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

Cryoprotectant

Non-penetrating

Cryoprotectant

Size Small Long chain polymers

Action On the freezing point Before freezing

Solubility High solubility in water at

low temperature and low

cellular toxicity (nonionic

molecule)

Soluble in water (large

osmotic coefficients,

increase the osmolality far

in excess of their molar

concentration)

Activity -Lowering concentration of

salts found in physiological

solutions ( below freezing

point, ice formation causes

concentration of these salts)

- They do this by lowering

the amount of ice present

for a given temperature, act

as second solvent for salt.

-Dehydrating the cell,

thereby reducing the

amount of water that the

cell needs to lose to remain

close to osmotic

equilibrium during freezing.

Results -Reduce magnitude of

injury and kinetic at which

damage accumulates when

-During rapid cooling, the

intracellular solute

concentration remains low

Ability

cell exposed to increased

extracellular solute

concentration at lower

temperature.

-Greatly mitigate slow

cooling injury.

-Little protection on rapid

cooling injury.

because the cell cannot lose

water fast enough to remain

in equilibrium with the

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

cooling injury.

-Little protection on slow

cooling injury.

REFERENCES

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