• Terminology

• Cryopreservation in nature
• History
• Foundation and Milestones
• Methods
 • Definition
• Cryopreservation in nature
Cryopreservat • History
ion- History • Traditional milestones
and • Foundation and discovery
Principles
Cryobiology-study of life at below temperatures

Cryopreservation-Preservation and storage of living systems in viable condition

Cryogenics branch of Physics causes and effects of extremely low temperatures- liquefied
gases

Cryonics Keeping clinically dead human or brain at extremely low temperatures

Vitrification preservation at extremely low temperatures without freezing

 Frozen wood frog
Freezes internally
Survives -8 °C

Tardigrades (water bears)

They survive Alaskan beetle (larvae)
Desiccation (1% water) Survive -60 easily.
-270 °C May survive -100°C
+ 150 °C
Complete vacuum
Extremely high pressure
10 days in outer space
Histo
ry

documented the effects of

freezing temperatures on
1683- The first documented
living animals in his
cryobiological study by Sir
monograph "New
Robert Boyle
Experiments and
Observations Touching Cold
Tradition • 1930 Walton and Hammond: J. Exp. Biol.:
Effect of temperature on rabbit sperm
al • 1938 Jahnel- Sperm cooled to -79 ºC
•
Mileston 1938 Luyet and Hoddapp- Biodynamica : Revival of
spermatozoa vitrified in liquid air
es •
•
1940 Shettles- froze sperm at -269 ºC
1945 A. Parkes- BJM-Human sperm at low
temperatures
 Foundation and
discovery
• C. Polge, A.U. Smith, and A.S. Parkes in 1948 -
discovery -glycerol would enable fowl spermatozoa to
Accidental
survive freezing to -70°C -cryopreservation.”

• How this discovery was made?

• mistake in the labelling -refrigerator
• fowl semen being frozen in a mixture of glycerol,
albumen,
and water
• rather solution of levulose.

•Audrey Smith-1961-The introduction of the glycerol

technique for freezing bull spermatozoa revolutionized the
cattle breeding industry
• 1949: Polge, Smith and Parkes: Nature: Revival of Spermatozoa after
Vitrification and Dehydration at Low Temperatures

• 1959: Lovelock and Bishop- discovered DMSO

• 1965: P Mazur- published the study on intracellular damage
 • 1949: Polge, Smith and Parkes: Nature: Revival of
Spermatozoa after Vitrification and Dehydration at
Low Temperatures
• 1952- Polge- First living animal from frozen sperm
• 1953 Bunge and Sherman: Nature: Fertilizing
Milestones capacity of frozen human spermatozoa.
• 1959: Lovelock and Bishop- discovered DMSO
• 1965: P Mazur- published the study on
intracellular damage
• 1972 Witthingham, Leibo and Mazur: Science.
Mouse embryo freezing
• 1985 Rall and Fahy : Nature: Ice-free
cryopreservation of mouse embryos at -196
degrees by vitrification.
 • Progress in gamete cryobiology advanced quickly
Milestones • post-thaw motility as a marker of retention of sperm function
• Bunge et al., 1954-the first birth -human cryopreserved spermatozoa
and • By-1950s- glycerol - to cryopreserve unfertilized oocytes (mouse:
Foundatio Sherman and Lin, 1959; sheep: Averill and Rowson, 1959) and rabbit
fertilized oocytes (Smith, 1953) with little success.
n
• 1960s- (reviewed in Mazur, 1970)-physiology of cell water movement and
solidification of liquid water -foundation for future cryobiology
• Asahina, 1961-Sea urchin oocytes -formulating these concepts
• Whittingham et al., 1972-successful preservation -fertilized mouse
oocytes
• Whittingham et al., 1972; Wilmut, 1972, Whittingham, 1977- stored
mature oocytes with dimethyl sulphoxide to sub-zero freezing of mouse
mature oocytes

• Whittingham 1977-demonstrated good cryosurvival, comparable

fertilization rates and the birth of live offspring
• Kasai et al., 1979; Parkening and Chang, 1977-subsequently used
for rat
• Critser et al., 1986; Parkening and Chang, 1977-hamster
• rabbit (Diedrich et al., 1986; Siebzehnruebl et al., 1989) and
SUCCESFUL CRYOPRESERVATION OF MAMMALIAN EMBRYOS YELDING
LIVE OFFSPRING

Species

• Mouse Whittingham et al. 1972

• Cattle Willmut & Rowson 1973
• Rabbit Bank & Maurer 1974
• Sheep Willadsen et al. 1974
• Rat Whittingham 1975
• Goat Bilton & Moore 1976
• Horse Yamamoto et al. 1982,
Eland Kramer et al. 1983
• Human Trounson & Mohr 1983
• Baboon Pope et al. 1984
• Marmoset Summers et al. 1986
• Macaque Monkey Balmaceda et al. 1987
• Cat Dresser et al. 1988
 • storage at -196C (liquid nitrogen)-viability independent of storage period
• Embryos
• Oocytes
• Ovarian tissue
• Stem cells etc
Cryopreserva
• Storage at temperatures above -196C-viability decreases below -135C
tion • microbial suspensions,
methods • tissue culture

•Freeze drying-convenient- easy storage, cold chain etc but viability and
mutagenic in bacteria, mouse- live births

• Vitrification-An alternative approach to cryopreservation.

• Conventional methods of cryopreservation-to accomodate
the consequences of ice formation
• Vitrification combining the use of concentrated solutions with
rapid cooling
• avoids the formation of ice.
• Samples reach low temperatures in a glassy state, which has the molecular
structure of a viscous liquid
 The Biological Chemistry of Water

• H2O is a complex and behaviourally anomalous molecule, possessing special properties -

polar, electrostatic, cohesive, solvent, buffering and thermal stabilizing characteristics.

• Its hydrogen atoms carry a slight net positive charge and act with unpaired “negatively
charged” electrons of oxygen to make water bipolar.

• The lone pairs of electrons on water’s oxygen atom and its two polarized hydrogens
enable
the creation of hydrogen bonds.

• How water molecules associate with one another is influenced by H-bonding. Neighbouring
molecules orient so that partially positively charged H atoms align with the partially
negatively charged oxygen.
 Cohesion and
adhesion
• Water molecules stay close to each other (cohesion), due to the collective
action of hydrogen bonds between water molecules.
• These hydrogen bonds are constantly breaking, with new bonds being
formed with different water molecules; but at any given time in a sample
of liquid water, a large portion of the molecules are held together by such
bonds.
• Water also has high adhesion properties because of its polar nature.
• On extremely clean/smooth glass the water may form a thin film because
the molecular forces between glass and water molecules (adhesive forces)
are stronger than the cohesive forces.
• In biological cells and organelles, water is in contact with membrane and
protein surfaces that are hydrophilic; that is, surfaces that have a strong
attraction to water.
 Water as a solvent

• Water is an excellent solvent

• Substances that mix well and dissolve in water are known as hydrophilic, while
those that do not mix well with water are known as hydrophobic
• When an ionic or polar compound enters water, it is surrounded by water
molecules (hydration).
• The relatively small size of water molecules allows many water molecules to
surround one molecule of solute.
• In general, ionic and polar substances such as acids, alcohols, and salts are
relatively soluble in water, and non-polar substances such as fats and oils are
not.
• An example of an ionic solute is table salt; the sodium chloride, NaCl,
separates
into Na+ cations and Cl− anions, each being surrounded by water molecules.
• The ions are then easily transported away from their crystalline lattice into
solution.
• An example of a nonionic solute is table sugar. The water dipoles make
hydrogen bonds with the polar regions of the sugar molecule (OH groups) and
allow it to be carried away into solution.
 Melting point

• The melting point of ice is 0°C at standard pressure; however, pure liquid
water can be supercooled well below that temperature without freezing
if the liquid is not mechanically disturbed.
• It can remain in a fluid state down to its homogeneous nucleation point
of about 231 K (−42 °C)
• Because of the way crystal is arranged, there is actually more empty space
between the molecules than there is in liquid water
• Ice less dense and hence floats
• When water converts into ice which has more orderly arrangement, and
minimal movement of the water molecules there is release of energy- this
energy is called latent heat
• Each time water changes state, energy is released or absorbed
• To melt one gram of ice-80 calories of heat. This plays an important role in
nucleation and supercoiling of freezing solutions and recrystallization during
thawing
 Physics of water
• Water moves from a region of low osmolarity to a region of high osmolarity
• Nucleation temperature is the temperature at which water forms ice crystals
• Solutes dissolved in the solution decreases the nucleation temperature of the solution
• Such solutions tend to supercool and form ice rapidly at low sub-zero temperatures
• Three physical states of matter-solid liquid and gas
• Eg; water Vapour/gas-water molecules are not bounded together, float around as single
molecules
• Liquid state-some of its molecules are bound to each other with hydrogen bonds, bonds
break and reform continuously
• cooled to solid state-molecules bonded in a crystalline state six sided-each molecules of
water connected to four others with hydrogen bonds
 DAMAGE BY ICE
• When a dilute aqueous solution is frozen, the ice that forms is essentially pure crystalline water

• Ice has negligible ability to dissolve solutes. Solutes are therefore rejected and concentrate in the dwindling
volume of unfrozen liquid

• In the presence of ice, composition is determined solely by temperature and is therefore independent of initial
composition.

• However, initial composition does control the amount of ice that forms and the factor by which the
concentration increases at a given temperature.

• It is important to realize that the increase in concentration of Nacl that occurs during the freezing of isotonic
saline is enormous, actually 32-fold at 21°C.

• The question then arises: Is it the ice, the elevated salt concentration, or both that damage cells during
progressive freezing?
• biological systems compartmentalized- intracellular spaces and extracellular space - semipermeable cell
membranes.

• whether ice forms inside or outside the cells or both?

• Freezing is a nucleation induced event

• probability of nucleation increases directly with the degree of super-cooling and volume.

• When cell cools below the equilibrium freezing point of the saline, a nucleation event will occur in the
extracellular compartment before cells have nucleated.
• further cooling -extracellular ice grow, concentration of the extracellular solution, and dehydration of the cells by
osmosis through their semipermeable membranes.

• Sufficient low cooling rate and high water permeability of the cells- the intracellular spaces will remain free of
ice.

• fundamental phenomenon was discovered and elucidated by Mazur.

• He provided quantitative analysis of the effect of cooling rate on water transport during progressive cooling, the
greater the degree of super-cooling, the greater the probability of freezing.
 Stress Encountered Cellular
1.
Response
Reduction in temperature 1. Membrane lipid phase changes,
2. Increase in solute concentration Depolymerisation of the cytoskeleton
3. Increase in ionic concentration 2. Osmotic shrinkage
4. Dehydration 3. Direct effects on membranes, including
solubilization of membrane proteins
5. Gas bubble formation
4. Destabilization of the lipid bilayers
6. Solution becomes extremely viscous
5. Mechanical damage to membranes and the
7. Changes in pH cytoskeleton
8. Cells become closely packed 6. Diffusion processes, including osmosis may
become limited
7. Denaturation of proteins etc.
8. Membrane damage
• the osmotic properties of a system are defined by flow of a solvent across a semipermeable membrane, which
permits the passage of the solvent, but not the solutes.

• Osmotic pressure is the pressure that must be applied to a solution to prevent the flow of a solvent across the
semipermeable membrane

• Isotonic: same concentration of solutes as surrounding solution across a semipermeable membrane

• Hypotonic: Solutions that cause cells to swell as the bathing solution has a lower solute content than the cell. `

• Hypertonic: Solutions that cause cells to shrink ,they have a higher solute level than the cell H2O, water has
unique physio-chemical properties essential for cell function.

• Cryobiological manipulations influence the different states of water placing it in an important and central role in
cryopreservation and storage stability.
 Thermodynamics
• Temperature is a measurement of the internal energy in a physical system.
• It is this internal energy in fluid systems that allows molecules to tumble, twist, dissociate
from one another, move from place to place in the fluid, and chemically react with other
molecules
• If Temperature reduced- less energy to drive molecular motions. Pure water-below certain
point-abruptly reorganise into a solid lattice-crystal-Freezing

• In other systems-temperature reduction causes more slowing of molecular motions, less

molecular mobility, slower chemical reaction rates, until a critical temperature is reached
below which there is insufficient energy for most mobile molecules to move in the fluid-at
this temperature, system loses its fluidity and becomes a solid liquid (glass)

• Vitrified-molecular rearrangements are arrested

 Slow freezing
• In slow-freezing, cells in a medium are cooled to below freezing point. At some stage, ice masses containing
pure crystalline water will form.
• What remains between the growing ice masses is the so-called unfrozen fraction, in which all cells and all
solutes are confined

• The concentrations of sugars, salts and cryoprotectant increase, while the volume of the unfrozen fraction
decreases. The increase in osmotic strength causes an efflux of water from the cells.

• Slow cooling is needed in order to allow sufficient efflux of water to minimize the chance of intracellular ice
formation.

• As cooling continues, the viscosity of the unfrozen fraction ultimately becomes too high for any further
crystallization. The remaining unfrozen fraction turns into an amorphous solid that contains no ice crystals.

• As ice forms during cooling, only water molecules comprise the ice crystals.

• As a result, all other components (salts, etc.) become concentrated in the remaining solution.
• As the solution concentration increases, the chemical potential of the water in the solution decreases.
When cells are frozen in suspension, the cells are sequestered in channels of concentrated unfrozen medium.

The high concentration of this unfrozen solution establishes an osmotic gradient across the cell membrane, and as a
result, water will flow out of the cell via exosmosis.

Below a cell’s equilibrium freezing point, the cytoplasm is in a supercooled state.

If the sample is cooled slowly enough, exosmosis occurs to a sufficient degree to keep the cells in a near-equilibrium
state with the extracellular solution.

Such a situation will preclude intracellular ice formation.

On the other hand, if the cooling rate is relatively rapid, water cannot leave the cell fast enough to maintain a near-
equilibrium state with the extracellular solution, and at some point equilibrium will be re-established by intracellular
ice formation

The formation of intracellular ice is usually (but not necessarily) fatal to cells.

Direct cryomicroscopic observation of intracellular ice formation in mouse oocytes and the correlation to cell
survival were some of the most convincing data to support the assertion that ice formation was the lethal cause of
cell death at supra-optimal cooling rates
 Principles of
cryopreservation
3 steps
Freezing Storage Thawing

Vitality & viability depends on rates of freezing and thawing

Cooling rate is too low Cooling rate-too high Cooling rate moderate
• cells get sufficient time to lose water • time for water to escape • only part of the freezable
out of the cell is very limited water leaves the cell
• maintains osmotic equilibrium with •
little or no water escapes • water in the cell
extra cellular solution
out of the cell, vitrifies/ forms ice crystals
• Cell death caused by • intracellular freezing takes which are tolerable if
excessive dehydration place, ice crystals are thawing is fast
formed, fatal to cells
• The rate at which water flows out of a cell is dictated by the cell membrane water
permeability.
• The permeability of cells to water is dependent upon several factors including
temperature and the presence of cryoprotectants.

• For example, in the absence of cryoprotectant, human sperm water permeability is 1.84
μm/min/atm at 22°C, but is reduced to 1.23, 0.84, 0.77 and 0.74 μm/min/atm in the
presence of propylene glycol, dimethylsulfoxide, glycerol, and ethylene glycol,
respectively

• Furthermore, water permeability can vary greatly across cell types. For example, water
permeability for human erythrocytes is higher than for human oocytes.

• Because intracellular ice formation is dependent upon the degree of supercooling, the
rate of cooling which results in intracellular ice formation differs widely across cell types.
The density of salt water depends on the dissolved salt content as well
as the temperature.
As the surface of salt water begins to freeze at −1.9 °C for normal
salinity seawater, the ice that forms is essentially salt-free, with about
the same density as freshwater ice.
This ice floats on the surface, and the salt that is "frozen out" adds to
the salinity and density of the sea water just below it
Attempts have been made to explain the mechanism(s) which cause intracellular
ice formation, and to date several theories have been put fourth.

• in order for ice formation to occur in cells above ~−30°C, extracellular ice must
be present, and the proximity of the cells and ice is important

• extracellular ice is not a necessary precondition for intracellular ice below

~−30°C

• intracellular ice formation usually happens immediately if the cells and the
surroundings are supercooled −15 to −20°C and extracellular ice is rapidly
initiated

• the nucleation temperature decreases substantially if the extracellular solute

concentration increases.
• As extracellular ice does not nucleate intracellular ice at low degrees of supercooling, an
intact plasma membrane effectively blocks the passage of ice into the cell.

• However, the plasma membrane is implicated in the major theories put fourth to explain the
initiation of intracellular ice formation above ~−30°C

• Mazur hypothesized that ice crystals can grow through membranes via protein pores like
aquaporins.

• ice can grow through channels which connect cells (i.e., gap junctions, the pore size in these
channels is much larger than those in aquaporins, making ice growth more likely.
• ice interaction with the plasma membrane causes a structural change to the inner
membrane surface, resulting in an increase in the efficiency of ice nucleation

• a different mechanism suggests that ice grows through the membrane after the formation
of a lesion as a result of the osmotic pressure gradient and resultant water efflux.

• A similar argument regarding the formation of a membrane lesion as a prelude to

intracellular ice formation had been proposed

• preventing ice formation in the cytoplasm (at least to a large degree) during
cryopreservation is critical.
 Permeating cryoprotectants

1. glycerol,
2. ethylene glycol (EG),
3. 1,2-propandiol (PROH) and
4. dimethylsulfoxide (DMSO)

• penetrate the cell membrane, although more slowly than water.

• stabilise intracellular proteins,

• reduce the temperature at which intracellular ice forms and
• decrease the impact of intra- and extra-cellular electrolytes.

• Glycerol- poor tissue penetration compared with DMSO, PROH and

EG.
• show high water solubility,
• rapid tissue penetration and
• induce less osmotic damage at high concentrations
 Dimethyl Sulfoxide (DMSO) - Dimethyl Sulfone (DMSO2)

dimethyl sulfone (DMSO2) -worse cryoprotectant, precipitates below freezing point

DMSO show the characteristic cryoprotective action of a penetrating cryoprotectant.
The mechanism of cryoprotective action of penetrating cryoprotectants due to their colligative properties

colligative properties- properties of solutions that depend on the ratio of the number of solute particles to the
number of solvent molecules in a solution, and not on the nature of the chemical species present

Colligative properties-
• vapor pressure lowering,
• boiling point elevation,
• freezing point depression, and
• osmotic pressure.
 Non-permeating cryoprotectants

• generally polymers that form extensive hydrogen bonds with water, reducing the water activity to a much greater extent

• Non-penetration cryoprotectants are thought to act by dehydrating the cell at high sub-freezing temperatures, thereby
allowing them to be rapidly cooled before the solution effects injury of slow cooling can lead to extensive damage.

• sugars,
• polymers and
• amphipathic compounds. (hydrophilic and lipophilic)
• Sugars -raffinose and lactose decrease the percentage of unfrozen water and/or decrease salt concentrations.
• Amphipathic compounds- glycine, proline and trehalose interact with membrane lipids and proteins to alter phase
transitions and hydration status.
•
.Propylene Glycol Ethylene Glycol Glycerol
Disaccharide- glucose and fructose - C12H22O11.
Raffinose-galactose, glucose and
fructose

Trehalose-2 glucose
Sucrose C12H22O11
It is a disaccharide, a molecule composed of
two monosaccharides: glucose and fructose.
Sucrose is produced naturally in plants

Ethylene Glycol (CH OH) 2 2

It is an odorless, colorless, sweet-

tasting, viscous liquid.

DMSO
(CH3)2SO
• Numerous compounds confer protection to spermatozoa from cold shock.

• include glycerol,
• phosphatidylserine,
• egg yolk,
• lecithin,
• milk, and
• albumin.

• The low density lipoprotein fraction of egg yolk is particularly effective at preventing cold
shock injury, with phosphatidylcholine being a particularly active component.
 Cryoprotectants: Beneficial Effects

• Cryoprotectants are defined in a functional manner as compounds that allow a higher degree
of cell survival during freezing in their presence than in their absence.

• it is likely that the effects of cryoprotectants are multi-factorial, and cryoprotectants of

different classes (e.g., alcohols, sugars, diols, amides, large polymers) may act by different
mechanisms.

• One of the earliest theories of the mechanism of action of cryoprotectants was developed
from a series of experiments investigating the protective action of glycerol on erythrocytes.

• the degree of damage could be explained by the increase in salt concentration due to ice
precipitation.
 Cryoprotectants: Beneficial Effects

• when cells are frozen in solutions containing glycerol, the temperature at which
hemolysis began was progressively lower as the amount of glycerol was
increased.

• By adding glycerol to a cryopreservation solution, the amount of water that

freezes at any given temperature will be reduced. As a consequence, the final
concentration of the salts in the remaining solution will also be reduced.

• Several other modes of action have been proposed for cryoprotectants. One
effect includes interacting with water molecules and altering the water
structure in a solution, and reducing the ability of water to join the ice phase.
 OSMOTIC EFFECTS OF CRYOPROTECTANTS

• The most effective cryoprotectants penetrate cell membranes but they do so more slowly than water, which
means that some osmotic imbalance is inevitable during the addition or removal of these compounds.

• Gross osmotic shock results in cell damage- lysis

• it is therefore logical to control changes in cell volume
• designing cryopreservation methods- measure the volume response to changes in external osmolality of a non-
permeating solute and to correlate each volume with subsequent structural and functional damage.

• The next step is to measure to estimate the water and solute permeability for the chosen cryoprotectant.

Two strategies for controlling volume during the addition and removal of penetrating solutes

• the first is to use low rates of change in concentration or, to change the concentration in several small steps

• Second, applicable only to the more critical removal phase, is to incorporate solutes that do not penetrate the
cells and therefore function as “osmotic buffers” by restricting the inflow of water as the concentration of
cryoprotectant is reduced.
• Cryoprotectants have also been shown to either directly interact with or be preferentially
excluded from biosurfaces (e.g., the surface of lipid bilayers or proteins).

• The apparent opposite nature of these modes of interaction seems to suggest opposite
effects. However, each mode of interaction can be beneficial to the stability of these
structures.

• trehalose and proline can prevent freezing-induced fusion of lipid vesicles.

• Many organisms living in climates where freezing temperatures are encountered have
evolved to include the metabolic production of cryoprotectants as a survival strategy.

• The cryoprotective properties of sugars and glycerol in plants were described by Nikolay
Maximov in the early 20th century.
• Inhibition of freezing at high sub-zero temperatures is one strategy among arthropods and fish, and is
accomplished by regulative supercooling.

• In many instances the freezing point of physiological solutions is regulated by thermal hysteresis proteins. The
separation of the melting and freezing temperature is usually referred to as thermal hysteresis, and the
temperature of ice growth is referred to as the hysteresis freezing point. The hysteresis is supposed to be the
result of an adsorption of antifreeze proteins to the crystal surface.

• The tertiary structure of these proteins allows them to directly interact with ice crystals due to polar residues
along the protein backbone.
• These proteins lower the freezing point of water without significantly altering the melting point.

• these proteins bind to ice crystals and alter the radius of curvature of the growing crystal

• Overall, these proteins restrict ice growth when the environmental temperature is slightly below the
equilibrium freezing temperature of the body fluids.
As an alternate strategy, organisms across many phylogenetic groups have developed mechanisms of regulating ice
formation in situ (appropriate place)
In naturally freeze-tolerant organisms, avoiding the formation of intracellular ice is managed by actively promoting
and regulating the formation of extracellular ice. This allows freeze-induced dehydration of the cells and prevents ice
from forming in the cytoplasm.

four requirements for the successful freeze tolerance in animals:

(1) ice must be confined to extracellular spaces and damage from ice crystals must be minimized;
(2) the rate of freezing must be slow and controlled;
(3) cell volume reduction beyond a minimum tolerable volume must be avoided; and
(4) mechanisms must be present to prevent damage from resulting ischemia (inadequate blood supply).

These requirements are often met through both behavioural and physiological adaptations.

Cryoprotectant synthesis (e.g., glucose production) in some organisms is initiated by freezing.

 Cryoprotectants: Detrimental Effects

• high glycerol concentrations contribute to the damage of cells frozen slowly. Similar results have also been
shown for dimethylsulfoxide.
•
• Injury from cryoprotectants is not limited to those which occur during freezing. Exposing cells to solutions
containing cryoprotectants prior to cooling can be damaging due to an osmotic effect.

• Many of the commonly used permeating cryoprotectants have lower plasma membrane permeability
coefficients compared to that of water.
• This relationship results in cells experiencing osmotically driven volume excursions during cryoprotectant
addition to and removal from the cell during the course of a cryopreservation procedure.
 Cryoprotectants: Detrimental Effects

• It has been shown in numerous cell types that damage to cells can occur as a result from
volume excursions alone.

• many studies have demonstrated a beneficial effect of prolonging the cryoprotectant

addition and/or removal process which reduces the associated volume excursions.

• Osmotic damage is often ascribed to the associated volume reductions.

• Cell volume response can be controlled during cryoprotectant addition and removal by
modifying the procedures for loading and unloading these compounds.
• True chemical toxicity is also a concern associated with the use of cryoprotectants.

• This is particularly true for vitrification methods as very high concentrations of these
compounds are necessary to achieve and maintain a vitreous state at practical cooling rates.

• The precise nature of the toxic effects of cryoprotectants remains, to a large degree,
uncertain.

• Cryoprotectants have been shown to alter cytoskeletal components in mammalian oocytes,

particularly the filamentous actin network and meiotic spindle.

• Toxicity is known to be a significant cause of cell death in oocyte cryopreservation studies.

• damage associated from exposure to 1,2-propanediol was not a result of the osmotic effects,
but a true chemical effect. Exposure to the same concentration (2.5mol/L) of ethylene glycol
was not detrimental to mouse oocyte survival.
 Toxicity

• they can interact with fewer water molecules.

• Such compounds are poorer glass formers

• Hence, weak glass forming cryoprotectants are less toxic.

• Using this new information, the investigators were able to predict and confirm that
substitution of 1,2-propanediol (a very good glass former) with ethylene glycol (a very
poor glass former) in a previously developed vitrification solution (VS41A) would be a
superior vitrification solution
 Cell Death at Sub-Optimal Cooling Rates

• Many of the factors which might contribute to cell damage as a result of freezing are interdependent.

• For example, ice crystal formation may have deleterious mechanical effects on cells in suspension or in tissues
and the amount of ice formed and the crystal structure is dependent upon cooling rate, warming rate, and the
presence of cryoprotectants.

• The cooling rate at which cell survival is highest is dependent upon other factors such as cryoprotectant
concentration and warming rate.

• In general, in the presence of a permeable cryoprotectant, cell water will crystallize at a slower cooling rate
compared to the cooling rate resulting in crystallization in the absence of an intracellular cryoprotectant .

• This effect is likely a result of several factors. One includes the reduction in the water permeability of cell
membranes in the presence of cryoprotectant.

• A second is likely a result of lowering the freezing point of the cytoplasm which causes a general
reduction in
the temperature at which a given driving force for water efflux is present.

• Because of the temperature dependence of water permeability, less water can move out of the cell in a given
Solution effects- has been coined to collectively describe the various forms of injury to cells cooled slowly enough to
preclude damaging intracellular ice formation.

• damage is physical and not a biological event , resulting either from the osmotic dehydration of the cells and the
resulting stress placed upon the cell membrane due to cell volume reduction, or a direct osmotic effect on the
membrane itself.

• Mazur and colleagues -showed a strong correlation between survival and the unfrozen fraction when the unfrozen
fraction was low (5–15%).

• They proposed that as the unfrozen fraction was reduced, the cells were damaged by mechanical effects of the ice
and/or close apposition with other cells.

• When the unfrozen fraction is increased, damage was less strongly dependent on that variable and more on the
salt concentration until the effects of the unfrozen fraction were lost.

• When the unfrozen fraction is not a damaging mechanism, the loss was attributed to the osmotic effect of the
solute (both during exposure and dilution)

• The molecular mechanisms of cryodamage, particularly the induction and the trigger for apoptosis is not
necessarily an immediate effect of the cryopreservation stresses, but can be delayed for several hours as the cells
try to recover from these stresses.
 Cooling and Cooling Injury/Cold Shock

• Even in the absence of ice, cold temperatures have profound effects upon cells.

• Injuries from rapid cooling are usually categorized as cold shock injuries.

• These types of injuries occur quickly after cooling, and are generally independent of the warming
rate

• At physiologic temperatures, the membrane is fluid such that molecular mobility is high and many of
the proteins and lipids are free to diffuse laterally within the bilayer

• As membranes are cooled, the lipids tend to transition from a liquid-like state to a gel-like state, with
the molecules being arranged in an orderly, crystalline fashion with a characteristic hexagonal
arrangement.

• this transition is a distinct change from the usual lipid arrangement, and can have significant effects
on membrane function.

• The temperature at which this transition occurs is dependent upon several factors, including the
length of the hydrocarbon chain in the lipid group, the presence and location of cis-unsaturated
bonds
• Rearrangements such as these can alter the selective permeability of
membranes, resulting in the loss of cell homeostasis.

• A reduction in anaerobic glycolysis and respiration, ATP levels, Cytochrome C

loss from the mitochondria, and release of numerous intracellular enzymes
have all been described.