CRYOPRESERVATION

MARIEL SUAREZ BERMUDEZ

MAT-SCIENCE
HISTORY
Theoreticians of cryopreservation was James Lovelock (born
1919).
Osmotic stress
Salt concentration
Christopher Polge, carried out cryopreservation of first fowl
sperm.
Later, in 1950s they tried it on humans where pregnancy was
obtained after insemination of frozen sperm.
However, the rapid immersion of the samples in liquid nitrogen
did not produce the necessary viability to make them usable after
thawing.
Importance of controlled or slow cooling to obtain maximum
survival on thawing of the living cells.
Cryoprotectants were introduced.
CRYOPRESERVATION
Cryo is Greek word. (krayos – frost)
It literally means preservation in “frozen
state.”
The principle - to bring plant cells or
tissue to a zero metabolism and non
dividing state by reducing the temperature
in the presence of cryoprotectant.
It can be done :
Over solid carbon dioxide (at -79 degree)
Low temperature deep freezer (at -80
degree )
In vapor phase nitrogen (at -150 degree)
In liquid nitrogen (at -196 degree)
CRYOPRESERVATION
Cryopreservation is a non-lethal storage of
biological material at ultra-low temperature. At the
temperature of liquid nitrogen (-196 degree)
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 degree) without affecting the cell
viability.
Liquid nitrogen- is most widely used material for
cryopreservation.

Why Liquid nitrogen ?

Chemically inert
Relatively low cost
Non toxic
Non flammable
Readily available
CRYOPRESERVATION

• The use of very low

temperatures to structurally
preserve intact living cells
and tissue

• Unprotected freezing is
normally lethal to cells
while controlled cooling can
be used to produce stable
conditions that preserve life
BENEFITS OF
CRYOPRESERVATION
• Generation of safety
stocks
• Saves time and money
• Preservation of cells
• Insurance against
phenotypic drift
• Standard for
experiments
WHY PRESERVATION
IS IMPORTANT ?
Until two decades ago the genetic resources were getting
depleted owing to the continuous depredation by man.
It was imperative therefore that many of the elite,
economically important and endangered species are
preserved to make them available when needed.
The conventional methods of storage failed to prevent
losses caused due to various reasons.
A new methodology had to be devised for long term
preservation of material.
MAJOR ADVANTAGES
ARE :
1. Once the material is sucessfully
conserved to particular temperature it can
be preserved indefinately.
2. Once in storage no chance of new
contamination of fungus or bacteria.
3. Minimal space required.
4. Minimal labour required
MECHANISM OF
CRYOPRESERVATION
The cryopreservation technique followed by the regeneration of
plants involves following steps :
1. Selection of material.
2. Addition of cryoprotectant.
3. Freezing.
4. Storage in liquid nitrogen.
5. Thawing.
6. Washing and reculturing.
7. Measurement of viability.
8. Regeneration of plants
1.SELECTION OF
PLANT MATERIAL :
Selection of proper plant material is important.
Two important factors depend on it such as (a)
nature and (b)density.
Any tissue can be selected for this purpose.
e.g. meristem, embryo, ovules, seeds etc..
The density should be high.
2. ADDITION OF
CRYOPROTECTANT
They are chemical which prevent cryo
destruction.
These are sucrose, alcohols, glycols, some
amino acid (proline), DMSO (dimethyl
sulfoxide).
Generally two cryoprotectant should be used
together instead of single one as they are more
effective.
CRYOPROTECTANTS
• Dimethyl sulfoxide
(DMSO) and glycerol
are the two most
widely used
cryoprotectants

• Aid in preserving cells

• Encourage
dehydration
• Minimize solution
effects
3. FREEZING
The sensitivity of cells to low temperature depends on the
plant species. There are four different types of methods :
Slow freezing method - In this method the rate of freezing is
slow (0.1-10°C / min). It is commonly used for animal
germplasm. • In this due to cooling below freezing point, extra
cellular crystals are formed not intracellular.
Rapid freezing method - This method is simple & easy. •
Freezing is done quickly so that there should be least change
or development of intracellular crystals.
Stepwise freezing method- In this temperature gets lowered
by -20°C to -40°C & allows protective freezing of the cells. •
Freezing stopped for 30 mins & then rapidly freezed in liq.N ;
this results in decline in temperature & good results are
obtained.
4. STORAGE
The maintenance of the frozen cells or
material at specific temperature is very
important.
In general the temperature is kept -70 to -
196 degree.
Prolong storage is done at temperature of -
196 degree in liquid nitrogen.
To prevent damage, continuous supply of
nitrogen is done.
5. THAWING
Usually carried out by plunging
the vials into warm water bath
with vigorous swirling.
As thawing occurs the vials are
transferred to another bath at 0
degree
6. WASHING AND
RECULTURING
The preserved material is washed
few times to remove the
cryoprotectant.
This material is then recultured in
a fresh medium.
7. MEASUREMENT OF
VIABILITY
There is possibility of death of cells
due to storage stress.
Thus viability can be found at any
stage.
It is calculated by formula : No of cells
growing / no of cells thawed * 100
8. PLANT
REGENERATION
The viable seeds are cultured on non
specific growth medium.

Suitable environmental conditions are

maintained.
APPLICATIONS OF
CRYOPRESERVATION
EMBRYO CRYOPRESERVATION
• It is used for embryo storage when in-vitro
fertilization has resulted in more embryo than is
currently needed.
• Pregnancies have been reported from
embryos stored for 16 yrs.
• The result has been uniformly positive with no
increase of birth defects or developmental
abnormalities.
OOCYTES CRYOPRESEVATION

• Human oocytes cryopreservation is a new

technology in which a woman’s eggs(oocytes)
are extracted, frozen & stored.
• Later, when a woman is ready to become
pregnant, the eggs can be thawed, fertilized &
transferred to the uterus as embryos.
SEMEN CRYOPRESERVATION

• Semen can be used successfully & indefinitely

after cryopreservation.
• The longest reported successful storage is 22
yrs.
• It can be used for sperm donation where
recipient wants the treatment in a different time
or place or as a means of preserving fertility.
TESTICULAR CRYOPRESERAVTION

• Cryopreservation of immature testicular

tissue is a developing method to avail
reproduction to young male who need to have
gonadotoxic therapy.
• Health offsprings have been obtained after
transplantation of frozen testicular cell
suspension or tissue pieces.
LABORATORY FREEZER
-ROBUST CELLS
LN2 CONTROLLED RATE FREEZE
-STEM CELLS, IVF, DNA,HUMAN
EMBRYO
GLACIER G50-
MAMALIAN CELLS
BLAST FREEZER
REFERENCES
https://www.kean.edu/~evassili/images/Cryopreservation%20
Chpt.%2020.ppt

https://www.atcc.org/~/media/PDFs/webinars/Presentations/2
016/ATCC%20Webinar%20Cryopreservation.ashx

https://s3.amazonaws.com/ppt-download/cryopreservation-
151023075253-lva1-app6891.pdf?response-content-
disposition
END..
Thank you for listening…