a mass equal to the mol. wt in g; molar solutions have 1 mol/l of

solution; molal solutions have 1 mol/1000 g of solvent

CPA In this context; cryoprotectant solution

Cryoprotectant,

non-permeating

Any of a range of compounds which do not readily cross the cell membrane but which none the less provide cells with

protection against cryoinjury. The ones most commonly used for cells include sugars, polymers and proteins

Cryoprotectant

Permeating

Any of a range of low mol. wt hydrophilic compounds which can hydrogen bond with water. The ones most commonly used

for cells because of their comparatively low toxicity are DMSO, ethylene glycol, glycerol and propylene glycol

Dimethylsulphoxide

(DMSO) (Me2SO)

Mol. wt 78.13. Antifreeze compound widely used for oocytes and embryos at a concentration of 1.5 mol/l (11% v/v) for slow

cooling, and >3.5 mol/l for rapid cooling. It is commonly used in combination with glycol-type compounds as the

combination interacts with water and biological materials in a slightly different way to glycols alone

EFS E = ethylene glycol, F = Ficoll, S = sucrose. This combination has been found to be useful for mammalian embryos of a

wide range of species. First developed by Kasai et al. (1990)

Ethylene glycol (EG) Also called 1,2-ethanediol, mol. wt 62.07. Antifreeze compound widely used for oocytes and embryos. It is commonly used

in combination with DMSO. It has relatively low toxicity, and usually, because of its small size, penetrates membranes rapidly

Formaldehyde A naturally occurring compound which at high concentrations (1 to 4%) is used as a ®xative. Has been found, at varying

concentrations, in batches of propanediol, thus it is advisable to screen or test propanediol before use

(see e.g. Mahadevan et al., 1998)

Glycerol Glycerine; also called 1,2,3-propanetriol; mol. wt 92.1. Antifreeze compound widely used for sperm and blastocysts. It has

low toxicity, but is slow to cross membranes which can contribute to osmotic shock

Glycol A chemical classi®cation of a group of chemicals which includes glycerol, propylene glycol (1,2-propanediol), ethylene glycol

(ethane diol), polyethylene glycol, butanediol (some of which may be methoxylated; Wowk et al., 1999). Most of these have

cryoprotectant properties but many are very toxic

Heat of solution The heat released when a cryoprotectant is dissolved, for example into water to make an aqueous solution

Hydrogen bonding A chemical bond between a hydrogen atom on one molecule and a nitrogen, oxygen, sulphur or halogen. The hydrogen atom

is usually is covalently bonded in its molecule to oxygen, nitrogen or sulphur

Molar (M, mol/l) A solution made up based on the mol. wt of a compound. For a compound such as sucrose (mol. wt 342) a 1 molar solution is

made by weighing out 342 g of sugar and adding liquid to make it up to a total volume of 1 l. (Not the same as molality or

mole fraction.)

Molecular weight

(MW, mol. wt);

formula weight (FW)

Mol. wt is the sum of the weights of each atom in each molecule of this compound, calculated from the atoms which it contains

(number and atomic weight). Water for example contains one oxygen atom (16 in the periodic table) and two hydrogen atoms

(1 in the periodic table). Its mol. wt is calculated as 16 + 1 + 1 = 18. For salts it is more common to give the formula weight

(e.g. anhydrous MgCl has a FW of 95.21, and MgCl hexahydrate has a FW of 203.3)

Polyethylene glycol A polymer of ethylene glycol available in a range of mol. wt. In¯uences membrane permeability and fusability. Has proved

bene®cial in some glycerol (Rall, 1987), DMSO (O'Neil et al., 1997) and DMSO + PG (Rall and Fahy, 1985)-based vitri®cation

solutions for several species including the mouse and cow (Ohboshi et al., 1997)

Polymer Large mol. wt compounds such as Ficoll, Dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol. Large mol. wt

proteins can have analogous effects. Can increase the viscosity and physical vitri®cation characteristics (Wowk and Fahy, 2002;

Shaw et al., 1997). Used in some solutions for slow cooling and vitri®cation (Rall, 1987; Kasai et al., 1990; Carroll et al., 1993;

Rall and Wood, 1994)

Propylene glycol See `Propanediol' (commonly abbreviated as PG or PROH)

Propanediol (PROH) 1,2-Propanediol, FW 76.1. Antifreeze compound very widely used for human embryos. It readily forms vitrifying solutions.

Commonly abbreviated as PG or PROH. A potential problem is that one of its natural breakdown products is formaldehyde

which has the potential to damage gametes (see e.g. Mahadevan et al., 1998)


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dictated by the composition of the solution but also other factors,

the most important being the cooling rate and the warming rate.

Thus, a solution which vitri®es in one protocol may form ice

crystals when used under other conditions; these crystals may

develop only during warming (de-vitri®cation) or during both

cooling and warming. Vitri®cation occurs most readily at high

cooling and warming rates. Because of the interactions between

ice formation and the cooling and warming conditions, it is

possible to have both vitrifying and ice-forming regions within the

same solution. A solution with a set composition but cooled and

warmed at different rates, or solutions with differing compositions

but cooled at a set cooling and warming rate, can as a result form

anything from a wholly transparent (vitri®ed, ice-free) and glass-

like state, through having some microscopic ice crystals (usually

seen as a `haze'), to being fully opaque in the presence of many

macroscopic ice crystals. However, even transparent solutions

may contain countless ice nuclei and ice crystals, because the ice

crystals only become detectable optically once they become larger

than the wavelength of light. Sensitive equipment such as

differential scanning calorimeters can measure the release and

uptake of energy associated with ice crystal formation and melting,

but even with this equipment it can be dif®cult to differentiate

between a fully vitri®ed ice-free state and a nearly vitri®ed state in

which only some ice nuclei or small ice crystals are present. If a

specimen is not uniformly cooled or the solution is not well mixed

before cooling, then it may form both vitri®ed and non-vitri®ed

regions. Thus vitrifying, partially vitrifying, de-vitrifying and ice-

forming conditions, although they are clearly different physically,

are part of a continuum rather than each having a clear boundary.

Unlike slow cooling solutions that expand during cooling because

ice occupies a larger volume than water, vitri®cation solutions

appear to remain the same volume or shrink. In the absence of ice

there is also no redistribution of salts, solutes or cells. These

attributes should in theory make solutions that remain vitreous

Table VII Continued

Proton bonding See `Hydrogen bonding'

PVA Polyvinyl alcohol. A polymer used to seal straws, but also to substitute for proteins. Recently shown to be highly effective

in promoting vitri®cation (reducing ice nucleation) (Wowk and Fahy, 2002)

PVP Polyvinylpyrrolidone. A polymer used to seal straws, but also in media, buffers and cryoprotectants to substitute for proteins.

Can be used to promote vitri®cation or to replace some penetrating cryoprotectant (Shaw et al., 1997). Some batches are toxic to

embryos even after dialysis against water

Raf®nose A sugar, mol. wt 594.5. Used to promote dehydration before, during and/or after cryopreservation. Normally does not cross the

cell membrane (non-permeating/impermeant) and of low toxicity to oocytes and embryos (Kuleshova et al., 1999b;

dela Pena et al., 2002). Used for mouse sperm cryopreservation

Solute concentration How much solute (salts, sugars, cryoprotectants, and other additives) a solution contains. Vitri®cation solutions commonly

have a high solute concentration (>40% wt/vol) (Fahy et al., 1987)

Solution control Oocytes, embryos or cells that have been exposed to the solutions used in a cryopreservation procedure but have not actually

been cryopreserved. Gives an indication of the toxicity of the solutions

Solution effect Damage caused in a slow cooling procedure by the non-frozen fraction (Mazur et al., 1972)

Solvent In cryopreservation, water is usually the solvent in which other chemicals are dissolved

Sucrose A sugar, mol. wt 342. Used to promote dehydration before, during and/or after cryopreservation. Normally membrane

impermeant and of low toxicity to oocytes and embryos

Sugars Monosaccharides (fructose, galactose, glucose); disaccharides (maltose, sucrose, trehalose, lactose); polysaccharides

(e.g. raf®nose) (Abas Mazni et al., 1989; McWilliams et al., 1995; Kuleshova et al., 1999b)

Total solute

concentration

How much solute (salts, sugars, cryoprotectants, and other additives) a solution contains. Vitri®cation solutions commonly have

a high solute concentration (>40% wt/vol)

Toxicity General term, referring to any reduction in viability or developmental potential in cryopreserved and solution control

embryos/cells, but not non-treated controls. Degree of toxicity assessed under any particular set of conditions will depend on

the particular criteria used in the assessment. Strongly in¯uenced by cryoprotectant type and concentration, temperature and

time of exposure, cell type and species. Unrelated to chill sensitivity and osmotic shock. Can affect both cytoplasm and the

nucleus (see e.g. Kono et al., 1988)

Trehalose A sugar, mol. wt 378.3. Used to promote dehydration before, during and/or after cryopreservation. Normally membrane

impermeant and of low toxicity to oocytes and embryos (Kuleshova et al., 1999b; Ergolu et al., 2002)

v/v (vol/vol) The composition of a cryoprotectant solution expressed by volume (volume of the additive/volume of the total solution).

Useful when compounds differ in density.

Vitri®cation solution Solutions which, on cooling, can form a solid, non-crystalline phase. Whether a solution vitri®es is governed by its

composition and the cooling and warming rate. Solutions may vitrify on cooling, but crystallize (de-vitrify) during warming.

Procedures which allow the solution to retain its glass-like state throughout cooling and warming are `true' vitri®cation

procedures. Procedures which allow any crystals (ice) to form in the solution at any stage during cooling and warming are

not vitri®cation procedures but can be classi®ed as rapid cooling procedures

VS1 The vitri®cation solution used by Rall and Fahy (1995), the 100% VS1 (~ 13 molal) contained 10% PROH (~1.5 mol/l),

20% DMSO (~3 mol/l) + 15% acetamide + 6% polyethyleneglycol

VS3/VS3a VS3 = 48% glycerol (6.5 mol/l) + 6% polyethylene glycol; VS3a = 48% glycerol (6.5 mol/l) + 6% bovine serum albumin

wt/wt (wt%) The composition of a cryoprotectant solution expressed by weight (weight of the additive/weight of the total solution).

Useful when compounds differ in density. The composition of a cryoprotectant solution can be expressed as its percentage

of the weight of the solution. Thus when 100 g of the ®nal solution contains 10 g of a chemical, this chemical constitutes

10 wt%. [Could also be written as 10% (wt/wt).]


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throughout cooling and warming less damaging to cells than ones

that form ice. Whether this is true or not remains unclear.

In the literature the term `vitri®cation' has on occasion been

applied to both wholly ice-free and ice-forming conditions. The

terms cooling, freezing, warming, melting and thawing have been

used relatively interchangeably whether ice is present or not. The

term `freezing' speci®cally relates to ice crystal formation whereas

`thawing' speci®cally refers to the melting of ice crystals. These

terms are therefore only relevant to solutions with ice. Under

conditions in which no ice forms, the terms `cooling' and

`warming' are more correct. Several papers also describe the use

of `ultrarapid' cooling procedures, but this may be thought of as a

catchphrase and not a cryopreservation procedure since this term

has been loosely and inconsistently applied to a range of protocols.

As it can be dif®cult to determine from a description of a procedure

whether the specimen actually vitri®es and remains vitri®ed, this

review includes rapid cooling (e.g. >20°C/min) procedures

irrespective of whether these would result in vitrifying, de-

vitrifying or non-vitrifying conditions.

Procedures/processing steps in cryopreservation forassisted reproductive technology

Most human IVF units use slow cooling rather than rapid cooling

procedures to store embryos. In the mouse there has, over the past

10 years, been a shift away from slow cooling procedures to

ethylene glycol (Kasai et al., 1990; Zhu et al., 1993; Kuleshova

et al., 1999b), glycerol (Rall and Wood, 1994; Dinnyes et al.,

1995; Zhu et al., 1996) or DMSO (Shaw et al., 1991a,b)-based

rapid cooling and vitri®cation procedures. For many mouse strains

Table VIII. Terminology for processing steps/procedures

Break point The temperature at which a slow cooling ramp is ended (most commonly between ±30 and ±80°C), and a more rapid cooling step (usually

3±300°C/min), usually to the ®nal storage temperature, starts

Cooling rate The known or estimated change in temperature over a set time. In slow cooling, the rate of cooling most commonly is 0.3 to 1°C/min from

±6 to ~±35°C, and then >10°C/min to ±196°C. With rapid protocols, cooling rates of between 200 and 20 000°C/min are most common

Cryoinjury Damage to cell components caused by cryopreservation. Virtually any component can suffer cryoinjury

Denaturation A change in the 3-dimensional conformation of a protein. Cryoprotectants such as DMSO can, if added too quickly to a solution

containing a protein, make it milky or opaque. It is likely that the protein becomes denatured by the DMSO. It is recommended that

the DMSO be added stepwise

Equilibration

temperature

The temperature of the solution in which cells are placed/equilibrated (see e.g. Shaw et al., 1992; Mukaida et al., 1998)

Equilibration

time

Length of time that the specimen is exposed to a given solution (see e.g. Shaw et al., 1992)

Equilibrium

cooling

A term used for cryopreservation protocols in which the water content of a cell remains in osmotic balance with the surrounding solution

during cooling in the presence of ice crystals. Exempli®ed by slow cooling procedures

Exposure time Length of time that the specimen is exposed to a given solution (see e.g. Shaw et al., 1992)

Holding time A step in a slow cooling programme. A period of time over which the temperature is held constant. (Also called soaking time.)

In-straw

dilution

Loading and sealing a straw so that it contains both the dilution solution and the cryoprotectant solution containing the cells. These

are separated by an air bubble. After warming the straw is shaken to move the air bubble, allowing the two solutions to mix

Latent heat Latent heat of crystallization is the heat (energy) released by water molecules when they change from a liquid to a solid (ice)

Loading

pattern

In this context this refers to the size and number of air bubbles/columns of solution within a straw. Semen is commonly loaded into a

straw with either no, or only a very small, airgap before being sealed. When embryos or oocytes are cryopreserved by slow cooling,

a small amount of air (e.g. 0.05 ml) is usually placed both in front of and behind the cryoprotectant solution containing the cells. This

prevents the cells from becoming lodged in the plug or sealed ends of the straw. A further amount of cryoprotectant or dilution solution

is also commonly placed beyond the air. If more than one solution is placed in a straw, it is important that the bubble or interface

separating them is not disturbed during the cooling and warming steps

Non-equilibrium

cooling

A term used for cryopreservation protocols in which cellular dehydration and cryoprotectant penetration are enforced by solutions with

a high osmolarity before cooling commences, and change very little during the cooling step. Exempli®ed by all rapid-cooling procedures

Plug There are usually two small pieces of material on either side of a water-absorbent polymer (e.g. PVP) at the end of cryopreservation

straws. The water-absorbent material swells and forms an impervious barrier when an aqueous solution is drawn into it. In slow cooling,

the straw is usually loaded in such a way that the plug becomes sealed by the ®rst aliquot or column of solution. The straw may in

addition be heat, or impulse sealed. A bubble of air between the specimen and the solution containing cells is thought to reduce the

likelihood of cellular damage by heat being transferred into the liquid

Ramp A step in a slow cooling programme, which starts at one temperature and ends at a different temperature. The cooling or warming rate

is constant between the two points e.g. 0.3°C/min

Rehydration Cells are normally dehydrated by cryopreservation and need to be re-hydrated before use. Cells are usually rehydrated and at the

same time have the cryoprotectant removed, by placing them in one or more solutions with successively lower osmolarity ending

with the return to a physiological solution (buffer or media) or transfer to a recipient

Rinse Straws are commonly rinsed with the cryoprotectant solution before being loaded with the cells to remove debris and possible

contaminants. We have noticed that conventional cryopreservation straws can change the pH of the rinse solution

Seeding Manual or automated initiation of ice formation. Usually as part of a slow cooling procedure. Is initiated for example by touching

the edge of the solution with a cold implement (e.g. forceps at ±196°C). The cryoprotectant is seeded at a high subzero temperature

(between ±4°C and ±9°C) to initiate dehydration of the specimen

Soaking time Alternative term to `holding time' (see above)

Ultrarapid A term which has (confusingly) been applied to a very wide range of cooling procedures and cooling rates. It is therefore best

considered as a `catch' phrase


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the in-vitro and in-vivo development of embryos cryopreserved by

these rapid cooling procedures is the same (statistically) as control

non-frozen embryos and signi®cantly better than for slow cooled

embryos. Highly ef®cient protocols have also been developed for

other species including the cow (Vajta et al., 1998, 2000), sheep

(Naitana et al., 1997; Martinez and Matkovic, 1998; Baril et al.,

2001; Zhu et al., 2001; Al-aghbari and Menio, 2002;

Papadopoulos et al., 2002; Isachenko et al., 2003) and rabbit

(Lopez-Bejar and Lopez-Gatius, 2002). The animal research

indicates that there is no single `correct' or single optimum

cryopreservation protocol because the outcome is in¯uenced by

many factors (see e.g. Table V for 8-cell mouse embryos). Thus

there is now a variety of different solutions, equilibration and

dilution strategies (volumes, times and temperatures), and cooling

and warming procedures that can give excellent results (for the

mouse >90% blastocysts, >60% fetuses). However, for each

effective combination there are many other combinations of

solutions, times and temperatures that give poor or erratic

outcomes (Table V). The slow cooling procedures used for

human embryos do generally give consistent results, but the

embryo survival rates (usually <80%) are not ideal. Early rapid

cooling trials for human oocytes and embryos (e.g. Trounson,

1986) obtained some promising results, but there was also

considerable concern as several early papers indicated that rapid

cooling could harm oocytes and embryos. The tangible bene®ts of

rapid protocols (cost savings and convenience) have, however,

ensured that some groups have persevered in trying to optimize

rapid protocols for human oocytes and embryos. Several very

signi®cant advances have recently been made in rapid cooling of

human embryos, and as a result we may now be at the point where

Table IX. Terms describing processes associated with cells (other than those already covered in previous tables)

Apoptosis Cell suicide. An active process in which the cell undergoes an orderly sequence of events leading to its disintegration.

Cryopreservation can trigger apoptosis in cells

Chill injury Damage to cells that occurs at temperatures >0°C (see e.g. Pollard and Leibo, 1994; Martino et al., 1996a)

Dilution effect The change in sperm motility when semen is diluted (this is often a sharp fall)

Extender Term used widely in the sperm cryopreservation literature. A solution added to semen. It can have a very simple composition

but most contain cryoprotectant, and additives such as oocyte yolk or milk powder

Free radical damage Most cellular components can be damaged by free radicals (unpaired oxygen atoms, free hydroxyl groups and hydrogen peroxide)

as these compounds are highly reactive and cause damage by oxidation or reduction. See `ROS' below

Lipid peroxidation Membranes and other lipid-containing cellular components are degraded by free radicals (see `ROS'). Removal of lipid stores

can greatly improve survival following cryopreservation (Ushijima et al., 1999; Nagashima et al., Dobrinsky 2001)

Metabolism The biochemical reactions occurring in a cell. Placing cells at temperatures below that of the normal body temperature perturbs

metabolic functions. The cell's capacity to fully recover after rewarming to body temperature may be affected by these changes

(see e.g. Hammerstedt and Andrews, 1997; Parks, 1997)

Oocyte Oocytes of all species so far tested are more dif®cult to cryopreserve than embryos of the same species (see e.g. Nakagata, 1989;

Van Blerkom, 1989; Carroll et al., 1993; Bernard and Fuller, 1996; Martino et al., 1996; O'Neil et al., 1997, 1998;

Paynter et al., 1997 a,b; 1999a,b; Stachecki et al., 1998, 2002; Trad et al., 1999; Kuleshova et al., 1999a; Papis et al., 2000;

Vajta, 2000; Chen et al., 2001, 2003; Hochi et al., 2001; Lane and Gardner, 2001; Ledda et al., 2001; Matsumoto et al., 2001;

Arav et al., 2002; Songsasen et al., 2002; Begin, 2003)

Parthenogenic activation Development of an unfertilized oocyte. This can be triggered by the chemicals and procedures associated with cryopreservation.

Parthenogenically activated oocytes can be haploid or diploid and develop to somite stage fetuses following transfer. Care is

therefore needed to not confuse them with normally fertilized oocytes (two-pronuclear and with two polar bodies)

Phase transition See de®nitions in Table VI for membrane phase transition and phase transition diagram, `Saturation' (below) and `Free radical

damage' (above)

Reactive oxygen species

(ROS)

Oxygen molecules are normally paired. If the two molecules are separated they form highly reactive charged ions that can

readily damage cellular components by oxidation. Not all cell types and components of the cell are equally vulnerable to ROS

injury (see `Metabolism', `Saturation'). Free radical scavengers (including cryoprotectants, sugars, vitamins such as vitamin C

and E, and enzymes such as superoxide dismutase) may help protect against damage by ROS

Saturation Hydrocarbon chains in fatty acids may have a variable number of double bonds. These chains are said to be saturated if there

are no double bonds, monounsaturated if there is a single double bond and polyunsaturated if there are multiple double bonds.

The number of double bonds affects two main properties of the fatty acid. Double bonds represent potential sites for

biochemical reactions, particularly with ROS or other tree radicals. They also limit the ¯exing of the carbon skeleton of the

fatty acid, which affects the melting temperatureÐthe greater the number of double bonds the greater the in¯exibility, and the

higher the temperature at which melting occurs (see e.g. Arav, 1996, 2000; Zeron et al., 2001, 2002)

Spindle

disassembly/reassembly

In metaphase II oocytes the chromosomes are held in place by very ®ne ®bres (the spindle). The spindle can break down in

response to cooling and cryoprotectants. On warming, the spindle will reassemble but may not do so correctly. This can lead

to the loss of chromosomes when the cell extrudes the polar body (see e.g. Chen et al., 2001, 2003)

Storage time At ±196°C it should be possible to store cells for considerable lengths of time without spoilage (decades, possibly centuries).

For human gametes and zygotes, legal constraints usually impose a shorter storage time than this

Thermal shock Damage caused by reduced temperature. Membrane shrinkage and rearrangement of membrane components are important,

particularly when this increases membrane permeability

Zona hardening Changes that increase crosslinking of the glycoproteins of the zona pellucida, making it more impenetrable. Important for

oocytes as it can reduce sperm penetration. Caused for example by cryoprotectants, sugars or cooling (see e.g.

Matson et al., 1997)


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a transition from slow to rapid cooling procedures will start to take

place also for the human.

Rapid cooling protocols are very inexpensive and easy to

establish, but it is important that the procedure is fully optimized

for the material which is to be preserved. The cheapest and

simplest rapid cooling procedures to set up are those which allow

the materials to be cooled in the vapour phase of liquid nitrogen or

in liquid nitrogen itself. Specimens placed deep within a stable

vapour phase cool at rates of between 10°C/min for large

specimens (e.g. goblets of straws containing sperm) and >300°C/

min (individual straws, or small samples). Specimens placed

directly into liquid nitrogen cool at rates of 2000°C/min (e.g. for

straws) to >20 000°C/min (e.g. for small grids and pulled straws).

Which cooling rate and which cryopreservation solution is best for

a given cell type is dependent on several parameters.

Many studies have investigated the effect of different cooling

and warming rates. Some early studies compared cooling rates of

between 5 and 2500°C/min and warming rates of between 10 and

2000°C/min. Moderate or high cooling and warming rates gave

more consistent and better results than the slowest cooling and

warming rates, but the magnitude of the effect differed markedly

depending on the composition and concentration of the solution.

Two glycerol-based vitri®cation solutions, VS3 and VS3a, gave

high (>80%) or very high (>90%) survival rates of mouse embryos

over most of the tested cooling and warming rates, and even the

combination of the very slowest cooling and warming rates did not

reduce survival to <75%. By contrast, vitri®cation solutions VS1

and VS2, and dilutions of VS1, were strongly in¯uenced by the

cooling and warming rate, in that the slowest cooling and warming

rates could kill all embryos. Solutions which formed ice during

cooling usually give suboptimal results, but in other respects the

results did not correlate closely with physical changes. Many

recent studies have used much higher (e.g. >20 000°C/min)

cooling and warming rates than in the earlier studies. The very

high cooling rates and warming rates have been achieved by (i)

reducing the cryoprotectant volume, (ii) modifying the container,

(iii) cooling in liquid nitrogen slush (±210°C), or (iv) cooling on

metal blocks. Increasing the cooling and warming rates is very

bene®cial for the lipid-rich oocytes and embryos of domestic

species (cow, pig), as it helps to overcome chill sensitivity (Pollard

and Leibo, 1994; Martino et al., 1996a,b). The bene®t of using

faster rates must, however, be balanced against the increased

likelihood of stress fractures (most materials shrink on cooling)

(Kasai et al., 1996; Poledna and Berger, 1996; Shi et al., 1998; Liu

et al., 2000; Arav et al., 2002). This is best exempli®ed by a study

in which repeated cooling and warming of mouse embryos at

intermediate cooling and warming rates gave signi®cantly better

survival than at high rates. Reducing the volume of cryoprotectant

reduces the likelihood of stress fractures (by reducing the

temperature difference across the specimen), but this needs to be

balanced against the increased likelihood of osmotic shock in the

®rst dilution solution.

Slow cooling (equilibrium) and rapid cooling (non-equilibrium)

procedures all comprise four basic steps: exposing the cell/tissue

to a cryoprotectant, cooling to the storage temperature (usually ±

196°C, the temperature of liquid nitrogen), warming and

cryoprotectant removal. These steps aim to reduce chill and

cryoinjury, e.g. intracellular ice formation, dehydration injury,

osmotic shock and toxicity when the cells are cooled to low

subzero temperatures.

Slow cooling

Slow cooling and rapid cooling procedures tend to differ in

cryoprotectant concentration and cooling rate, and it is possible

(but not neccessarily optimal) to use either high and low

cryoprotectant concentrations and high and low cooling rates

with either procedure. Thus the principal difference between the

two lies in when and how the specimen becomes loaded with

antifreeze compounds and dehydrated. Equilibrium, or slow

cooling procedures usually use solutions containing 1±2 mol/l

concentrations of cryoprotectant and reduce the likelihood of

intracellular ice formation by initiating extracellular ice crystal

formation (seeding) at a high subzero temperature (e.g. ±6°C). The

rate of cooling is then used to govern how quickly these

extracellular ice crystals grow. The best results are thought to

occur at the fastest cooling rate at which a balance (equilibrium) is

maintained between water loss (by diffusion) from the cell and the

incorporation of this water into the extracellular ice crystals

surrounding the cell (for a fuller description of the mathematics

and principles of equilibrium cooling procedures see Mazur,

1990). The cooling rate used for oocytes and embryos (between

0.3 and 1°C/min) re¯ects their size, permeability and diffusion

characteristics and is signi®cantly slower than for somatic cells,

erythrocytes or sperm (1 to >100°C/min). Even though equilibrium

protocols use relatively low concentrations of cryoprotectant, it is

still necessary to control damage from cryoprotectant toxicity and

osmotic shock by selecting the most appropriate cryoprotectants as

well as the time, temperature, and number of addition and removal

steps. A small number of equilibrium slow cooling procedures are

very widely used for human preimplantation embryos at all stages

of development. These procedures generally result in 70% survival

of embryos and the pregnancy rate and development to term

following transfer are similar to those of non-frozen embryos. It

would be highly desirable to have a slow cooling protocol that is

100% ef®cient for human oocytes and embryos, but it is not clear

whether this will ever be possible. There are groups who have

consistently obtained excellent slow cooling outcomes (>90%

continued development) for mouse embryos. Mouse oocytes and

embryos are more tolerant of cryopreservation than most other

species because of their smaller size, low intracellular ice

nucleation temperature and low chill sensitivity. Oocytes and

early embryos of domestic species such as the pig and cow are by

contrast much more chill sensitive than those of the human.

Despite the lack of animal models, there have recently been

changes to the protocols for both human oocytes (Fabbri et al.,

2001) and blastocysts (Gardner et al., 2003) which improve the

post-thaw outcomes. Further advances are therefore possible. Thus

the main disadvantages of slow cooling are that the equipment can

be expensive to purchase and maintain and that each `run'

(equilibration and cooling) takes >1 h.

Rapid cooling

Rapid cooling procedures began to be developed in the 1970s (e.g.

Wilmut, 1972; Elliott and Whelan, 1977; Leibo et al., 1978).

These are non-equilibrium procedures in that the water loss

(dehydration) and cryoprotectant gain by the cell does not reach an

equilibrium with the surrounding solution before cooling com-


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mences. Early studies investigated a very wide range of cooling

rates, warming rates, containers, cryoprotectants and cryoprotec-

tant additives and formed the basis for subsequent work (Rall et al.,

1980, 1983; Takeda et al., 1984; Krag et al., 1985; Scheffen et al.,

1986; Williams and Johnson, 1986). Rall and Fahy (1985) were the

®rst to apply vitrifying conditions to embryos. This approach,

which was based on the pioneering work of Lujet, reasoned that

because ice formation is a major contributor to freeze±thaw injury,

cryopreservation conditions which prevented any ice from forming

should be bene®cial. The concept is attractive, which may explain

why vitri®cation appears to receive more attention/mention than

other rapid cooling procedures.

Whether procedures in which the solutions remain vitreous

throughout the cooling and warming procedure are better or worse

than those in which solutions vitrify during cooling but de-vitrify

during warming is not wholly clear. In the mouse, conditions in

which ice forms during both the cooling and warming step of a

rapid cooling procedure are usually damaging (Tables IV, V),

while those rapid cooling procedures in which the specimen

remains vitri®ed or only forms ice (de-vitrify) during warming can

both give either excellent or poor results (Tables IV, V). In this

case the best outcome (developing fetuses) was achieved when de-

vitrifying rather than vitrifying conditions were used. It is possible

that the more concentrated solution gave this result by being more

toxic or by causing greater loss of bound water. Hochi et al. (2001)

showed that with a single vitrifying solution the fastest cooling and

warming rates did not necessarily give the best results.

Vitri®cation and a rapid warming rate is thus not a guarantee of

success. Rather it is now evident that embryo/oocyte survival is

dependent on optimization of the whole cryopreservation proced-

ure (see e.g. Kasai et al., 1980, 1990, 1996; Rall and Fahy, 1985;

Rall, 1987; Szell and Shelton, 1987; Shaw et al., 1991a,b; Ali and

Shelton, 1993a,b; Mukaida et al., 1998; Vajta et al., 1998; Vajta,

2000; Arav et al., 2002). Adding the cryoprotectant in several

steps, starting with a low concentration, can be bene®cial as this

ensures that the cells gain enough cryoprotectant to protect them

from dehydration injury without increasing toxic effects. Early

vitri®cation procedures aimed for relatively full equilibration with

the ®nal cryoprotectant solution, but it now appears that a short

exposure time to the ®nal (cryoprotectant) solution is better

because less cryoprotectant enters the cell, thereby reducing both

toxicity and cryoprotectant removal problems. Providing that the

osmolarity of the ®nal solution is high, and it contains some

cryoprotectant, even a very short exposure time (<1 min) appears

to remove enough water from a cell (by osmosis) to minimize

intracellular ice formation. Although vitri®cation and other rapid

cooling procedures can be made without any polymers, proteins or

sugars, it does appear that these compounds can be bene®cial in

both vitrifying and de-vitrifying procedures. Polymers such as

Ficoll, Dextran, polyvinylpyrrolidone, hylauronic acid, polyvinyl

alcohol, polyethylene glycol, or proteins such as bovine serum

albumin (BSA) or sugars such as sucrose or trehalose, raise the

total solute concentration of a solution without increasing the

toxicity of the solution (providing that the additive is not itself

embryotoxic). The large polymers do not penetrate a normal cell

membrane but may markedly increase the viscosity of the solution

and can contribute to, or modify, the formation of a stable glass-

like state (vitri®cation) (see e.g. Sutton et al., 1992; Shaw et al.,

1997; Kuleshova et al., 2001) with the result that less penetrating

(toxic) cryoprotectant needs to be added to the solution. The

sugars, in addition to contributing to glass formation, also aid

dehydration by exerting an osmotic effect on the cells, and can

help to stabilize membranes (Anchordoguy et al., 1987; Crowe

et al., 1988). Together they can greatly reduce the amount of toxic

penetrating cryoprotectant needed to protect the cell from

cryoinjury and the length of time needed to dehydrate the cells

before cooling and restore the cell to an iso-osmotic solution after

warming. Even very small amounts of additive can have a

dramatic effect on the vitri®cation properties of a solution (Wowk

and Fahy, 2002). Rall (1987) and Rall and Wood (1994) showed

that adding either polyethylene glycol or BSA promoted the

vitri®cation of their glycerol-based solution. Adding BSA could

reduce the likelihood of ice formation in glycerol based solutions

cooled at 5 or 200°C/min from 100% to 0.

Processing steps with the potential to compromise cell viability

Both slow and rapid cooling procedures can kill or damage cells

(oocytes and embryos). Unfortunately even effective procedures

can be compromised by inexperienced users or inappropriate

handling. The most common errors which we see among

inexperienced users are failure to pre-plan, failure to adhere to

the speci®ed temperatures, times and volumes and the failure to

load, cool, warm and dilute as speci®ed. They may also

inadvertently compromise the specimens by causing partial

warming when they lift, hold, move or inspect cold specimens

(or the canes/goblets/or canisters that they are in). In the steps

before cooling it is important that the pre-equilibration and ®nal

cryoprotectant solutions are made up as directed, used in the

speci®ed quantities and not diluted. Thus the cells (oocytes/

embryos) should be moved with a minimal volume of attendant

solution (e.g. <1 ml). For oocytes and embryos rapidly cooled in

large volumes, e.g. >10 ml, it appears that the best outcomes are

obtained if they are always handled gently (i.e. slowly and gently

expelled into the diluent and from there gently moved on in a

generous amount e.g. 20 ml of ¯uid) as the cells are very fragile and

susceptible to osmotic shock. All steps should be performed

rapidly and smoothly, in particular the insertion and removal of the

specimen into/out of vapour or liquid nitrogen. Specimens cooled

and warmed in larger volumes (>10 ml) appear to bene®t from

steps aimed at minimizing fractures, e.g. by holding the specimen

very deep in the vapour phase for several minutes before and after

nitrogen storage (Kasai et al., 1996).

Equipment/materials

To perform cryopreservation requires materials, equipment and

the knowledge how to use them. In addition to standard

embryology equipment (microscopes, incubators etc.), the only

additional materials needed by a functional IVF laboratory to

perform rapid, ultrarapid or vitri®cation procedures are a large

vessel in which to store samples (liquid nitrogen or vapour

storage), small containers for the individual oocytes or embryos, a

waterproof pen (or equivalent for labelling), safety equipment

(gloves, goggles and long forceps) and a container (Dewar) for

bench-top work (see Appendix 1).


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Containers for oocytes and embryos

It is possible to drip or deposit a drop of cryoprotectant solution

containing cells, oocytes or embryos directly, as a drop, into liquid

nitrogen (Landa and Tepla, 1990; Papis et al., 2000), but this

technique is rarely used, and instead most workers place their

specimens within a specialized holder or container. A number of

different containers can be used for rapid cooling and vitri®cation

of oocytes and embryos. These include: (i) whole plastic straws,

e.g. 0.25 or 0.5 ml (e.g. Rall and Fahy, 1985; Kasai et al., 1990;

Shaw et al., 1991a,b; Vanderzwalmen et al., 2002); (ii) pulled

plastic straws (Vajta et al., 1998; Lopez-Bejar and Lopez-Gatius,

2002; Chen et al., 2003); these are made by stretching conven-

tional straws to give them a smaller diameter and thinner walls

(pulled straws); (iii) plastic straws which have been cut in half

(Beni¯a et al., 2000; Vandervorst et al., 2001); (iv) nylon loops

(Lane et al., 1999; Yeoman et al., 2001; Begin et al., 2003;

Mukaida et al., 2003); (v) copper or gold electron microscope

grids (Martino et al., 1996b; Chen et al., 2001); and (vi) pipette

tips (Liebermann et al., 2002; Hochi et al., 2003), nylon mesh

(Matsumoto et al., 2001), glass capillaries (Kong et al., 2000;

Hochi et al., 2001), pieces of aluminium foil (J.M.Shaw,

unpublished data), `cryotop' minimum volume ®lament (Hochi

et al., 2003), solid metal surfaces (Begin et al., 2003), vials

(Nakagata, 1989) and tiny drops placed on another surface (e.g.

Misumi et al., 2003) (Table X). Many of these have only been used

for rapid cooling of oocytes or embryos of animals. Each type has

speci®c characteristics which in¯uence the total size and

cryoprotectant volume, cooling rate warming rate, likelihood of

loosing the specimen, risk of fracture, risk of explosion, risk of

contamination, ease of use, ease of labelling, ease of storing, ease

of sterilization, loading cost and availability. Straws are the most

widely used container type. They come in many varieties,

including different sizes, compositions (plastics, polymers) and

colours. The risk of a straw shattering, breaking or leaking is

in¯uenced by its composition and how it is sealed. Straws for

agricultural use (semen preservation) are generally low cost and

supplied in large packets. Straws for human use are generally of

higher quality and supplied pre-sterilized in small packets. All can

be used for slow cooling and rapid/direct cooling protocols, but

some types of plastic become unsuitable for rapid cooling

procedures if they are sterilized by irradiation (Shaw et al., 1988).

Nitrogen slush

Most direct cooling protocols can be performed without any large

or costly items of equipment using normal liquid nitrogen or

nitrogen vapour. However, there are claims that using liquid

nitrogen slush, which gives a faster cooling rate, can give better

outcomes than ordinary liquid nitrogen. We do not know if this

applies to the human oocytes or embryos. Liquid nitrogen slush is

made by applying a vacuum to liquid nitrogen. A vacuum lowers

the boiling point of the liquid nitrogen and raises the evaporation

rate. As the nitrogen boils off, evaporative cooling makes the

temperature fall. As the temperature of the remaining liquid

nitrogen falls, the viscosity of the remaining liquid appears

greater, and it boils off less readily around specimens immersed

into it. It is possible to buy equipment designed to make liquid

nitrogen slush.

Storage vessels

The function of a storage vessel is to hold specimens (cryopre-

served by either rapid or slow protocols) at a low subzero

temperature. Most groups use liquid nitrogen storage vessels

(variously referred to as tanks, Dewars or ¯asks) which keep

specimens at ±196°C, but very low temperature electrical freezers

(±130 or ±150°C), or nitrogen vapour tanks (ideally as close to ±

196°C as possible) can also be used. Within each of these three

categories there are many types of vessel, differing in their overall

dimensions, storage volume (e.g. 20±500 l), storage capacity

(number of samples), type of storage (commonly vials, straws,

canes, goblets or boxes), and running costs. Running costs are

governed by the insulation, neck diameter, use, and the age of the

tank (nitrogen consumption rates in a new tank lie between 0.1 and

1 l/day). The vessel con®guration should be matched to the user's

requirements. Most IVF units cryopreserve oocytes or embryos

individually, or in small groups, in some form of small container

(e.g. straws, grids, loops, or vials) which in turn is placed on a cane

or in a goblet (the goblet may then be af®xed to a cane). Canes or

goblets are then most commonly inserted into a canister in a large

storage vessel. An alternative is to place specimens in boxes which

are held in a rack. Boxes are more widely used for vials and cell

lines than for oocytes, embryos or sperm. Most equipment and

accessories (e.g. racks for boxes, canisters for canes or goblets) for

liquid nitrogen storage are available from general scienti®c

suppliers (e.g. Sigma Aldrich), and other outlets e.g. BOC, CIG,

and manufacturers (e.g. MVE, Taylor Wharton). Electrical

freezers and vapour storage units are also available from scienti®c

suppliers.

The main advantage of liquid nitrogen storage over vapour

tanks or others is that the specimens are at a very low and constant

temperature. The main concerns, over and above the safety issues

associated with any procedures using liquid nitrogen, are that: (i)

the specimens will warm up if the level of nitrogen is not

monitored and maintained (Siegel-Itzkovich, 2003); (ii) older

vessels (>5 years) will use increasing amounts of liquid nitrogen

and may suffer outright failure (loss of all nitrogen within a few

hours); (iii) more than one specimen is housed in each canister,

thus they may be partially warmed each time a specimen in the

same canister is accessed; (iv) with time, labelling may smudge or

become indistinct; (v) specimens and contaminants can escape into

the liquid nitrogen; (vi) liquid nitrogen can enter the specimen

container, which may cause the container to explode if it is

removed rapidly from the liquid nitrogen, and the possibility of

contaminants entering with the nitrogen. Movement of pathogens

between samples (cross-contamination) has resulted in clinical

disease once, when hepatitis C virus from a broken blood bag was

transmitted by the liquid nitrogen to other blood bags (Tedder

et al., 1995; Hawkins et al., 1996). Experimental cross-contam-

ination was not seen for vials (Kyuwa et al., 2003) but could occur

with open straws (Beni¯a et al., 2000; Bielanski et al., 2000;

Letur-Konirsch et al., 2003). There are no reports of transmission

of pathogens between specimens by Units performing assisted

reproduction, but steps to reduce the possibility of cross-contam-

ination are recommended (Tomlinson and Sakkas, 2000;

Kuleshova and Shaw, 2000). Storage in freezers or in nitrogen

vapour eliminates the possibility of cross-contamination and

specimens ¯oating into the tank, but there is a higher risk of


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accidental warming/thawing of specimens during activities such as

checking for specimen ID, because the storage temperature is

closer to that at which ice crystals can grow. To prevent warming

at times of power failure, electric freezers may come with a liquid

nitrogen back-up.

Conclusions

Vitri®cation and other rapid cooling protocols provide an alter-

native to slow cooling. These cooling protocols now generally use

short equilibration times (down to a few seconds), fast cooling

rates (>200°C/min) and no expensive equipment other than the

storage tanks for the cryopreserved specimens. Rapid cooling

procedures use the same cryoprotectant chemicals as slow cooling

protocols, but a high total solute concentration (cryoprotectants

and additives) is used to ensure that suf®cient dehydration and

cryoprotectant permeation occurs before cooling to ±196°C

commences. There are now many different rapid cooling protocols

differing in how much and which chemicals are used in the

solutions, how and when the cryoprotectant is added and/or

removed (number of steps, equilibration temperatures and times)

and how the specimen is cooled and warmed (volume, rate,

container). In species in which rapid cooling procedures have been

optimized, cryopreservation causes almost no loss (e.g. Shaw et al.,

1991b, 2000a; Dinnyes et al., 1995; Vajta, 2000; Kuleshova et al.,

2001; Zhu et al., 2001). Optimizing any cryopreservation proced-

ure (slow or rapid) for human oocytes or embryos would be a

signi®cant advance, which would greatly bene®t human IVF

clinics.

Further information on cryopreservation can be obtained from

the websites of companies and universities e.g. the IMV site (http://

www.cryobiosystem-imv.com/Cryobiology/CONSCBS.htm), and

Xytex (www.xytex.com/cryobiology).

Acknowledgements

We thank Dr G.Shaw, Prof. D.MacFarlane and Prof. D.Galloway forhelpful input. This review was prepared while in receipt of fundingfrom Monash IVF, and the NH&MRC (J.M.S.).

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Appendix 1

Table X. Terminology for equipment/hardware which can be used for cryopreservation

`Cryotop' A very ®ne strip onto which oocytes or embryos can be loaded. The strip is then immersed into liquid nitrogen or nitrogen

vapour to give a very fast cooling rate (Hochi et al., 2003)

Ampoule Ampoules made of glass were used by many early cryopreservation studies. They were usually ¯ame-sealed, or sealed with

plastic caps. They have usually been replaced by plastic vials, or other freezing containers, in part because of the risk of

explosion with attendant spread of glass shards. Mainly used now for slow cooling

Box Cardboard or plastic boxes are commonly used as storage devices to hold straws or vials within a tank. Not as widely used

in assisted reproduction as goblets or canes. Mainly used in slow cooling of cell lines

Cane Commonly used storage device. Usually a ~30 cm long aluminium strip with four to six paired projections designed to grip

straws and goblets. A code (usually the patient ID) written on the cane facilitates retrieval of particular specimens in a tank.

Alternative storage devices are boxes and goblets

Canister Storage device for canes and/or goblets that ®ts inside a storage vessel. Liquid nitrogen tanks commonly have six to 10 canisters

each one with a diameter that matches the opening (neck). The canisters are usually `parked' inside the tank along the wall, but

have an attached handle which allows them to be moved. Specimens (usually canes or goblets) can only be placed in a canister

when it is moved to the position directly under the tank opening. The canister can then be raised out of the tank via the tank

opening.

Capillary Glass capillaries are brittle when used for cryopreservation but were used by Hochi et al. (2001) to show interactions between

cooling and warming rates. They found for 7.2 mol/l ethylene glycol and 1.0 mol/l sucrose that `capillaries of 2000, 1400,

1000, 630 and 440 mm diameters provided cooling rates of 2000, 3000, 5000, 8000 and 12 000 °C/min and warming rates of

5000, 8000, 17 000, 33 000 and 62 000°C/min, respectively.' They observed that a moderate warming rate (3000°C/min) gave

the highest post-thaw survival of bovine oocytes irrespective of the warming rate

Closed pulled straw Describes a new way of loading embryos into an open pulled straw (OPS, see below) for rapid cooling which prevents liquid

nitrogen from coming into direct contact with the cryoprotectant bead containing the oocyte/embryo. Cooling rate would be

approximately the same as for OPS, i.e. >20 000°C/min

Cryoloop, nylon loop,

loop

A thin nylon loop. Oocytes and embryos can be suspended in a cryoprotectant ®lm within the loop of nylon. Specialized vials

have been developed to handle and store the loops. They achieve a very high cooling rate (Lane et al., 1999; Yeoman et al.,

2001). Device initially developed for crystallography

Dewar Originally a double-walled silvered glass vessel in which the space between the walls is evacuated, and used for storage of

liquids at low temperature. Invented by the Scottish chemist Sir James Dewar. Now used more generally for any similar container

in which liquid nitrogen can be held. Can refer to large storage tanks or small (e.g. 1 l) containers for short-term use, e.g. for

bench-top work and to transport samples between rooms. Most commonly made from stainless steel. `Thermos'-type domestic

Dewars with glass walls should never be used, even for short-term work as they can explode because the caps have no pressure

vent, and the walls are not suf®ciently thick to cope with materials as cold as liquid nitrogen

Flexipet A pipette tip which can be used as the container in which oocytes or embryos are cryopreserved. Rapid cooling only

(Liebermann et al., 2002)

Forceps Paired metal blades for holding specimens deep within the vapour phase or retrieving specimens from liquid nitrogen. Long

handles or blades, or specialized tools such as the `cryoclaw' are less likely to result in frost bite. Also used in

ice-nucleation/seeding. May damage straws if not equilibrated to their temperature or if used with too much pressure

Freezing machine(s)/

biological

freezer

For controlled rate cooling and warming of specimens. Different types include: (i) platform cooling devices and equivalent

where specimens are gradually being lowered into nitrogen vapour (vapour cooling); (ii) gradient freezers which gradually

move the specimen along an increasingly cold surface; (iii) chamber freezers in which controlled amounts of nitrogen vapour

are injected into the specimen chamber; (iv) uncontrolled or passive cooling devices which are placed for example in a ±80°C

freezer or on dry ice; (v) chamber freezers in which the outside of the chamber is at ±196°C and the specimen's temperature

is regulated by controlled warming of the chamber wall. Machines differ in: size, portability, durability, ¯exibility

(range of options),

repeatability and controllability (e.g. over rates/times, number of programs etc.), purchase price, maintenance costs, number

and size of specimens, nitrogen consumption, lowest controllable temperature, seeding procedure, ease of loading/unloading,

ease of use, permanent record output and dependence on a reliable power supply and decibel output

Grid Very thin 5 mm diameter copper or gold mesh upon which oocytes and embryos can be placed; the ¯uid is then blotted away

from below, and the grids rapidly cooled. They can achieve a very high cooling rate (Martino et al., 1996a,b). Initially developed

for electron microscopy. Cooling rate; in nitrogen ~ 9000°C/min and up to 30000°C/min in nitrogen slush.

Heat sealer Piece of equipment (e.g. autoclave bag sealer or plastic bag sealer) used to close and seal plastic straws

Impulse sealer Piece of equipment used to close and seal CBS straws (Cryo Bio System by IMV Technologies, France)

Insulation Material surrounding a storage tank to minimize heat transfer through the walls.

Inventory Sometimes used to refer to the hardware placed within storage vessels to hold the specimens

Liquid nitrogen Nitrogen below its boiling point (±196°C). Often abbreviated LN2 or LN2

Liquid nitrogen slush When the liquid nitrogen is at a temperature lower than its boiling point it becomes more viscous. The most common strategy

by which nitrogen slush is made is by applying a vacuum to liquid nitrogen. This increases the evaporation rate, which in turn

lowers the temperature due to evaporative cooling


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Table X Continued

Loading A term describing the process of transferring oocytes and embryos to the container in which they are to be cryopreserved (e.g.

oocytes are loaded into straws). To reduce the risk of contamination during storage, the loading procedure should avoid all

contact between the cryoprotectant solution (with the cells) and all surfaces that come in direct contact with liquid nitrogen or

other specimens. Failure to avoid this contact could result in contaminationÐfor example viruses have been transferred

between samples carried in the liquid nitrogen

Minimum drop size A vitri®cation method in which nanolitre-sized drops can be cooled at up to 130 000°C/min (Arav et al., 2002)

Nitrogen Air contains almost 80% nitrogen gas. It is liquid below its boiling point of ±196°C. Note the dangers associated with

asphyxiation by nitrogen gas, and frost burns from the liquid

Nitrogen vapour Usually referring to the cold nitrogen gas that accumulates above the surface of liquid nitrogen. Specimens can be held deep

within the vapour phase as part of a cooling protocol. This gives a less abrupt cooling rate than plunging directly into liquid

nitrogen (see vapour cooling) due to the higher temperatures and the lower thermal transfer between nitrogen vapour

compared to transfer from LN2. This reduces the likelihood of physical damage (cracks and stress fractures) and may give as

good or better results than plunging into liquid nitrogen. However, some cell types (e.g. early pig embryos) appear to

bene®t from the highest possible cooling rates. Specimens are also commonly held in the vapour phase as the ®rst event in the

thawing step, to reduce the risk of specimens exploding due to trapped nitrogen, and to reduce this likelihood of stress

fractures (see stress fractures) (at very low temperatures (e.g. <±196°C) most material is brittle and is easily damaged by

non-uniform sudden temperature changes. Cooling and warming is best performed deep within a stable layer of vapour

where the temperature is <±130°C. To establish such a stable, cold, vapour layer the liquid nitrogen level must be well below

the rim of the container (>15 cm) and left to stand for >10 min without being disturbed by air currents

Open pulled straw

(OPS)

A type of container used to rapidly cool oocytes and embryos. Basically a plastic straw which has been narrowed and thinned

by stretching, and then cut at its thinnest point to give two `OPS' straws. These are usually loaded by capillary action. A straw

which is pulled to narrow its diameter so that it will take up ~2 ml of cryoprotectant. When immersed deeply into the vapour

phase or just let to touch the surface of liquid nitrogen the OPS gives a cooling rates of >20 000°C/min (Vajta et al., 1998).

Slower rates for OPS straws have, however, been measured (5300°C/min in nitrogen and 10 300°C/min in slush)

(http://www.agri.gov.il/AnimalScience/Reproduction/Rep-Arav1.html)

Opening size The diameter of the opening in a storage tank. A larger opening allows bigger canisters/inventory to be used, but also

compromises the insulation rating, reducing the static holding time

Passive cooling

device

A container which, when placed at a speci®c temperature (e.g. ±80°C), cools at a known rate, e.g. 1°C/min. A non-controlled

cooling method used for slow cooling exempli®ed by the isopropyl alcohol ®lled `Mr frosty' (Nalgene) for cooling vials

Seal Usually used in the context of straws. To prevent liquid nitrogen entering or the contents leaking out, straws can be sealed

with heat (thermosoldering), PVA or PVP powder, identi®cation plugs, ball bearings, or ultrasonically with an impulse sealer

Sintered disk A porous disk, for example made of brass, which can be applied to the end of nitrogen hoses to control the ¯ow and reduce

the likelihood of personal injury

Static holding time The length of time taken for a liquid nitrogen storage vessel which is not used or re-®lled to go from full to empty. Commonly

measured in weeks. Old or damaged tanks may have a greatly reduced static holding time (can be as little as hours)

Static nitrogen

consumption

The amount of nitrogen which boils off from a storage vessel which is not in use. Commonly 0.1±1 l a day. The rate of loss

can be

much higher in vessels which are opened frequently

Straw Traditionally supplied as 0.25 or 0.5 ml plastic straws. Now available in a range of materials and con®gurations. Materials

include polyvinyl chloride (PVC), polyethylene terephthalate glycol (PETG) and high-security ionomeric resin (IR). Straws

may or may not be sealed after inserting specimens (see seal). The likelihood of leakage and breakage in liquid nitrogen is

lowest for soft straws (type `CBS', Cryo Bio System by IMV Technologies, France) sealed with an `impulse sealer', but we are not

aware of any instance where these have been used for rapid cooling procedures. Straws which have been stretched and then cut at the

narrowest point have a smaller diameter and thinner walls and are widely used for rapid cooling (see open pulled straws and closed

pulled straws). Cooling rates of ~200°C/min (in vapour), >2000°C/min (plunging in nitrogen) (Kasai et al., 1996) and 4000 °C/min

for plunging in nitrogen slush.

Warming rate is ~2500 °C/min (Rall and Fahy, 1985).

Tank Usually refers to the container into which specimens are placed for long-term storage. Three main categories: liquid nitrogen

tanks, nitrogen vapour tanks and ultralow temperature (<±130°C) electrical freezers. Can also be referred to as a vessel or a Dewar

Vapour shipper A specialized container to maintain specimens at low subzero temperatures during transport. They can be shipped in cars and

aeroplanes. They are pre-cooled with liquid nitrogen, but the nitrogen is then emptied out fully and specimens inserted. Their

well-insulated design allows a low subzero temperature to be maintained for days after the nitrogen has been emptied out.

Must be kept upright at all times during transport

Vial Container, usually of 1±5 ml capacity. Used mainly for slow cooling of cells or gonadal tissue. Only vials which are made to

withstand cryopreservation should be used to reduce the risk of explosions. The size should be chosen to ®t the canes,

freezing machine and specimen. Vials differ in volume, the con®guration of the base, the shape inside (U- or V-shape), internal

or external thread, the writing area, the type of O ring, and whether the lids can be colour-coded. They are bulky but easy to

load, and have a large surface on which to write ID details. Some manufacturers make vials with bar-coding printed on them.

Vials have been used for some mouse rapid cooling protocols but fast cooling rates can only be achieved if specimens are

dripped into vials precooled to a low subzero temperature. Vials take a long time to warm which means that they are not

suitable for all cryoprotectant solutions. Their thermal inertia makes accidental thawing unlikely

Volume In relation to liquid nitrogen storage vessels, the amount of liquid nitrogen that they will hold (commonly between 20 and 120 l)

Waterproof pens Water vapour condenses on cold surfaces, so it is very important that all information on surfaces which are going to become

cold is written with pencil or indelible waterproof pens or using specialized labelling devices. As most waterproof ink is

soluble in alcohol it is important that all information is copied or noted before the surface is wiped with alcohol (for example

if the surfaces are wiped with alcohol to sterilize them before opening them)


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Appendix 2

Table XI. Safety issues associated with the use of liquid nitrogen/low temperature preservation (issues are equally relevant to rapid and slow coolingprocedures)

Asphyxia Death from asphyxia has occurred in several laboratories around the world where liquid nitrogen is in routine use. Nitrogen gas is

odourless and colourless. It forms 80% of the air we breathe; the remaining 20% is mainly the essential oxygen. Nitrogen gas is

released from liquid nitrogen as it evaporates, and if ventilation is inadequate, it can displace the air, creating a hypoxic or anoxic

environment where there is a high risk of asphyxia. The risk is highest in enclosed, poorly ventilated rooms. A person can become

unconscious after just a few breaths of 100% nitrogen gas. A litre of liquid nitrogen produces ~1600 l of nitrogen gas (at room

temperature and pressure). Liquid nitrogen storage vessels should therefore be stored in well-ventilated rooms. Ideally that room should

have a regularly serviced oxygen meter/alarm. Travelling in a lift (elevator) together with a large nitrogen tank is not recommended.

Liquid nitrogen should not be transported in cars where nitrogen vapour can access the passenger compartment and cannot (legally) be

shipped by air. Specialized vessels (see vapour shippers/dry shippers) which keep specimens at low subzero temperatures without

giving off nitrogen gas are suitable for transport by car and by air

Burns Tissue damage similar to heat-burns can be caused by exposure to extremely low temperatures, for example contact with liquid

nitrogen at a temperature of <±196°C. Skin exposed to this temperature will blister in the same way as a burn. A full face visor,

goggles or glasses should be worn when handling/dispensing liquid nitrogen because a splash to the eye could burn the cornea;

appropriate personal protective equipment should be worn to protect the rest of the body. See also `Frostbite'

Cold tolerant

containers

Most containers become brittle at cryogenic temperatures. Only use those speci®cally designed for cryopreservation. Containers such

as Eppendorf tubes are not designed to tolerate low subzero temperatures and easily shatter/explode (see below)

Explosions Explosions easily happen if great care is not taken when using liquid nitrogen. The most common cause is entry of liquid nitrogen

into a container (such as an unsuitable sample tube). This easily happens due to the reduction of pressure inside the vessel when

the air inside is cooled by placing in liquid nitrogen. When the vessel is rewarmed, the nitrogen boils and the resulting gas pressure

causes the container to explode. This is particularly common with containers not designed for use with liquid nitrogen. Flasks must

never be of the `thermos' type (silvered glass walls with a vacuum) as these can explode, ®lling whole laboratories with glass shards;

use instead stainless steel ¯asks or polystyrene boxes with appropriate venting to prevent build up of internal pressure

Frostbite Skin exposed to liquid nitrogen or any other surface cold enough to cause the cells in the skin to freeze will hurt, or if the injury is bad

enough it will blister. Injury is most commonly caused by cold metal surfaces, or ice, due to their high thermal conductivity. See

also `Burns'

Gloves Used to protect the hands/®ngers from frost bite/burns. The best sorts extend up the forearms, e.g. leather welder's gloves, or special

gloves (e.g. Sigma `cryogloves'). Gloves are not designed to allow the user to insert their hands into liquid nitrogen. The user should

use long-handled forceps or tongs to hold specimens within the vapour phase or retrieve specimens from within the liquid nitrogen

Goggles, visors Used to protect the eyes/face in case of explosions or liquid nitrogen splashes.

Nitrogen level

monitor

For tanks as a back-up to provide a warning about low nitrogen levels. Sophisticated versions can be linked to a phone dial-up facility.

These devices are not failsafe and should be tested according to the manufacturer's recommendations, and routine physical checks of

nitrogen levels made at appropriate intervals

Oxygen meter Used in liquid nitrogen storage facilities to provide warning of lowered oxygen levels in the air. Note that oxygen meters may need

regular servicing and routine testing to remain functional

Safety valves Storage vessels for nitrogen (not the specimen storage tanks) hold and dispense nitrogen under pressure. Safety valves are required to

ensure that the pressure stays within safe limits. Pressurized vessels should be safety-tested (pressure-tested) at least every 10 years

Spills A small spill of nitrogen is generally harmless unless it is to a particularly vulnerable part of the body (e.g. the eye). Larger spills can

cause serious injury. Clothes that become soaked by liquid nitrogen should be removed or at least held away from the body.

Alternatively the affected clothing can be ¯ushed/soaked with water (e.g. using a safety shower) until the trapped LN2 is all vaporized

Transport/shipment For safety reasons, liquid nitrogen should not be transported by car or air. Vapour shippers can be transported by both cars and aeroplanes

Ventilation When liquid nitrogen is used/held in enclosed spaces, the ventilation must be suf®cient to prevent nitrogen gas accumulation

(see `Asphyxia')

Weight Liquid nitrogen weighs approximately the same as water. Large storage vessels can be too heavy to move, in particular for small

persons. Canisters, or racks, within the storage vessels, particularly those of large capacity, can be very heavy. Tall tanks

exacerbate the problem. Use of appropriate lifting techniques, trolleys and other equipment must be considered


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Documents

slow cooling procedures

ultrarapid cooling

period of slow cooling

cryopreservation tables

different procedures

optimum rate

thawing rate

rate ofcooling