Cryopreservation is the use of very low temperatures to preserve living cells and tissue. Cryoprotectants like glycerol and DMSO are used to reduce ice formation and allow cells to withstand freezing. They work by penetrating cells, lowering the freezing point, and minimizing solution effects. Glycerol is often used for most cell types due to lower toxicity, while DMSO is preferred for larger cells. Cryoprotectants must be soluble, able to penetrate cells, and minimally toxic at high concentrations required for effective cryopreservation.

1. CRYOPRESERVATION
CRYOPRESERVATIVES

2. CRYOPRESERVATION
• Cryopreservation is the use of very low
temperatures to preserve structurally intact
living cells and tissue
• Cryoprotectants, simply by increasing the
total concentration of all solutes in the
system, reduce the amount of ice formed at
any given temperature; but to be biologically
acceptable they must be able to penetrate
into the cells and have low toxicity.

3. • Ice can be avoided by vitrification ,
the production of a glassy state that
is defined by the viscosity reaching a
sufficiently high value to behave like
a solid, but without any
crystallization.
• Toxicity is the major problem in the
use of vitrification methods.

4. CRYOPROTECTANTS
cryoprotectants are the agents
used in cryopreservation in order
to protect the living entities from
cryoinjuries.

5. • glycerol,
• dimethyl sulfoxide
• ethanediol
• Propanediol
• polyvinylpyrolidone
• sorbitol,
• dextran and
• trehalose.
DIFFERENT CRYOPROTECTANTS

6. ACTION OF CRYOPRESERVENTS
• Cryoprotective agents serve several functions during the
freezing process.
• Freezing point depression is observed when DMSO is
used which serves to encourage greater dehydration of
the cells prior to intracellular freezing.
• Cryoprotective agents also seem to be most effective
when they can penetrate the cell, delay intracellular
freezing and minimize the solution effects.1 The choice of
a cryoprotective agent is dependent upon the type of cell
to be preserved.

7. glycerol DMSO are agent of choice for cryopreservation
• most cells, glycerol is the agent of choice
because it is usually less toxic than DMSO
• DMSO is more penetrating and is usually the
agent of choice for larger, more complex cells
such as protists.
• DMSO and glycerol are generally used in
concentrations ranging from 5-10% (v/v), and
are not usually used together in the same
suspension with the exception of plant cells.

8. • examine the sensitivity of the cells to increasing
concentrations of the cryoprotective agent to determine
the optimum concentration.
• Glycerol and DMSO should be sterilized prior to use,
Glycerol may be sterilized by autoclaving for 15 minutes
at 121°C and 15 psig.
• Glycerol should be protected from light during storage.
• DMSO must be sterilized by filtration using a 0.2 micron
nylon syringe filter or a Teflon® PTFE syringe filter .
• Cryoprotective agents should be prepared in single-use
volumes to minimize the risk of contamination and
moisture introduction with repeated use from one
container.

9. (1).the compound must be highly soluble in
water and remain so at low temperatures in
order to produce a profound depression of
the freezing temperature.
(2).it must be able to penetrate into the cells.
(3).it must have a low toxicity so that it can be
used in the high concentrations that are
required to produce these effects. Many
compounds have these properties
EFFECTIVENESS OF DIFFERENT CRYOPROTECTANTS

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