Cryopreservation

Cryopreservation
Cryopreservation is a non-lethal storage of

biological material at ultra-low temperature.
At the temperature of liquid nitrogen (-196
°C) almost all metabolic activities of cells are
ceased, and the sample can then be
preserved in such state for extended periods.
However, only few biological materials can be

frozen to (-196 °C) without affecting the cell
viability.
1
INTRODUCTION
In recent years with tremendous increase in population:
Forest
Land resources
Population of medicinal plants
Aromatic plants species
2
• Liquid nitrogen is most widely
used material for
cryopreservation.
• Dry ice can also be used.
• Why Liquid nitrogen ?

• Chemically inert
• Relatively low cost
• Non toxic
• Non flammable
• Readily available
3
STORAGE
THAWING
DETERMINATION OF
SURVIVAL OR VIABILITY
4
• The frozen cells/tissues are
kept for storage at
temperature ranging from
-70 to -196°C.
STORAG • Temperature should be
sufficiently low for long term
E storage of cells to stop all the
metabolic activities and prevent
biochemical injury.
• Long term storage is best done at
-196°C.
5
• It is done by putting ampoule
containing the sample in a warm
water bath (35 to 40°c).
• Frozen tips of the sample in
tubes or ampoules are plunged
THAWING into the warm water with a

vigorous swirling action just to
the point of ice disappearance.
• It is important for the survival of
the tissue that the tubes should
not be left in the warm water
bath after ice melts .
6
• Regrowth of the plants from stored
tissues or cells is the only test of
survival of plant materials.
• Various viability tests include
DETERMINATION Fluorescien diacetate (FDA)
OF SURVIVAL: staining , growth measurement by
cell number , dry and fresh weight.
• Important staining methods are:
• Evan’s blue staining.
7
EVAN’S BLUE
STAINING
• One drop of 0.1% solution of Evan’s

blue is added to cell suspension on
a microscope slide and observed
under light microscope.
• Only non-viable cells (dead cells)
stain with Evan’s blue.
• % of viable cells = Number of
fluorescent cells × 1oo total no
of cells(viable + non-viable).
8
APPLICATIONS OF
CRYOPRESERVATION:
Maintenance
Freezing of
of disease- Seed Bank
cell cultures.
free stock
Storage of
Gene Bank rare
germplasm.
9
• Collection and preservation of
cells & tissues
• Addition of cryoprotectants
• Vitrification.
• Cryoprotective dehydration.
Techniques: • Freezing
• Slow freezing method.
• Rapid freezing method.
• Step wise freezing method.
• Storage
• Thawing
Collection Of Cells:
What can cryopreserved?
• In general, cryopreservation is easier for thin samples and small
clumps of individual cells, because these can be cooled more
quickly and so require lower doses of toxic cryoprotectants.
• Therefore, the goal of cryopreserving human livers and hearts
for storage and transplant is still some distance away.
• Nevertheless, suitable combinations of cryoprotectants and
regimes of cooling and rinsing during warming often allow the
successful cryopreservation of biological materials, particularly
cell suspensions or thin tissue samples like.
1. Semen . 6. Embryo (2,4 or 8 cell).

2. Blood . 7. Plant (Seed & Shoot).
3. Stem cell.
4. Umbilical cord blood.
5. Egg (Oocytes).
Addition of There are two potential
Cryoprotectant: sources of cell damage during
cryopreservation.
1. Formation of large ice

crystals inside the cell.
2. Intracellular concentration of
solutes increase to toxic levels
before or during freezing as a
result of dehydration.
• Cryoprotectants acts like

antifreeze, they lower freezing
• temperature and increase

viscosity.
Benefits Of Vitrification:-
Vitrification: It is the process in which ice formation can’t take place

•
because the aqueous solution is too concentrated to permit
ice crystals nucleation, instead, water solidified into an
amorphous ‘Glassy’ state.
• Rapid cooling also promotes vitrification.
Cryoprotective Dehydration:-
• If cells are sufficiently dehydrated, they may be able to

withstand immersion in liquid nitrogen. Dehydration can be
achieved by growing in presence of high concentration of
osmotically active compounds like sugars, polyols and / or
air desiccation in a sterile flow cabinet or over silica gel.
• Dehydration reduces the ice formation, increases the
osmotic pressure of the intracellular solution (the
cytoplasm) which depresses its freezing temperature.
• Various cryoprotectants used are glycerol,
dimethylsulphoxide, mannitol, propylene,
choline etc.
A. Slow Freezing Method(SFM):-
• The tissues is slowly frozen with
decrease in temperature
• of -0.5°C to -5°C/ min from 0°c-
100°c, and then transfer to
liquid nitrogen.
B. Rapid Freezing Method(RFM):-

• The material is plunged into liquid
Freezing:
nitrogen decreases in temperature
from -300°C to -1000°C/min or more,
the quicker the freezing is done
the smaller the ice is crystal.
C. Stepwise-Freezing Method:-
• In this method slow freezing down
to -20°C to -400°C ,a stop for a
period of approximately 30 min
and then additional rapid freezing
to -196°C is done by plunging in
liquid nitrogen.
• Storage of frozen material at the correct
temperature is as important as freezing.
• In general the frozen cells/tissues are kept
for storage at a
• ranging from -70°C to -196°C.
• However, with temperature above -130°C ice
crystal grow may occur, inside the cells which
reduces viability of cell. Storage is ideally done
in liquid nitrogen refrigerator at -150°C in vapor
Storage:
phase or at -196°C in the liquid phase.
• The ultimate objective of storage is to stop all
the
• cellular metabolic activities and maintain their
viability for long term.
• For long term storage temperature at -196°C in
liquid
• nitrogen is ideal.
• A regular & constant supply of
liquid nitrogen to refrigerator
is essential.
It is done by putting the ampoule containing the sample in a
warm water (35°to 45°C )bath.
By this approach, rapid thawing [at the rate of (500

to&750°C/min)
occurs, and the protects and this protects the cell from
damaging effect of ice crystal formation.
Thawing: As the thawing occurs ( ice completely melts the ampoule are
quickly transferred to water bath at temperature 20 to 25°C .
This transfer is necessary since the cells get damaged if left for
long in warm (37°C - 45°C) water.
For cryopreserved material(cells/tissues) where the water

content has been reduced to an optimal level before freezing,
the process of thawing become less critical.
Benefits and Disadvantages:
Benefits:
 It is useful in the breeding of dairy cattle, pigs & dogs.

 The cryopreserved blood can stored for many
years & transfuse to required person.
 The cancer & tumor cells can be preserved for
further research.
 Stem cells, Umbilical cord blood , skin cells are
preserved for further use.
 It is helpful for endangered animals & plants
(medicine & fragrant).
 It is quite safe for “IVF” because the donor is retested
for HIV .
Disadvantages:
A cell bank is a facility that
stores cells of specific
genome for different
purposes.
Cell
Banking The first person accredited
with making a cell bank for
widespread use was Kral, a
Czechoslovakian scientist
who created his cell bank
collection in the late 1890s.

Cryopreservation

Uploaded by

Copyright:

Available Formats

Cryopreservation

Uploaded by

Document Information

Original Description:

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Cryopreservation

Uploaded by

Copyright:

Available Formats

Cryopreservation

Cryopreservation is a non-lethal storage of

However, only few biological materials can be

Population of medicinal plants

Aromatic plants species

• Why Liquid nitrogen ?

THAWING into the warm water with a

• One drop of 0.1% solution of Evan’s

1. Semen . 6. Embryo (2,4 or 8 cell).

1. Formation of large ice

• Cryoprotectants acts like

• temperature and increase

Vitrification: It is the process in which ice formation can’t take place

• If cells are sufficiently dehydrated, they may be able to

B. Rapid Freezing Method(RFM):-

By this approach, rapid thawing [at the rate of (500

For cryopreserved material(cells/tissues) where the water

 It is useful in the breeding of dairy cattle, pigs & dogs.

You might also like