cryopreservation of oocytes and gametes

1. Submitted by:
Biplab Roy Karmakar
M.Sc. 3rd Semester
Dept. of Life Science & Bioinformatics
Paper- 301 (Z)

2. What is Cryopreservation?
Cryo (Greek) = krayos. Meaning- frost.
Literally cryopreservation is preservation in “frozen state”.
Cryopreservation is the long-term preservation of the
biological materials at sub-zero temperature where all the
metabolic activities are arrested keeping its viability
intact, to be used in future.

3. In practice, the frozen cells are stored at a temperature of -196°C in
liquid nitrogen (N2).
At low temperatures, any enzymatic or chemical activities which
might cause damage to the biological material in question is effectively
stopped.
Cryopreservation methods seek to reach low temperatures without
causing additional damage caused by the formation of ice during
freezing.
Traditional cryopreservation has relied on coating the material to be
frozen with a class of molecules termed as Cryoprotectants, which
protects the biological materials from freezing damage.
Today, the most common procedures are cryopreservation of
embryos, spermatozoa and some somatic cells and tissue.

4. TYPES OF CRYOPRESERVATION:
Cryopreservation at -196˚C.
Cryopreservation at above -196˚C.
Freeze Drying.
Vitrification.

5. Cryopreservation at -196˚C
 The biological materials are stored at -196˚C (liquid
nitrogen temperature).
The viability of storage by this process is independent of the
period of storage.
The stresses associated with this cryopreservation are not
mutagenic.

6. Cryopreservation at above -196˚C:
 Storing biological materials in a conventional freezer is more
convenient than liquid nitrogen.
 Freezers are available which maintain temperatures down to
-130˚C.
However, unless biological material is stored at temperatures
below -135°C the viability decreases during long-term storage.
 This may be appropriate with microbial suspensions
or with mammalian tissue cultures where large numbers of
cells are frozen and some loss of viability may not cause
practical problems, storage at these temperatures would not
be acceptable with embryos, oocytes etc.

7. Freeze Drying:
 Freeze Drying is the process of preserving the biological
materials by rapidly freezing it and then subjecting it to high
vacuum which removes ice by sublimation.
 It is convenient as the cost and maintenance associated
with frozen storage can be avoided.
 However, evidently, the freeze drying is highly mutagenic
and that viability of the cells would be expected to decrease
with storage time.
 These two factors precludes the use of freeze dried
spermatozoa in IVF.

8. Vitrification:
 Vitrifications is the process which, by combining the use of
concentrated solutions with rapid cooling, avoids the formation of
ice.
 Samples reach low temperatures in a glassy state, which has the
molecular structure of a viscous liquid and is not crystalline.
This method has the potential advantages of being rapid to carry
out and does not require controlled rate cooling
apparatus.
 Although good survival has been demonstrated, however is still
experimental, as the additives used are potentially cytotoxic and
tendency of vitrified solutions to devitrify during storage and
thawing leads to lose the viability.

9. EFFECT OF FREEZING ON CELLS:
 During freezing, it is essential for cells remove a particular
amount of water from cells osmotically to avoid intracellular
freezing.
 At slow rates of cooling, cells try remain in equilibrium with
external solution because of the protective effect of cell membrane,
thus exosmosis takes place and outside of the cell becomes
hypertonic, resulting in equilibrium disturbance.
 As cooling rate is increased, water gets less time to move out of
the cell, becomes increasingly supercooled, and intracellular ice
formation occurs that is lethal to the cells.
 So optimum rate of cooling, results from the balance of these two
phenomena.

10. CRYOPROTECTANTS:
 A cryoprotectant or cryoprotective additives is a substance used to
protect biological materials (cells, tissues) from freezing damage.
 Properties: Low Molercular weight, Non toxic, and can permeate
cells.
 Cryoprotectants achieve their protective effects by increasing the
unfrozen fraction at a given temperature & by reducing ionic
concentration.
 Commonly used Cryoprotectants: Glycerol, Dimethyl sulfoxide
(DMSO), propanediol, etc.
 Protective efficiency of these compounds vary from cell to cell types.
Eg.: Human embryo best frozen by propanediol while human
blastocysts by glycerol.

11. THAWING & POST THAW HANDLING:
 Thawing is the change from a frozen solid state to liquid by gradual
warming.
 Cryoprotective additives gets diluted and intracellular ice are melted.
 Thawing is done by:
a. Materials cryopreserved are hold in air for 40 secs, during which
temperature rises to approx. -50˚C.
b. Then they are transferred to water bath held at 30˚C for 1 minute.
 Cells after freezing and thawing, will contain cryoprotective
additives and upon exposure to normal growth medium tends to
expand.
 To prevent swelling, shrinkage is induced by using wash out
solutions containing hypertonic sucrose (0.2 M).

12. APPLICATIONS & TREATMENT IN INFERTILITY
With relation to assisted human reproduction, cryopreservation refers to
the process of freezing human gametes, embryos, ovarian and testicular
tissues, for use in Assisted Reproduction Treatment (ART).
 It enables the reproductive cells collected or fertilized in one treatment
cycle, to be used for fertilization and/or implanted into female’s uterus in a
future treatment cycle.
 Couples undergoing ART cycles typically cryopreserve gametes collected
or embryos produced in one treatment cycle.
 Individuals whose fertility is threatened, such as they will undergo
chemotherapy or radiotherapy, may preserve their gametes or gonadal
tissues through cryopreservation.
 Frozen gametes, embryos and tissues can be donated for research
purpose, if a couple has successfully conceived and consents to this use of
their embryos.

14. REFERENCE
1. Ozkavuksu. S. & Erdemli. E., (2002) “Cryopreservation: Basic Knowledge
And Biophysical Effects”, Journal of Ankara Medical School, Vol.24, No.4, pp.
187-196.
2. “Basic Principles of Cryopreservation”, Cryoconservation of animal genetic
research, pp. 83-94.
3. Wolf. D. P., (2008), “Gamete and Embryo Cryopreservation”, GLOWM.
4. Web URL: http://www.myvmc.com/treatments/cryopreservation/#c1