2. Introduction
Cryopreservation is the technique of freezing cells and tissues at
very low temperatures (sub-zero temperatures, typically -196oC)
at which the biological material remains genetically stable and
metabolically inert, while minimizing ice crystal formation.
Any biological activity including the biochemical reactions that
would cause cell death are effectively stopped.
This technique is used to freeze vital and useful tissues like blood,
stem cells, sample of tumors and histological cross sections,
oocytes, sperm, embryos, ovarian tissue and plant parts for study.
Traditional cryopreservation has relied on coating the material to
be frozen with a class of molecules termed cryoprotectants.
3. History
Ernest John Christopher Polge, an English biologist, was the first person to solve the mystery of
how to preserve living cells at very low temperatures in 1949. He accidently discovered the
cryoprotective properties of glycerol on fowl sperm.
During early 1950s, James E. Lovelock suggested that increasing salt concentrations in a cell as
it dehydrates to lose water to the external ice might cause damage to the cell. He also proposed
that the mechanism of action of cryoprotectants.
In 1953, the research work of Jerome K. Sherman led him to successfully freeze and thaw human
sperm.
In 1983, Alan Trounson, was credited for successfully achieving a pregnancy after freezing early
human embryos one to three days after fertilization.
4. Cryopreservation procedures
There are two main types of cryopreservation procedures: equilibrium (slow programmable) freezing
and non-equilibrium or ultra-rapid freezing (vitrification).
1. Equilibrium (Slow programmable) freezing:
The first step of the slow-freeze procedure is to expose the cell to cryoprotectant in a gradual step-wise fashion to
slowly allow equilibrium of the cell with the cryoprotectant while releasing water. Once the cells have been cleared
of the majority of cellular water, they are placed in a container of some kind, such as plastic or straw, a glass
ampoule or a plastic vial.
The volume of the liquid surrounding the cells for slow freezing is typically less then a teaspoon and may only be a
few drops. The pre-labeled container is filled, sealed and put away in a programmable freezer, which slowly
decreases the temperature of the container over a period of minutes or hours to very low temperatures.
When the container reaches temperatures between -30oC and -85oC, the container holding the cells can be directly
plunged into liquid nitrogen to complete the cooling to -196oC.
5. 2. Non-equilibrium or ultra freezing (Vitrification):
This procedure uses higher concentrations of cryoprotectants coupled with an almost instantaneous
freezing rate achieved by plunging cells directly into liquid nitrogen.
Vitrification bypasses the ice-crystal formation phase and moves the water directly into a glass-like
phase.
For vitrification, the cells are usually placed on the tip of a straw and excess cryoprotectant is
removed, leaving just enough so that the cell clings to the container by surface tension, prior to
plunging in liquid nitrogen.
Because of the rapid freezing, the duration of exposure to cryoprotectants is much less.
Warming of the cell to return it to normal metabolic functioning must also be incredibly rapid.
6.
7. Cryoprotectants
Cryoprotectants are substance that is used to protect biological tissue from freezing
damage.
Cryoprotectants operate simply by increasing the solute concentration in cells.
However to be biologically viable they must
1. Easily penetrate cells
2. Not be toxic to cells
8. Types of cryoprotectants
Based on their ability to diffuse across cell membranes, two types of
cryoprotectants are there :
Penetrating cryoprotectants
Non-penetrating cryoprotectants
9. Penetrating cryoprotectants
Penetrating cryoprotectants are so called because they penetrate the cell membrane and
enter the cytosol.
They are exclusively small molecules.
They form hydrogen bonds with water to prevent ice crystallisation.
They act by replacing water and therefore controlling cell size changes as well as
preventing intracellular ice formation and prevent excessive dehydration during cell
cryopreservation.
Common penetrating cryoprotectants are DMSO (Dimethyl sulfoxide), glycerol, ethylene
glycol.
10. Non-penetrating cryoprotectants
This type of cryoprotectants do not penetrate the cell membrane.
They are larger molecules, usually polymers such as polyethylene glycol or saccharides such as sucrose.
Non-penetrating cryoprotectants 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.
They inhibit ice growth by the same mechanism as penetrating cryoprotectants, but they do not enter
cells.
They help to dehydrate the cell prior to cryopreservation by altering the osmotic balance and also help to
prevent damage to cells during recovery from cryopreservation by preventing solutes, particularly larger
protoplasmic elements, from escaping the cell too rapidly.
11. Risks involved in cryopreservation techniques
Phenomena which can cause damage to cells during cryopreservation mainly occur during
the freezing stage, and include:
1. Solution effects: As ice crystals grow in freezing water, solutes are excluded, causing them to become
concentrated in the remaining liquid water. High concentrations of some solutes can be very damaging.
2. Extracellular ice formation: When tissues are cooled slowly, water migrates out of cells and ice forms in the
extracellular space. Too much extracellular ice can cause mechanical damage to the cell membrane due to
crushing.
3. Dehydration: Migration of water, causing extracellular ice formation, can also cause cellular dehydration. The
associated stresses on the cell can cause damage directly.
4. Intracellular ice formation: While some organisms and tissues can tolerate some extracellular ice, any
appreciable intracellular ice is almost always fatal to cells.
12. Applications of cryopreservation
1. Embryo storage for research
2. Fertility preservation
3. Efficiency of assisted reproduction
4. Reduce the implantation of multiple
embryos
5. Biodiversity conservation
6. Conservation of plant germplasm
