THE MODE OF ACTION OF NON-PENETRATIVE CRYOPROTECTANT IN

CRYOPRESERVATION OF PLANT GENETIC RESOURCE.

INTRODUCTION:
Embracing the science of plant biotechnology has lead to high yielding cultivars
from cultivated plants and their wild relatives. Many conservation programs have been
adapted in order to preserve the reservoirs of genetic diversity to overcome the threat.
One of it is the cryopreservation methodology which involves storage of plant genetic
resource at ultra low temperature and it is becoming or it has already become a very
important tool for the long term storage of plant genetic resource using a minimum of
space and maintenance. Availability and the development of safe, cost-effective, reliable
cryogenic protocols and subsequent plant regeneration without genetic change are basic
requirements for plant germplasm conservation.
Cryopreservation is a method of storing living cells at ultra low temperature,
usually in liquid nitrogen at typically -196°C. At this temperature any biological activity
including biochemical reactions that would eventually cause the cell to die is paused.

Development of Cryopreservation
Cryopreservation is a four step process that involves:
- Adding cryoprotective agents to cells before cooling
- Cooling the cells to low temperature at which the cells are stored
- Warming the cells
- Removing the cryoprotective agents from the cells after thawing

The process can be successful only if intra-cellular ice crystal formation (IIF) is
avoided i.e since it causes irreversible damage to the cell membranes by destroying their
semi-permeability. Pre freezing method is widely used as the routine method for
cryopreservation to reduce hardy cultured cells and tissues with prior application of
cryoprotectants.
Cultured cells and tissues to be cryopreserved are generally sensitive to freezing.
Thus, they are first treated with cryoprotectants, so that they can survive pre freezing to
-30 or -40°C before being immersed into liquid nitrogen (LN2). Less cryoprotectant is
needed inside cells than in the extracellular space because of dehydration (which drives
water from cells into the extracellular space).
However in order to prevent crystal formation without an extreme reduction of
cellular water, the samples have to undergo vitrification.
Vitrification is a physical process where an aqueous solution transforms into an
amorphous that is free from any crystalline structure and for a cell to be able to vitrify it
requires:
i. Rapid freezing
ii. Concentrated cellular solution

How freezing injures cells

Water expands when it freezes, but contrary to popular belief it is not expansion of
water that causes injury, it is the purification of water during freezing that causes injury.
Water freezes as a pure substance that excludes all else. It is this exclusion process that
causes injury. Instead of remaining a solvent that allows the molecules of life to freely
mix within it, water that freezes gathers itself up into crystals pushing everything else out.
Freezing causes damage by two distinct mechanisms. The first is mechanical damage as
the shape of cells is distorted by ice crystals. The second is damaged caused by chemical
and osmotic effects of concentrated solutes in the residual unfrozen water between ice
crystals. This is so called “solution effects” injury.

Properties of cryoprotectants and how they protect the cell

After a brief description of how freezing injures the cell, it is essential to know
how cryoprotectants protect cells and their common properties.
Not all chemicals that dissolve in water are cryoprotectants. In addition to being
water soluble, good cryoprotectants are effective at depressing the melting point of water,
they do not precipitate, form eutectics or hydrate and they are relatively non-toxic to cells
at high concentration. All cryoprotectants form hydrogen bonds with water.
For applications outside cryobiology, cryoprotectants are sometimes called
“antifreeze”. Common examples are glycerol, ethylene glycol, propylene glycol and
dimethylsulfoxide (DMSO). A cryoprotectant concentration of about 5% to 15% is
usually all that is required to permit survival of a substantial fraction of isolated cells
after freezing and thawing from liquid nitrogen temperature. In simple explanation,
growing ice compacts cells into smaller and smaller pockets of unfrozen liquid as the
temperature is lowered. The presence of cryoprotectants makes these pockets larger at
any given temperature than they would be if no cryoprotectant were present. Larger
unfrozen pockets for cells reduces damage from both forms of freezing injury,
mechanical damage from ice and excessive concentration of salt.

Cryoprotective Agents/ Additives

Many chemicals have now been identified as having a cryoprotective action, by
adding these cryoprotective additives to a cell suspension the survival following freezing
and thawing can be substantially increased. Cryoprotective additives are chemicals that
reduce the injury of cells during freezing and thawing. The agents protect the cells by
modifying the freezing behavior of cells, specifically affecting the rate of water transport
across cell membranes and ice crystal growth.
They are separated into two broad classes based on their ability to diffuse across
cell membranes hence penetrating cryoprotectant (for chemicals that will diffuse
through the plasma membrane and equilibrate in the cytoplasm). Penetrating
cryoprotectants are small molecules able to cross cell membranes. The role of penetrating
cryoprotectants is to reduce ice growth and reduce cell dehydration during freezing. In
vitrification, the role of penetrating cryoprotectants is to completely prevent ice formation
while non-penetrating cryoprotectant (here chemicals do not enter the cytoplasm) are
large molecules, usually polymers added to cryoprotectant solutions. They inhibit ice
growth by the same mechanisms as penetrating cryoprotectants, but they do not enter
cells. Polyethylene glycol (PEG) and polyvinylpyrrolidone (PVP) are common examples
non-penetrative cryoprotectants. Non-penetrating cryoprotectants are usually less toxic
than penetrating cryoprotectants at the same concentration. They reduce the amount of
penetrating cryoprotectnats needed by mimicking outside the cell the cryoprotective
effects of proteins inside the cell. It has also been recently discovered that using non-
penetrative cryoprotectants to increase the tonicity (osmotically active concentration) of
vitrification solutions can prevent a type of injury called chilling injury.

The mode of action of Non-Penetrative Cryoprotectant

Non-penetrative cryoprotectants are generally long chain polymers that are
soluble in water and have large osmotic coefficients (they increase the osmolality far in
excess of their molar concentration). They are not able to move across cell membranes.
The basic feature of non-penetrating cryoprotective agents is the ability of their
hydrophilic groups to bind water, giving rise to a phenomenon often described as “bound
water”. Osmotic dehydration can be obtained through the application of non-penetrating
cryoprotective substances such as sugar, sugar alcohols and high molecular weight
additives such as polyethylene glycol (PEG).
The efficacy of non-penetrating cryoprotective agents lies in their ability to
enable the cell membrane to leak solute reversibly under osmotic stress. The success of
such an approach presumably depends upon the achievement of a freezing rate which is
rapid enough to limit solute leak to low-molecular-weight solutes, prevent loss of the
larger and more essential protoplasmic elements and yet not be so fast as to produce
intracellular freezing. It will also be essential that the cell have an opportunity to heal and
restore its normal solute content. Also the concentration of penetrating cryoprotectant
(hence toxicity) can also be reduced by the use of non-penetrating cryoprotectants such as
large molecular weight polymer (e.g polyvinylpyrolidone or PEG) or sucrose. Non-
penetrating cryoprotectants are too large to diffuse into cells, but they assist with
vitrification of water (and inhibition of devitrification) in the extracellular space.
Non-penetrating cryoprotectives are thought to act by dehydrating the cell before
freezing, thereby reducing the amount of water that the cell needs to lose to remain close
to osmotic equilibrium during freezing. The cytoplasm does not super cool to the same
extent and therefore intracellular ice becomes less likely at a given cooling rate. They
provide little protection from slow cooling injury.
Some common and structural examples of non-penetrating cryoprotectants are:
Hydroxy-ethyl-starch (HES) Polyvinyl Pyrrolidone (PVP)

Polyethylene Oxide (PEO)

From the above compounds or examples, it could be seen that they are generally
polymers that form extensive hydrogen bonds with water, reducing the water activity to a
much greater extent than would be predicted by their molar concentration (they do not
obey Raoult's law). Also since non-penetration cryoprotectants are thought to act by
dehydrating the cell at high sub-freezing temperatures, it allows them to be rapidly cooled
before the solution effects injury of slow cooling can lead to extensive damage.
Below is a graphical representation on the effect of hydroxyl-ethyl starch on the
survival of cells:

FIG. I
FIG. I: Survival of cells in hydroxyethyl starch (HES) cooled at 1°C/min to various
temperatures before rapid warming or rapid cooling to -196°C followed by rapid
warming. (McGann 79)

NOTE:

Hydroxyethyl starch is a polysaccharide that is commonly used as a non-

penetrating cryoprotectant. It is a large molecule that can only be taken up by cells
through endocytosis. The upper curve shows that it has no effect on slow cooling injury,
however, it shows a significant effect on rapid cooling injury. The recovery at -20°C is
almost completely due to slow cooling injury even though the sample was plunged into
liquid nitrogen.
Tabular difference between penetrating and non-penetrating cryoprotectant

Penetrating Non-penetrating
Cryoprotectant Cryoprotectant

Size Small Long chain polymers

Action On the freezing point Before freezing

Solubility High solubility in water at Soluble in water (large

low temperature and low osmotic coefficients,
cellular toxicity (nonionic increase the osmolality far
molecule) in excess of their molar
concentration)

Activity -Lowering concentration of -Dehydrating the cell,

thereby reducing the
salts found in physiological amount of water that the
solutions ( below freezing cell needs to lose to remain
point, ice formation causes close to osmotic equilibrium
concentration of these salts) during freezing.
- They do this by lowering
the amount of ice present
for a given temperature, act
as second solvent for salt.

Results -Reduce magnitude of -During rapid cooling, the

injury and kinetic at which intracellular solute
damage accumulates when concentration remains low
cell exposed to increased because the cell cannot lose
extracellular solute water fast enough to remain
 concentration at lower in equilibrium with the
temperature. extracellular solution. Thus,
delays the effects of having
a highly concentrated
intracellular solution to
lower temperatures (where
it has a less damage effects).

-Greatly mitigate rapid

Ability -Greatly mitigate slow cooling injury.
cooling injury.
-Little protection on slow
-Little protection on rapid cooling injury.
cooling injury.

REFERENCES

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