This document provides an overview of cryopreservation, which involves preserving biological material such as cells, tissues, organs, and embryos at ultra-low temperatures, typically in liquid nitrogen. It discusses the history, principles, mechanisms, and applications of cryopreservation. Key aspects covered include the use of cryoprotectants to prevent freezing damage to cells, various freezing and thawing methods, long-term storage in liquid nitrogen, and viability testing after thawing to regenerate plants or animals from preserved material. Cryopreservation has important applications in biobanking, conservation of endangered species, and preservation of disease-free agricultural crops.

1. CRYOPRESERVATION:
Presented By:
Naveen K L
1st (Sem) M pharma
Dept of Pharmacology
Srinivas college of pharmacy
Mangalore

2. INTRODUCTION:
 Cryo is Greek word. (krayos- frost)
 It literally means preservation in “ frozen state.”
 It’s a process where tissues, organelles, cells, extracellular
matrix, organs or any biological constructs susceptible to
damage caused by unregulated chemical kinetics are
preserved by cooling to very low temp 196˚ using liquid
nitrogen .
 Cryopreservation is long term storage technique with very low
temperature to preserve the structurally intact living cells &
tissues for extended period of time at a relatively low cost.
 The science pertaining this activity known as “Cryobiology”

3. CRYOPRESERVATION:
 Cryopreservation is a non-lethal storage of
biological material at ultra-low temperature. At the
temperature of liquid nitrogen (196˚) almost all
metabolic activities of cells are ceased and the
sample can be preserved in such state for extended
periods.
 however only few biological material can be frozen
without affecting the cell viability.

4. WHY PRESERVATION IS IMPORTANT ?
 Until two decade ago the genetic resource 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 method of storage failed to
prevent losses caused due to various reason .
 A new methodology had to be devised for long
term preservation of material.

5. 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 temperature in 1949. he
accidently discovered the cryoprotective properties of
glycerol on fowl sperm.
 During 1950s James E. Lovelock suggested that
increasing salt concentration in a cell as it dehydrated
due to lose of water results in cell damage and he also
proposed that the mechanism of action of
cryoprotectant.
 In1953, research work of Jerome K. Sherman led him to
successfully freeze and thaw human sperm.
 In1983, Alan Trounson, was credited for Sucessfully
achieving a pregnancy after freezing early human
embryos one to three days after fertilization.

6. STORAGE METHODS:
 Cryopreservation - generally in liquid nitrogen.
 Cold storage – in low and non freezing
temperature
 Low pressure – reducing the atmospheric pressure
partially.
 Low oxygen storage – involve reducing the oxygen
level but maintaining the pressure.

7. PRINCIPLE:
 To bring plant cells or tissues to a zero metabolism
and non - dividing state by reducing the
temperature in the presence of cryoprotectant.
 Done by :
 Over solid CO2 (at 79˚)
 Low temperature deep freezer (at 80˚)
 In vapor phase N2 (AT 150˚)
 In liquid N2 (at 196˚)

8. MECHANISM OF CRYOPRESERVATION:
 Selection of plant material.
 Pregrowth
 Addition of cryoprotectant.
 Freezing.
 Storage in liquid nitrogen.
 Thawing.
 Washing and Reculturing.
 Measurement of viability.
 Regeneration of plant.

10. SELECTION OF PLANT MATERIAL:
 Morphological and Physiological conditions of plant
material influences the ability of explant to survive
during cryopreservation.
 Cells or tissues from the late lag phase or log
phase.
 Different types of tissues can be used as follows:
 Ovules
 Anther or pollen
 Embryos
 Endosperm
 Protoplast

11. FACTORS TO BE CONSIDER IN SELECTION OF
PLANT MATERIAL…….
 Tissue must be selected from healthy plants.
 Small.
 Young.
 Rich in cytoplasm.
 Meristic cells can survive better than the larger.
 Highly vacuolated cells.
 Water content of cell or tissue used for
cryopreservation should be low freezable water,
tissue can withstand extremely low temperature.

12. PREGROWTH:
 Pregrowth treatment protect the plant tissue against
exposure to liquid nitrogen.
 Pregrowth involves the application of additives
known to enhance plant stress tolerance.
E.g.
Abscisic acid
Praline
Trehalose

13. ADDITION OF CRYOPROTECTANT:
 A cryoprotectant is a substance that is used to protect
biological tissues from freezing damage (due to ice
formation) or prevent cryodestruction.
 they acts like antifreezing.
 They lower freezing temperature.
 Increase viscosity and prevents damage to the cells.
E.g.
Sucrose, Alcohols, Glycols, Amino acid (Proline),
DMSO (dimethyl sulfoxide)
* Generally two cryoprotectant should be used together
instead of single.

14. FREEZING:
 Freezing is a phase transition where a liquid
turns into a solid when its temperature is
lowered below its freezing point.
 The sensitivity of cells to low temperature
depends on the plant species.
Methods of freezing:
Slow freezing
Rapid freezing
Step down freezing

15.  SLOW FREEZING METHOD:
* The tissue is slowly frozen with decrease in temp of -0.5 ˚C to
-5˚C /min and transfer into liquid nitrogen .
* Slow cooling permits flow of water from the cells to the outside ,
thereby extracellular ice formation instead of lethal intracellular
freezing.
* employed for cryopreservation of meristems of peas , potato ,
cassava.
o RAPID FREEZING METHOD:
* The material is plugged into liquid nitrogen with decrease in
temp from -300 ˚C to -1000 ˚C /min or more
* This method is technically simple and easy to handle.
* employed for cryopreservation of shots tips of potato ,
strawberry , brassica species .
o STEPWISE FREEZING METHOD:
* in this method freezing is down to -20 ˚C to -400 ˚C , a stop for a
period of approximately 30min and then additional rapid
freezing to -196 ˚C is done by plugging in liquid nitrogen.

16. STORAGE:
 Storage of frozen material at correct temp is as
important as freezing.
 The frozen cells / tissues are kept for storage at temp
ranging from -70 to -196 ˚C.
 Temperature should be sufficiently low for long term
storage of cells to stop all the metabolic activities are
ceased and prevent the biochemical injury.
 Long term storage done at -196 ˚C…… & constant
supply of liquid nitrogen essential.
 Temp above -130 ˚C ice crystal grow may occur inside
the cells , which reduces the viability of cells , storage is
ideally done at liquid nitrogen refrigerator at -
150 ˚C In vapour phase or -196 ˚C in liquid phase.

17. WHY LIQUID NITROGEN IDEAL FOR
CRYOPRESERVATION ?
 Also called as cryogenic fluid.
 Chemically inert.
 Relatively low cost.
 Non toxic.
 Non inflammable.
 Readily available.

18. THAWING:
 Process in which its carried out by plugging the
vials containing sample into warm water bath (35 to
40 ˚C) with vigorous swirling (15 sec).
 After thawing process vials are transfer into a
another bath at 0 ˚C.
 Its imp for the survival of tissue that the tubes
should not be left in the warm water bath after ice
melts .

19. WASHING AND RECULTURING:
 The preserved material is washed few minutes to
remove the cryoprotectant.
 The material is then recultured in a fresh medium.
 When low toxic or non-toxic cryoprotectants are
used , then the culture should not be washed but
simply recultured.

20. MEASUREMENT OF VIABILITY:
 Regrowth of the plants from stored tissues or cells
is the only test of survival of plant materials.
 Various viability tests include Fluorescien diacetate
staining (FDA), Growth measurement by cell
number, dry and fresh weight.
 Imp staining methods are …..
* Triphenyl Tetrazolium chloride.
* Evan’s blue staining.

21. TRIPHENYL TETRAZOLIUM CHLORIDE ASSAY :
 Cell survival is measured by amount of red
formazan product formed due to reduction of TTC
assay which is measured by spectrometrically.
 Only the viable cells which contain the enzyme
mitochondrial dehydrogenase which reduces TTC
to red formazan will be stained and dead cells will
not take up the dye.

22. 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 = Num of fluorescent cells ×100
total Num of cells (viable + non viable )
o Individual cell viability assayed with Evan’s blue dye and
fluorescein diacetate

23. PLANT REGENERATION
 The ultimate purpose of cryopreservation of
germplasm is to regenerate the desired plant. For
appropriate plant growth and regeneration, the
cryopreserved cells/tissues have to be carefully
nursed, and grown. Addition of certain growth
promoting substances, besides maintenance of
appropriate environmental conditions is often
necessary for successful plant regeneration.

25. APPLICATION:
 Cryopreservation of Embryo, Ovarian tissue,
Oocytes, Semen, Testicular tissue.
 Ideal method for long term conservation of material.
 Disease free plant can be conserved and
propagated.
 Recalcitrant seeds can be maintained for long time.
 Endangered species can be maintained.
 Pollens can be maintained to increase longitivity.

26. REFERENCE:
 Culture of animal cells, a manual of basic
technique by R. Ian Freshney, 5th edition
Pg no 321-329.
 www.biologydisscussion.com germiplasm
conservation and cryopreservation article by
Nandkishore Jha